(Human Health Handbooks) Caroline Hollins-Martin, Olga Van Den Akker, Colin Martin, Victor R. Preedy-Handbook of Diet and Nutrition in The Menstrual Cycle, Periconception and Fertility-Wageningen Pers

of
diet and
nutrition in the
Handbook
menstrual cycle,
cycle, periconception and fertility

d iet and nutrition in the menstrual
periconception
and fertility
ISBN 978-90-8686-212-2 edited by:

Human Health Handbooks no. 7
ISSN 2212-375X
Caroline J. Hollins-Martin
Olga B.A. van den Akker
WageningenAcademic
Colin R. Martin
P u b l i s h e r s Victor R. Preedy
WageningenAcademic
P u b l i s h e r s
Handbook of diet and nutrition in the
menstrual cycle, periconception and
fertility
Handbook of
diet and nutrition in
the menstrual cycle,
periconception and fertility
Edited by:
Caroline Hollins-Martin
Olga van den Akker
Colin Martin
Victor R Preedy
Human Health Handbooks no. 7

ISSN 2212-375X
WageningenAcademic
P u b l i s h e r s
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Table of contents
Preface 13
Setting the scene

1. D
iet and nutrition in fertility: an overview including special requirements with ageing 17
J.H. Ford
2. Fertility in women 33
H. Daabis and T.A. Gelbaya
3. P sychophysiological changes in the menstrual cycle phases 47

M.K.B. Lustyk, E.A. Shilling, D.L. Reaves and C.M. Hokama
Puberty, menarche and the menstrual cycle

4. A
ge at menarche: international perspectives 67
M. Celik
5. Menarcheal onset and body composition 79

S. Kirchengast
6. Calcium intake and premenstrual syndrome 95

B.B. Takashima-Uebelhoer and E.R. Bertone-Johnson
7. D
iet and eating changes in premenstrual syndrome 109
N. Erbil
8. V
itamin D and other nutrients in the treatment of premenstrual syndrome 121
F. Cioni and F. Ferraroni
9. Perimenstrual chocolate craving: from pharmacology and physiology to cognition and culture 137
J.M. Hormes
10. The influence of body mass index and socioeconomic status on pubertal development 155
A. Bereket and Z. Atay
Handbook of diet and nutrition in the menstrual cycle, periconception and fertility7
Table of contents
11. Neuroimaging menstrual cycle associated changes in appetite 169

D.A. Van Vugt and R.L. Reid
12. Fluctuations of appetite and food intake during the menstrual cycle 189
S.A. Tucci
13. Dietary strategies contributing to low energy availability in exercising women: underlying
physiology and impact on the menstrual cycle 207
B.R. Hill and N.I. Williams
14. Iodine deficiency: female menstrual cycle to conception 223

F. Vermiglio, M. Moleti and F. Trimarchi
15. The menstrual cycle and lipid levels 239

S.L. Mumford, K.C. Schliep, A. Prasad and E.F. Schisterman
16. Menstrual patterns in adolescents with eating disorders 255

A. Deltsidou
Conception
17. Role of micronutrients in the periconceptional period 273
I. Cetin, M. Massari and F. Parisi
18. Prevalence and predictors of periconceptional folic acid use 293

R.P. Deane and D.J. Murphy
19. Lower fertility associated with periconceptional obesity and underweight 313
E. Derbyshire
20. Preconception care and barriers to addressing overweight and obesity: a focus on
weight loss advice and weight loss strategies 327
M. Dekker Nitert, H.L. Barrett, S. de Jersey, K. Matusiak, H.D. McIntyre and
L.K.Callaway
21. Herbal medicine and fertilisation 343

L. Stocker and Y. Cheong
22. Diet containing endocrine-disruptors and reproductive health 359

D. Balabani and A. Krivograd Klemeni
8 Handbook of diet and nutrition in the menstrual cycle, periconception and fertility
Table of contents
23. Anemia, Roux-en-Y gastric bypass and conception 373

R.M.Y. Nomura, A.M.K. Igai, R. Pulcineli Vieira Francisco and M. Zugaib
Fertility-infertility
24. Vitamin D and assisted reproductive technology 387
A. Mahdavi and H. Hosseini Jadda
25. The impact of obesity on ovulation and early pregnancy: a focus on ovarian function,
fertilization, implantation and early embryo development 401
A. Gonzalez-Bulnes and S. Astiz
26. Nutraceutical approaches in female infertility: setting the rationale for treatments tailored
to the patients phenotype and based on selected molecules 413
E. Papaleo, V.S. Vanni and M. Candiani
27. Folate and female infertility: folate-metabolizing pathway in folliculogenesis, infertility

treatment, and implantation 431
S. Altme, M. Laanpere, C. Campoy and A. Salumets
28. Dietary intolerance and endometriosis: an immunological link in the pathogenesis of an

enigmatic disease? 449
Y. Muscat Baron
29. Gastro-intestinal symptoms in women with pelvic endometriosis 471

Y. Muscat Baron
30. The role of vitamin D for conception, polycystic ovary syndrome, endometriosis and the
menstrual cycle 489
M. Grundmann and F. Von Versen-Hynck
31. Polycystic ovary syndrome and the metabolic syndrome 505

T.J. Hunter and R. Hart
32. Diabetes mellitus and polycystic ovary syndrome: implications for diet and nutrition 521
N. Mendoza and M.J. Cancelo
33. Hypocaloric diets in overweight and obese patients with polycystic ovary syndrome 533
S. Palomba, A. Falbo and G.B. La Sala
Table of contents
Index 555
About the editors 565
Preface
The reproductive cycle in women is rather complex and can be considered to begin with epigenetic
programming and ending with post-menopause. Intervening steps involve a variety of processes
including for example the development of the sex organs, menarche, episodic endocrine cycles,
menstruation, ovulation and conception. The optimal conditions for these processes results in a
fertile individual in which conception, pregnancy and birth of an offspring can arise. However
all of these processes can be influenced by diet and nutrition and vice versa. Body composition
also has an impact on the menstrual cycle and fertility. These factors in turn also influence body
composition per se. For example, the menstrual cycle has a marked influence on diet selection
and food consumption. Similarly, either food deprivation, dietary excess or obesity can result in
marked changes in the menstrual cycle with a concomitant effect on fertility. The complex web of
diet, nutrition, body composition, menstruation, ovulation and fertility can only be understood
and untangled is there is a comprehensive source of scientific material. Ideally such text should
be within a single source that is structured in such a way as to treat all these aforementioned
factors as being dependent on each other in multiple ways. The Handbook of diet and nutrition
in the menstrual cycle, conception and fertility achieves this by having a structured focus with
the following main parts:
1. setting the scene;
2. puberty, menarche and the menstrual cycle;
3. conception;
4. fertility-infertility.
Coverage includes micronutrients in general, calcium, iodine, vitamin D, folic acid and folate,
herbal medicine, nutraceuticals, chocolate, fertility, ageing, psychophysiological changes,
menarche, body composition, premenstrual syndrome, treatment of premenstrual syndrome,
body mass index, socioeconomic status, neuroimaging, appetite, dietary strategies, exercise,
lipid levels, eating disorders, obesity and underweight, barriers, weight loss advice, endocrine-
disruptors, anaemia, gastric bypass, ovarian function, embryo development, folliculogenesis,
infertility, implantation, dietary intolerance, endometriosis, gastrointestinal symptoms, polycystic
ovary syndrome, metabolic syndrome, diabetes, hypocaloric diets and many other area relevant
to the interrelations between diet, nutrition, the menstrual cycle, conception and fertility.
Contributors are authors of international and national standing, leaders in the field and
trendsetters. Emerging fields of science and important discoveries relating to the menstrual cycle,
periconception and fertility will also be incorporated in the Handbook of diet and nutrition
in the menstrual cycle, conception and fertility. This represents one stop shopping of material
related to the menstrual cycle, periconception and fertility and will be essential reading for
endocrinologists, cardiologists, nutritionists, dietitians, paediatricians, pharmacologists, health
care professionals, research scientists, general practitioners as well as those interested in womens
health in general.
The editors
Setting the scene
1. Diet and nutrition in fertility: an overview
including special requirements with ageing
J.H. Ford
Centre for Regional Engagement and Community Health, University of South Australia, GPO Box
2471, Adelaide, South Australia 5001, Australia; judy.ford@unisa.edu.au
Abstract
Conception, term pregnancy and birth of a live-born infant can occur under minimal nutrition in
younger women although extreme under nutrition or over-exertion limits fertility. Alternatively,
excessive nutrition and obesity is associated with polycystic ovarian syndrome, an increasing
cause of infertility in developed nations. Undernourished babies are at risk of very low birth
weight, disability and/or infant mortality and very low birth weight is associated with early onset
of chronic disease and shortened lifespan. Protein may be the most critical peri-conception
nutrient but many minerals and vitamins are critical for optimal development. Reproduction in
primates demonstrates that increasing birth weight is associated with decreasing lifetime fertility
of a species. In humans increased birth weight is also associated with decreased male fertility in
all countries where suitable data is available. Since increased median birth weight is associated
with increased longevity, balancing loss of male fertility may be a desirable outcome of natural
selection. Ageing in females is associated with loss of fertility and increased risk of unfavourable
outcomes. Since ageing is associated with changes in fatty acid metabolism, it is proposed that
suitable supplementation of fatty acids and mitochondrial enzymes might improve outcome in
healthy women aged 38 and older.
Keywords: female fertility, male fertility, female ageing, birth weight, fatty acid metabolism
C. Hollins-Martin et al. (eds.) Handbook of diet and nutrition in the menstrual cycle, periconception and fertility
17
Human Health Handbooks no. 7 DOI 10.3920/978-90-8686-767-7_1, Wageningen Academic Publishers 2014
J.H. Ford
Summary points
Young females are fertile even when their diet is minimal. Only really severe lack of nutrition or extreme
expenditure of energy limits fertility.
Excessive nutrition has a greater impact on both male and female fertility than under-nutrition. Infertility is
increased in both obese males and females.
Fertility does not ensure quality reproduction. Poor nutrition is associated with low birth weight and infant
mortality. Low birth weight predisposes to shortened lifespan and early onset of chronic diseases such a renal
and cardiovascular disease.
Across all nations, the number of children born to each woman is inversely proportional to longevity i.e. the
longer the life expectancy, the less children are borne to each woman.
On a population basis, male fertility is declining as birth weight is increasing. Research needs to be done to
determine whether birth weight is physiologically related to male fertility.
At about age 38, a womans fertility reduces dramatically. This is observed as either infertility or the
spontaneous abortion of (usually) developmentally abnormal embryos.
Fatty acid metabolism changes at the same approximate age as female fertility declines. It is likely that
supplementation with specific fatty acids especially oleic acid may improve age-related infertility.
1. Diet and nutrition in fertility
Abbreviations
BMI Body mass index
CoQ10 Co-enzyme Q10
DHA Docosahexaenoic acid
DHEA Dehydroepiandrosterone
EPA Eicosapentaenoic acid
FSH Follicle stimulating hormone
GLA -linolenic acid
PCOS Polycystic ovarian syndrome
PUFA Polyunsaturated fatty acids
SCD-1 Stearoyl-coenzyme A desaturase 1
1.1 Reproduction and natural selection in humans

Reproduction is arguably the most important function in life. Without the ability to reproduce
itself a species will die out after a single generation. In animals cross species hybrids are usually
infertile and only plants seem to be able to survive with polyploidy. Throughout the plant and
animal kingdoms there is an inverse relationship between the number of progeny produced
and the likelihood of the progeny to survive to the age of reproduction. Since natural selection
functions through reproduction, the nutritional requirements for any species fertility need only
be sufficient to allow about two of the progeny to survive to adulthood. So unless there is some
critical biological role for individuals past the age of reproduction, death after reproduction does
not affect the population in biological terms.
In humans, like most animals we observe this inverse relationship between number of live-
born children and survival to reproductive age. As societies become better nourished and more
sophisticated the rate of births per woman decreases (Table 1.1). Usually we would regard this
decrease in fertility with increasing longevity as being the result of active decision making but
could it be that as in many plants, where increased nutrition leads to vegetative growth rather
than flowering, that human fertility also decreases with excessive nutrition?
1.2 Reproduction in women of different ages

In all women there is a dramatic reduction in fertility that occurs in the late thirties. On average
this reduction occurs at about age 38 and appears to be concordant with follicle number
reaching a low threshold, after which the rate of further follicle loss rapidly accelerates. The age-
related results of reproductive outcome in our prospective study called the PALS (Pregnancy
and Lifestyle Study) are shown in Figure 1.1. This same age is known to be associated with a
dramatically increased risk of infertility, miscarriage and of births of infants with trisomy 21
(Down syndrome). It has recently been shown (Ford and Tavendale, 2010) that highly significant
J.H. Ford
Table 1.1. Life expectancy of women and births per female by country.1
Country Life expectancy female Births per female
Chad 46.7 4.93

Somalia 47.78 6.25
Mali 48.38 6.35
Sudan 50.5 4.17
Niger 51.39 7.52
Uganda 51.66 6.65
Republic of Congo 52.9 5.59
Papua New Guinea 63.4 3.07
Pakistan 64.1 3.39
Tonga 68.2 3.55
Egypt 69.6 2.94
Malaysia 70.6 2.64
Argentina 73.71 2.29
Cuba 80.1 1.45
USA 80.9 2.06
Denmark 81.1 1.74
United Kingdom 82.3 1.91
Australia 84.4 1.77
Italy 84.5 1.40
Monaco 93.7 1.51
1 Statistics for life expectancy and the births per female are derived from the World Fact Book 2011 (CIA, 2011).
The life expectancy figures for the countries with high infant mortality do not adjust for deaths under one year of
age.
changes in fatty acid metabolism occur at this same approximate age and the authors proposed
that these changes might be responsible for the ultrastructural changes in mitochondria that
occur in the granulosa cells of older women.
Because of these critical age-related changes, it is necessary to consider the nutritional

requirements of younger and older women separately.
0.8
0.7
0.6
0.5
Adverse outcome
0.4
0.3
0.2
0.1
0
0 10 20 30 40 50
Maternal age (years)
Figure 1.1. Adverse outcome (infertility or spontaneous abortion) as a proportion of all outcomes of women of
different ages. Horizontal axis is maternal age at commencement of the program. Data from the PALS Pregnancy
and Lifestyle Study.
1.3 What is the minimum diet for fertility in young women?

1.3.1 Nomadic tribes: insights into diet and fertility
Studies of the daily lives of nomadic tribes are understandably very difficult and are thus not
totally accurate. Nevertheless such studies are likely to give the best insight into the minimal
nutritional requirements for human fertility. The different rates of fertility in three non-nomadic
and two nomadic populations in the Sudan have been reported in detail (Henin, 1969). One of the
two nomadic populations, the Baggara people, grow millet and drive their herds of cattle. They
rarely eat their animals but drink the milk, eat millet and have no vegetable intake other than
Weika, which is dried okra (Abelmoschus esculentus). Very occasionally their diet is supplemented
with mutton and game that they have killed during their roaming.
Despite the near subsistence diet, 81.8% of women (n=332) in the 30-39 year old age group
had given birth to one or more live-born children. This is very similar to the rate for the second
nomadic population, the Blue Nile nomads (81.6%) but considerably less than the non-nomadic
groups Gezira and Mangil, who had fertility rates of 95.7% and 96.5% respectively in the same
age groups. A third non-nomadic group had infertility rates (11.9%) that approached the rates of
infertility in the nomadic groups.
Table 1.2 shows the approximate nutrient content of the three staple foods consumed by the
Baggara people. We do not know how much food was consumed on a daily basis but if we assume
that about two serves of each food was consumed, the diet would be very low in calories and fat
intake but would meet the current recommendations for almost all of the nominated nutrients.
J.H. Ford
Table 1.2. Nutrient sources of key components of the diet of nomadic Baggara people (in percentage of daily
requirement).
Nutrient Okra, 100 g Millet, 1 cup cooked Milk, 1 cup
Protein 4% 12% 16%

Fibre 9% 9% 0%
Fat 0% 0% 12%
Omega 6 36 mg 835 mg 293 mg
Omega 3 0.8 mg 48.7 mg 183 mg
Cholesterol 0% 0% 4%
Calories 1% 10% 7%
Carbohydrate 1% 14% 4%
Vitamin A 12.5% 0.00% 5%
Vitamin C 22% 0% 0%
Vitamin K 44% 1% 1%
Vitamin E 2.5% 0% 1%
Vitamin D 0% 0% 24%
Vitamin B6 16.5% 9% 5%
Vitamin B12 0% 0% 18%
Thiamin 17% 12% 7%
Niacin 6% 12% 1%
Riboflavin 4.5% 8% 27%
Folate 22% 8% 3%
Pantothenic acid 5% 3% 9%
Choline 7.4 mg 19.5 mg 43.5 mg
Calcium 8% 1.00% 29%
Magnesium 14% 19% 7%
Phosphorous 9% 17% 23%
Sodium 0% 0% 4%
Potassium 3% 3% 10%
Iron 10% 6% 0%
Copper 10% 14% 1%
Manganese 43% 24% 0%
Zinc 5.5% 11% 7%
Selenium 1% 2% 12%
Betaine 0% 0% 1.5 mg
Only choline, vitamins A and E and iron seem to be well below the recommended levels although
several others, including folate, are low.
The nutritional content of okra given in Table 1.2 does not include the seeds but it would seem
very likely that the nomadic people would consume the whole of the okra plant, including the
seeds, which are very high in oleic acid, in minerals and polyphenols (Al-Wandawi, 1983). Since
previous studies have shown that there is not a specific requirement for Vitamin E in reproduction
but that its role is entirely associated with its antioxidant activity (DRAPER et al., 1964), it would
seem that okra seeds are the likely source of the alternative antioxidants and of some fatty acid.
1.3.2 Energy expenditure versus calorie intake and fertility
In comparison to the settled tribes, who have about 15% higher fertility, the nomadic people are
more likely to suffer from poor sanitation, venereal disease and malaria. Despite this, it is probably
their lowered body fat, due to malnutrition, hard work and continuous movement that causes the
drop in infertility. Given the life conditions, the greater than 80% fertility seems remarkably high
and attests to the fact that fertility can be achieved in young women when minimal nutritional
requirements are met. However, like other women, fertility drops markedly in the late thirties
age group. The mean age of the mother at the birth of her last child is 36.96 for the settled Gezira
women and 36.36 for the nomadic Baggara women (Henin, 1969).
1.3.3 Fertility versus offspring short and long-term health and survival
Giving birth to a child by no means ensures that the child will survive to adulthood. In the
disadvantaged countries of the world, there are high rates of infant mortality due to starvation
and infectious diseases. Moreover, in addition to childhood mortality, there are also high rates
of chronic disease in adulthood; in 1990 the numerical contribution of developing countries to
the global burden of cardiovascular disease was 2.8 times higher than of the developed countries
(Reddy, 1998).
There is now considerable evidence supporting the hypothesis (Barker, 1995) that low birth
weight is a cause of later life coronary heart disease. Low birth weight is a major cause of low
nephron number and kidney disease (Luyckx and Brenner, 2005) and this is related to both
kidney disease per se and to hypertension related illnesses. Studies in rats have demonstrated that
maternal protein restriction specifically suppresses the development of the renin-angiotension
system (Woods et al., 2001).
Whilst malnutrition, especially protein deficiency is a common cause of low birth weight
in developing countries, low birth weight also occurs in developed countries especially in
association with low socio-economic status. In the latter group, low birth weight has often been
largely attributed to smoking, drug use and whole of life stress but our own prospective study
where couples volunteered for the study prior to conception, peri-conception low intake of
protein was clearly identified as a major predisposing factor to very low birth weight, especially
in conceptions in winter (Ford, 2011). Carbohydrate and fat sources can also influence the effects
of low protein diets in rats (Armitage et al., 2004) however protein intake is probably of major
importance. A detailed study of the diets of 557 pregnant women in Adelaide showed that the
J.H. Ford
proportion of energy derived from protein early in pregnancy was correlated with both birth and
placental weight (Moore et al., 2004).
1.3.4 Does poor nutrition cause spontaneous abortion?
The rates of spontaneous abortion in nomadic populations are not reported but when estimates are
given for similar populations they seem to be extremely low. This could be that the spontaneous
abortions occur before the pregnancies are recognised or because they are indeed very low. In
our own prospective study of 585 couples who were attempting to become pregnant, we did
not detect any associations between nutrition and spontaneous abortion. The factors that were
associated with abortion were both male and female age and various exposures, especially male,
to occupational and environmental chemicals (Ford et al., 1994). This is not to say that individual
nutrients are not essential to normal foetal development but rather that only fairly minimal
nutrition is necessary for fertility and progression to birth.
It is interesting that apart from age, our study identified exposures that are associated with
industrialization as the major factors associated with spontaneous abortion. Such factors are
absent in undeveloped rural environments.
1.3.5 Infertility in malnourished women
The comparison of the fertility rates in nomadic and sedentary populations in Sudan strongly
suggests that apart from disease, the relationship between food intake and activity is the
major determinant of female fertility. This same relationship is found in western populations
where there is a substantial difference in fertility between the general population and females
undertaking intense exercise. Studies of the percentage of women with menstrual irregularities
were summarised (Warren and Perlroth, 2001), which were as low as 1.8% in one study of the
general population and as high as 79% in one study of ballerinas. Fertility seems to require female
body fat of about 22% and in most cases fertility can be restored in athletes with either a gain in
fat of 1-2 kg or a 10% decrease in exercise load.
1.3.6 Excess calories as a cause of infertility in developed countries?
So if severe under-nutrition is a cause of infertility in the poorest of nations, is over-nutrition a

cause of infertility in developed countries?
PCOS is a major cause of female infertility in developed countries and affects one in ten women
of reproductive age (Gambineri et al., 2002). It is characterised by raised levels of luteinizing
hormone relative to FSH and higher levels of androgens. Obesity is a major contributing factor
in PCOS with 44% of obese women having PCOS compared with 9% of those of normal weight.
Despite this substantial effect of excess nutrition on female infertility, it is still not sufficient to
explain the relationship between increasing longevity and decreasing fertility. Moreover since
obesity itself is associated with shortened lifespan there must be a major, as yet unrecognised
factor that is reducing fertility in the healthy, non-obese population.
1.4 Parental investment in size of offspring and decreasing fertility

Examination of all mammals shows that there is an inverse relationship between fertility rate per
year and body size in kilograms (Walker et al., 2008). These authors have also shown that within
primates, there is an inverse relationship between fertility rate and weaning size and that the
inverse relationship is particularly strong within the apes. This relationship has been referred to
a parental investment in offspring and is a quantity-quality trade-off.
The authors also demonstrate that across sixteen different human natural fertility populations,
there is an inverse relationship between fertility rate and offspring size at ages 5 and 10. Much of
this relationship is attributed to a greater time spent nurturing the offspring, including the period
of lactation but there is also an increased size of offspring at the time of birth, possibly because
of the reduced demands on the nutrient balance of the women with increased birth intervals.
The inverse relationship between longevity and number of children/woman shown in Table
1.1 can now be reconsidered in the light of this further relationship. But the question needs to
be asked as to whether the reduced fertility is entirely due to choice or is there an associated
physiological reduction in the ability to conceive? The answer to this important question has
implications for how health interventions are undertaken in disadvantaged populations.
Two clear examples of active decisions to reduce fertility can be seen in the introduction of the
contraceptive pill and in a political decision taken by the Chinese Government. In China in 1978
there was a law invoked to reduce population size by reducing by law the number of children per
couple. The one child per couple law was taken to alleviate social, economic and environmental
problems and this law which applies to most of the Chinese population appears to be having
significant effect on population size. In a similar time period in many other countries there has
been a large drop in the number of births to younger women with the introduction of the oral
contraceptive pill. The ability to protect against an unplanned pregnancy has an obvious outcome
of reduced pregnancy rates.
1.5 I s male reproductive potential responsible for the reduction of fertility with
increased longevity?
Although this book has a focus on nutrition and female fertility, this cannot be understood
without considering the role of the male.
There is no evidence to suggest that female fertility decreases with increased body weight unless
body weight increases to obese and beyond. Indeed most studies, including our own, suggest that
J.H. Ford
slightly overweight women are more fertile than normal weight women. This then suggests that
the control of fertility might lie in male bodyweight.
There is good evidence that a males BMI has a major impact on sperm quantity and quality.
The total number of normal-motile sperm cells differed with the BMI groups (Kort et al., 2006):
normal weight was 18.6106 cells, overweight was 3.6106 cells and obese was 0.7106 cells.
As well as an effect on normal motile sperm numbers there was also a lesser but statistically
significant effect of increasing body weight on decreasing sperm chromatin integrity.
In our prospective study of reproduction known as the PALS Pregnancy and Lifestyle study, we
also found an effect of male BMI on fertility. We did not analyse sperm but couples where the
male was obese were more likely to be infertile for the nine months that they tried to conceive in
our program (Table 1.3). Underweight men appeared to be more fertile than average but we had
insufficient underweight and obese subjects to test these results statistically.
To my knowledge there has been no study relating body weight at birth to adult male fertility
but population data suggests that this might be so. In Figure 1.2, the distribution of birth weights
data is shown for the USA, Hong Kong and Denmark (United Nations statistics from http://data.
un.org) in the years that the reported semen analyses were made (Carlsen et al., 1992). Although
it has to be recognised that both sets of data are population data and that if this concept is correct
that the semen analyses should reflect the mans own birth weight rather than the current birth
weight, there is a very obvious inverse relationship between sperm count and birth weight that is
reflected in the modal birth weights.
Further research needs to be undertaken to demonstrate that the relationship reflects a mans
individual birth-weight, nevertheless the fact that each of the statistics, firstly a males increasing
current BMI and secondly the trend of a countrys increasing birth weight each correlate inversely
with sperm count, it is more than likely that nutrition in utero determines a mans future fertility.
Table 1.3. Male body mass index and infertility.1
Body mass index Pregnancy Nine months infertility Total
Less than 18.5 9 0 (0%) 9

Normal: 18.5 to 25 261 54 (17.1%) 315
Overweight: >25 to 30 160 32 (16.7%) 192
Obese: >30 19 6 (24.0%) 25
Total 449 92 (17.0) 541
1 Data from the PALS Pregnancy & Lifestyle Study suggests an association between low male weight and enhanced
fertility versus obesity and decreased fertility. Couples were involved in a prospective study with no intervention.
0.8
Hong Kong USA Denmark
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
<2,500 2,500 - 3,499 3,500 - 4,499 4,500
Birth weight (g)
Figure 1.2. Birth weight by country. Birth weight is reported as a proportion of total births in years in which
sperm numbers were reported: Hong Kong (1985), USA (1982) and Denmark (1990). Data was obtained
from the United Nations website. Mean concentration 106 cells per ml were: Hong Kong 83.0, USA 66.0 and
Denmark 58.6.
It appears that the bigger the male baby (within the normal birth weight cohort), the lower will
be his future fertility and the longer will be his expected lifespan.
1.6 P hysiological changes in adipose tissue in women in their late thirties and
changed nutritional requirements
Examination of the adipose levels of fatty acids in a very large database of fertile women aged
between 25 and 48 years showed that there are highly statistically significant changes that
occur with age (Ford and Tavendale, 2010). Principal component analysis identified three key
factors that were significantly associated with aging, which reflected changes in the activity of
the desaturase enzymes in each of the omega 3, omega 6 and omega 9 pathways. We identified
Factor 1 as reflecting delta-9-desaturase or commonly called stearoyl-CoA desaturase-1 or SCD-
1, Factor 2 as delta-5-desaturase and Factor 4 as delta-6-desaturase. The changes of the two key
factors with age are shown in Figure 1.3 and it is obvious that the age-related changes in fatty
acid metabolism occur in the same age range as the changes in reproductive outcome shown in
Figure 1.1.
J.H. Ford
0.6
0.4
0.2
0.0
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
-0.2
-0.4
-0.6
-0.8
Factor 1 Factor 2
-1.0
Female age (years)
Figure 1.3. Plot of the mean values of Factors 1 and 2 with female age. Factor 1 reflects the loss of activity of
delta-9 desaturase (stearoyl-CoA desaturase-1) and Factor 2 reflects the loss of activity of delta-5-desaturase.
Female age is shown across the middle of the graph with positive values for the factors above the zero (age) line
and negative below. Full explanation of this analysis can be found in Ford and Tavendale (2010).
Each of these factors showed significant changes in enzyme activity at age 38, the same approximate
age at which follicle numbers are very low and the rates of infertility, spontaneous abortion and
the conceptions of infants with Downs syndrome all increase dramatically.
In this and other papers we argued that the changes in fatty acids provided an explanation for
the extensive changes in ultrastructure observed in the granulosa cells of women in the older age
group. Furthermore these changes in fatty acids are likely to have a range of effects that affect the
fidelity of both meiotic and mitotic cell divisions. To date I know of no nutritional studies that
have been undertaken to address the changes in fatty acids and the following are suggestions for
interventions that might have beneficial effects.
1.6.1 Fatty acid supplementation
Animal studies show that dietary supplementation with PUFA can influence some the diverse
functions of male and female fertility in animals but the treatments have not yet been sufficiently
controlled to provide any clarity (Wathes et al., 2007). In humans, studies of diets rich in omega-3
PUFA show beneficial effects on age-related changes in mitochondrial membranes in lymphocytes
(Mebarek et al., 2009) and myocardial cells (Pepe, 2005). However to date there are no studies on
the effect of omega-3 or GLA enriched diets on age-related reproduction in humans or in rodent
models of human reproductive aging. One study of omega-3 supplementation in five week old
mice showed some adverse effects including increased rates of oxygen radical production and
reduced normal embryo development (Wakefield et al., 2008) but the diet used in this study was
dramatically altered and may well have been deficient in n-6 PUFA.
The results of the fatty acid alterations that occur with female aging suggest that any trial diet
should pay attention to all three omega-3, omega 9 and omega-6 fatty acids and in the latter,
especially to n-6 GLA.
1.6.2 Supplementation with mitochondrial antioxidants: l-carnitine, coQ10
Since mitochondrial function is seriously disturbed in the ageing ovary, supplementation with
antioxidants might have beneficial effects. In the studies of repeated induction of ovulation in mice
(Miyamoto et al., 2010), l-carnitine supplementation greatly reduced both the loss of oocytes and
the reduction in quality of the remaining pool. Since l-carnitine both reduces endothelial reactive
oxygen species and increases the release of the vasodilator prostaglandin I2 (Bueno et al., 2005), it
is likely that supplementation in women would also be beneficial. The effectiveness of l-carnitine
given as a supplement in male infertility was reviewed. L-carnitine showed beneficial effects on
many fertility parameters but all studies showed loss of participants because of unacceptable side
effects (Zhou et al., 2007).
In a preliminary study, in which 52 week old mice were supplemented with three mitochondrial
nutrients CoQ10, r-alpha lipoic acid and resveratrol; CoQ10, but not the other two supplements,
was associated with increased oocyte numbers (Bentov et al., 2010). Since CoQ10 has been used
extensively as a supplement in other age-related disorders in humans and has no noted adverse
effects (Chinnery et al., 2006), it seems reasonable to advocate its trial by older women who are
trying to improve their oocyte quality.
1.6.3 DHEA-S supplementation
In a multi-centre trial of DHEA supplementation in women undergoing IVF who were either
aged 41 or older or younger but had elevated FSH, the spontaneous abortion rate was reduced in
all age groups, but by between 50 and 80% in women aged 40 and older (Gleicher et al., 2009).
The authors point out, however, that conceptions only occurred in a small minority of cycles.
1.6.4 Endurance and aerobic interval training
Studies of the expression of genes involved in fatty acid metabolism following endurance training
in rodents showed that SCD-1 and other related lipogenic genes were up-regulated in skeletal
muscle (Ikeda et al., 2002) but the changes were restricted to only some muscles and did not affect
liver SCD-1 (Dobrzyn et al., 2010). Nevertheless, since in 50 year olds with metabolic syndrome,
aerobic interval training enhanced endothelial function, excitation-contraction coupling in
muscle and lowered lipogenesis in adipose tissue (Stensvold et al., 2010), exercise training might
be considered as an adjunct to other interventions.
J.H. Ford
1.6.5 Suggestions for future work in counteracting ovarian ageing
Since the decline of the ovarian environment is exponential from about age 37, women who are
likely to defer reproduction could be encouraged to consider supplementation with antioxidants
to reduce oocyte atresia. Supplementation with l-carnitine in mice and either genistein or
resveratrol in rats each showed significant preservation of oocytes. Once women reach the point
of experiencing the first changes in fatty acid metabolism, it is likely that the fatty acid balance
might be stored by supplementation of the now deficient fatty acids, omega-3 EPA and DHA (per
fish oil), oleic acid (per olive oil) and GLA (per evening primrose oil or borage oil) in addition to
antioxidants. The longer the time since the fatty acid changes were initiated (usually a function of
aging), the greater will be the dysfunction of membranes and mitochondrial function. We might
expect that mitochondrial enzymes might need to be administered such as CoQ10 and the
results are likely to be better if this supplementation is administered in conjunction with fatty
acids. As has already been demonstrated, supplementation with DHEA will reduce the risk of
miscarriage in older women (aged 41 or older) who become pregnant. DHEA probably restores
the fatty acid balance induced by aging.
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2. Fertility in women
H. Daabis1 and T.A. Gelbaya2
1London Fertility Centre, Cozens House, 112A Harley Street, London W1G 7JH, United Kingdom;
2Leicester Fertility Centre, University Hospitals of Leicester, Leicester Royal Infirmary, Leicester
LE1 5WW, United Kingdom; tarek.gelbaya@uhl-tr.nhs.uk
Abstract
Fertility is the natural capability of producing offspring. The human oocytes pass through a long
process of folliculogenesis that commences in the intrauterine life and continue throughout
puberty and reproductive age until the follicle pool is depleted and menopause ensue. Puberty
follows maturation of the hypothalamic-pituitary-ovarian axis, which is the corner stone of female
reproduction. In addition, an intact functioning genital tract is essential for natural conception.
Female fertility is affected by a variety of factors including chronological age, ovarian reserve,
life style and environmental factors. Fertility is a word used to describe the couples and not the
individuals reproductive ability. A subfertile couple should therefore be seen and investigated
together. Investigations include semen analysis, assessment of ovulation and ovarian reserve,
Chlamydia screening and assessment of Fallopian tubes patency. In this chapter we are going to
discuss physiology of female reproduction, factors affecting fertility in women, prognosis and
investigation of subfertility in women. The detailed management of infertility is outside the scope
of this chapter.
Keywords: fertility, fecundity, reproduction, subfertility, ovulation, endometriosis
33
Summary points
Fertility is the ability to conceive within two years of unprotected sexual intercourse. Fecundability is the
probability of achieving a pregnancy within a single menstrual cycle.
Infertility is the inability to conceive after two years of regular unprotected sexual intercourse. With the
advancement of fertility treatments and assisted reproductive techniques the word subfertility is used in
favour of infertility.
Intact hypothalamic pituitary ovarian axis and genital tract are fundamentals of female fertility.
The prognosis of future fertility depends on: female age, ovarian reserve, previous pregnancy, duration and
cause of infertility.
Fertility assessment includes tests for: ovulation, ovarian reserve, tubal patency, and peritoneal factors.
More and more immunological testing are being used in current management of infertility.
Abbreviations
AFC Antral follicle count
AMH Anti-Mllerian hormone
ART Assisted reproductive technique
BMI Body mass index
GnRH Gonadotrophic releasing hormone
HCG Human chorionic gonadotrophin
HFEA Human Fertilisation and Embryology Authority
HSG Hysterosalpingography
IVF In vitro fertilization
LH Luteinizing hormone
MRI Magnetic resonance imaging
PCOS Polycystic ovary syndrome
PCT Postcoital test
PID Pelvic inflammatory disease
SHBG Sex hormone binding globulin
WHO World Health Organisation
2.1 Introduction
Fertility is the ability to conceive within two years of unprotected sexual intercourse. Fecundability
is the probability of achieving a pregnancy within a single menstrual cycle, and fecundity is the
probability of achieving a live birth within a single cycle. The fecundability of a normal couple
has been estimated at 20% to 25%, although clearly the chance of conception drops throughout
the first year to less than 10% after 7 cycles, and only 3% during the 12th cycle (Cramer et al.,
1979). In the general population (which includes people with fertility problems), it is estimated
that 84% of women would conceive within one year of regular unprotected sexual intercourse.
This rises cumulatively to 92% after two years and 93% after three years (Te Velde et al., 2000).
Infertility is defined by the WHO as inability to conceive after two years of regular unprotected
sexual intercourse. Primary infertility describes a couple who have never conceived while
secondary infertility follows a previous pregnancy (Gnoth et al., 2005).
With the advancement of fertility treatments and the availability of ART, the word subfertility
is used in favour of infertility or sterility. Very few couples have absolute infertility, which
can result from congenital or acquired irreversible loss of functional gametes or the absence
of reproductive structures in either partner (Habbema et al., 2004). In these specific instances,
couples should be counselled regarding their options of adoption, the use of donor gametes, or
surrogacy.
Pregnancy is the only way to prove fertility of a man or a woman, even if both are known to have
normal fertility investigations. Therefore, fertility is a word used to describe the couples and not
the individuals reproductive ability (Table 2.1). However, the focus of this chapter is on womens
and not mens fertility. Detailed management of infertility is outside the scope of this chapter.
2.2 Female reproductive physiology

In humans, approximately 6-7 million of primordial follicles are present at 20 weeks of intrauterine
life. The total number of primordial follicles drops to about 1-2 million at birth and 300,000
at puberty as a result of the ongoing follicular atresia. At puberty the hypothalamic-pituitary-
ovarian axis is activated, and the menstrual cycle starts. The process of folliculogenesis is a
dynamic process, which is controlled by a complex network of endocrine and autocrine factors
leading to the monthly development and release of a single oocyte (ovulation) in approximately
two thirds of women. At the menopause the follicle pool becomes practically exhausted by the
continuing folliculogenesis (Himelstein et al., 1976).
An intact hypothalamic-pituitary-ovarian-uterine axis is the cornerstone of a healthy female

reproductive system. The hypothalamus secretes GnRH in a pulsatile fashion into the portal
circulation, where it travels to the anterior pituitary gland. The sudden drop in serum progesterone
and inhibin-A by the end of the luteal phase of the menstrual cycle, and just before the onset of
menstruation, stimulates the release of hypothalamic GnRH, which in turn stimulates the pituitary
gonadotrophs to secrete FSH. The rising FSH stimulates a cohort of ovarian follicles to grow,
differentiate and secrete increasing amounts of oestrogen and inhibin-B. Oestrogen stimulates the
growth and proliferation of the functional layer of the endometrium (uterine lining). The rising
levels of oestrogen and inhibin-B inhibit the release of FSH leading to reduction in the serum
FSH level. Subsequently all the growing ovarian follicles but the leading one undergo atresia. The
leading follicle acquires more FSH receptors during development and becomes more responsive
Table 2.1. Relative prevalence of the aetiologies of infertility (%).
Male factor 25-40

Both male and female factors 10
Female factor 40-55
Unexplained infertility 10
Approximate prevalence of the causes of infertility in the female
Ovulatory dysfunction 30-40
Tubal or peritoneal factor 30-40
Unexplained infertility 10-15
Miscellaneous causes 10-15
to the lower concentration of serum FSH. The leading follicle releases sustained concentrations
of oestrogen leading to a surge in pituitary LH secretion.
Ovulation and the subsequent development of corpus luteum are triggered by the LH surge. The
corpus luteum secretes oestrogen and progesterone leading to luteinisation of the endometrium in
preparation for implantation. If pregnancy occurs, the trophoblast secretes HCG, which mimics
the action of LH sustaining the corpus luteum. The corpus luteum continues to secrete oestrogen
and progesterone supporting the decidualised endometrium and allowing the pregnancy to
continue until the placenta is formed at 8-9 weeks gestation. If pregnancy does not occur, serum
LH concentration drops leading to degeneration of the corpus luteum in 10-12 days, drop in the
serum oestrogen and progesterone, and the commencement of menstruation.
The length of the menstrual cycle is 21-35 days in two thirds of adult women, counted from the
first day of menstruation to the first day of next menstruation. The menstrual cycle is divided into
2 phases: the follicular and luteal phases. The follicular phase varies from 10 to 14 days long, and
this variability is responsible for most variations in the total cycle length. The sudden increase
in the local concentrations of prostaglandins and proteolytic enzymes leads to perforation of
the mature follicles wall and the release of the oocyte. After ovulation, the corpus luteum forms
from the remaining follicular shell and regulate the luteal phase. The luteal phase is 14 days long
and ends by the onset of menses. Menstruation and endometrial shedding follow hormonal
deprivation and uterine ischaemia. Anovulatory irregular cycles are more common at the
extremes of reproductive life (Fehring et al., 2006).
2.3 Factors affecting female fertility

2.3.1 Endocrine factors
Ovulation dysfunction is usually indicated by absence of menstruation (amenorrhoea),

infrequent menstruation (oligomenorrhoea) or frequent menstruation (polymenorrhoea),
accounting for 21% of causes of infertility. Women with regular menstrual cycles usually have
normal ovulatory cycles (Collins, 1988). Physiological anovulation occurs after menarche due to
incomplete activation of the hypothalamic-pituitary axis, and before menopause due to depletion
of primordial follicles. The WHO classified anovulation into three classes (Table 2.2). PCOS is
the commonest cause of anovulation, and is diagnosed in the presence of two out of the three
criteria: oligoovulation or anovulation, clinical or biochemical evidence of excess androgen and
polycystic ovaries detected by ultrasonography (the Rotterdam ESHRE/ASRM-sponsored PCOS
consensus workshop group, 2004). PCOS is the commonest cause of subfertility and affects up
to 8% of women of reproductive age. The insulin resistance in women with PCOS is greatest in
women with severe oligomenorrhoea or amenorrhoea. The tissue selective insulin resistance
theory suggests the ovaries preserve their insulin sensitivity leading to ovarian over response to
the circulating insulin. Ovarian steroidogenesis and ovarian androgen synthesis are subsequently
Table 2.2. WHO classification of ovulatory disorders.
Incidence Biochemistry Causes
Class I: Hypothalamic pituitary 10% Low FSH/LH/oestrogen Kallmanns syndrome

failure (hypothalamic amenorrhoea Normal prolactin Hypothalamic dysfunction (weight
or hypogonadotrophic loss, strenuous exercise)
hypogonadism) Pituitary tumours, head injury,
cranial irradiation
Hypopituitarism
Class II: Hypothalamic pituitary 85% Normal FSH/LH/ Predominantly PCOS
dysfunction oestrogen
Class III: Hypergonadotropic 4-5% High FSH/LH Premature ovarian failure or
hypogonadism (ovarian failure) Low oestradiol menopause
Turner syndrome
Gonadal dysgenesis
Galactosaemia
Chemotherapy
Radiotherapy
increased as well as adrenal androgen synthesis. In addition, the free serum androgen rises due
to the inhibition of the hepatic synthesis of SHBG by insulin.
Hyperprolactinemia impairs ovulation and requires cranial MRI to exclude pituitary

macroadenoma or other intracranial pathology. Hypogonadotrophic hypogonadism also requires
MRI to rule out central space-occupying lesions. Hypothyroidism may cause subfertility and
recurrent implantation failure, in addition to the ovulatory and menstrual abnormalities (Arojoki
et al., 2000).
2.3.2 Tubal factor
Tubal factors include damage or obstruction of the fallopian tubes, most likely due to previous
PID, previous pelvic or tubal surgery, peritonitis or endometriosis. The incidence of tubal
infertility has been reported to be 12%, 23%, and 54% after one, two, and three episodes of PID,
respectively. Most of these women are presumed to have had subclinical Chlamydia infections
(Macmillan et al., 2000). Still, about one half of patients with documented tubal damage have no
identifiable risk factors for tubal disease.
2.3.3 Endometriosis
Endometriosis is the presence of endometrial glands and stroma outside the uterus. Clinical
presentations of women with endometriosis include dysmenorrhoea, chronic pelvic pain,
dyspareunia (pain during sexual intercourse), heavy menstrual loss and subfertility. Clinical
evidence linking the presence of endometriosis to infertility exists, but the mechanisms remain
unclear. In severe endometriosis, distorted pelvic anatomy may explain the association. In cases
of minimal or mild disease with normal tubo-ovarian relationships, endometriosis is believed to
affect fertility via associated elevations in a variety of cytokines, including tumour necrosis factor
(De Ziegler et al., 2010).
2.3.4 Uterine factors
Uterine pathologies are diagnosed in approximately 15% of couples seeking fertility treatment.
Subfertility can be associated with; uterine anomalies, endometrial polyps, submucous fibroids
and intrauterine adhesions. Congenital uterine anomalies may be associated with subfertility
and pregnancy loss (Homer et al., 2000). Large fibroids and fibroids distorting the cavity or
obstructing the tubes can be associated with subfertility and pregnancy loss (Oliveria et al.,
2004). Endometrial polyps can be diagnosed by transvaginal ultrasonography or vaginal
sonohysterography. Endometrial assessment by transvaginal sonography is an important factor
in successful IVF programs. Future studies of endometrial growth factors may explain recurrent
implantation failure and recurrent pregnancy loss and help their management. However
endometrial biopsy has no role in the routine evaluation of infertility or early pregnancy loss
(Coutifaris et al., 2004).
2.3.5 Life style factors
Regular sexual intercourse every 2 to 3 days optimises the chance of pregnancy. Timing the
intercourse to coincide with ovulation causes stress and is not recommended. Female or male
sexual dysfunction can interfere with the couples natural fertility.
Obesity can interfere with ovulation, conception, and success of fertility treatments. Co-morbidity
is common with obesity, in particular hypertension cardiovascular disease, insulin resistance,
diabetes, and back pain (Farquhar and Gillett, 2006). Weight loss often requires multidisciplinary
efforts, including effective exercise programs and the involvement of an experienced dietician.
Low BMI disrupts the hypothalamic function and may cause anovulation, infertility, pregnancy
loss, and preterm labour. Low BMI may be associated with rigorous athletic training, malnutrition,
and anorexia nervosa. Weight gain in these women is likely to restore regular menstruation and
ovulation, and improve the chance of conception (Knuth et al., 1977).
A balanced diet provides the daily vitamins requirements. Dietary supplementation with folic
acid 0.4 mg daily before conception and up to 12 weeks gestation reduces the risk of having a baby
with neural tube defects. A higher dose of 5 mg per day is recommended for women who have
previously had an infant with a neural tube defect or who are receiving anti-epileptic medication
(Expert Advisory Group, 1992).
The department of health recommends no more than one or two units of alcohol once or twice
per week and avoiding episodes of intoxication for women seeking fertility treatment. Smoking
active or passive is likely to reduce female fertility and general health. There is no consistent
evidence of an association between consumption of caffeinated beverages (tea, coffee and colas)
and fertility problems (Klonoff-Cohen, 2002).
Couples are advised to avoid exposure to hazards that can reduce male or female fertility. Exposure
of the ovaries to radiation may cause early menopause. Some prescriptions, over-the-counter and
recreational drugs interfere with male and female fertility. The effectiveness of complementary
therapies for fertility problems has not been properly evaluated.
2.4 Prognostic factors of female fertility

2.4.1 Age
Chronologic age is the strongest determinant of reproductive success in both spontaneous and
assisted cycles as it is a predictor of ovarian reserve (Chuang et al., 2003). The decline in womens
fecundability begins in the early 30s and accelerates during the late 30s and early 40s. The effect
of womens age on fertility is clearly demonstrated in donor insemination cycles. Figure 2.1
illustrates the average birth rate for donor insemination in UK in the years 2007-2008 published
by the HFEA. The live birth rate for women under the age of 35 is 15%. The live birth rate decline
to 11.4% for women aged 35-37, 8.2% for women ages 38-39, 5.9% for women aged 40-42 and
0.7% for women aged 43-44. Oocyte donation programs also provide insight into the physiology
of declining fertility in older women. When embryos produced from oocytes retrieved from
younger women were transferred into older women, the pregnancy rates among the older women
approximated those of the younger women, and variations in pregnancy rates were directly
dependent on the age of the donors rather than that of the recipients. These observations strongly
support that it is the age of the oocyte, rather than the age of the endometrium, that accounts
for the age-related decline in female fertility. Another factor contributing to decreased fecundity
among older reproductive-age women is the increased risk of miscarriage in this population due
to the increased incidence of chromosomally abnormal conceptuses (Heffiner, 2004).
2.4.2 Ovarian reserve
Ovarian reserve refers to the number of the resting primordial follicle population that determines
the number of growing follicles and the quality of oocytes. Ovarian reserve also predicts how the
ovaries respond to exogenous gonadotrophins and the number and quality of embryos available
for transfer per IVF cycle (Biasoni et al., 2011). Approximately 10% of women have a low ovarian
35
2007
Live birth rate per cycle (%)

30
25 2008
20
15
10
5
0
Under 35 35-39 40-42 43-44 Over 44
Womans age at the start of cycle
Figure 2.1. Average birth rate for donor insemination in the UK in 2007-2008.
reserve by their mid-30s, whereas others respond well to exogenous gonadotrophins and achieve
pregnancies despite their advanced age.
2.4.3 Previous pregnancy
Women who had a previous successful pregnancy are more likely to conceive than women who
have never been pregnant.
2.4.4 Duration of infertility
The fecundibility of couples is inversely proportional to the duration of involuntary subfertility.

The success rate of ART is also related to the duration of subfertility especially in couples with
unexplained subfertility. In addition, couples who have been trying to conceive for longer
duration are more likely to be older.
2.4.5 Cause of infertility
Long unexplained infertility, particularly after recurrent implantation failures with IVF carries
the worst prognosis. Although controversial, there is some evidence to suggest that IVF is less
successful in women with moderate-severe endometriosis than in women with tubal factor
infertility.
2.5 Tests of female fertility

2.5.1 Rubella susceptibility
Women who are susceptible to rubella by screening test should be offered rubella vaccination
and advised not to become pregnant for at least 1 month following vaccination. Enquiry about
the timing and result of the most recent cervical smear test avoids delay in fertility treatment.
2.5.2 Assessment of ovulation
Ovulation occurs 34 to 36 h after the onset of the LH surge and about 10 to 12 h after the LH
peak. Regular menstrual cycles in the range 26 to 36 days are usually indicative of ovulation.
Women with regular menstrual cycles and more than 1 years infertility can be offered a blood test
to measure serum progesterone in the mid-luteal phase of their cycle (day 21 of a 28-day cycle)
to confirm ovulation. For women with irregular cycles, this test may need to be performed later
in the cycle (e.g. day 28 of a 35-day cycle) and repeated weekly until the next menstrual cycle
starts, unless the bleeds are so infrequent that ovulation induction therapy will be needed in any
case. Serum progesterone values ranging from 16-28 nmol/l, as the lowest limit are indicative
of ovulation (Wathen et al., 1984). Studies that evaluated the use of basal body temperature
or urinary LH kits to time intercourse did not report improvement in the chance of natural
conception. However, for the minority of couples who find it difficult to have frequent sexual
intercourse, the prediction of ovulation using LH kits can be useful.
2.5.3 Assessment of ovarian reserve
Because of the imperfect correlation between chronological age and ovarian biological age,
different tests have been developed to assess ovarian reserve. Some tests have become part of the
routine work to predict the response in subfertile women undergoing ART (Chuang et al., 2003).
AMH is synthesized in the pre-antral and small antral follicles by the granulose cells and inhibits
the initiation of primordial follicle growth. The serum level of AMH declines with age and
becomes undetectable by the menopause. The greatest amount of AMH is produced by the antral
follicles measuring <6 mm. AMH can be regarded as a significant and independent predictor
of excessive and poor ovarian response to gonadotrophin stimulation (Tremellen et al., 2005).
A strong correlation exists between AMH and AFC, a good predictor for ovarian reserve. AFC
can be accurately measured during a trans-vaginal baseline scan, early in the follicular phase and
prior to commencement of exogenous gonadotrophin therapy for IVF. Early follicular FSH has
been used for many years to assess ovarian reserve. It is only predictable of poor ovarian reserve
at high serum levels. Serum FSH levels vary widely from a menstrual cycle to another, thus AMH
or AFC are more reliable.
The ideal marker for ovarian reserve is one that can be performed in a basal state with high
sensitivity and specificity for identifying patients who will have live pregnancy outcomes. On-
going efforts to identify such a prognostic test are warranted so patients can be counselled
regarding various treatment options (Biasoni et al., 2011).
2.5.4 Screening for Chlamydia and other vaginal infections
Chlamydia trachomatis is present in 11% of the sexually active population aged 19 years or less
(Macmillan et al., 2000). It is a major cause of pelvic inflammatory disease, leading to chronic
abdominal pain, ectopic pregnancy and tubal factor infertility. Asymptomatic chlamydial
infection may go unrecognised and untreated. Although the prevalence of C. trachomatis among
subfertile women in the UK is only 1.9%, uterine instrumentation carried out routinely as part of
the infertility investigation may reactivate or introduce upper tract dissemination of endocervical
Chlamydia infection, resulting in iatrogenic pelvic inflammatory disease. Clinical pelvic infection
following HSG has been reported in up to 4% of cases and in 10% of patients with tubal disease.
Prophylactic antibiotics are effective in reducing this and should be considered. Both doxycycline
and azithromycin are effective prophylaxis and treatment for Chlamydia. There is evidence that
screening for and treating cervical Chlamydial infection can reduce the incidence of pelvic
inflammatory disease in women at increased risk of Chlamydia.
2.5.5 Assessment of tubal, paratubal, and peritoneal factors
Laparoscopy is the golden technique for diagnosing tubal and peritoneal disease, allowing
assessment of the external architecture of the tubes, visualization of the fimbria and tubo-ovarian
relationship, in addition to tubal patency. Identified abnormalities, including tubal obstruction,
pelvic adhesions, and endometriosis, can be treated at the time of diagnosis (Corson et al., 2000).
HSG has a sensitivity of 85% to 100% in identifying tubal occlusion. Unilateral fill or spill is
uncommon. Sonohysterography with contrast media offers a much less invasive method of
diagnosing fallopian tubal obstruction while maintaining a sensitivity and specificity similar to
that of laparoscopic chromotubation.
2.5.6 Assessment of cervical mucus
PCT is the classic test for evaluation of the potential role of cervical factor in infertility. The
most common cause of abnormal PCT is incorrect timing of the test within the menstrual cycle,
leading to the production of cervical mucus that is suboptimal for sperm penetration. The PCT
lacks uniform criteria for assessment, methodology, and reproducibility. Additionally the use of
PCT as a standard tool in infertility patients is further impaired by the poor correlation between
pregnancy outcome and the PCT results (Oei et al., 1998).
2.5.7 Assessment of uterine cavity
Sonohysterography appears to be superior to HSG in the detection of uterine malformations,

correctly identifying 90% of abnormalities in subfertile patients. Office hysteroscopy has been
proven to have superior sensitivity and specificity in the evaluation of the endometrial cavity
(Brown et al., 2000).
2.5.8 Immunological tests
The association of antiphospholipid antibodies, particularly anticardiolipin antibodies and the

lupus anticoagulant, with recurrent pregnancy loss led to the investigation of a role for these
antibodies in infertility. These antibodies are more prevalent in the infertility population. These
findings do not support a role for the routine testing of antiphospholipid antibodies in the
infertility evaluation (Hornstein et al., 2000).
2.5.9 Unexplained infertility
The laboratory assessment of an infertile couple is relatively simple and should be performed
rapidly to establish a diagnosis and initiate appropriate therapy. Evaluation of the male partner by
semen analysis in an accredited laboratory skilled in andrology testing is essential. A controversial
issue in the management of unexplained infertility concerns the potential role for laparoscopy
in the evaluation of this condition. The main reason for performing diagnostic laparoscopy in
women with unexplained infertility and normal HSG findings is the possibility of identifying
and treating endometriosis. In view of the available evidence, it is reasonable to initiate fertility
treatment empirically in women with unexplained infertility and normal HSG findings without
assessment of the pelvis by diagnostic laparoscopy (Tanahatoe et al., 2003).
The best overall approach to patients with unexplained infertility involves a discussion of
the options of empiric therapy or diagnostic laparoscopy and allowing the patient to actively
participate in decisions regarding the course of treatment.
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sensitivity index is strongly related to circulating AMH and may be used to predict ovarian response to
exogenous gonadotropins in IVF. Reproductive Biology and Endocrinology 9, 112.
Brown, S.E., Coddington, C.C., Schnorr, J., Toner, J.P., Gibbons, W. and Oehninger, S., 2000. Evaluation of outpatient
hysteroscopy, saline infusion hysterosonography, and hysterosalpingography in infertile women: a prospective,
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Chuang, C.C., Chen, C.D., Chao, K.H., Chen, S.U., Ho, H.N. and Yang, Y.S., 2003. Age is a better predictor of
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Collins, J.A., 1988. Diagnostic Assessment Of The Infertile Female Partner. Current Problems in Obstetrics,
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Farquhar, C.M. and Gillett, W.R., 2006. Prioritising for fertility treatments--should a high BMI exclude treatment?
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Fehring, R.J., Schneider, M. and Raviele, K., 2006. Variability in the phases of the menstrual cycle. Journal of
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Gnoth, C., Godehardt, E., Frank-Herrmann, P., Friol, K., Tigges, J. and Freundl, G., 2005. Definition and prevalence
of subfertility and infertility. Human Reproduction 20, 1144-1147.
Habbema, J.D., Collins, J., Leridon, H., Evers, J.L., Lunenfeld, B. and Te Velde, E.R., 2004. Towards less confusing
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Homer, H.A., Li, T.C. and Cooke, I.D., 2000. The septate uterus: a review of management and reproductive outcome.
Hornstein, M.D., Davis, O.K., Massey, J.B., Paulson, R.J. and Collins, J.A., 2000. Antiphospholipid antibodies and
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3. Psychophysiological changes in the menstrual
cycle phases
M.K.B. Lustyk1,2, E.A. Shilling1, D.L. Reaves1 and C.M. Hokama1
1School of Psychology, Family, & Community, Seattle Pacific University, 3307 Third Ave. W.,
Suite 107, Seattle WA 98119, USA; 2School of Nursing, Department of Psychology, University of
Washington, P.O. Box 351525, Seattle WA 98195-1525, USA; klustyk@spu.edu
Abstract
The menstrual cycle is characterized by dramatic fluctuations in ovarian steroid levels. Beyond
reproductive effects, ovarian steroids actuate changes in well-being and are implicated in
stress processes that influence physical and mental health. In this chapter, studies explicating
psychophysiological responses to stress provocation in laboratory settings are considered along
with those that reveal interrelationships among cyclical hormones, stress processes, and risk
factors for disease. We begin by reviewing hormone release patterns across cycle phases and
the bi-directional relationships with the autonomic nervous system and hypothalamic-pituitary-
adrenal axis. The bi-directional model is supported by research demonstrating cycle phase
variations in responses to laboratory stressors. Studies generally reveal greater reactivity and lower
cardiac vagal control during the luteal phase of the menstrual cycle compared to the follicular
phase. Three explanations are offered for this luteal phase increase including the cardioprotective
effects of estrogen, withdrawal effects of plummeting hormones during the late luteal phase, and
premenstrual symptomatology. Finally, we consider neuroendocrine processes associated with
increased stress reactivity as risk factors for mental health conditions that plague women. We
conclude with recommendations for clinicians to consider treatment plans that are sensitive to
the effects of cycle phase on stress.
Keywords: stress, hormones, women, cortisol, cardiac vagal control, cardiovascular reactivity
47
Summary points
The menstrual cycle is characterized by fluctuations in ovarian steroid hormone levels that vary by cycle
phase.
Ovarian steroid hormones communicate with the autonomic nervous system and hypothalamic-pituitary-
adrenal axis modulating a womans response to stress.
Estrogen and progesterone affect a womans physical and psychological well-being including: cardiovascular,
skeletal, neuromuscular, and neurochemical systems that regulate cognition, affect, stress, and appetite.
The neuroactive effects of progesterone on -aminobutyric acid receptors contribute to the anxiolytic effects
of elevated progesterone during the early-mid luteal phase and withdrawal of anxiolytic effects during the
late luteal phase.
Gender differences in psychophysiological responses to stress exist with women of reproductive age,
generally revealing lower responses than men.
In general, studies that show a cycle phase effect on psychophysiological responses to laboratory stressors
find greater reactivity during the luteal phase of the menstrual cycle compared to the follicular phase.
Increased cortisol levels and stress reactivity are implicated as risk factors for a number of ailments including
cardiovascular disease, depression, generalized anxiety disorder, anorexia nervosa, and substance use
disorder.
Research has shown that women are more successful at quitting smoking if attempts to quit begin during the
follicular phase compared to the luteal phase of the cycle.
In general, women are greatly underrepresented in clinical trials, and when women are included, research
designs do not systematically investigate cycle phase factors or gender differences in outcomes.
To better promote positive health outcomes in women, research should recognize and control for cycle phase
factors in outcomes so that clinicians can develop and implement appropriate treatment plans.
3. Psychophysiological changes in the menstrual cycle phases
Abbreviations
ACTH Adrenocorticotropic hormone
ANS Autonomic nervous system
CL Corpus luteum
CRH Corticotropin-releasing hormone
CVD Cardiovascular disease
GABA -aminobutyric acid
GnRH Gonadotropic-releasing hormone
HDL High density lipoproteins
HPAA Hypothalamic-pituitary-adrenal axis
HPG Hypothalamic-pituitary-gonadal
LDL Low density lipoproteins
PMDD Premenstrual dysphoric disorder
PMS Premenstrual syndrome
PNS Parasympathetic nervous system
SNS Sympathetic nervous system
TNF Tumor necrosis factor
3.1 Introduction
Much research supports the link between stress and health in women. Of particular note
is the intimate link between heightened cardiovascular reactivity and heart disease risk. As
such, explicating the stress processes in women, including the complexities of cardiovascular
reactivity during different menstrual cycle phases, remains an important goal for womens health
researchers. Herein we provide an overview of research aimed at this goal, along with a review
of scientific concepts addressed in our discussion. We start with an overview of the menstrual
cycle and neurobiological stress processes, followed by a review of research investigating
psychophysiological changes in the menstrual cycle. We conclude with a discussion of clinical
implications of this research.
3.1.1 The menstrual cycle: a brief overview
Ovarian steroid levels demonstrate dramatic fluctuations over the menstrual cycle. These
fluctuations occur in two phases within each cycle. The first day of menstrual bleeding marks
day one of the first phase. The second phase follows ovulation and concludes with the onset of
menstruation.
The two phases of the menstrual cycle can be further characterized by time, what is occurring
in the uterus, or what is occurring in the ovary. With regard to time, ovulation indicates the
transition between the two cycle phases, such that the first portion of the cycle is known as the
pre-ovulatory phase and the second portion of the cycle is known as the post-ovulatory phase.
From the perspective of uterine changes, the first portion of the menstrual cycle is identified as
the proliferative phase due to concurrent proliferation of endometrial cells lining the uterus.
From this same uterine perspective, the second portion of the menstrual cycle is called the
secretory phase due to the influx of fats, glycogen, and other secretory products that fill the
endometrial cells. Finally, in terms of what is occurring within the ovary, the first or follicular
phase references the ovarian follicle cells that secrete hormones during this time. Following
ovulation, the formation of the CL marks the transition to the luteal phase, during which the CL
takes over as the endocrine secreting tissue within the ovary.
The transitions between cycle phases are marked by drastic fluctuations in gonadotrophic
and gonadal steroid hormone levels (Figure 3.1). Specifically, the cyclical rise and fall of the
gonadotrophic hormones, FSH and LH, initiate the subsequent release of the ovarian steroids,
estrogen and progesterone. Such fluctuations are enabled by the HPG axis (illustrated in Figure
3.2), a communication pathway that connects the hypothalamus, pituitary gland, and ovary(ies).
The HPG axis utilizes complex neuro-hormonal feedback mechanisms to regulate the release
of estrogen and progesterone from the ovarian follicle and CL during the different cycle phases.
The anterior pituitary is cued to emit gonadotropic hormones (e.g. FSH) by GnRH from the
10
9 Estrogen
Progesterone
8
Hormone levels in arbitrary units
LH
7 FSH
6
5
4
3
2
1
0
1 4 7 10 13 16 19 22 25 28
Days of a 28 day menstrual cycle
Figure 3.1. Hormone levels across the menstrual cycle. Fluctuations in levels of the gonadotrophic hormones
lutenizing hormone (LH) and follicle stimulating hormone (FSH) and the ovarian steroid hormones estrogen
and progesterone occur across the menstrual cycle. LH cues the release of estrogen and progesterone from the
ovarian follicles and the corpus luteum. As shown here, in a 28-day menstrual cycle, the follicular phase begins
on day 1 and concludes on day 14 with ovulation. The luteal phase follows which commences on day 15 and
concludes on day 28.
Figure 3.2. The Hypothalamic-pituitary gonadal axis (HPG) is a communication pathway that connects the
hypothalamus, pituitary gland and gonads. HPG is under tight neuro-hormonal feedback in women, allowing for
the fluctuating levels of estrogen (E) and progesterone (P) released from the ovarian follicle and corpus luteum
during different cycle phases. GnRH = gonadotropin releasing hormone, LH = lutenizing hormone, FSH = follicle
stimulating hormone.
hypothalamus. This tonic HPG axis regulation results in the constantly fluctuating levels of
hormones that characterize the female reproductive cycle.
Keeping with our two-phase model, we can view the starting point of the cycle as occurring when
the HPG axis is activated at the onset of the follicular phase (i.e. day one of menses). At this onset,
estrogen and progesterone are at very low levels which signal the hypothalamus to release GnRH,
resulting in the release of LH and FSH. The increase in FSH stimulates the growth of the ovarian
follicles, and the increase in LH stimulates the production and release of estrogen by the follicles.
As estrogen levels increase, a negative feedback mechanism is enacted which tightly regulates the
release of further GnRH. In the days preceding ovulation, the ripened Graffian Follicle releases
large amounts of estrogen which override the negative feedback resulting in LH and FSH surges
that signal ovulation. After the surge in gonadotropic hormones, LH and FSH levels drop off and
remain low for the rest of the cycle.
Ovulation marks the transition from the follicular to luteal phase of the menstrual cycle. During
the luteal phase, the CL becomes the ovarian source of estrogen and progesterone. As the CL
becomes fully functioning, estrogen and progesterone levels rise. As shown in Figure 3.1, mid-
way through the luteal phase, both steroid hormones reach peak levels, and towards the end
of the luteal phase, both steroid hormones rapidly decline. The plummeting of estrogen and
progesterone during the late luteal phase initiates menstrual bleeding, signaling the completion
of one menstrual cycle and the start of another.
In addition to the roles of estrogen and progesterone in a womans monthly reproductive cycle,
these gonadal hormones have vast effects on a womans physical and psychological well-being.
With regard to physical well-being, estrogen and progesterone have effects on cardiovascular,
skeletal, neuromuscular, and cognitive brain systems. In terms of psychological well-being,
estrogen and progesterone have profound effects on neurochemical systems that regulate affect,
stress, and appetite (interested readers are referred to an excellent review by McEwen, 2002).
Herein we provide an overview of studies that investigate the roles of cycle phase and associated
hormonal fluctuations in psychophysiological stress responses. Most of the research studies cited
are laboratory investigations aimed at systematically studying ANS and HPAA processes that
vary by cycle phase. In a psychophysiology laboratory, the ANS and HPAA can be provoked with
laboratory stressors in systematic and well-controlled ways, which allow for reliable quantification
of biomarkers of stress processes including those implicated in cardiovascular health, such as
cardiac vagal control.
Given the intimate link between stress and cardiovascular health in women, understanding the
complexities of cardiovascular reactivity during different menstrual cycle phases remains an
important goal for womens health researchers. Cognitive and physiological stressors are ever
present in the daily lives of women. As such, explicating the processes affected by stress provocation
in a laboratory setting is an important first step towards understanding the interrelationships
among cyclical hormones, psychophysiological stress, and risk factors for disease in women.
Presently, cardiovascular disease is the leading cause of death among American women (Schiller
et al., 2012), which further emphasizes the importance of studying the association between stress
and cardiovascular reactivity. Before delving into our discussion of laboratory studies assessing
cycle phase effects on stress processes, we provide a brief overview of the ANS and HPAA
responses during stress to facilitate readers understanding of the data reported.
3.1.2 Autonomic nervous system and hypothalamic-pituitary-adrenal responses to stress
As part of the peripheral nervous system, the ANS controls visceral functions including regulatory
or homeostatic processes in physiological systems. The ANS is organized into two divisions: the
SNS and the PNS. Depending on the organs acted upon, the SNS and PNS can have antagonistic
effects. For example, as illustrated in Figure 3.3, the SNS increases heart rate while the PNS slows
heart rate.
The modulatory effects of the PNS at the level of the heart exemplifies the adaptive responses
that allow humans to self-regulate during stress. When utilized, these adaptive processes can
attenuate the sympathetically-driven fight-or-flight response which may serve a person well in
socially stressful situations where fighting or running away are not adaptive. Keeping with
this contextually driven model of adaptive stress responses, many argue that the fight-or-flight
Parasympathetic:
Sympathetic: Heart rate
Heart rate Respiration
Respiration Salivation and digestion
Salivation and digestion
Figure 3.3. Branches of the autonomic nervous system. During stress, the sympathetic branch of the autonomic
nervous system causes the pupils to dilate and the heart and lungs to accelerate in function while digestive
(e.g. inhibits salivation) and reproductive functions (e.g. inhibits genitals) are suppressed. With parasympathetic
activation, these processes are reversed such that pupils constrict, the heart and lung functions slow, and digestive
and reproductive processes are no longer inhibited.
response is the default and most beneficial response when one perceives a serious (e.g. physical)
threat.
In the face of such serious threats, the PNS disengages allowing the sympathetic nervous system
to take hold and mobilize resources to deal with the threat. Part of this process is the activation of
the HPAA. Akin to HPG activation, as illustrated in Figure 3.4, HPAA activation is hierarchical
starting with the hypothalamus secreting CRH into the specialized portal system that connects
the hypothalamus with the adjacent pituitary gland. CRH stimulates the secretion of ACTH
from the anterior portion of the pituitary gland. ACTH enters the general circulation ultimately
reaching the adrenal cortex and actuating the release of cortisol into the blood stream. Cortisol,
sometimes simply called the stress hormone, circulates throughout the body causing cells to
mobilize energy resources needed for the fight-or-flight response. Cortisol, in turn, provides
negative feedback to the hypothalamus, and as such, the HPAA represents a tightly regulated
physiological stress process.
Cortisol release is also regulated by diurnal and metabolic factors, as well as by higher brain
processes. The release pattern is driven by the intrinsic circadian rhythm of a hypothalamic
region that lies outside the HPAA. As shown in Figure 3.5, the diurnal secretion of cortisol peaks
in the morning and gradually declines through waking hours.
Cortex perceives the stressor
Hypothalamus releases CRH
Pituitary releases ACTH
Adrenal cortex releases cortisol
Figure 3.4. Schematic representation of the hypothalmic-pituitary-adrenal axis. When a threatening stressor is
perceived, the hypothalamus releases corticotropic releasing hormone (CRH) into the hypothalamic-hypophyseal
portal system causing adrenocorticotropic hormone (ACTH) to be released from the anterior pituitary. This in turn
causes the adrenal cortex to release the glucocorticoid cortisol into the bloodstream.
Sleeping Awakening 1:00 3:00 Evening
Figure 3.5. Diurnal rhythm of cortisol release. Driven by the circadian rhythm of the hypothalamus, the diurnal
pattern of cortisol secretion evinces peak levels with awakening and a gradual decline over the course of the day
reaching the lowest levels at night.
Cortisol release is also modulated throughout the day by metabolic factors such as blood glucose
levels. For example, when blood glucose levels are low, the cortisol response to a stressor is low
or absent. Likewise, when blood glucose levels are high, the cortisol response to a stressor is
high (Hucklebridge et al., 1998). Additionally, cortisol secretion is highly responsive to feedback
from the limbic system and prefrontal cortex during stress. Ultimately, in response to cortisol,
SNS responses are amplified, which can in turn increase HPAA reactivity. This bi-directional
relationship is also present between cortisol and the gonadal steroids. For example, cortisol affects
both uterine and ovarian functioning by increasing estrogen resistance and inhibiting estrogen
release respectively (for review see Lustyk and Gerrish, 2010).
Ovarian steroids are intrinsically involved in the regulation of HPAA and ANS activity, and as
such, one would expect menstrual cycle phase effects on these systems to exist. Indeed, several
laboratory studies reveal cycle phase variations in stress responses to acute laboratory stressors.
An overview of findings from these studies is provided in the next section.
3.2 Cycle phase differences in stress responses

Gender differences in psychophysiological responses to stress exist with women of reproductive
age generally revealing lower responses than men. This sexually dimorphic effect is diminished,
however, when the females studied are pre-pubescent or post-menopausal. Given the apparent
hormonal implications of such findings, researchers have attempted to understand gender
differences in psychophysiological stress responses, stress-related behaviors, and coping
by examining gender differences in ANS and HPAA responses to acute laboratory stressors.
Some excellent reviews have been published that elucidate these differences (e.g. Kudielka
and Kirschbaum, 2005). Our purpose in this section is to briefly outline what is known about
psychophysiological responses to laboratory stressors during different menstrual cycle phases in
women without necessarily considering comparisons to a control group of men.
In general, studies that show a cycle phase effect on psychophysiological responses to laboratory
stressors find greater reactivity during the luteal phase of the menstrual cycle (Kirschbaum et
al., 1999; Lustyk et al., 2010; Manhem et al., 1991; Sato et al., 1995; Tersman et al., 1991) and
lower cardiac vagal control (indexed by high-frequency heart rate variability; Bai et al., 2009;
Sato et al., 1995). Yet there is some inconsistency with Miller and Sita (1994), finding greater
cardiovascular reactivity during the follicular phase, and Stoney et al. (1990) as well as Weidner
and Helmig (1990) failing to show cycle phase effects on stress responses. Specific hemodynamic
and neuroendocrine findings are summarized in Table 3.1.
Recently, Lustyk et al. (2010) completed a systematic investigation of cardiovascular and

neuroendocrine responses to laboratory stressors in freely-cycling women. One purpose of
their study was to determine if the discrepant findings with regard to cycle phase effects on
stress processes were due to the varied stressors employed in published reports. Using both a
cognitive and a physical stressor, Lustyk and colleagues investigated a large sample (n=78) of
women during both the follicular and luteal cycle phases. All of the women were healthy and not
taking hormones known to affect the menstrual cycle or medications known to affect the stress
response. Their results revealed both significant cycle and stressor effects. Specifically, women
showed significantly greater heart rate, systolic blood pressure, and cortisol reactivity during
the luteal phase compared to the follicular phase (Figures 3.6 and 3.7). While heart rate and
blood pressure responses were not dependent upon the type of stressor used, cortisol reactivity
was stressor-dependent with women showing greater reactivity during the luteal phase to the
cognitive stressor compared to the physical stressor (Figure 3.7). Still, all of the physiological
stress responses observed by Lustyk and colleagues were greater during the luteal phase compared
to the follicular phase, and these findings align with the majority of results published in other
studies as previously cited. Thus, collectively considered, the luteal phase is associated with
exacerbated responses to stress provocation. Across studies, the purported reasons for the
exacerbated luteal stress responses are fairly consistent and include favorable cardioprotective
effects of estrogen during the follicular phase, luteal hormone plummeting, and/or increased
premenstrual symptomatology.
Table 3.1. Cycle phase effects on psychophysiological responses to laboratory stressors and tonic heart rate
variability.
Psychophysiological measure1 Response to laboratory Reference

stressor
Heart rate reactivity luteal > follicular Childs et al., 2010

Blood pressure reactivity luteal = follicular
Cortisol reactivity luteal = follicular
Catecholamine reactivity luteal > follicular
Progesterone reactivity luteal < follicular
Allopregnanolone reactivity luteal > follicular
Self-reported anger luteal > follicular
Self-reported depression luteal > follicular
Self-reported anxiety luteal > follicular
Cortisol reactivity luteal > follicular Kirschbaum et al., 1999
Heart rate reactivity luteal > follicular Lustyk et al., 2010
Blood pressure reactivity luteal > follicular
Cortisol reactivity luteal > follicular
Heart rate reactivity luteal > follicular Manhem et al., 1991
Catecholamine reactivity luteal = follicular
Heart rate reactivity luteal = follicular Miller and Sita, 1994
Blood pressure reactivity luteal < follicular
Self-reported anger luteal < follicular
Low-frequency heart rate variability luteal > follicular Sato et al., 1995
High-frequency heart rate variability luteal < follicular
Heart rate reactivity luteal = follicular Stoney et al., 1990
Heart rate reactivity luteal = follicular Tersman et al., 1991
Blood pressure reactivity luteal > follicular2
Cortisol levels luteal > follicular
Heart rate reactivity luteal = follicular Weidner and Helmig, 1990
High-frequency heart rate variability luteal < follicular Xiaopen et al., 2009
1 Changes in hemodynamic, neuroendocrine and self-reported mood in response to laboratory stressors during
the follicular and luteal phases of the menstrual cycle that are discussed in the text are highlighted here. Baseline
levels of high and low frequency components of heart rate variability during the follicular and luteal phases are
also tabulated.
2 Findings pertain to cold pressor testing only.
a Heart rate
82
74
Heart rate (BPM)
66 Follicular
Luteal
58
50
Baseline: Stressor Recovery 1: Recovery 2: Recovery 3:
0-30 min 0-10 min 10-20 min 20-30 min
b Systolic blood pressure c Diastolic blood pressure

120 74
115 70
110 66
mmHg
mmHg
105 62
100 58
95 54
90 50
Baseline: Stressor Recovery 1: Recovery 2: Recovery 3: Baseline: Stressor Recovery 1: Recovery 2: Recovery 3:
0-30 min 0-10 min 10-20 min 20-30 min 0-30 min 0-10 min 10-20 min 20-30 min
Figure 3.6. Heart rate and blood pressure responses during the follicular and luteal phases of the menstrual
cycle collapsed across stressor type. (a) Points represent mean heart rate in beats per minute (BPM); (b) Points
represent mean systolic blood pressure in millimeters of Mercury (mmHg); (c) points represent mean diastolic
blood pressure in mmHg. For (a) thru (c) vertical lines depict standard errors of the means. Baseline constituted
the first 30 min of recording; stressors were on average 9 min long as detailed in the methods section; 30 min of
recovery followed the stressor and are depicted in the above graphs in 10 min intervals. Counting menses onset
as day 1, the follicular laboratory session occurred during days 5-9 post-menses onset and treating ovulation as
day zero, the luteal laboratory session occurred during days 7-10 post-ovulation. Figure adapted with permission
from Lustyk et al. (2010).
3.2.1 Cardioprotective effects of estrogen
The cardioprotective effects of endogenous estrogen during the reproductive years are well
documented. Ironically, however, two large-scale clinical trials investigating the cardiovascular
benefits of hormone replacement therapy for post-menopausal women found either no benefit
(Hulley et al., 1998) or detrimental effects resulting in trial termination (Anderson et al.,
2004). Still, deleterious effects of exogenous treatments do not rule out beneficial functions of
endogenous systems. Moreover, more recent animal research points to beneficial cardiac effects of
estrogen replacement. For example, estrogen deficiency in rats is associated with elevated cardiac
TNF (a proinflammatory cytokine) levels which are improved with estrogen replacement (Xu
et al., 2006). Thus, attenuation of the inflammation response may be one beneficial mechanism
of estrogen effects on the cardiovascular system. Other beneficial cardiac-related effects of
estrogen include: (1) the promotion of HDL, more generally known as good cholesterol while
inhibiting the formation of LDL, or what is often referred to as bad cholesterol; (2) vascular
effects including the promotion of acute vasodilation as well as general vesicular resiliency; and
0.22
Follicular CP
Follicular PASAT
0.20 Luteal CP
Luteal PASAT
0.18
g/dl
0.16
0.14
0.12
20 min into baseline 10 min post-stressor 20 min post-stressor
Figure 3.7. Salivary cortisol responses during the follicular and luteal phases of the menstrual cycle displayed
by laboratory stressor type. Points represent mean salivary cortisol values in g/dl; vertical lines depict standard
errors. Counting menses onset as day 1, the follicular laboratory session occurred during days 5-9 post-menses
onset and treating ovulation as day zero, the luteal laboratory session occurred during days 7-10 post-ovulation.
Values fall within the expected salivary free cortisol range for adult females, ages 21-50 samples during 1:00-
3:00 pm as reported by Salimetrics (www.Salimetrics.com), the lab contracted to perform cortisol assays in the
present study. PASAT = paced auditory serial addition test; CP = cold pressor. Figure adapted with permission
from Lustyk et al. (2010).
(3) antioxidant effects which reduce potential damage from free radicals. These protective effects
are thought to underlie the gender differences in risk factors associated with CVD and explain
why pre-menopausal women fare much better than men when it comes to heart disease.
Briefly, for men, the primary predictor of CVD is elevated HDL, whereas for women, high blood
pressure and diabetes are primary predictors (Vitale et al., 2007). With high blood pressure
serving as a primary risk factor for CVD in women, understanding factors associated with
hypertension are of paramount importance. As pointed out earlier, accentuated stress responses
are one such factor, hence the interest in laboratory stress testing during the various cycle phases
when estrogen waxes and wanes. Not only can exogenously administered estrogen attenuate
the stress response (Lindheim et al., 1994), the follicular phase of the cycle, which is estrogen
dominant, is associated with less hemodynamic reactivity to stressors. While estrogen peaks
again during the luteal phase, the luteal phase is progesterone dominant, and as such, estrogen
effects may be opposed by progesterone. For example, progesterone is associated with enhanced
HPA activity (Roca et al., 2003). However, when it comes to preventing dramatic changes in
blood pressure, progesterone works in conjunction with estrogen, and as such, mid-luteal high
levels of ovarian steroids may prevent increased hemodynamic reactivity to laboratory stressors.
Conversely, increased hemodynamic and other measures of psychophysiological reactivity
observed during the late luteal phase are often explained via hormone withdrawal responses
and/or associated premenstrual symptomatology.
3.2.2 Plummeting hormones and withdrawal
As can be seen in Figure 3.1, during much of the luteal phase, both estrogen and progesterone
reach relatively high levels, with progesterone levels surpassing estrogen levels. The effects of
elevated estrogen and progesterone are vast, affecting most, if not all, systems of a womans body.
Of particular note, elevated progesterone has both diuretic and anxiolytic effects.
The neuroactive effects of progesterone on GABA receptors contribute to the anxiolytic effects
of elevated progesterone during the luteal phase. GABA-ergic circuits within the central nervous
system are inhibitory in nature, and these circuits are found throughout the limbic system and
are implicated in emotion regulation processes. There are subclasses of GABA receptors and the
one relevant to this discussion is the GABAA receptor. Herein we will refer to this simply as the
GABA receptor. With regard to ligand-binding processes, progesterone and the progestogenic
metabolites allopregnenalone and pregnenalone serve as ligands to the steroid-binding site on
GABA receptors. As this ligand-binding interaction results in inhibitory post-synaptic potentials,
the result within a neural circuit is attenuated firing or signaling. This attenuation within limbic
circuits that regulate negative emotions, such as circuits involving the amygdala, is implicated in
the anxiolytic effects of progesterone observed during the mid-luteal phase.
However, when progesterone plummets during the late luteal phase, women can suffer a
withdrawal effect. Withdrawal refers to symptoms that oppose endogenous effects of a hormone.
Thus, during the late luteal phase when progesterone levels plummet, women may experience
anxiety-like symptoms due to progesterone withdrawal. This withdrawal effect has been used
in the literature to explain the increased psychophysiological responses to stress provocation
during the luteal phase compared to the follicular phase when progesterone is steady and at very
low levels (Figure 3.1). Moreover, this withdrawal may also explain the hemodynamic responses.
Given that the opposite effect of vascular dilation is constriction, progesterone withdrawal during
the late luteal phase likely contributes to exaggerated blood pressure responses during stress
provocation. However, the withdrawal effects on vascular resiliency would also likely affect
baseline levels of blood pressure, which is not consistently observed. As such, other factors are
likely operating to affect accentuated hemodynamic reactivity during the luteal phase.
3.2.3 Increased premenstrual symptomatology
One such factor is premenstrual symptomatology. The majority of freely cycling women of
reproductive age experience some degree of negative symptomatology during the late luteal
phase of the menstrual cycle (for discussion see: Lustyk and Gerrish, 2010). While symptom type
can vary between women and within one woman from cycle to cycle, they most likely include
affective and somatic changes such as depression, anxiety, breast tenderness, and headaches.
Symptom severity also varies across women and cycles ranging from molimina, which refers to
the subclinical level of premenstrual symptomatology experienced by most women, to PMDD,
a mental health condition characterized by the most severe symptomatology with concomitant
impaired quality of life and disease burden.
Yet, in studies where premenstrual symptomology effects on stress provocation have been
systematically investigated, results have been inconsistent. For example, Girdler et al. (1998)
found that peripheral resistance and norepinephrine reactivity in response to a cognitive stressor
was greater in women prospectively diagnosed with PMDD compared to healthy control women
irrespective of the cycle phase of testing. However, women with PMDD evinced lower cardiac
output, stroke volume, and cortisol responses compared to controls. While the findings of Girdler
and colleagues suggest that symptoms may be the primary predictor of stress responses over and
above cycle phase, Woods et al. (1994) observed greater electromyogram and skin conductance
responses to cognitive-based laboratory stressors administered during the luteal cycle phase
in women meeting criteria for PMS compared to less symptomatic women. These cycle phase
differences align with those observed by Epperson et al. (2007) in a study of women prospectively
diagnosed with PMDD who evinced a significantly greater acoustic startle response during the
luteal phase compared to the follicular phase.
Still, generalizations from clinical samples are limiting. The severity of symptoms among those
diagnosed with PMDD or PMS exceeds that of subclinical samples, which makes up the majority
of women, and therefore may not best explain the luteal phase increases in stress responses of
freely-cycling, healthy women. On the other hand, studying stress processes in women and the
role that menstrual cycle phase plays in those processes can inform treatment plans for various
mental and physical conditions that affect women.
3.3 Stress and mental health conditions

Given the complex interactions among stress and cycle phase in women, it is not surprising that
both stress and ovarian hormones are associated with the etiology and symptom expression of
various mental health conditions affecting women. For example, both elevated cortisol and low
levels of estrogen and progesterone have been implicated in the development of depression in
women. More specifically, the likelihood of developing melancholic depression has been linked to
low levels of estrogen in women (Kammerer et al., 2006). These hormonal factors may be affected
by stressful life events. According to the Diathesis-Stress Model of Depression, stressful events
can activate depressogenic cognitive structures in vulnerable individuals (Ingram et al., 1998).
Thus, the presence of stressful life events, which raise cortisol levels, has the potential to trigger
depression in women. When considered with the potential for heightened reactivity to stressors
during the luteal cycle phase, which repeats monthly, certain women may be more vulnerable
to stressful life events and thus be more likely to suffer from depression. This carries therapeutic
implications for depressive patients, as treatments could potentially target luteal phase hyper-
reactivity. Given that depression is associated with an increased risk for developing myriad
physical maladies including diabetes, dyslipidemia, coronary heart disease, and stroke (Wiltink
et al., 2011), evidence supporting unique avenues for increased therapeutic efficacy is noteworthy.
Along with depression, prevalence rates for general anxiety disorders and eating disorders also
favor women, and again, hormonal factors and stress are implicated in symptom expression.
For example, Ziora et al. (2011) found women with anorexia nervosa to have significantly lower
estradiol and elevated urinary cortisol levels than both healthy control and obese research
participants. Again, studying stress processes that may inform symptom expression or provide
an understanding of predictive mechanisms would allow for targeted cycle phase approaches to
interventions.
A cycle phase approach in an intervention study was successfully implemented to treat women
with substance use disorder. Carpenter et al. (2008) randomized women to start treatment for
smoking during either the follicular or luteal phase of their menstrual cycle. Given that the
premenstruum is associated with more frequent and intense cravings, they hypothesized that
women who started treatment during the follicular phase, when ovarian hormones are relatively
low and steady and cravings are less, would fare better at their quit attempts than those with a
luteal start. Results revealed significantly greater abstinence rates for women with a follicular
start to treatment compared to women with a luteal start. Given that cravings are a form of stress
for substance abusers, these findings suggest that controlling for cycle phase and stress during
treatment implementation allows for more favorable outcomes. These findings argue in favor of
treatment tailoring approaches for clinical interventions and more generally counter the One size
fits all model of therapeutics in which gender specifics are ignored.
3.4 Conclusions and future clinical implications

Clearly, the phase of a womans menstrual cycle can help or hinder her response to stressful life
events. Some clinicians are beginning to recognize this and to incorporate this knowledge into
treatment plans. However, a recent report from the IOM (Institute of Medicine, 2010) found
that women are still largely underrepresented in clinical trials, and when women are included
in clinical trials, data analysis often fails to take cycle phase of testing or gender differences into
account. Future research should recognize the biopsychosocial differences between men and
women, in particular the impact of ovarian hormones on stress reactivity. In doing so, specific
treatment plans can be developed for more favorable outcomes in women.
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Puberty, menarche and
the menstrual cycle
4. Age at menarche: international perspectives
M. Celik
Department of Family Medicine, Faculty of Medicine, Kahramanmaras Sutcu Imam University,
Kahramanmaras, Turkey; drmcelik@ksu.edu.tr
Abstract
From both social and medical perspectives, menarche is often considered the central event of
female puberty as it signals the possibility of fertility. The aim of this report is to review the
menarcheal age in different countries and consider the factors that influence it. We also review
the implications of early or late menarcheal age on a young womans life. The improvement
of socioeconomic conditions during the 20th century resulted in an earlier onset of puberty
in children, indicated by the fall of the age at menarche (AAM). However, there are reports
from industrialised countries that this trend has been levelling off. There is high variability due
to the many factors (i.e. neuroendocrin, genetic, socioeconomic and environmental) that act
synergistically on the onset of menstruation. Menarcheal age has important health implications,
as early menarche is associated with more cardiovascular incidents and other ailments, including
cancer mortality, especially of the breast. Late menarche is associated with osteoporosis and
an increased fracture risk. Moreover, early menarche has been related to anxiety symptoms,
depression, premature intercourse and violent behaviour. In general, mean ages at menarche
13.0 years are recorded in central-southern Europe, Russia, China, Japan, many countries of the
Americas, Australia and New Zealand, while higher mean menarcheal ages are found in Africa,
many Asian countries and many developing and geographically disadvantaged countries (i.e.
those at high altitudes). The AAM in Asian countries is similar to that of Mediterranean girls.
More and bigger studies involving a larger population base and considering the effecting factors
are required to predict menarcheal ages more accurately.
Keywords: age at menarche, menstruation, international
67
M. Celik
Summary points
From both social and medical perspectives, menarche is often considered the central event of female puberty,
as it signals the possibility of fertility.
Age at menarche (AAM) was first calculated in the mid 19th century in Denmark. There are three methods
for assessing AAM: the status quo, the recall or retrospective and the prospective method.
There is high variability, due to multiple factors (i.e. neuroendocrin, genetic, socioeconomic and environmental)
acting synergistically on the onset of menstruation.
It is thought that during the 20th century, the dramatic improvement of socioeconomic conditions and the
general health of the population in industrialised countries resulted in an earlier onset of menarche in children
(secular trend).
Early menarche is associated with increased body mass index, insulin resistance, total number of metabolic
syndrome components (and hence increased cardiovascular risk), slightly higher bone mineral density, risk
of breast cancer, risk of ovarian cancer and risk of preeclampsia.
Early menarche leads to earlier sexual intercourse and is a risk factor for adolescent depression, elevated
social anxiety symptoms and a higher risk of substance use in mid-adolescence.
Late menarche is associated with osteoporosis and has an impact on school, work and social status.
In general, mean AAM of 13.0 years are recorded in central-southern Europe, Russia, China, Japan, many
countries of the Americas, Australia and New Zealand, while higher mean menarcheal ages are found in
Africa, many Asian countries and many developing and geographically disadvantaged countries, i.e. those
at high altitudes. AAM of girls in Asia is similar to Mediterranean girls.
More studies involving a large population base and considering all effecting factors are required to predict
accurate menarcheal age.
Abbreviations
AAM Age at menarche
BMI Body mass index
CI Confidence interval
4.1 Introduction
Menarche is the onset of menses or the first time females experience menstruation. It is the
culmination of a series of physiological and anatomic processes of puberty. The first sign of
puberty in females is the breast bud formation, the onset of breast development. Next is the
appearance of pubic hair and then finally, menarche. From both social and medical perspectives,
menarche is often considered the central event of female puberty, as it signals the possibility of
fertility (Karapanou and Papadimitriou, 2010). AAM was first calculated in the mid 19th century
in Denmark (Manniche, 1983). There are three methods for assessing AAM: the status quo, the
recall or retrospective and the prospective method (Cameron, 2002).
4.2 Determinants of age at menarche

There is high variability due to multiple factors acting synergistically on the onset of
menstruation. Karapanou and Papadimitriou (2010) reviewed the recent developments and the
current knowledge in the neuroendocrinology of pubertal onset and the factors, genetic and
environmental, which influence menarcheal age. According to this review, the AAM depends
on a genetic component and certain important environmental characteristics. For example, a
hot climate causes greater precocity, whereas high altitude, isolation and a rural environment
are responsible for delayed menarche. No less important are the girls general living conditions,
including nutrition and health status, physical activity, sibship size, BMI and birth order,
educational level, income and the profession of the parents. In addition, the presence of sisters,
especially older ones, in the household while growing up was associated with delayed menarche
(Ellis and Garber, 2000). Other stresses like acute/chronic illnesses (Van de Berghe et al., 1998) or
war (Tahirovie, 2000) suppress the hypothalamic-pituitary-gonadal axis and delay pubertal onset.
4.3 Secular trend of ages at menarche

A secular trend as defined in the Epidemiology dictionary is a change in the distribution of an
outcome in a population during a specified time frame, usually a long period of time, generally
years or decades (Last, 2001).
Mean is considered the most important indicator of female puberty because of the ease
and reliability of its determination. During the 20th century, the dramatic improvement of
M. Celik
socioeconomic conditions and general health of the population in industrialised countries

resulted in an earlier onset of puberty in children. From 1830 to 1980, the mean AAM decreased
in Western Europe by about 3-4 months per decade, settling at around 12.5-13.0 years in these
countries (Ulijaszek, 1998). Meanwhile, a recent study from rural south Mexico, presumably a
poorer region of a poor country, found that the median age of menarche decreased from 14.8
years in 1978 to 13.0 years in 2000, a decrease of 1.8 years over a period of 23 years (Malina et al.,
2004). In south China, a significant secular trend in the age of onset of puberty and menarche
was observed between studies conducted in 1962, 1978 and 1993. In the most recent study, the
mean age of menarche was 12.38 years (Huen et al., 1997).
Wyshak (1983) observed a stasis in the mean AAM in the USA, which was confirmed by Malina
(1990), in samples referring to 1940, 1950 and 1960, in which the value remained around 12.8
years. Recent reports also suggest that the decline in the age at the onset of menses may be slowing
in US females. McDowell et al. (2007) reported a decline of approximately 2.5 months in the
mean age of menarche for US females between 1963-1970 and 1988-1994, and a further decline
of approximately 2.3 months (from 12.53 to 12.34 years) between 1988-1994 and 1999-2002.
However, there are conflicting indications, such as those of Nichols et al. (2006) who confirmed a
decrease of the mean menarcheal age for women born between 1919 and 1949 (13.1 to 12.7 years)
in three American states, but an increase to 13.0 years for those born between 1960 and 1969.
There are reports from industrialised countries that the menarcheal age has been levelling off or
showing an upward trend (Dan and Roberts, 1993; Lindgren et al., 1991). It was reported that
the secular trend in menarcheal age had ended or slowed down in some European countries,
including Great Britain, Iceland, Norway, Denmark, Hungary, Belgium and Poland (Juul et
al., 2006; Karapanou and Papadimitriou, 2010). In Denmark, 1,100 schoolgirls were studied
between 1991 and 1993 and were found to have a mean age of menarche of 13.42 years (Juul et
al., 2006), showing no decrease when compared with data collected in 1964. Similarly, a cross-
sectional study conducted in the Netherlands in 1997 found that the mean age of menarche (13.15
years) was unchanged from a 1980 study and only slightly lower than in 1965 (Mul et al., 2001).
Moreover, Prebeg et al. (1994) recorded a slight inversion of the trend in Croatia, from 12.87 to
12.95 in Split (1977-1982) and from 12.7 to 12.82 in Zagreb (1982-1991). A secular trend of ages
at menarche is shown in Figure 4.1.
4.4 Health effects of early menarche

Early menarche is associated with increased BMI, insulin resistance, the total number of metabolic
syndrome components and hence an increased cardiovascular risk (Feng et al., 2008; Remsberg et
al., 2005). There are also links to slightly higher bone mineral density (Chevalley et al., 2009) and
a risk of breast cancer (Velie et al., 2005), ovarian cancer (Lakshman et al., 2009) and preeclampsia
(Abetew et al., 2011). Early menarche leads to earlier sexual intercourse and is a risk factor in
adolescent depression (Kaltiala-Heino et al., 2003), elevated social anxiety symptoms (Blumenthal
et al., 2009) and substance use in mid-adolescence (Dick et al., 2000; Stice et al., 2001).
12.8
12.7
Mean age at menarche (years) 12.6
12.5
12.4
12.3
12.2
1960 1962 1964 1966 1968 1970 1972 1974 1976 1978 1980 1982 1984 1986 1988 1990
Birth year
Figure 4.1. A secular trend example. Moving average of age at menarche in one-year birth year groups in
44,487 women born 1960-1991 (Morris et al., 2011).
4.5 Health effects of late menarche

A cross sectional study of a group of Colombian university students demonstrated that AAM was
positively associated with the practice of at least 2 h of daily physical activity (Chavarro et al.,
2004). Menarche, on average, occurs later in athletes, including ballet dancers, than in the general
population, with the exception of swimmers, suggesting that intense exercise delays puberty
(Malina, 1983). Conversely, girls with a delay in puberty and the onset of menstruation feel that it
has an impact on school, work or social status, and would prefer to accelerate their growth spurt
through treatment (Crowne et al., 1991). A summary of the health implications of early or late
menarche is shown in Table 4.1.
Table 4.1. Health effects of early or late menarcheal age (Karapanou and Papadimitriou, 2010).
Early menarche Late menarche
Abdominal type obesity Osteoporosis

Insulin resistance Adolescent depression
Glucose intolerance Social anxiety symptoms
Cardiovascular risk
Coronary heart disease
Increased bone mineral density
Increased cancer mortality
M. Celik
4.6 Ages at menarche in different countries

A review by Parent et al. (2003) reported that studies carried out in northern Europe (where the
population is more ethnically homogeneous than in the USA) between 1983 and 2000 all placed
the mean age of menarche between 13.1 and 13.5 years. However, studies from the Mediterranean
countries of France, Italy, Spain and Greece between 1995 and 2002 found mean ages of menarche
of between 12.0 and 12.6 years. It is not clear whether the warmer climate or the presence of a
higher proportion of ethnic minorities is responsible for this striking difference. Similarly, in
the Asian countries of Japan, Hong Kong and Thailand, the range of mean ages of menarche
lay between 12.1 and 12.6 years, with a more recent study from Bangkok (Thailand) placing
the mean age of menarche at 12.1 years (Mahachoklertwattana et al., 2002). The influence of
socioeconomic status, a likely marker for better health and nutrition, was illustrated in a study
from India that reported the mean age of menarche at 12.1 years for well-off girls, and 15.4 years
for underprivileged girls (Parent et al., 2003).
In general, mean ages at AAM 13.0 years have been recorded in central-southern Europe,
Russia, China, Japan, many countries of the Americas, Australia and New Zealand, while higher
mean menarcheal ages are found in many African and Asian countries and many developing and
geographically disadvantaged countries (i.e. those at high altitudes). AAM in Asia is generally
similar to Mediterranean girls; mean menarcheal age in Hong Kong (Huen et al., 1997) and Japan
(Nanao and Hasegawa, 2000) is 12.38 and 12.2 years respectively, and in Greece (Papadimitriou
et al., 2008) and Spain (Salces et al., 2001) 12.27 and 12.34 respectively.
Table 4.2 is drawn from the paper by Thomas et al. (2001), which included the mean and/or
median AAM in many countries worldwide. The data was grouped by geographical macro areas
(the five continents and the Middle East) for easier interpretation by Danubio and Sanna (2008),
and the present study added data from other countries or more recent studies.
4.6.1 Age at menarche in the USA
In the study of Chumlea et al. (2003), the median AAM for all US girls was 12.43 years. Non-
Hispanic black girls start to menstruate earlier than non-Hispanic whites and Mexican American
girls, and this difference is significant when compared with the ages at which 10%, 25% and 50%
of white girls had started menstruating. However, the median (mean) AAM for all US girls has
not changed significantly in 30 years, with a shift of approximately four months only in that
period. Only 10% of all US girls, regardless of race, start to menstruate before approximately 11
years of age. Girls are not gaining reproductive potential earlier than in the immediate past with
regards their menstrual status. Non-Hispanic black girls, however, are 5.5 months younger at
menarche than 30 years ago (Chumlea et al., 2003).
Table 4.2. Mean (or median*) age at menarche in the different countries.
Continent Country Age at Continent Country Age at

menarchea menarchea
Europe Greece 12.0/12.3b Middle East Israel 13.3

Spain 12.3 Yemen 14.4
Italy 12.5/12.4c Asia Thailand 12.3/12.1i
Hungary 12.9 China 12.4*
Belgium 13.0* Hong Kong 12.4
Denmark 13.0 Japan 12.5
Russia 13.0 Indonesia 13.0
Switzerland 13.0 Sri Lanka 13.5
France 13.1 Philippines 13.6
Iceland 13.1 Korea 13.8j
Poland 13.1 Malaysia 14.2
Sweden 13.1 India (Punjab) 14.3
Finland 13.2 Bangladesh 15.8
Norway 13.2* Nepal (high altitude) 16.2*
Britain 13.3/12.7d Africa Congo-Brazza 12.0
Turkey 13.3/13.1e South Africa (black) 12.4k
Ireland 13.5 Egypt 13.2
Romania 13.5 Zimbabwe 13.5
East Germany 14.0* Zambia 13.7
Czechoslovakia 14.6 Congo-Kinshasa 13.8
Americas Mexico 12.4/13.0f Morocco 13.8*
Argentina 12.6 Sudan 13.8
Dominican Rep. 12.6 Ghana 14.0
Venezuela 12.7 Algeria 14.3
USA 12.8/12.4g Kenya 14.4
Canada 12.72h Cameroon 14.6
Colombia 12.8 Somalia 14.8
Chile 13.0* Nigeria 15.0*
Cuba 13.0 Tanzania 15.2
Jamaica 13.1 Senegal 16.1
Peru 13.2 Oceania Tahiti 12.8*
Guatemala 13.8 New Zealand 12.9
Nicaragua 14.0 Australia 13.0*
Papua NG 15.8
a Data adapted from Thomas (2001), Danubio and Sanna (2008).

b Papadimitriou et al. (2008); c Rigon et al. (2010); d Morris et al. (2010); e Adal & Ko (2011); f Malina et
al. (2004); g Chumlea et al. (2003); h Al-Sahab et al. (2010); i Mahachoklertwattana et al. (2002); j Cho et al.
(2010); k Jones et al. (2009).
M. Celik
4.6.2 Age at menarche in South Africa
In a study by Jones et al. (2009), menarcheal age was estimated for 287 (188 black, 99 white) urban
South African girls born in Soweto-Johannesburg in 1990. The median menarcheal age for blacks
was 12.4 years (95% CI 12.2-12.6) and 12.5 years (95% CI 11.7-13.3) for whites. Data from six
studies of menarcheal age, including the current study, were analysed to examine the evidence
for a secular trend between 1956 and 2004 in urban South African girls. There was evidence of
a statistically significant secular trend for blacks, but not for whites. The average menarcheal age
for blacks decreased from 14.9 years (95% CI 14.8-15.0) in 1956 to 12.4 years (95% CI 12.2-12.6)
in the current study, an average decline of 0.50 years per decade. Less data was available for
whites, but the average menarcheal age decreased from 13.1 years (95% CI 13.0-13.2) in 1977 to
12.5 years (95% CI 11.7-13.3) in the current study, an average decline of 0.22 years per decade.
The decreasing AAM and the current lack of difference between blacks and whites is probably
reflective of the continuing nutritional and socioeconomic transition occurring within South
Africa (Jones et al., 2009).
4.6.3 Age at menarche in Canada
Al-Sahab et al. (2010)s study was based on female respondents aged 14 to 17 years during Cycle
4 (2000/2001) of the National Longitudinal Survey of Children and Youth (NLSCY). The total
number of girls analysed in this study was 1,403, weighted to represent 601,911 Canadian girls.
The estimated mean and median AAM was 12.72 years (standard deviation=1.05) and 12.67
years, respectively (Al-Sahab et al., 2010).
4.6.4 Age at menarche in Turkey
Ekerbicer et al. (2007) conducted a study in Kahramanmaras, in the east Mediterranean region
of Turkey, mean AAM was 13.04 years (95% CI 13.01-13.06), and median AAM was 13.00
years (95% CI 12.97-13.03). In another study, Adal and Ko (2011) used data taken from the
Turkey Demographic and Health Survey 2008. The mean AAM was estimated as 13.30 (95% CI
13.26-13.35), and estimated as 13.17 years (95% CI 12.95-13.38) for the youngest birth cohort
(1989-1993), as opposed to 13.44 years (95% CI 13.37-13.52) for the cohort born in 1959-1968.
Regression analysis indicated a decrease of 1.44 months per decade, providing evidence of a
secular trend in menarcheal age in Turkey.
4.6.5 Age at menarche in Mexico
Malina et al. (2004) studied the status quo menarcheal status of girls between 9 and 18 years of
age in 1978 (n=101) and 2000 (n=238), and retrospective ages at menarche of adult women 19+
years of age in 1978 (n=228) and 2000 (n=246) were obtained via interview. The median ages at
menarche of adolescents were 14.81.2 years (0.24 year, 95% CI 14.2-15.4) in 1978 and 13.01.0
years (0.10 year, 95% CI 12.7-13.3) in 2000. AAM has declined by 1.8 years over about 23 years, at
a rate of 0.78 years/decade (95% CI 0.65-0.91 year/decade). The estimated rates of secular decline
in AAM in adult women vary between 0.38 and 0.42 years/decade (0.260.56 year/decade).
4.6.7 Age at menarche in Britain
Morris et al. (2010) investigated factors that might affect menarcheal age in 81,606 women
participating in the Breakthrough Generations Study (BGS). Mean AAM was 12.71.5 years
(7-20 years).
4.6.8 Age at menarche in Korea
Cho et al. (2010) studied the Korean National Health and Nutrition Survey 2005, for which 3,562
women born between 1920 and 1985 were enrolled to identify secular trends in the AAM, and
620 girls born between 1986 and 1995 were recruited to evaluate the factors influencing the age
at onset of menarche. Mean AAM decreased from 16.901.25 years for women born between
1920 and 1925 to 13.791.37 years for those born between 1980 and 1985, indicating a downward
trend of 0.68 years/decade (95% CI 0.64-0.71) in AAM.
4.6.9 Age at menarche in Italy
In the study by Rigon et al. (2010) of 3,783 girls, the mean AAM was 12.41.3 years, and the
median age was 12.4 years (95% CI 12.34-12.46). For girls from northern Italy, the median AAM
was 12.44 years (95% CI 12.38-12.49), and for girls from southern Italy, the median AAM was
12.10 years (95% CI 12.00-12.28).
4.6.10 Age at menarche in France
Early menarche was assessed in the Health Behaviour in School-aged Children cross-sectional
survey. Data was collected in 2006 using an anonymous self-reported standardised questionnaire
from a nationally representative sample of 1,072 15-year-old girls in school classrooms.
Median and mean AAM were 13.0 years (interquartile range 12.0, 13.8) and 12.8 years (SD
1.2) respectively. In total, 57 girls were considered as early-matured (5.3% of the 15-year-old
population) (Gaudineau et al., 2010).
4.7 Conclusions
The literature identifies many things, such as neuroendocrin, genetic and environmental factors,
that affect the AAM. It seems there has been a decrease in the age of menarche over time from
generation to generation (secular trend). During the 20th century, the dramatic improvement
of socioeconomic conditions and general health of the population in industrialised countries
resulted in an earlier onset of menarche in children. From 1830 to 1980, the mean AAM decreased
in Western Europe by about 3-4 months per decade, settling at around 12.5-13.0 years in these
M. Celik
countries. In general, mean ages at menarche 13.0 years are recorded in central-southern
Europe, Russia, China, Japan, many countries of the Americas, Australia and New Zealand, while
higher mean menarcheal ages are found in Africa, many Asian countries and many developing
and geographically disadvantaged countries (i.e. at high altitudes). AAM in Asia is similar to
Mediterranean girls. More large-scale studies that consider the range of effecting factors are
required to predict accurate menarcheal ages.
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5. Menarcheal onset and body composition
S. Kirchengast
University of Vienna, Department of Anthropology, Althanstrasse 14, 1090 Vienna, Austria;
sylvia.kirchengast@univie.ac.at
Abstract
A strong association between menarcheal onset and body composition is still assumed since a
long time. In general, obesity and/or a higher amount of adipose tissue were seen as triggers of
pronounced menarcheal onset. Based on this assumption during the 1970s the so called Frisch
or critical weight/fat hypothesis was postulated, indicating body fat is the key factor signaling
that the body is ready for successful reproduction. During the 1980s this hypothesis was declared
to be obsolete, and the skeletal maturation was pointed out as key factor for menarcheal onset.
Nevertheless the association between weight status, body fat and female reproductive function
was still discussed intensively. The discovery of the hormones leptin and ghrelin offered new
proximate possibilities to explain the pathways of this association. From an evolutionary point
of view the linkage between body fat and reproductive maturation in human females may be
explained as a mechanism for ensuring reproductive success even under less favorable phases of
decreased food supply and energy shortages.
Keywords: age at menarche, body composition, fat distribution, energetics, leptin
79
S. Kirchengast
Summary points
The timing of menarcheal onset is of an important trait in the framework of human life history.
The timing of menarcheal onset and body composition are related.
Obesity and a higher amount of body fat enhance sexual maturation in girls.
Key factors signaling the readiness of the girls body for reproduction are skeletal maturation, in particular
pelvic breadth and a sufficient amount of adipose tissue.
The hormones leptin and ghrelin are discussed to signal energy status to the hypothalamic and initiate in this
way sexual maturation.
Abbreviations
BMI Body mass indice
GnRH Gonadotropin releasing hormone
HPG Hypothalamic-pituitary-gonadal axis
LH Lutenizing hormone
5.1 Introduction
Life history of recent Homo sapiens is characterized by sexual maturation in the first half of
the second decade of life. Sexual maturation reflects the transition from prereproductive to
reproductive phase, which is the result of pubertal transition and development. In girls sexual
maturation is achieved about two years earlier than among boys (Kirchengast, 2003). This
sex difference in reproductive development was already reported by ancient Greek or Roman
scientists such as Aristoteles or Soranus (Tanner, 1981). Aristoteles thought correctly that females
develop more quickly than males, but he explained this accelerated development of human
females incorrectly as a result of their lack of heat. Today this marked sexual dimorphism in
reproductive maturation is interpreted as an evolutionary adaptation to the different reproductive
strategies of human females and males. Although sexual maturation is a milestone in female
and male development, much more attention was traditionally paid on female reproductive
maturation. This is mainly due to that female reproductive maturation is not only characterized
by puberty associated somatic changes such as breast development, pubic hair growth and peak
height velocity, but also cumulates in the first spontaneous menstrual bleeding, the menarche. The
menarche is clearly a landmark of pubertal events among girls and is interpreted as the first visible
sign of a girls fecundity. Therefore in many cultures the first menstrual bleeding has signified
the passage from childhood to womanhood and is treated as a typical rite the passage. Since the
age at menarche is a marked single event, it is relatively easy to determine using retrospective or
status quo methods. Consequently the timing of menarche was documented since ancient times
and consequently is the best studied adolescent event until today.
5.2 Age at menarche: recent variation and historical trends

The first menstrual bleeding occurs between the ages of 8 and 19 years. The great majority of
girls in industrialized countries however experience menarche between the ages 10 and 15 years.
Therefore in industrialized countries an age at menarche before 9 or later than 16 years is classified
as a pathological variation. Nevertheless age at menarche shows a wide range between different
populations. Girls, who live under less favorable conditions such as in rural India or Papua New
Guinea may experience menarche much later (Worthman, 1999).
S. Kirchengast
It has to be stated however, that this early occurrence of the first menstrual bleeding in Europe
and other westernized Industrial countries is the result of a marked secular trend which is
documented for the last 170 years (Figure 5.1). From 1840 to 1980 a marked decrease (about 4
months per decade) of the age at menarche was observed for many European countries. During
the first half of the 19th century the mean age at menarche was about 16.5 years in Europe. In
the 1970s it settled at age of 12.5 to 13 years. In the 1980s this secular trend in menarcheal age
had slowed down or ended in many European countries such as Denmark, Iceland, Norway,
Great Britain, Belgium, Hungary and Poland. A similar trend was reported for the United States
(Danubio and Sanna, 2008).
The enormous variation of age at menarche among recent populations lead to the question, which
factors influence age at menarche or with other words, which factors are responsible for puberty
onset which results in menarche. In order to answer these questions we have to start with a short
description of the neuroendocrinological regulation of puberty.
5.3 Neuro-endocrine regulation of menarche

From an endocrinological point of view female sexual maturation is mainly linked to the
maturation of the hypothalamus-pituitary gonadal axis. The hypothalamus secretes GnRH, which
stimulates the anterior pituitary to secrete the gondotropines LH and FSH. The gonadotropines
stimulates the gonads (testes and ovaries) to produce androgens and estrogens mainly testosterone
and estradiol. The hypothalamus-pituitary-gonad axis is regulated by a negative feed-back
mechanism (Figure 5.2). The GnRH pulse generator, which is active during fetal period and early
neonatal period, is suppressed during infancy and childhood. Throughout childhood GnRH,
LH and FSH are secreted the levels however, are extremely low. At the age of about 8 years the
adrenarche occurs characterized by an increased secretion of androgens by the adrenals. This
18
16.6 16.5 16.2
16 15.8 15.7
15.1 14.6
14 13.4
12.7 12.5
Average age at menarche
12
10
8
6
4
2
0
<1850 18501870 18701890 18901910 19101920 19201940 19401960 19601980 19802000 >2000
Figure 5.1. Decrease in age at menarche since 1850: an example of a secular trend.
Hypothalamus
GnRH
Negative feedback
mechanism
Anterior pituitary
FSH LH
Androgen synthesis in theca cells

Follicle development Estrogen synthesis in the granulosa cells
Figure 5.2. Hypothalamus-pituitary-gonad axis.
endocrine event is the forerunner of pubertal development, which starts with the reactivation of
the GnRH pulsue generator and the hypothalamic pituitary gonad axis at the end of iuvenal stage
(Karapanou and Papadimitriou, 2010) (Figure 5.3). Pubertal transition itself is an event of short
duration, which includes beside hormonal alterations, somatic and behavioral changes. LH and
FSH levels increase markedly with a special increase of the night time secretion. During early
puberty this night time secretion expanded into day time hours. As a result the hypothalamus
pituitary gonad axis matures and estradiol and testosterone levels increase markedly. These
changes occur in both sexes, in girls this hormonal change stimulates not only follicle maturation
but also the endometrium. Finally the hormonal stimulation culminates in the first menstrual
bleeding, the menarche (Karapanou and Papadimitriou, 2010).
Fetal period and early Infancy and childhood Puberty

neonatal period GnRh pulse generator Reactivation of the
GnRh pulse generator is suppressed GnRh pulse generator
is active
Figure 5.3. Activation of the hypothalamus-pituitary-gonad axis.
S. Kirchengast
5.4 Factors influencing age at menarche

The factors that regulate the timing of endocrine changes which induce the onset of puberty
remain elusive up to now. It can be assumed that beside genetic disposition, environmental and
metabolic factors are critical regulators of the hypothalamus pituitary gonad axis (Karapanou
and Papadimitriou, 2010).
5.4.1 Intrinsic factors
Genetic disposition is seen as the main intrinsic factor of timing of sexual maturation, although
the specific genetic determinants are largely unknown (Karapanou and Papadimitriou, 2010).
Dvornyk and Waqar-ul Haq (2012) identified about 50 candidate genes for determining age at
menarche, however, specific genes have not been identified consistently. On the other hand it is
well documented that girls tend to experience menarche at the same age as their mothers and
sisters. This fact, but also the results from twin studies Tanner staging during puberty all display
greater concordance between monozygotic than dizygotic twins were interpreted as evidence
for a strong genetic influence on the timing of sexual maturation (Karapanou and Papadimitriou,
2010) (Figure 5.4).
5.4.2 Extrinsic factors
Environmental or extrinsic factors influencing the onset of sexual maturation are mainly
discussed to explain the well documented secular trends towards an earlier onset of sexual
maturation during the last 170 years. Recently one prominent hypothesis to explain the decrease
in menarcheal age since the 1990s is that the exposure to endocrine disruptors, i.e. chemicals that
interfere with steroid hormone activity, causes an earlier activation of HPG-axis (Euling et al.,
2008). The long term decline in age at menarche since 1840 is mainly attributed to improvements
in hygiene, but first of all nutrition during this time period (Euling et al., 2008). During the
last few decades the increasing obesity rates among prepubertal, children are also discussed as
relevant factors for decreasing age at menarche (Ahmed et al., 2009). Consequently body weight
Intrinsic factors
Genetic disposition
Hormonal situation
Age at menarche
Extrinsic factors
Nutrition
Infections
Hygiene
Energetics
Figure 5.4. Intrinsic and extrinsic factors influencing age at menarche.
and body composition, in particular body fat are discussed as one of the main extrinsic factors
influencing the timing of menarche and female reproductive function (Figure 5.4).
5.5 Body composition during childhood and adolescence

Homo sapiens diverge from other mammals by depositing large quantities of adipose tissue in utero.
Consequently humans are one of the fattest species on record at birth. This high amount of body
fat serves as insulation to compensate human hairlessness, but it is mainly interpreted as an energy
buffer during phases of increased energy demands such as infection or nutritional interruptions.
During infancy and childhood the amount of adipose tissue reduces but after adrenache body
composition changes. At this time sex differences in body composition are observable which
enhance during pubertal transition (Kirchengast, 2003). During pubertal transition both sexes
experience rapid increase in adipose tissue, although this increase is much higher among girls,
while boys experience a significant higher increase in lean body mass (Siervogel et al., 2003).
Puberty however, seems not only to induce typical changes in body composition, a typical kind
of body compositions seems to be essential for pubertal onset (Figure5.5).
5.5.1 Body composition and age at menarche
An association between body composition and reproductive function was has been recognized
at least since ancient times. The famous Greek physician Hippokrates (460-377 BC) noted in his
essay on the Scythians the girls get amazingly flabby and podgy. People of such constitution cannot
be prolific. Fatness and flabbiness are to blame. The womb is unable to receive the semen and they
menstruate infrequently and little.
However, descriptions of a negative association between body fat and reproductive function
were rare until the beginning of the 20th century, when Marshall and Peel (1908) described a
strong relationship between excessive fatness and sterility in animals. Stein and Leventhal (1935)
Stature
Body weight
Body fat
Girls Gynoid fat distribution
Lean body mass
Bone mass
Pubertal transition
Stature
Body weight
Lean body mass
Boys Bone mass
Body fat
Android fat distribution
Figure 5.5. Somatrometric factors and body composition during pubertal transition.
S. Kirchengast
pointed out the strong relationship between excessive overweight and reproductive failure among
patients suffering from PCOS.
Contrary much more attention was paid on the adverse effects of malnutrition and underweight on
female reproduction. An increased body weight and a higher amount of body fat were mainly seen
as promoters of sexual maturation. As early as 1610 the Tyrolean physician Hippolyt Guarinoni
(1571-1654) described his observations about age at menarche in his largest book Die Grewel
der Vewstung des menschlichen Geschlechts: The peasant girls in general menstruate much later
than the daughters of the townsfolk or the aristocracy, and seldom before the seventeenth, eighteenth
or even twentieth year. The townsfolk have usually borne several children before the peasant girls
have yet menstruated. The cause seems to be that the inhabitants of the towns consume much more
fat foods and drink and so their body become soft, weak and fat and come early to menstruation, in
the same way as a tree which one waters too early produces earlier but less well formed fruit than
another. Guarinoni was the first who associate body weight or body composition clearly with
menarcheal onset.
A positive association between body fat and sexual maturation and reproductive success was also
observed by several African ethnicities, such as the Annang in Nigeria, who practice traditional
fattening of prepubertal girls in so called fattening rooms to enhance sexual development and
increase fertility (Brink, 1989).
During the second half of the 20th century and increasing number of studies focused on the
relationship between body fat and sexual maturation. According to Dunger et al. (2006) Tanner
observed in 1955 from the Harvard Growth Study that early maturation, based on age at peak
height velocity, was associated with a higher weight/height ratio and Garn and Haskell (1959)
documented the impact of increased weight gain and body fat acquisition during childhood on
earlier menarcheal onset (Dunger et al., 2006). Especially the association between overweight and
obesity during childhood and earlier pubertal development was considered.
The most prominent hypothesis concerning the association patterns between body weight or
body composition and menarcheal age was published by the US American biologist Rose Frisch.
Together with her coworkers Roger Revelle and Janet Ward McArthur, Rose Frisch postulated the
existence of a critical weight cut-off. In their study of early and late maturing girls they suggested
that there may be a critical body weight of 48 kg required for normal pubertal development and
the onset of regular menstrual cycles (Frisch and Revelle, 1970, 1971; Frisch and McArthur,
1974). This critical weight of 48 kg was based on the extensive review of longitudinal growth data
of 181 healthy girls. Frisch and her coworkers observed that the mean weight at age at menarche
was constant at about 48 kg for girls who experienced menarche younger than 12 years, between
12 and 13 years, between 13 and 14 years and older than 14 years. In contrast to body weight,
stature height increased progressively with increasing age at menarche (Kaplowitz, 2008). This
association between improved nutritional status and decreasing age at menarche resulted in the
introduction of the so called Frisch hypothesis or hypothesis of critical body weight during the
early seventies (Frisch and Revelle, 1970, 1971; Frisch and McArthur, 1974).
Later Frisch modified her theory and a critical fat mass (Figure 5.6). According to this critical
fat mass hypothesis a minimum level of body fat (i.e. stored, easily mobilized energy) is required
for the onset and the maintenance of regular menstrual cycles (Frisch and McArthur, 1974). In
particular 17% body fat are absolutely necessary for the onset of menarche and 22% of body
fat are needed for maintenance of reproductive capacity (Frisch, 1994). Rose Frisch explained
this strong influence of body fat on sexual maturation and subsequent regular cycles with the
high energetic costs of gestation and lactation in humans. A successful pregnancy requires about
50,000 kcal additionally to normal metabolic requirements. Lactation requires between 500
and 1000 kcal additionally per day. 16 kg body fat is an equivalent of 144,000 kcal (Figure 5.7).
This energy store may enable the female body to maintain pregnancy and lactation even under
less favorable conditions when energy supply is reduced. From an evolutionary viewpoint this
explanation makes sense because our ancestors did not live in the Garden of Eden, and shortfall
of nutritional supply belonged to the common experiences during evolution and closer history
of Homo sapiens. Therefore body fat evolved to the key factor signaling the female that the body
has accumulated enough energy storages to reproduce successfully.
The critical body weight/fat hypothesis was corroborated by the well documented fact that girls
or women who are underweight or too lean, because of injudicious dieting or excessive physical
activity such ballet dancers and elite athletes experience primary or secondary amenorrhoea or
Minimum body weight

(48 kg) Indicating sufficient Menarcheal onset
Minimum body fat energy stores
(17%)
Figure 5.6. The Fritsch hypothesis postulating a minimum body weight as essential for menarcheal onset.
Lactation
Pregnancy Successful
500 to 1000 kcal
50,000 kcal reproduction
additionally per day
Figure 5.7. Energetic requirements for successful reproduction.
S. Kirchengast
a reduction of the reproductive capability (Frisch, 1994). Nevertheless, the original hypothesis
that menarcheal onset was associated with the attainment of a critical weight or fat mass was
plagued by faulty statistical analyses, methodological problems of body composition assessment
and extended critique in the literature (Ellison, 1990).
During the eighties the Frisch hypothesis was declared to be obsolete. Ovarian function was
related to energy balance and skeletal maturation, in particular pelvic breadth rather than a critical
body weight or amount of adipose tissue (Ellison, 1990). Two evolutionary trends resulted in two
contrasting sets of selection pressures on the female pelvis. On the one hand encephalization, the
trend towards increasing cranial capacity required increasing cranial dimensions of the fetus, on
the other hand the evolution of biped locomotion, favored narrower pelves by biomechanical
reasons. As a consequence birth became an enormous problem for human females. A too narrow
or inmatur pelvis makes birth impossible and results consequently in the death of mother and
child. Therefore Ellison suggested that skeletal maturation is the key factor signaling that the
body is ready for reproduction. Sexual maturation in girls mean that the girls reproductive
phase starts and for a successful reproduction including a safe birth process a minimum pelvic
breadth is absolutely necessary. There is no doubt that sexual maturation and growth are strongly
interrelated. Both are based on genetic disposition and are influenced by extrinsic parameters
such as somatic or psychic stress factors, which may result in delayed sexual maturation and
growth retardation. Sufficient growth is essential for girls to achieve reproductive maturity.
Therefore in girls the first spontaneous menstrual bleeding occurs shortly after the peak height
velocity took place. Consequently sexual maturation in girls is strongly related to skeletal growth,
especially of the bony pelvis (Figure 5.8). It could be shown that a minimum pelvic breadth of
24 cm is necessary that menarche takes place (Ellison, 1990). Furthermore the secular trends
towards an earlier sexual maturation, which was documented for Europe and the USA since the
mid-19th century is accompanied with an increase in body height. The strong association between
sexual maturation and skeletal development however, does not contradict the impact of energy
stores, i.e. adipose tissue, on sexual maturation in girls. Sufficient energy supply is also required
for an undisturbed growth process. Malnutrition and underweight result in growth retardation
and a delay of sexual maturation. On the other hand it is well documented that overweight an
Minimum pelvic
Peak height velocity breadth of 24 cm
before menarcheal Sexual maturation
makes vaginal birth
onset possible
Figure 5.8. The impact of skeletal growth on female reproductive maturation.
obese children are taller than their normal weight counterparts. Therefore the effects of skeletal
maturation and body fat on reproductive maturation in girls may be two sides of the same coin.
However, up to now the association between body composition parameters and pubertal onset
is still discussed controversially, although the discovery of hormones such as leptin and ghrelin
offered completely new explanations for the pathways of body fat pubertal hormonal transition
(Kaplowitz, 2008). While some authors found no support of the hypothesis that a critical body
weight/fat is absolutely necessary for menarcheal onset (Sherar et al., 2007), other authors
introduced a new aspect concerning the relationship between body fat and age at menarche
(Lassek and Gaulin, 2007). Lassek and Gaulin (2007) analyzed body fat, fat distribution and age
at menarche among girls aged 10 to 14 years from the National Health and Nutrition examination
survey (NHANES III) and showed that fat distribution rather than total body fat may be related to
menarcheal onset. In particular the found a strong relationship between lower body fat, i.e. body
fat at the gluteofemoral region and an earlier age at menarche. Even girls with a low amount of
total body fat started to menstruate when they had stored relatively more fat in fat depots at the
hips, thighs and buttocks. Furthermore the authors documented a negative association between
age at menarche and high waist as well as thigh circumference, but a positive association between
age at menarche and waist circumference, even among young women who had already completed
linear growth. The positive association between age at menarche and waist circumference may be
seen as a result of increased androgen secretion among girls with increased abdominal fat depots.
This observation that lower body fat depots enhance sexual maturation support the critical fat
hypothesis because fat depots in the lower body region are excellent energy stores even during
phases of increased somatic stress such as pregnancy and lactation. These fat depots remain
stable even under conditions of negative energy balance. Even young amenorrhoeic women
suffering from severe under nutrition as a result of anorexia nervosa show fat depots at the lower
body region. Nevertheless other investigators reported opposite observations. Guo and Ji (2011)
found a strong association between high waist circumference and early menarche. However these
authors did not distinguish between abdominal and lower body fat distribution. Centralized
or upper body fat distribution was described to be typical of postmenopausal women, women
suffering from PCOS or excessive obese women of reproductive age. Therefore centralized fat
distribution was mainly associated with reduced fertility. The relationship between earlier age at
menarche and high waist circumference may be explained by the positive association between
waist circumference and general overweight. Overweight and obesity are also still discussed as
relevant factors for the timing of sexual maturation.
5.5.2 Weight status and menarcheal onset
Another recent phenomenon which provokes a new discussion concerning body weight or body
fat and menarcheal onset relationships, is the association between the increasing prevalence of
childhood obesity and decreasing ages of menarche among US American girls (Himes, 2006).
Therefore the debate whether body composition and weight status are main extrinsic factors in
determining the timing of pubertal transition in girls still goes on. A large study of US girls, the
Pediatric Research in Office Settings (PROS) yielded that 48% of African-American girls and
S. Kirchengast
15% of white girls showed initiated breast and/or pubic hair development at the age of 8 years
(Herman-Giddens et al., 1997). The study was based on a large number of girls (>17,000) and
prompted enormous response in mass media, but also in scientific literature because the estimates
of the ages of pubertal onset were markedly earlier than had been reported previously (Himes,
2006). A new secular trend in age at menarche associated with increasing obesity prevalence was
feared. Between the 1960s and 2000 the prevalence of childhood obesity increased significantly
in the majority of industrialized countries. During this time period the average age at menarche
decreased by more than 3 months among white girls and 5.5 months among Afro-American
girls in the United States (Kulie et al., 2011). Furthermore sexual maturation among such
young girls represents a psychosocial but also a health problem. On the one hand such early
sexual maturation enhances adolescent risk taking behavior, the risk of extremely early sexual
contacts and teenage pregnancies, on the other hand it is well documented that women with a
history of earlier menarche have an increased risk adult obesity, diabetes type II, cardiovascular
diseases and breast and colon cancer. Nevertheless the data reported by Herman-Giddens et
al. (1997) remain controversial. The PROS study was neither nationally representative nor was
the assessment of breast development methodological correct. The discussion regarding the
relationship between weight status and sexual maturation continued. While several authors
found no significant association patterns between weight status, in particular obesity and advance
pubertal development (Himes et al., 2009), others reported a strong correlation between weight
status and age at menarche (Bralic et al., 2012). Overweight or obesity during childhood seems
to enhance pubertal development in girls. Obese girls experience the onset of puberty, i.e. their
first menstrual bleeding at a significantly younger age than their normal-weight or under-weight
counterparts (Kulie et al., 2011). On the other hand underweight or a lack of adipose tissue mass
was described to be associated with delayed menarcheal onset. This was true ballet dancers and
elite athletes, but also of populations suffering from chronic malnutrition (Simondon et al., 1997).
5.5.3 Long term effects of menarche and body composition relation patterns
Another important aspect of the association patterns between body composition and sexual
maturation are the long term effects of early or late maturation on adult body composition and
weight status. Data from several retrospective studies of large scale indicated that early maturing
girls, i.e. menarcheal onset before the age of 12 years have a higher BMI during adulthood
compared to late maturing girls (Rachon and Teede, 2010). A large population based study, the
Norfolk cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC),
containing data of 13,308 mainly white European women, demonstrated that earlier age at
menarche increased the risk of adult type II diabetes and adult obesity (Lakshman et al., 2008).
However the causality between early menarche and obesity during adulthood is much debated.
It has been suggested that overweight or obesity during infancy or early childhood are the key
factors contributing to earlier sexual maturation and obesity during adult life (Figure 5.9).
Only few studies considered the impact of age at menarche on long term development of body
composition. Frisancho and Flegel (1982) reported an association between menarcheal age and
adult fat distribution. In particular they found an association between early maturation and
Early age at menarche
Increased risk of Increased risk of Increased risk of Reduced risk of

adult type II adult obesity breast cancer oestoporosis
diabetes
Figure 5.9. Long term effects of early age at menarche.
centralized fat distribution patterns among adult women. This association could not be proved
by Kirchengast et al. (1998), while in contrast a significant impact of age at menarche on body
weight, BMI and the amount of fat tissue could be proved. According to the results of this study
early-maturing females were shorter, but heavier and exhibited a higher amount of fat tissue than
late-maturing ones. This association was mainly found among premenopausal women. After
menopause the associations between age at menarche and body composition weakens.
5.6 R easons of the association patterns between age at menarche and body
composition
The association between body compostion and menarcheal onset can be viewed from a
physiological or proximate or an evolutionary or ultimate viewpoint.
5.6.1 Proximate or physiological explanations
As pointed out above the hallmark of pubertal transition is the reactivation of the hypothalamic
pulsatile secretion of GnRH, which activates the HPG and the production of sex hormones.
For a long time it was unclear which factors influence these neural networks, in particular how
information about the developmental status and energy stores is passed to the hypothalamus.
Rose Frisch tried to explain this effect by the fact that adipose tissue acts as a source of sex
hormones, this explanation however, was not satisfying (Frisch, 1994). The discovery of
the adipocyte-derived protein leptin in 1994, provided a molecular basis for the interaction
between energy stores and pubertal onset. The demonstration that leptin synthesis is directly
related to the amount of adipose tissue, thus energy stores, that it is involved in the control
of energy homeostasis, and that leptin modulates several neuroendocrine systems, including
HPG turned leptin into the paradigm of a hormone involved in the control of metabolism and
sexual development (Martos-Moreno et al., 2010). Leptin turned out to be the key factor in the
association between body fat and age at menarche. While leptin levels in prepubertal children
are low, leptin levels increase during pubertal transition in both sexes, however this increase
is much higher among girls. Furthermore girls show higher pulse amplitude in the circadian
rhythm of leptin secretion in comparison to boys (Martos-Moreno et al., 2010). Furthermore
the positive correlation between leptin and pubertal transition is more pronounced in females.
S. Kirchengast
It can be assumed that higher amounts of adipose tissue enhance leptin concentrations. From a
physiological point of view leptin can be considered to be the main peripheral signal providing
information about body fat to the hypothalamic. In this way the endocrine regulation of the body
composition age at menarche relationship may be explained. Beside leptin the gastric hormone
ghrelin, which is mainly produced by oxyntic cells in the gastric mucosa in the proximal intestine,
was related to pubertal onset. Ghrelin levels increase during the first two years of life, when the
GnRh pulse generator acitivity declines. Ghrelin correlates negatively with body fat and decreases
during pubertal transition when the amount of body fat increases. The decreasing ghrelin
levels during pubertal transition may signal the increase in energy stores to the hypothalamic.
Therefore a putative role of ghrelin as a signal of energy insufficiency, in determining the onset
and progression of puberty is suggested (Martos-Moreno et al., 2010) (Figure 5.10).
5.6.2 Ultimate or evolutionary explanations
According to Theodosius Dobzhansky nothing in biology makes sense despite in the light
or evolution. Therefore from a bioanthropological viewpoint it is absolutely necessary to
provide an evolutionary interpretation of the association patterns between body composition
and menarcheal onset. As pointed out above body fat represents the most important energy
store. Sufficient energy stores are of special importance for female reproductive fitness because
gestation and lactation each have enormous energy costs. About 50,000 kcal additionally to
normal metabolic requirements are needed to complete a pregnancy successfully. To insure the
infants nutritional needs successful lactation is essential. Lactation however, requires between
500 and 1000 kcal additionally per day. Today well-nourished females have no problems to meet
these energetic demands, but our ancestors experienced periods of food shortages and starvation
resulting from climatic change, drought and uneven hunting and foraging success. Under these
conditions sufficient energy stores were absolutely necessary to reproduce successfully. Too low
Estradiol Leptin
Source: adipose tissue Source: adipocytes
increasing levels indicate increasing levels indicate
a sufficient amount of a higher amount
adipose tissue of adipose tissue
Energetic status
Ghrelin
Source: gastric mucosa of
the intestine decreasing
levels indicate increasing
amount of adipose tissue
Figure 5.10. Hormonal indicators of energetic status during pubertal transition.
energy stores might reduce reproductive success markedly. Energy deficiency during childhood
and prepubertal period may result also in delayed growth and too narrow pelvic dimensions.
Both make successful birth impossible and would result in maternal death. Therefore sufficient
energy supply and energy stores enhance reproductive fitness and an association between body
composition in particular body fat and sexual maturation evolved.
5.7 Conclusion
The results of numerous studies plead for an association between body composition, in particular
body fat, and timing of menarchel onset. Some studies reported a positive impact of fat tissue
on pubertal transition and on age at menarche. Up to now however, the explanation of the
association patterns between body composition and menarcheal onset are still under discussion.
The major problem is, that all studies that demonstrate a relationship between age at menarche
and body fat or body weight do not answer the question whether increased fat mass predisposes
to earlier menarche or earlier menarche and hormonal transition lead to an estrogen induced
increase in body fat. Furthermore it is still not clear whether obesity enhances menarcheal onset
or early menarche promotes obesity.
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of menarche. Annals of Human Biology 34, 673-677.
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Towne, B., 2003. Puberty and body composition. Hormone Research 60, 36-45.
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McKenna, J.J. (eds.) Evolutionary medicine. Oxford University Press, Oxford, UK, pp. 135-163.
6. Calcium intake and premenstrual syndrome
Division of Biostatistics and Epidemiology, Department of Public Health, School of Public Health
and Health Sciences, University of Massachusetts, 715 North Pleasant Street, Amherst, MA
01003-9304, USA; biki@schoolph.umass.edu; ebertone@schoolph.umass.edu
Abstract
Premenstrual syndrome (PMS) affects up to 20% of women of reproductive age, substantially
reducing quality of life. Levels of impairment due to PMS may be as severe as those of other
major dysphoric disorders and close to that of major depressive disorder. While the aetiology
of PMS is largely unknown, previous literature has suggested that calcium deficiency and
dysregulation of calcium-relating hormones during the luteal phase of the menstrual cycle
may play a role in PMS. Several randomized trials have reported calcium supplementation
to significantly reduce the severity and occurrence of premenstrual symptoms. In particular,
a large randomized trial observed a 48% reduction in total luteal symptom score among 248
women given calcium supplementation of 1,200 mg per day for 3 months. More recently, a sub-
study within the prospective Nurses Health Study II suggested that women with high dietary
intakes of calcium (median, 1,283 mg daily) had a significant 30% lower risk of developing PMS
compared to women with very low intakes (median, 529 mg daily). Further, in 2007, a large
prospective study observed that the metabolism of calcium and vitamin D across the menstrual
cycle is noticeably different between women with and without premenstrual dysphoric disorder.
Given that calcium supplementation and/or high dietary calcium intake has been consistently
associated with improvements in menstrual symptom in research studies, clinicians should
consider recommending calcium supplementation for women experiencing PMS.
Keywords: menstrually related disorders, micronutrients, oestrogen, calcium supplementation
95
Summary points
Women with premenstrual syndrome (PMS) may be suffering from calcium dysregulation during the luteal
phase of the menstrual cycle.
Cyclic fluctuations of oestrogen are correlated with changes in calcium concentrations.
Several randomized clinical trials suggest calcium supplementation may be an effective treatment for PMS.
Results from a large observational study in the prospective Nurses Health Study II cohort suggest high dietary
calcium intake may lower risk of incident PMS.
Based on the findings of large studies, clinicians should consider recommending supplementation of 1,200
to 1,600 mg of calcium daily to women experiencing PMS.
Abbreviations
PTH Parathyroid hormone
25(OH)D 25-hydroxyvitamin D
1,25(OH)2D 1,25-dihydroxyvitamin D
6.1 Introduction
PMS is defined by the recurrence of physical and psychological symptoms during the luteal phase
of the menstrual cycle. While it is estimated that 85-90% of women of reproductive age experience
some level of menstrual symptoms, 8-20% report moderate to severe symptoms associated with
substantially reduced quality of life and meet the clinical criteria for PMS (Deuster et al., 1999;
Dickerson et al., 2003; Sternfeld et al., 2002). Approximately 3-8% of women experience severe,
psychologically debilitating symptoms and significant inability to function, thereby meeting the
criteria for PMDD (Halbreich, 2004). Recent reports suggest that PMS may be underreported
and that the actual prevalence is likely higher, estimated at 20% among women of reproductive
age (Halbreich, 2004).
PMS symptoms begin on average between the ages of 25 and 35, but can develop at any time
during the reproductive years (Dickerson et al., 2003). Half of women seeking treatment for PMS
are suffering from major medical, psychological, and martial problems (Chuong and Burgos,
1995). Over 200 different physical and psychological symptoms have been associated with PMS.
Among psychological symptoms, irritability, tension, and depression are the most prominent.
Among physical symptoms, breast pain, abdominal bloating, and a sense of weight gain occur
most commonly. Common behavioural symptoms include lethargy and food cravings (Deuster
et al., 1999). Levels of impairment due to PMS and PMDD may be as severe as those from other
major dysphoric disorders and are comparable to that of major depressive disorder (Halbreich
et al., 2003).
The aetiology of PMS remains uncertain, largely because multiple physiologic mechanisms likely
underlie the diverse nature of menstrual symptoms. Recently it has been suggested that PMS
may be associated with calcium dysregulation and vitamin D deficiency during the latter half of
the menstrual cycle (Thys-Jacobs, 2000). Further, there are similarities between PMS symptoms
and those of hypocalcemia, including anxiety, depression, and fatigue (Thys-Jacobs, 2000; Thys-
Jacobs and Alvir, 1995). A number of randomized trials have shown calcium supplementation
to significantly reduce the occurrence and severity of physical and emotional premenstrual
symptoms (Penland and Johnson, 1993; Thys-Jacobs et al., 1989, 1998). High dietary calcium
intake may also reduce a womans risk of initially developing PMS. These relations are reviewed
below.
6.2 Calcium and the menstrual cycle

6.2.1 Menstrual cyclicity of reproductive and calcium-regulating hormones
Altered sensitivity to cyclic fluctuations in reproductive hormones during the menstrual cycle
is likely central to the occurrence of PMS. Symptoms are confined to the luteal phase of the
cycle when both progesterone and oestradiol levels are high, and abate after on the onset of
menses when reproductive hormone levels fall. Furthermore, symptoms are often improved
by treatments suppressing ovulation, including oral contraceptives and gonadotropin releasing
hormone agonists (Halbreich, 2003).
Oestrogen fluctuations during ovulation and the luteal phase of the menstrual cycle have been
observed to correlate with changes in calcium and calcium regulating hormones levels (Buchanan
et al., 1986; Gray et al., 1982; Muneyvirci-Delale et al., 1998; Thys-Jacobs, 2000; Thys-Jacobs and
Alvir, 1995; Tjellesen et al., 1983).
Calcium is an essential cation that plays an important role in intracellular and extracellular
events. Calcium levels are tightly regulated within a narrow physiologic range. Among its many
functions, intracellular calcium influences the synthesis of neurotransmitters implicated in PMS,
including serotonin, and changes in extracellular calcium could thus plausibly result in emotional
dysregulation and mood symptoms in PMS (Gardner et al., 2007; Rapkin, 1992).
The major hormones regulating plasma calcium levels are PTH, 25(OH)D and 1,25(OH)2D
(Figure 6.1). PTH is produced by the parathyroid glands and is controlled by extracellular calcium
concentration. A drop in plasma calcium level stimulates PTH secretion, while a rise in plasma
calcium decreases PTH secretion. 25(OH)D is the major circulating metabolite of vitamin D. It
must be hydroxylated in the kidney and in target tissues to form the biologically active metabolite
1,25(OH)2D. 1,25(OH)2D stimulates calcium absorption by the gastrointestinal tract. Hence,
vitamin D deficiency results in decreased calcium absorption and low plasma calcium (Gardner
et al., 2007). To maintain appropriate plasma calcium levels, the Institute of Medicine (USA)
recommends that women between the ages of 19 and 50 consume 1000 mg of calcium daily, along
with 600 IU of vitamin D (Institute of Medicine, 2010).
In 1978, Pitkin and colleagues assessed changes in calcium and calcium regulating hormone
levels across the menstrual cycle of seven healthy women (Pitkin et al., 1978). They observed that
levels of ionized calcium dropped prior to ovulation, while PTH and 1,25(OH)2D levels increased
throughout the follicular phase, both peaking in relation to low ionized calcium concentrations.
Peak PTH levels were 30-35% above early follicular and late luteal values. In subsequent studies,
Gray et al. (1982) and Tjellesen et al. (1983) reported that 1,25(OH)2D levels at ovulation were
nearly double those in the early follicular. However, neither observed changes in serum total
calcium level.
Plasma calcium level
PTH secretopm
(from parathyroid glands)
Plasma PTH rises
Calcium reabsorption Bone reabsorption

25(OH)D
1-hydroxylase
1,25(OH)2D
Calcium urinary 1,25(OH)2D

excretion
Calcium absorption
Plasma calcium
Figure 6.1. Hormonal control of plasma calcium.
6.2.2 Calcium regulating hormones and PMS
Ample evidence suggests that the fluctuation of calcium and its regulating hormones may differ
in women experiencing PMS and PMDD compared to symptom free women. Thys-Jacobs and
Alvir (1995) compared patterns in seven women with a documented history of PMS to those
of 5 asymptomatic women (Thys-Jacobs and Alvir, 1995; Thys-Jacobs, 2000). In both groups,
there was a significant decline in serum total and ionized calcium at mid-cycle, at the time of the
oestradiol peak (Figure 6.2). Among the asymptomatic women, PTH and 25(OH)D did not vary
significantly across the menstrual cycle. However, among women with PMS, PTH levels rose 30%
at mid-cycle. Significant differences were also observed between the groups for total calcium,
intact PTH, and 25(OH)D levels (Figure 6.3).
Thys-Jacobs et al. (2007) expanded upon this important initial work in 2007 in a prospective
study of 68 women with PMDD and 47 controls. Participants completed two months of daily
self-administered premenstrual symptom questionnaires and one month of hormonal evaluation.
Control participants had no prior medical history of PMDD or PMS. Total serum, ionized, and
urine calcium, PTH, and 1,25(OH)2D levels varied significantly over the menstrual cycle in both
groups. During menses, ionized calcium levels were significantly lower in women with PMDD
compared with controls (1.1660.072 vs. 1.1820.087 mmol/l; P=0.027). Similarly, urine calcium
excretion during the late follicular, mid-cycle, and early luteal phases was significantly lower in
a b
1,400 1.34
1,200 1.32
Ionized calcium (mmol/l)

1.30 P<0.05
1000
Estradiol (pmol/l)
Control
800 1.28
P<0.05 Control 1.26 P<0.03*
600 PMS 1.24 PMS
400 1.22 P<0.05 P<0.05
P<0.05
200 1.20
0 1.18
Early follicular LH maximum Late luteal Early follicular LH maximum Late luteal
Figure 6.2. Mean levels of (a) estradiol and (b) ionized calcium in seven women with postmenstrual syndrome
(PMS) and five asymptomatic women. * Indicates P-value comparing controls vs. women with PMS; other P-values
are for the change from one menstrual phase to the next within PMS cases or within controls (from early follicular
to luteinizing hormone (LH) maximum, or from LH maximum to late luteal phase) (Adapted from Thys-Jacobs,
2000).
a b
160 60
Intact parathyroid hormone (ng/l)
1,25-dihydroxyvitamin D (pmol/l)
140 50
PMS P<0.01
120 40
P<0.05* PMS
100 Control P<0.05*
30
Control
80
20
60
Early follicular LH maximum Late luteal Early follicular LH maximum Late luteal
c
140
120
25-hydroxyvitamin D (ng/l)
Control
100
80 P<0.01* P<0.01* P<0.05*
60
PMS
40
20
0
Early follicular LH maximum Late luteal
Figure 6.3. Mean levels of (a) 1,25-dihydroxyvitamin D, (b) intact parathyroid hormone, and (c) 25-hydroxyvitamin
D in seven women with postmenstrual syndrome (PMS) and five asymptomatic women. * Indicates P-value
comparing controls vs. women with PMS; other P-values are for the change from one menstrual phase to the
next within PMS cases or within controls (from early follicular to luteinizing hormone (LH) maximum, or from LH
maximum to late luteal phase) (Adapted from Thys-Jacobs, 2000).
women with PMDD. Luteal phase 1,25(OH)2D levels were lower in PMDD cases vs. controls, and
continually increased from the follicular phase to the late luteal phase (45.027.52 vs. 50.633.75
pg/ml; P=0.032). Although the prevalence of vitamin D deficiency was similar among both
PMDD and control groups, markers of within-cycle metabolism of calcium and 1,25(OH)2D
were significantly different between groups. The authors hypothesized that women in the control
group had greater access to stored calcium than those in the PMDD group.
Similar patterns of fluctuation in calcium-regulating hormones in women with PMS were

recently observed by Dullo and Vedi (2008). However, not all studies of these relations have
been consistent. Studies by Baran et al. (1980) and Muse et al. (1986) did not observe significant
changes in serum PTH, calcitonin, and 1,25(OH)2D across the menstrual cycle among healthy
young women.
6.2.3 Trials of calcium supplementation and PMS
The interesting findings of observational studies comparing calcium levels in women with and
without PMS led directly to clinical trials testing the effects of calcium supplementation on
menstrual symptoms. A number of randomized trials on calcium supplementation have shown
a significant reduction of premenstrual symptom severity and occurrence.
In 1989, Thys-Jacobs and colleagues conducted a randomized, double blind, crossover placebo-
controlled trial. 78 women were initially screened for inclusion, with 60 included in the trial
at baseline. Women were eligible if their symptom scores during the late luteal phase of the
menstrual cycle were at least 50% greater than those during the intermenstrual (i.e. post
menstruation) phase. Each participant completed a baseline daily self-assessment questionnaire,
recording the occurrence and severity of 14 symptoms (nervousness, irritability, crying, mood
swings, depression, fatigue, violent tendencies, abdominal bloating, headache, breast fullness,
increased appetite, abdominal cramps, back pain, and craving for sweets) on a four-point scale
(absent=0, mild=1, moderate=2, or severe=3) over one menstrual cycle. A participant met the
study definition of PMS if she suffered from cyclically recurring symptoms during the luteal phase
of the menstrual cycle, but was symptom-free following menses. Women with an active major
depression or a history of psychosis were excluded. Daily dietary assessments were completed
over a week to calculate each participants mean dietary calcium intake.
Each participant received treatment over 6 menstrual cycles, consisting of 3 menstrual cycles
of daily calcium supplementation (1000 mg of elemental calcium in the form of calcium
carbonate) and three menstrual cycles of placebo. Half of the participants received the calcium
supplementation first; the other half received the placebo first. Symptom scores were measured
at baseline, throughout the six cycles, and for one additional menstrual cycle off medication. A
retrospective assessment was also performed asking participants whether their overall symptoms
had improved during the first or second treatment of the study. Participants with less than 70%
overall compliance were excluded from analysis, and 27 women of the initial 60 dropped out
before treatment crossover. The remaining 33 women (mean age 34.6 years) were included in
the final analysis.
Compared to baseline, luteal phase symptom scores were 50% lower after three months of
calcium supplementation (3.33 vs. 6.71). In contrast, luteal phase symptom scores were only
20% lower after three months of placebo treatment (comparing treatments: P=0.011) (Figure 6.4).
Significant improvement in symptom scores with calcium supplementation compared to placebo
was also reported during the menstrual phase (P=0.032) but not during the intermenstrual phase
(P=0.935). The retrospective assessment of overall symptoms also demonstrated improvement
with calcium supplementation: 73% of participants reported fewer symptoms during the
calcium treatment, 15% reported fewer during placebo, and 12% indicated no preference. For
the four symptom factor scores evaluated, calcium treatment significantly reduced nervousness,
irritability, crying, mood swings, depression and violent tendencies (factor 1; P=0.045); fatigue,
abdominal bloating, headache and breast fullness (factor 2; P=0.003); and abdominal cramps and
back pain (factor 3; P=0.036) during the luteal phase. Calcium had no effect on symptom factors
during the intermenstrual phase.
This clinical trial was the first to demonstrate the benefits of calcium supplementation in
reducing symptoms of PMS. However, nearly all study participants reported a baseline calcium
intake of less than 1000 mg/day. It is thus unclear whether the observed improvements in
symptoms were the result of correcting underlying calcium deficiency, and whether women
with sufficient background calcium intake would experience similar improvement in response
to supplementation.
Subsequently, Thys-Jacobs et al. (1998) replicated the initial findings in a larger randomized
multicentre trial. 720 participants from 12 outpatient centres were recruited. Participants aged
18-45 years were screened over two menstrual cycles for moderate to severe cyclically recurring
symptoms using the PMS diary (Thys-Jacobs et al., 1995) to document 17 core symptoms and 4
symptoms factors. To be included in the treatment component of the study, symptom intensity
had to be at least 50% higher during the luteal phase as compared to the 7 days after menses for
at least two of the three menstrual cycles evaluated. In addition, participants had to suffer from
a b
8 10
7 9
8
6
Total mean menstrual scores
Total mean menstrual scores
Placebo 7
5 Placebo
6
4 5
P=0.011 P=0.032 Calcium
3 Calcium 4
3
2
2
1 1
0 0
Baseline Treatment Baseline Treatment
Figure 6.4. Total mean symptom scores for (a) the luteal and (b) the menstrual phases at baseline and after
treatment with placebo and calcium. P-values are for the change in total mean symptom scores between baseline
and after 3 cycles of calcium treatment vs. placebo (Adapted from Thys-Jacobs et al., 1989).
mood swings, depression-sadness, tension-irritability, anxiety-nervousness, anger-aggression,

or crying spells during the luteal phase. A total of 497 premenopausal women met the inclusion
criteria and were included in the study (mean age 32.8 years).
Participants were randomized into one of two treatment groups, with one group receiving 1,200
mg/day of calcium for three menstrual cycles (n=248), while the second received a placebo
(n=249). All participants were required to keep a PMS diary and record daily occurrence of
symptoms, symptom severity, and their compliance with their assigned treatment. By the third
cycle of treatment, an overall 48% reduction in total symptom score compared to baseline was
observed among those assigned to calcium supplementation (P<0.001) (Table 6.1). Scores for
all four symptom factors were all significantly reduced by the third treatment cycle of calcium
compared to baseline, with the negative affect symptom factor decreasing by 45% (P<0.001)
compared to 28% for placebo. After three cycles, scores for 17 of the 19 individual symptoms
evaluated were significantly improved with calcium supplementation as compared to placebo.
Overall, results from this trial provided strong evidence that calcium supplementation may be
an effective treatment for PMS.
Additional evidence suggests that calcium may improve menstrual symptom experience among
women whose symptoms are not severe enough to meet diagnostic criteria for PMS. Penland and
Johnson (1993) examined the relationship between dietary intake of calcium and manganese and
Table 6.1. Mean total menstrual symptom scores and symptom factor scores during the luteal phase for calcium
supplementation vs. placebo treatment groups at baseline and after three treatment cycles (Adapted from Thys-
Jacobs et al., 1998).
Four symptom factors Symptom score Symptom score P-value1

at baseline after three cycles
All symptoms Calcium2 0.900.52 0.430.40 <0.001

Placebo 0.920.55 0.600.52 NS
Symptom factor 1: negative affect Calcium 0.990.59 0.46 0.47 <0.001
Placebo 1.040.66 0.65 0.64 NS
Symptom factor 2: water retention Calcium 0.960.58 0.510.46 <0.001
Placebo 0.970.60 0.690.58 NS
Symptom factor 3: food cravings Calcium 0.970.76 0.450.63 <0.05
Placebo 1.020.76 0.600.75 NS
Symptom factor 4: pain Calcium 0.740.63 0.300.40 <0.001
Placebo 0.690.58 0.500.52 NS
1 Comparison of mean symptom score after 3 cycles vs. baseline

2 Numbers of participants: calcium group at baseline = 231; calcium group at 3rd treatment = 212; placebo group
at baseline = 235; placebo group at 3rd treatment = 228.
menstrual symptoms. In contrast to the previously described clinical trial in which participants
met clinical criteria for PMS, this study investigated the effect of supplementation on menstrual
symptoms among 10 healthy women with normal menstrual cycles (mean age 27.2 years). Each
participant underwent a physical and psychological examination, including a standard metabolic
panel from blood and urine samples and a psychiatric diagnostic interview. Menstrual symptoms
were assessed using the Menstrual Distress Questionnaire, a retrospective self-report of the
presence and severity of symptoms throughout the menstrual cycle. Participants were each
assigned to four sequential 39-day dietary periods, for a total of 169 days of treatment: (1) 587
mg calcium lactate and 1.0 mg manganese sulphate per day; (2) 587 mg calcium lactate and 5.6
mg manganese sulphate per day; (3) 1,336 mg calcium lactate and 1.0 mg manganese sulphate
per day; and (4) 1,336 mg calcium lactate and 5.6 mg manganese sulphate per day. Higher
calcium supplementation was associated with general reduction in symptoms (P0.05), and with
a reduction in pain during the menstrual phase (P=0.034) and of water retention during the
premenstrual phase (P=0.041). Reducing participants calcium intake from 1,336 mg to 587 mg
resulted in significantly more undesirable behavioural changes (P=0.010), poorer concentration
(P=0.047), and greater water retention (P=0.041) during the premenstrual phase. Mean symptom
scores were at the low end in this study because participants were not experiencing the degree of
distress observed in women with PMS.
6.2.4 Calcium and vitamin D intake and risk of incident PMS
Although several randomized studies suggested that calcium supplementation might significantly
reduce the occurrence and severity of premenstrual symptoms, few studies have assessed whether
high calcium intake in asymptomatic women could prevent PMS from developing. To assess the
relation of dietary calcium intake with PMS onset, Bertone-Johnson et al. (2005) conducted a
case-control study nested within the prospective Nurses Health Study II cohort.
The Nurses Health Study II (NHS2) is a cohort of 116,678 female registered nurses in the United
States. Participants were between the ages of 27 and 44 years at the beginning of follow-up in
1991, at which time 6,000 women currently free from PMS were invited to participate in the
PMS sub-study. Of these women, 3,430 women reported a new clinician-made diagnosis of PMS
over 10 years of follow-up, while 2,570 did not report PMS. To confirm PMS case status, all
participants were asked to report whether they had experience any of 26 different symptoms
most months of the year for at least several days each month during the previous 2 years, using a
2-page questionnaire based on the Calendar of Premenstrual Experiences (Mortola et al., 1990).
The diagnosis of PMS was confirmed in 1,057 women reporting PMS, while among those not
reporting PMS, 1968 were validated as non-cases.
NHS2 participants completed 131-item food frequency questionnaires in 1991, 1995, and 1999
(Willett et al., 1985), which were used to measure intake of calcium from food sources and
from supplements, along with other nutrients. Every two years, NHS2 participants also provided
detailed information on their medical history and health-related behaviours, such as use of oral
contraceptives, menstrual and pregnancy history, and smoking status.
After adjustment for age, smoking, body mass index, and other factors, results suggested that
intake of calcium, especially from food sources, was inversely related to PMS risk. Compared
to participants with the lowest calcium intake (median in quartile 1 = 529 mg/d), participants
with the highest intake (median in quartile 5 = 1,283 mg/d) had a 30% reduction in risk of PMS
(relative risk = 0.70; 95% confidence interval = 0.50-0.97) (P for trend = 0.02) (Table 6.2). Risk of
PMS was somewhat lower among women reporting high total calcium intake, but results were
not statistically significant. Interestingly, use of calcium supplements was not related to the risk of
developing PMS, though in this population calcium supplementation was relatively uncommon.
As milk was the main source of dietary calcium among study participants, the relation of milk
Table 6.2. Multivariable relative risks of premenstrual syndrome by intake of calcium 2-4 years before reference
year, Nurses Health Study II Premenstrual Syndrome Sub-Study (1991-2001)1 (Adapted from Bertone-Johnson
et al., 2005).
Level of calcium intake P-value

for trend
Quintile 1 Quintile 2 Quintile 3 Quintile 4 Quintile 5
Total calcium intake

Median, mg/d 563 749 930 1180 1563
Case:control ratio 188:330 216:404 206:374 235:455 210:401
Relative risk2 1.00 0.95 0.90 0.84 0.80 0.13
95% CI Referent 0.72-1.25 0.66-1.21 0.61-1.14 0.58-1.10
Calcium from food sources only
Median, mg/d 529 684 812 997 1283
Case:control ratio 177:321 214:367 221:400 234:390 209:486
Relative risk2 1.00 1.11 0.96 1.03 0.70 0.02
95% CI Referent 0.84-1.46 0.72-1.28 0.76-1.39 0.50-0.97
Calcium from supplements Non-users Supplement users
Dose, mg/d 0 1-399 400-899 900

Case:control ratio 661:1280 231:401 113:198 50:85
Relative risk2 1.00 1.03 1.13 1.13 0.35
95% CI Referent 0.81-1.30 0.85-1.50 0.75-1.71
1 Reference year is equal to year of diagnosis (for cases) or randomly chosen year corresponding to the possible
year of diagnosis (for controls).

2 Adjusted for level of other factors before the reference year, including age, year of diagnosis, number of full-term
pregnancies, BMI, smoking status, tubal ligation, duration of oral contraceptive use, antidepressant use, and intake
of vitamin B6, vitamin D and potassium from foods and supplements. Calcium from food sources was adjusted for
calcium from supplements and vice versa.
intake to PMS was assessed separately. Results suggested that women consuming 2 or more
servings per day of skim or low fat milk had a 46% lower risk of developing PMS compared
to those consuming skim or low fat milk once per week or less (P for trend <0.001) (Figure
6.5). However, intake of whole milk was not related to lower risk. These results were among the
first to suggest that dietary intake of calcium-rich foods may lower a womans risk of initially
developingPMS.
6.3 Conclusion
Results from multiple studies suggest that calcium deficiency, dysregulation of calcium
metabolism and functions of calcium-regulating hormones may contribute to premenstrual
syndrome. Women with PMS have been observed to suffer from large fluctuations of calciotropic
hormones across the menstrual cycle compared to asymptomatic women. The findings of several
randomized trials and prospective studies have suggested that calcium supplementation may
be effective in treating PMS, with higher doses (i.e. 1,200 to 1,600 mg daily) providing greater
benefit than lower doses. Calcium supplementation appears to impact multiple symptom factors,
including negative affect, water retention, food cravings and pain. Additional findings from
observational studies suggest that women with diets high in calcium (>1,200 mg daily) may have
a lower risk of developing PMS than those not meeting current recommended daily allowances
for calcium. Currently, the American Congress of Obstetricians and Gynaecologists recommends
1.4
1.2
1.0
0.8
Relative risk
0.6
0.4
P trend <0.001
0.2
0.0
1/wk 26/wk 1/d 23/d 4/d
Frequency of intake
Figure 6.5. Multivariable relative risks and 95% confidence intervals of premenstrual syndrome by dietary
intake of skim or low-fat milk 2-4 years before the reference year. Reference year is equal to year of diagnosis
for premenstrual syndrome cases or a randomly-chosen year corresponding to the possible years of diagnosis
for controls. Relative risks were adjusted for age, year of diagnosis, number of full-term pregnancies, body mass
index, smoking status, tubal ligation, duration of oral contraceptive use, antidepressant use, and intake of vitamin
B6 and potassium from foods and supplements (Adapted from Bertone-Johnson et al., 2005).
eating calcium rich foods as treatment for PMS. Given that calcium and vitamin D may also
reduce the risk of osteoporosis and some cancers, clinicians should consider recommending these
nutrients for younger women experiencing menstrual symptoms.
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7. Diet and eating changes in premenstrual
syndrome
N. Erbil
Department of Nursing, School of Health, Ordu University, 52200 Ordu, Turkey;
nerbil@odu.edu.tr
Abstract
The purpose of this review was to investigate diet and eating changes in premenstrual syndrome
(PMS). Dietary modifications have been suggested as a treatment for PMS, but the nature of
the dietary changes has typically been vague, inconsistent between authors, and with little
empirical basis. The study review has determined that a poor or inadequate dietary intake and
vitamin and mineral deficiency have a negative effect on the symptoms of PMS. Also, the review
presents results on the role of neurotransmitters in PMS. Evidence suggests that diet can affect
neurotransmitter metabolism, but randomized controlled and placebo controlled research needs
to explore this further.
Keywords: eating changes, diet, premenstrual syndrome
109
N. Erbil
Summary points
The symptoms of premenstrual syndrome (PMS) are characterized by somatic, appetitive, cognitive and
behavioral changes.
The hormonal fluctuations associated with the menstrual cycle may influence appetite control and eating
behavior.
Physiological and psychological features of the menstrual cycle and PMS could influence the expression of
appetite through mechanisms and processes which influence the control of food intake.
Changes in appetite and eating behavior have been documented in women suffering from PMS, with an
increased food intake occurring during the luteal phase.
Dietary changes can improve womens general health and self-esteem, their tolerance to premenstrual
changes, and they can also reduce the impact of PMS on daily life.
Although dietary changes for the relief of PMS symptoms have not yet proven effective in well-controlled
studies, clinical experiences support the beneficial effects of changes in diet for some women.
7. Diet and eating changes in premenstrual syndrome
Abbreviations
APA American Psychiatric Association
ACOG American College of Obstetricians and Gynecologists
DSM-IV Diagnostic and statistical manual of mental disorders IV
DSM-V Diagnostic and statistical manual of mental disorders V
GABA Gamma-aminobutyric acid
NMES Non-milk extrinsic sugar
PMT Premenstrual tension
SSRIs Serotonin reuptake inhibitors
7.1 Introduction
The symptoms of PMS are characterized by somatic, appetitive, cognitive and behavioral changes.
These occur cyclically during the late luteal phase of the menstrual cycle and resolve quickly at
or within a few days of the onset of menstruation (Braverman, 2007). Premenstrual symptoms
are experienced by up to 95% of all women of reproductive age. PMS occurs in about 5% of
those women (Ismail and OBrien, 2005). The terminology of premenstrual disorders has become
complex. The acronym PMS is most often used in the UK; PMT is a lay term; PMDD is the
extreme, predominantly psychological, end of the PMS spectrum, estimated to occur in 3-9% of
women (Ismail and OBrien, 2005). Although more than 200 symptoms have been associated with
PMS, the symptoms that classically characterize the syndrome include depression, irritability,
mood swings, breast tenderness, bloating, changes in appetite and food cravings (Freeman, 2003).
As a result, millions of women experience disturbing symptoms that interfere with personal
relationships, social activities, and job performance (Tempel et al., 2001). PMS is seen in women
of reproductive age and is not present before puberty, during pregnancy or after menopause
(Ismail and OBrien, 2005).
Due to a lack of laboratory tests for diagnosis of PMS, diagnosis is usually based upon a patients
symptoms, signs and stories. Diagnosing PMDD is presently done with the use of scales which
have been developed. Prospective symptom rating charts are used for this purpose (Indusekhar
et al., 2007). The World Health Organizations International classification of diseases uses ICD-9
code 625.4 for PTS and lists PMS and PMDD under this heading. There is no separate diagnostic
code for PMS or PMDD (Mortola et al., 1990). The organizations that have published definitions
include the American Psychiatric Association and the American College of Obstetricians and
Gynecologists (APA, 2000; ACOG, 2000). According to the current diagnostic criteria proposed
by the ACOG, PMS is diagnosed if at least one mental and one somatic symptom occur in
moderate or intense severity (Borenstein et al., 2005). According to the APA, certain criteria
N. Erbil
contained in the DSM-IV are used for diagnosis of PMDD. The criteria for a diagnosis of PMDD
have been changed in the DSM-V diagnostic system expected to be published in 2013, and
PMDD will be placed under the depressive disorders section. According to the DSM-IV criteria
of the APA and purposed DSM-V criteria of the APA in order to diagnose PMDD, the more
severe form of PMS, at least 5 symptoms should be present, including 1 severe mental symptom
(APA, 2012). The cyclical symptoms appear mostly during the luteal phase of the menstrual cycle
and disappear shortly after the beginning of menstruation. The key diagnostic characteristic is
that the symptoms must be absent between the end of menstruation and ovulation (Indusekhar
et al., 2007).
The hormonal fluctuations associated with the menstrual cycle may influence appetite control
and eating behavior (Dye and Blundell, 1997). Conditions associated with the menstrual
cycle and PMS may predispose women to changes in appetite control (Figure 7.1), (Dye and
Blundell, 1997). Previous studies have investigated energy and macronutrient intake over the
menstrual cycle in women with and without PMS. Cross et al. found that nutrient analysis
of the diet diaries of women with PMS showed a significant increase in total energy and all
macronutrients premenstrually when compared to nutrient intake postmenstrually, (Cross
et al., 2001). Furthermore, study participants reported a rise in energy and fat intake, but all
other macronutrients and NMES were similar between the phases (Cross et al., 2001). Trout
et al. (2008) showed that there were no differences in proportions of macronutrients or total
kilocalories by cycle phase, despite a marked difference in food cravings between cycle phases,
with increased food cravings noted in the luteal phase among women with PMS. Both-Orthman
et al. (1988) demonstrated a significant increase in appetite in both women with PMS and the
control group, however appetite was greater in women with PMS and this correlated with the
Homeostatic Progesterone
system
Oestrogen
Brain Food supply
M
Metabolism
Food intake Environmental
adaptation
Nutritional
supply
Weight concern
(dieting)
Biological forces Eating pattern Cultural forces
Macronutrient
composition Structure
Figure 7.1. The fundamental elements influencing the control of food intake. The diagram illustrates how
hormonal fluctuations underlying the menstrual cycle exert direct and indirect effects on the homeostatic system
controlling food intake and pattern of eating (Dye and Blundell, 1997).
self-rating of mood. Erbil et al. (2010) indicated that young girls had changes in appetite (68.3%)
in the premenstrual period. Another study determined a significant premenstrual increase in
carbohydrate intake in women with PMS (Wurtman et al., 1989).
This chapter investigates the diet and eating changes in PMS.
7.2 Etiology of PMS

Although the factors associated with PMS have been well documented, the exact cause of
PMS remains unknown (Keye and Keye, 1998; Walker et al., 1998). However, the following
imbalances may play a role in PMS: the effects of hormonal disturbances, especially a low level
of progesterone in the luteal phase; disturbed function of aldosterone activity leading to sodium
and water retention; imbalance of the hypophyseal-pituitary-adrenal axis leading to inadequate
secretion of adrenal hormones; disturbed secretion of adrenal hormones; disturbed secretion of
neurotransmitters with functional hyperprolactinemia; dietary deficits of calcium, magnesium,
and pyridoxine; alcohol; carbohydrate tolerance disturbances; obesity; and environmental factors
such as stress (Milewicz and Jedrzejuk, 2006).
Alterations in neurotransmitters including endorphins, GABA, and serotonin have all been
implicated, and women with PMS/PMDD are felt to be more sensitive to normal cyclical
hormonal fluctuations (Steiner and Pearlstein, 2000). The GABA transmitter system is a major
inhibitory system in the central nervous system (Indusekhar, 2007). Women with PMS may
have an alteration in the GABA receptor complex response. A review of the literature has
determined both reduced GABA receptor sensitivity and reduced plasma GABA in the luteal
phase (Braverman, 2007).
Another theory links low levels of the neurotransmitter serotonin with PMS. Estrogen and
progesterone are known to alter the human brains serotonin receptor concentration and
serotonin metabolism (Tempel, 2001). Serotonin, which controls when a person feels full after
eating, can also cause a drop in appetite. In addition, it controls ones psychiatric status and
improves mood, regulates body temperature and cardiovascular function, and has effects on
sexual behavior (Mahmoodi et al., 2011). One study demonstrated that blood serotonin levels
in women with PMS were significantly lower than in controls during the last 10 days of the
menstrual cycle. It has been postulated that serotonin deficiency in women with PMS enhances
sensitivity to progesterone. The efficiency of SSRIs in the treatment of PMS/PMDD indirectly
supports the influence of serotonin in the etiology of PMS (Indusekhar, 2007).
Thys-Jacobs and Alvir (1995) observed mid-cycle fluctuations of calcium-regulating hormones

and increases in the parathyroid hormone in women suffering from PMS. When compared to
control subjects, increases in the PTH with transient, secondary hyperparathyroidism in women
who suffered from PMS were also observed. These data support the idea of a transient calcium
deficiency in women suffering from PMS (Thys-Jacobs and Alvir, 1995).
N. Erbil
7.3 Eating behavior during the menstrual cycle

Physiological and psychological features of the menstrual cycle and PMS could influence the
expression of appetite through mechanisms and processes which influence the control of food
intake (Figure 7.1) (Dye and Blundell, 1997). These variations influencing food intake have been
related to changes in female steroid hormones, especially in the estrogen and progesterone ratio
that occur during the cycle (Gil et al., 2009; Johnson et al., 1994).
Increased consumption of carbohydrates is one of the characteristic features of PMS in some

women. It has been hypothesized that this change in food intake may lead to an increase in
the serum ratio of tryptophan to other large neutral amino acids, which may in turn lead to a
serotonin-mediated improvement in mood (Bussel, 1998).
Energy and macronutrient intake over the menstrual cycle have been investigated in women with
and without PMS. Previous studies have reported increases of one or more macronutrients as
well as total energy intake during the luteal phase of the menstrual cycle (Gil et al., 2009; Martini
et al., 1994). A previous study evaluated the midfollicular and midluteal dietary intakes of 18
women during between four and six ovulatory menstrual cycles, and records were collected 6-8
d after menstrual onset and 6-8 d after ovulation, respectively (Martini et al., 1994). The same
study reported that intakes of energy, protein, carbohydrates, and fat were significantly higher in
the luteal phase than in the follicular phase of the menstrual cycle, and there were no differences
in the proportion of macronutrients between the two phases. The average energy intake was
significantly higher on weekends than on weekdays. Furthermore, the menstrual phase did
not significantly affect intakes of most of the micronutrients. However, intakes of vitamin D,
riboflavin, potassium, phosphorus, and magnesium were significantly greater in the luteal phase
than in the follicular phase. There were no statistical differences between follicular or luteal
intakes of any of these micronutrients when expressed relative to energy (Martini et al., 1994).
Cheikh Ismail et al. (2009) investigated energy and nutrient intakes during different phases of
the menstrual cycle in adult females. They showed that mean energy intake was significantly
lower in the menstrual phase compared to the pre-menstrual phase (P=0.002), but not during
the post-menstrual phase. Intakes of macronutrients and micronutrients were higher in the pre-
menstrual phase compared to the menstrual and post-menstrual phases. Intakes of carbohydrates,
protein and fat were significantly higher in the pre-menstrual phase than in the menstrual phase
(P=0.008, P=0.001 and P=0.013, respectively). Similarly, intakes of vitamin C and B vitamins
were significantly higher in the pre-menstrual phase than in the menstrual phase (P=0.019 and
P=0.05, respectively) (Cheikh Ismail et al., 2009).
Johnson et al. (1994) found that energy intake among women increased 685.9 kJ/day (P<0.05)
from the follicular/ovulatory to the luteal phase. The percentage of energy intake as protein
and carbohydrate was relatively constant over the three menstrual cycle phases, but the intake
from fat increased 2% (P<0.05) during the luteal phase, representing a 9.2 g/day increment.
Variations in energy and fat intake over the menstrual cycle were not attributable to differences
in energy expenditure through exercise or dietary restraint, and appear to be related to changes
in the estrogen/progesterone ratio (Johnson et al., 1994). Both-Orthman et al. (1998), using a
daily questionnaire to measure appetite and mood over a period of 2 to 9 months, indicated a
significant increase in appetite in both women with PMS and controls. However, the effect was
greater in women with PMS and this correlated with self-rating of mood.
Wurtman et al. (1989) monitored the food intake of 19 women with PMS and 9 controls for
48 h pre-and postmenstrually in an inpatient setting and reported a significant increase in
carbohydrate intake in women with premenstrual PMS. In this study, women with PMS and the
controls were admitted as inpatients and their food consumption was monitored for 48 h both
premenstrually and postmenstrually. The meals and snacks available to the subjects had either
a high or low carbohydrate content. There was no change in protein intake and fat intake rose
in proportion to total energy intake. No change in food intake was seen in the control group
(Wurtman et al., 1989).
Bryant et al. (2006) found that women with PMS did not increase their consumption of energy
or macronutrients in the premenstrual phase of their menstrual cycle. They demonstrated that
women with PMS consumed slightly fewer kilojoules during the premenstrual phase (8,286 kJ,
95% CI 7,667-8,906) compared to the postmenstrual phase (8,381 kJ, 95% CI 7,861-8,902). In
contrast, women in the control group consumed more energy during the premenstrual phase
than during the postmenstrual phase (8,577 kJ, 95% CI 7,881-9,272; 8,169 kJ, 95% CI 7,427-8,907,
respectively). Differences were not significant between cycle phases for women with PMS or those
in the control group. Similar patterns were found for total fat, carbohydrate, protein, non-starch
polysaccharides and percentage energy from protein; that is, compared with the postmenstrual
phase, premenstrual intakes were similar for the PMS group but higher for the control women.
Exceptions were with NMES and alcohol, which were both consumed in greater amounts in the
premenstrual phase in women with PMS. Significant correlations were observed between the
severity of symptoms and the change in the consumption of these nutrients (Bryant et al., 2006).
Johnson et al. (1995) indicated that reports of pain, water retention, mood swings, behavior
changes, and arousal were significantly higher in the perimenstrual with low levels of estrogen
and progestrone when compared to follicular and luteal phases. Also, during the perimenstrual, a
higher energy intake of carbohydrates was associated with higher ratings of mood swings. Lower
energy intake of protein was associated with higher ratings of well being. Overeating and dieting
behavior were related to greater water retention, autonomic reactions, and appetite.
Cross et al. (2001) provided evidence that there is a premenstrual increase in energy and all
macronutrient intake in overweight women with PMS symptoms. Nutrient analysis of the
diet diaries of the women with PMS showed a significant increase in total energy and all
macronutrients premenstrually when compared to nutrient intake postmenstrually. Women who
did not meet the criteria for PMS showed a significant increase in energy and fat intake but not
in the other macronutrients. When adjusted for energy, data collected from women with PMS
showed a premenstrual significant increase in fat, carbohydrate and simple sugars. There was a
N. Erbil
significant decrease in protein premenstrually. Women not meeting the PMS criteria showed
no significant difference between pre- and postmenstrual intakes when adjusted for energy.
Analysis according to food categories in women with PMS showed a significantly greater intake
of energy premenstrually and all macronutrients for cereals, cakes and desserts and high-sugar
foods. In women with PMS there was a significantly greater number of premenstrual episodes of
eating (Cross et al. 2001). Gold et al. (2007) found that fat intake was negatively associated with
cravings and bloating, and fiber intake was positively associated with breast pain; alcohol intake
was negatively associated with anxiety and mood changes and headaches.
7.4 Dietary changes in premenstrual syndrome

Dietary modifications have been suggested as a treatment for PMS, but the nature of the dietary
changes has typically been vague, inconsistent between authors, and with little empirical basis.
In a review, Bussel (1998) reported that PMS appears to have a decreased blood level of serotonin
during the luteal phase. Tryptophan is a precursor of serotonin and its uptake by the brain is
increased by carbohydrate consumption. A high-carbohydrate diet is believed to increase
serotonin production and diminish negative effects in patients with PMS, seasonal affective
disorder and carbohydrate craving obesity (Bussel, 1998). It is believed that refined carbohydrates
should be kept to a minimum as they may cause a rise in insulin secretion. This in turn leads to
increased fluid retention, which also leads to increases in the urinary excretion of magnesium
(Bussel, 1998). Another review study was noted that a possible deficiency in circulating serotonin
levels could be relieved by increasing tryptophan presence through increased ingestion of
carbohydrates or other foods high in tryptophan, resulting in a reduction of mood symptoms. A
reduction in intake of refined sugars and salts will reduce liquid retention. A reduction in intake
of coffee, tea, coke, and chocolate will reduce breast discomfort (Campagne and Campagne,
2007). A low-fat intervention diet to prevent premenstrual symptoms has been shown to reduce
breast swelling, tenderness, and nodularity (Boyd et al., 1988).
Changes in appetite and eating behavior have been documented in women suffering from PMS,
with an increased food intake occurring during the luteal phase. Moreover, in women with PMS,
a major effect of this phase of the menstrual cycle on appetite has been documented, and a high
correlation with self-ratings of mood, particularly depression, has been described (Verri et al.,
1997).
Stewart et al. (1993) reported that adopting a healthy diet could significantly reduce PMS
symptoms. A healthy diet would be low in fat but would also supply an adequate intake of
lean animal proteins. A higher quantity of fresh fruit and vegetables would also be ideal, with
consumption of tea, alcohol, salt, sugar and refined carbohydrates kept to a minimum (Stewart
et al. 1993). Based on evidence of its effectiveness in reducing PMS symptoms, Abraham (1994)
advocated a low-fat, high-carbohydrate diet as a first approach in the treatment of PMS.
Osofsky et al. (1998) recommended reducing the intake of refined sugars, white flour, coffee,
tea, chocolate and alcohol. Based on extensive clinical experience, but with little empirical
justification, Abraham (1984) provides more specific dietary recommendations by advocating
limits on the intake of refined sugar, dairy products, and fat and increases in the amount of
carbohydrates and vegetables.
Sayegh et al. (1995) reported that the ingestion of appropriately designed carbohydrate-rich
preparations during the late luteal phase of the menstrual cycle may relieve some premenstrual
disturbances of mood and appetite. Results of their study indicated possible improvements in
cognition, thus providing a benign intervention for women with moderate to severe PMS. In
addition, three different intervention studies related to relief of PMS symptoms were carried out
by Mahmoodi et al. (2011) and they investigated participants whose diets had been enhanced
with carbohydrate-rich supplements or a carbohydrate-rich diet and protein-rich supplements.
Study findings revealed that consumption of additional carbohydrates, either in supplement form
or within a carbohydrate-rich diet, is effective in relieving PMS symptoms (Mahmoodi et al.,
2011).
Dante and Facchinettis review (2011) indicated that some herbal remedies seem useful for the
treatment of PMS. However, more randomized controlled trials are required to account for the
heterogeneity of the PMS (Dante and Facchinetti, 2011).
7.5 Conclusion
Although dietary changes for the relief of PMS symptoms have not yet proven effective in well-
controlled studies, clinical experiences support the beneficial effects of changes in diet for some
women (Tempel, 2001). Dietary changes can improve womens general health and self-esteem,
their tolerance to premenstrual changes, and they can also reduce the impact of PMS on daily
life (Bussel, 1998). Nutritional information and education should also be available and offered to
women. It is effective and inexpensive and puts the woman in control of her own treatment (Bussel,
1998). Nutritional education should emphasize a well-balanced diet consisting of frequent small
meals high in complex carbohydrates, of fruit, and vegetables (Keye and Keye, 1998). It has been
suggested that certain foods should be avoided, such as coffee, tea, coke, and chocolate. These have
all been shown to increase the well-documented PMS symptoms of insomnia, headaches, breast
tenderness, irritability and anxiety. Furthermore, sodium and salty foods should be avoided to
reduce fluid retention (Campagne and Champagne, 2007; Tempel, 2001).
Health professionals should be aware of PMS patients possible use of dietary supplements as
well as all prescribed medications so they may offer patients the proper counsel about possible
benefits, risks and drug interactions.
N. Erbil
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8. V itamin D and other nutrients in the treatment
of premenstrual syndrome
F. Cioni1 and F. Ferraroni2
1Diabetology and Metabolic Disorders Unit, University Hospital of Parma, Via Gramsci
14, Parma, Italy; 2Consultorio Familiare UCIPEM, Strada N. Bixio 14, Parma, Italy;
federicocioni@mattioli1885.com
Abstract
Premenstrual syndrome (PMS) is a heterogeneous pattern of physical and psychological
disturbances that typically appear during the luteal phase of the menstrual cycle and end with
the menstruation or in the days immediately following the menstrual cycle. It is a disorder
characterized by a multifactorial etiopathogenesis which is extremely frequent in women of
fertile age, especially in their third decade of life. The triggering factor seems to be represented
by hormonal fluctuations linked to the menstrual cycle, yet other causes, that are in part still to
be defined, come into play. Great importance today is given to nutrients, in particular vitamin D
and calcium, but also essential fatty acids, some vitamins (vitamin B6), carbohydrates (especially
simple sugars), proteins, lipids, some minerals (magnesium and manganese), caffeine and
alcohol. The role of vitamin D in reducing the risk of PMS is still today object of study and
seems to be primarily correlated with the modulation of levels of calcemia, as well as of some
neurotransmitters and sexual steroids. Evidence suggests that improvement of calcium balance,
through the intake of milk derivatives or dietary vitamin D and calcium supplementation,
improves PMS symptoms. This confirms the importance of diet and lifestyle intervention in
the clinical management of the PMS. In fact, a comparative analysis of the literature available
on this topic suggests that a balanced diet, rich in minerals and vitamins together with a correct
glycaemic index, associated with regular physical activity leads to clinical benefits in women
affected by PMS. Regarding dietary supplementation, today available evidence justifies vitamin
D and calcium supplementation at doses respectively of 400/800 UI/d and of 1,200 mg/d and, to
a lesser degree, magnesium (200/250 mg/d) and vitamin B6 (40 mg/d) supplementation.
Keywords: premenstrual syndrome, vitamin D, calcium, vitamin B6, magnesium
121
Summary points
Premenstrual syndrome is a pattern of physical and psychological symptoms that typically appear during
menstrual cycle and is probably characterized by a multifactorial etiopathogenesis.
Among the causes and/or the triggering factors of premenstrual syndrome, some importance today is given
to nutritional intake of vitamin D and calcium, but also of essential fatty acids, some vitamins (in particular
vitamin B6), carbohydrates (especially simple sugars), proteins and lipids, some minerals (magnesium and
manganese), caffeine and alcohol.
Diet and lifestyle intervention can at last support pharmacological treatment in the clinical management of
premenstrual syndrome.
Today available evidence justifies vitamin D and calcium supplementation at doses respectively of 400/800
UI/die and of 1,200 mg/die.
Magnesium (200/250 mg/die) and vitamin B6 (40 mg/die) supplementation can be proposed at a lesser
degree of evidence.
8. Vitamin D and other nutrients in the treatment of PMS
Abbreviations
DSM IV Diagnostic and statistical manual of mental disorders IV
EFA Essential fatty acids
FGF-23 Fibroblast growth factor 23
PG Prostaglandins
8.1 Premenstrual syndrome

PMS, together with dysmenorrhea and the PMDD, is part of the menstrual cycle disorders, a
diversified pattern of symptoms and disorders that recognise, both at clinical and pathogenetic
level, cyclic fluctuation of the sexual steroids as a triggering factor. Dysmenorrhoea is a menstrual
alteration characterised by general or local disorders and pain of variable entity generally
localised in the pelvic area and abdomen that should not be confused with PMS. Mild forms
of this disorder can be found in almost half of the women of fertile age, whilst 10% of cases are
so intense that they can become temporarily invalidating. Usually pain is not to be attributed
to precise causes, but in certain cases it can be a symptom of endometriosis, adenomyomatosis,
fibromyomatosis or other well-definable pathologies (French, 2005).
PMS consists of a heterogeneous group of psychological and physical symptoms, often non-
specific, that typically appear during the luteal phase of the menstrual cycle and usually subside as
the menstruation tapers off or in the days immediately following the menstrual cycle. Psychological
symptoms include depression, irritability, anxiety, crying outbursts, insomnia, confusion, lack of
interest in social relationships, and food cravings. The most frequent physical symptoms include
oedema of extremities, breast swelling and tenderness, headaches, abdominal bloating and in
severe cases, real abdominal pain (Table 8.1). Diagnosis of PMS is not that simple as a thin line
exists between a temporary disorder and a real pathology that can sometimes be invalidating
and greatly influenced by individual levels of tolerance. The proposed diagnostic criteria are
based on the presence of at least one of the above symptoms in the 5 days before menses, in three
consecutive menstrual cycles, with disappearance of symptoms within 4 days after menses and in
the absence of specific pathologies that could otherwise justify the clinical picture. The intensity
of the disorder must significantly influence the patients quality of life (ACOG, 2001). Many
women of fertile age sense the arrival of their menstrual cycle with a series of disorders of variable
entity such as strong emotional tension, lack of concentration, migraine attacks: this complicates
the actual definition of PMS prevalence. According to several studies, the percentage of women
that suffer from full-blown PMS in its most severe form varies between 5 and 8% in women of
fertile age, reaching 20% and as much as 32% if one considers also milder forms (Borenstein et
Table 8.1 Signs and symptoms of premenstrual syndrome.
Physchological Neurovegetative Behavioral Alterations of the Pain

hydrosaline balance
Depression Lassitude Demotivation Edema Abdominal cramps

Sadness Lethargy Social isolation Bloating Headache
Anxiety Insomnia General pain
Irritability Food cravings
Mood swings
al., 2003). Such a high prevalence suggests that actually a certain degree of discomfort linked to
the menstrual cycle could be considered a physiological condition.
In the most severe cases of PMS the psychological symptomatology is more pronounced, with
dramatic mood swings that justify a clinical diagnosis of PMDD, a psychiatric disorder classified
by the IV edition of Diagnostic and Statistical Manual of Mental Disorders (DSM IV), that can
heavily interfere with the normal daily working activities and social life (Biggs and Demuth,
2011).
PMS is a complex disorder characterized by a multifactorial etiopathogenesis for which many

causes have been suggested, including alterations of certain hormonal axes (principally estrogen/
progesterone, but also aldosterone/renin) and of central-nervous-system neurotransmitter
interactions (especially through serotonin and GABA). The role played by fluctuations in sex
hormone concentrations appears to be confirmed by numerous observations that highlight the
absence of symptoms during non-ovulating cycles, their disappearance after ovariectomy or
with treatments using ovulation-inhibiting drugs and, on the contrary, their re-appearance after
hormonal replacement therapy (Yonkers et al., 2008). The problem related to the pathophysiological
definition of the somatic symptoms is even more controversial, so that the discussion remains
open as to whether there is a prevalence, in these patients, of the psychological component (in
terms of reduced tolerance to the discomfort) or whether an actual peripheral response of specific
target tissues comes into play. Evidence is still contradictory and, if on one hand it does not
confirm the presence of water retention or an increase in weight that can be measured in presence
of a diffuse sensation of abdominal bloating and breast swelling and tenderness described by
many patients, on the other the treatment with bromocriptine, a dopamine D2 receptor agonist
or with SRI, seems to have at least palliative effects also on somatic symptomatology (Yonkers et
al., 2008). Awaiting further confirmation the above seems, however, to be in line with what has
been observed in some functional syndromes such as fibromyalgia, where the central and somatic
components are inextricably correlated (Nilsen et al., 2007).
In terms of evolutionary medicine it is possible to hypothesize that the changes in lifestyle that
have come about over the past 50 years in developed societies, with a dramatic decrease in the
number of pregnancies, duration of breastfeeding and problems of malnutrition, have significantly
reduced the periods of amenorrhea during the lifetime of women, therefore exposing them far
more than their ancestors, to long periods of cyclic fluctuations of estrogens and progesterone
(McDonald et al., 1991). In addition, it should be also considered, as we will see, the effect of
unhealthy lifestyles, unhealthy dietary behaviors and insufficient physical activity.
Returning to the etiopathogenesis of the somatic disorders associated with PMS, it has been
hypothesized that a high concentration of estrogens in the blood during ovulation or during the
luteal phase inhibits bone reabsorption and reduces calcaemia leading to an increase in capillary
permeability that would explain symptoms such as depression, cramps, migraine, abdominal
bloating and breast swelling and tenderness (Figure 8.1). This could lead to a redistribution of
the bodys water content from the intracellular compartment to the extracellular one, rather
than an actual increase. A study (Thys-Jacobs et al., 2007) carried out on a group of women with
PMDD in phase I of their cycle confirmed this interesting hypothesis, showing reduced levels of
the serum ionic calcium of affected women in comparison to controls; in the following phases,
from day 7 to day 15 of the menstrual cycle, also calciuria seemed to be reduced. Finally, during
the luteal phase (from day 15 to day 28 of the menstrual cycle), also low serum concentrations
of calcitriol were recorded (the metabolically active form of vitamin D). The authors concluded
that a reduced availability of calcitriol, with subsequent inability to block the reduction of plasma
concentrations of calcium during ovulation or the luteal phase (Figure 8.2) of the menstrual cycle,
could act as a factor triggering the onset of PMDD. These data support other observations that
underline the importance of dietary imbalances among the possible concurrent causes and/or
Symptoms:
Depression
Tension
Cramps
Hematic concentration of estradiol
400 Migraine
E2 pg/ml
200
E2
Ca++
Ca++
0
1 14 28
Days of menstrual cycle
Figure 8.1. Relationship between hematic estradiol and symptomatology of premenstrual syndrome during the
menstrual cycle (modified from Thys-Jacobs, 2000).
55
Controls
50
45
25(OH)D3 (ng/ml)
40
35 *=P<0.05
**=P<0.01
30 Women with premenstrual syndrome
*
25 ** **
20
15
early follicular phase ovulation phase late lutheal phase
Phases of the menstrual cycle
Figure 8.2. Difference in values of 25(OH)D3 in women with premenstrual syndrome and in controls in the
various phases of the menstrual cycle (modified from Thys-Jacobs, 2000).
factors triggering PMS (Abraham and Rumley, 1987; Yonkers et al., 2008). This explains the role
of lifestyle intervention as important therapeutic tool that can be used in the clinical management
of PMS.
8.2 Vitamin D
Vitamin D is a hormone primarily involved in the calcium and phosphorous metabolism but
with several other important metabolic functions. In human, usually 80% of the daily need
of vitamin D is guaranteed by UV-B solar radiation (wave length between 290 and 315 nm)
on 7-dehydrocholesterol, whilst the remaining 20% is assured through the diet. In order to
carry out its metabolic functions, vitamin D must be converted in its active form, the 1,25
dehydroxycholecalciferol or 1,25(OH)2 or vitamin D3, through a series of metabolic pathways that
involve both the liver and the kidneys. Especially in cases of renal insufficiency, this mechanism can
be impaired thus justifying the use of active metabolites of vitamin D. The metabolism of vitamin
D is regulated by plasma concentrations of parathormone, serum concentrations of calcium and
phosphorous and by other factors such as FGF-23. In particular, calcium exerts its action both
directly and indirectly through PTH: an increase in calcaemia inhibits the conversion of 25(OH)
into 1,25(OH)2. An evaluation of the circulating concentrations of vitamin D is normally carried
out by measuring the hepatic metabolite 25-hydroxyvitamin-D: serum concentrations of around
30 ng/ml or more are generally considered as being normal. One talks of insufficiency when
concentrations are below 29 ng/ml (72 nmol/l) and of risk of intoxication when concentrations
exceed 150 ng/ml (374 nmol/l) (Holick, 2007). Concentrations below 20 ng/ml (<50 nmol/l) are
considered as severe deficiency (Adami et al., 2011).
Vitamin D, together with PTH is one of the main regulators of the plasma concentrations of
calcium ions. E.g. we should remember that for vitamin D concentrations lower than 29 ng/ml,
the intestinal calcium absorption is reduced by 85-90% and that of phosphorous by 40% (Heaney
et al., 2003). If the periods of pregnancy and breastfeeding are excluded, the organs involved in
calcium exchanges from and to the extracellular fluid are essentially three: the intestine, the bones
and the kidneys. The functions of vitamin D are exerted at all these three levels: at the intestinal
level vitamin D increases calcium absorption, through a satiable active transport mechanism
that takes place principally in the duodenum and jejunum. With a normal calcium dietary
intake, the absorption takes place almost exclusively through this pathway, as ionized calcium
concentrations in the ileum never exceed the degree of concentration necessary to activate
the second mechanism of intestinal absorption, that is passive. The kidney is a central organ
in the regulation of the calcium homeostasis and its intervention in the calcium-phosphorous
metabolism is under hormonal control, especially through PTH: the role of vitamin D is to
increase the tubular reabsorption of calcium and excretion of phosphorous. Since 98% of calcium
filtered through the glomerulus is recovered through renal reabsorption, even a slight reduction
(1%) of the tubular reabsorption could explain an increase in calciuria of up to 50%. Finally, at
the level of the skeletal system the vitamin D controls bone calcium deposition regulating the
balance between osteoblast and osteoclast formation.
Besides the crucial role played by vitamin D in the regulation of the calcium metabolism, over
the past years, numerous extra-skeletal functions of this vitamin have been described. Vitamin
D receptors are also expressed in the muscle: muscle speed and strength improve in presence of
optimal concentrations of circulating vitamin D (Bischoff-Ferrari et al., 2006). At metabolic level,
vitamin D modulates the secretion of and the sensitivity to insulin. At cardiocirculatory level,
it acts at different concentrations regulating arterial pressure through a down-regulation of the
renal production of renin and of endothelial homeostasis factors. Moreover, vitamin D modulates
the immune response and reduces the risk of certain types of tumor (breast, colon and prostate
cancers) (Holick, 2007).
The role of vitamin D in reducing the risk of PMS is still under study and seems primarily correlated
with the modulation of calcaemia concentrations, as well as of certain neurotransmitters and
sexual steroids. Evidence suggests that the improvement of calcium balance through the intake
of milk derivatives or supplements or calcium carbonate supplements leads to an improvement
of PMS symptoms: the real entity of this effect remains however to be defined (Penland and
Johnson, 1993; Thys-Jacobs et al., 1989, 1998, 2007). Actually, it has been shown that it might
be more important to reduce the loss of calcium: this objective can be achieved by normalizing
the protein intake (an excess of proteins of animal origin increases the renal loss of calcium),
avoiding dietary excess of sodium, caffeine and tobacco intake, practicing regular physical
activity and guaranteeing a correct intake of vitamin D with a regular exposure to sunlight or
intake of vitamin supplements. A correct calcium intake seems to be a necessary, but probably
insufficient, condition to prevent PMS. Evidence in fact confirms the importance of vitamin D
supplementation at a posology of 400 UI/d, once an adequate dietary intake of calcium has been
achieved (1,200 mg/d). Among these data, we would like to mention a pilot study carried out on
186 women aged between 18 and 30, of which 44 were affected by PMS. After being stratified on
the basis of their daily vitamin D intake from food, more or less than 100 IU/d, the women that
took vitamin D doses greater than 100 mg/d showed a statistically significant lower prevalence of
PMS. Concentrations of vitamin D measured during the luteal phase of the menstrual cycle were
not correlated with the incidence of PMS. The authors concluded that it was likely that there was
a link between vitamin D and PMS, however further studies are needed to evaluate in particular
vitamin D concentrations during the early follicular and luteal phase of the menstrual cycle
(Bertone-Johnson et al., 2005). Some years later another study published by the same Authors,
showed the efficacy of an adequate dietary calcium intake (1,200 mg/d) associated with correct
vitamin D supplementation (400 UI/d), necessary in order to achieve a correct intestinal calcium
absorption, in the treatment and prevention of PMS (Bertone-Johnson et al., 2010). 3025 women
aged between 27 and 44 years were enrolled in the study; 1057 of them were diagnosed as affected
by PMS with more than ten years of follow-up and 1968 were not diagnosed as affected by PMS,
neither having a menstrual symptomatology. Blood concentrations of calcium and vitamin D
were measured upon enrolment and then every 4 years and the study lasted ten years. After
having considered parameters such as age, pregnancies, smoking abuse and other risk factors, it
was shown that women with a higher intake of vitamin D (median, 706 IU/d) had a lower relative
risk of PMS compared to those with poor vitamin D intake (median, 112 IU/d) (P=0.01). Also
the amount of calcium introduced through the diet strongly correlated with PMS: by comparing
women with low intake (median, 529 mg/d) with those with greater intake (median, 1,283 mg/d)
it emerged that the relative risk was lower for the latter. This suggests that a high intake of vitamin
D and calcium may reduce the risk of PMS, as well as that of osteoporosis and of other neoplasias
and thus justifies calcium and vitamin D supplementation especially in young women affected
by PMS.
8.3 Other nutrients involved in the management of PMS

Research carried out over the past years suggests that, besides calcium and vitamin D, a wide
variety of foods can play a direct or indirect role in the pathogenesis of menstrual cycle disorders
in general and in particular of PMS: among them we can find EFA, some vitamins (in particular
vitamin B6), dietary intake of carbohydrates (especially simple sugars), proteins and lipids, some
minerals (magnesium and manganese), caffeine and alcohol (Table8.2).
Clinical evidence suggests that the dietary choice directly influences dysmenorrhea: in fact, it
has been shown that a vegetarian diet with a very low fat content, at least in some patients,
can cause a marked reduction in menstrual pain (Barr et al., 1994). This presumably happens
because the reduction of the intake of dietary fat, especially of animal origin, leads to a reduced
concentration of estrogens in the blood. Foods of vegetable origin, such as cereals, pulses, fruit
and vegetables (unlike the food of animal origin) contain dietary fiber such as bran, that tends to
drag the estrogens out of the organism: the estrogens extracted by the liver from the blood stream
are then sent through the biliary tract to the digestive system where they bind to the fiber. The
more vegetables there are in the diet, the greater the amount of fiber intake: without an adequate
Table 8.2. Principal nutrients that can influence the clinical manifestations of dysmenorrhea and premenstrual
syndrome.
Calcium
Vitamin D
Phytoestrogens
Essential fatty acids
Vitamin B6
Carbohydrates, lipids, proteins
Magnesium
Manganese
Caffeine
Alcohol
amount of fiber, the estrogens secreted into the digestive system are once again reabsorbed into
the blood circulation. Moreover, certain foods commonly used in vegetarian diets have beneficial
effects: for example, soy contains phytoestrogens, vegetable estrogens that, although not very
powerful, are able to move the natural estrogens from their physiological receptor ligand sites
(Bryant et al., 2005). Besides the single observations concerning an important reduction of
dysmenorrhea thanks to vegetarian diets with low fat content, vegetarian women also report
fewer disturbances during ovulation (Abraham and Rumley, 1987).
The dietary intake of EFA (linoleic acid, progenitor of the omega-6 fatty acids, and alpha-linolenic
acid, progenitor of the omega-3 fatty acids) influences the production of PG, substances that
are involved in the regulation of inflammatory processes, in the modulation of pain, muscle
contraction, vasoconstriction and coagulation. In particular, an imbalance of the omega-3/
omega-6 fatty acids ratio from suggested values of 1:4-1:6 to 1:10 as normally happens in the
diet of industrialized countries, determines an excessive amount of proinflammatory PGs with a
potentially negative effect even on the symptoms of PMS. In fact, it is not by chance that subjects
with a diet particularly rich in omega-3 fatty acids tend to present a more modest menstrual
symptomatology (Deutch, 1995). It is also interesting to note an inverse relationship between
the concentration of omega-3 fatty acids and the risk of postpartum depression: evidence seems
to confirm the correlation between an unbalanced ratio of omega-3/omega-6 fatty acids in the
diet and the prevalence of post-partum depression, confirming the importance of EFA in the
regulation of mood not only during pregnancy (Jadoon et al., 2011; Da Rocha and Kac, 2012),
even though actually the recent meta-analyses have scaled down the role of EFA in the treatment
of severe forms of depression (Bloch and Hannestad, 2011). In any case, available evidence seems
at least to justify a modulation of the dietary intake of EFA, if not their use at a pharmacological
dose in the treatment of PMS (Horrobin, 1983).
Among the possible concurrent causes or causes worsening PMS, worthy of mention is the
vitamin pattern, whose involvement at various concentrations has long been object of discussion.
In some clinical studies it was shown that pyridoxine (vitamin B6) can reduce the symptom
of pain (Bernstein, 1990). Vitamin B6 seems to act by modulating the perception of pain but
not the cause that triggers it. Other studies have shown that concentrations of vitamin B6 but
also of B12 and folic acid are associated with depression and mood (Hvas et al., 2004; Karakula
et al., 2009). Vitamin B6 itself is involved, together with other vitamins (thiamine (vitamin
B1), riboflavin (vitamin B2), niacin (vitamin PP), folate (vitamin B9) and vitamin B12) in the
synthesis of serotonin, whose imbalance is potentially involved in the pathogenesis of PMS. These
observations could represent the rationale behind the efficacy of vitamin B6 in the treatment of
PMS, but available clinical evidence appears rather contradictory. Several PRCT studies confirm
its efficacy when administered alone (Wyatt et al., 1999), as a dietary supplement or in association
with magnesium (Fathizadeh et al., 2010), whereas others (Chocano-Bedoya et al., 2011) have
highlighted a reduced risk of PMS only in women that have a high dietary intake of thiamines
and riboflavin and not of vitamin B6, B12, niacin and folate. Even though the available studies
probably do not allow us to draw a final conclusion on the actual efficacy of the administration
of dietary supplements containing vitamin B6 or other vitamins, in any case they are sufficient to
suggest that better caution should be used in their dietary intake.
As far as carbohydrates are concerned, evidence shows that an intake of complex carbohydrates
can lead to a certain benefit thanks to the modulation of cerebral neurotransmitters such as
serotonin that regulate the tone of mood (Freeman et al., 2002; Sayegh et al., 1995). A study carried
out in 2008 by Japanese researchers (Murakami et al., 2008) on a group of 640 female students
aged between 18 and 22, highlighted the existence of an inverse, independent and statistically
significant relationship between the glycaemic index of meals eaten by the women involved the
study and the incidence of PMS. As a confirmation of the complex relationship between dietary
behavior and menstrual cycle, however, the opposite is also true: i.e. it was observed that women
affected by PMS have greater difficulty in following a balanced diet. In particular in a study
carried out on 88 women affected by PMS an increased intake of fat, carbohydrates and simple
sugars and a concurrent reduction in protein intake during the premenstrual phase were both
observed (Cross et al., 2001).
A study showed that manganese intake, evaluated also in association with calcium intake,
correlated with the symptoms of irritability and dysmenorrhea of PMS (Penland and Johnson,
1993), but there was not sufficient evidence to support the idea of providing supplementation of
this mineral in the diet of women affected by PMS.
For what concerns the role of magnesium there are some studies in which it was observed that a
dietary supplementation of magnesium reduced the entity of several PMS symptoms, including
water retention especially if magnesium was administered in association with polivitaminic
complexes (Facchinetti et al., 1991; Holick, 2007; Walker et al., 1998). This is probably related to
the myorelaxant effect of magnesium (De Souza et al., 2009) and confirms previous observations
that highlighted in the erythrocytes of women affected by PMS lower magnesium concentrations
in comparison to controls (Shamberger, 2003).
Caffeine seems to worsen PMS: several studies showed a direct correlation between caffeine
intake and entity of PMS symptoms (Chou, 1992; Rossignol, 1985) that could justify a reduction
of the intake of this xanthine alkaloid in patients.
Finally, as far as alcohol consumption is concerned, though there is no evidence confirming the
existence of a direct correlation between alcohol consumption and onset of PMS, it has been
hypothesized that an early and prolonged consumption of alcohol to a certain extent can increase
the risk of PMS (Bertone-Johnson et al., 2009).
8.4 Intervention on diet and use of vitamin D in the premenstrual syndrome

On the basis of what has been said, it appears clear that the clinical management of PMS can
benefit from a lifestyle based approach. This holds true both for fully blown cases in which a
pharmacological therapy remains the first line treatment, both in borderline cases, in which
diagnosis can appear elusive.
An intervention on diet and lifestyle is recommended not only for the primary effect it can have
on the specific symptomatology but also because in these patients it can be frequently observed
an incorrect dietary behavior (Cross et al., 2001), that can lead to an increase in weight and
ultimately trigger a vicious circle that in the end influences negatively the subjects wellness.
In these cases the diet should be normocaloric or slightly hypocaloric according to the weight of
the patient, with a balanced intake of the principal nutrients and particularly rich in vegetables,
with the objective of increasing fiber intake and reducing the glycaemic index of meals. Vice versa,
the number of meals does not seem to have particular clinical significance (Yonkers et al., 2008).
Intake of carbohydrates, especially simple ones, should be monitored. Consumption of caffeine
and probably of all xanthine alkaloids should be reduced or temporarily suspended, as well as
alcohol consumption. A regular aerobic physical activity, especially if done outdoors during the
day is, as always, recommended (Steege and Blumenthal, 1993).
Magnesium is present in nearly all foods even if at different concentrations. Greater amounts are
found in pulses, wholegrain cereals and in dried fruit. Green leafy vegetables and bananas are
good sources of magnesium whilst other common fruit, meat, fish and milk are other sources of
minor importance.
Vitamin B6 is found in milk and milk derivatives, fish, cereals, potatoes, cheese, spinach, beans,
and carrots. A balanced diet is able to guarantee an adequate intake.
Serotonin is a neurotransmitter synthesized from tryptophan, an essential amino acid found

in several foods: chocolate, oats, bananas, dates, peanuts, milk and dairy products. However,
without vitamins B3, B6 and C, tryptophan cannot be converted into serotonin. Vitamin B3 is
found in wheat, barley, pulses, tomatoes, milk, cheese, fish, carrots, potatoes, while vitamin C is
found in fresh fruit and vegetables (especially in citrus fruits, kiwi, peppers, broccoli). Once again
a balanced diet guarantees an adequate intake of these substances.
As far as other possible nutrients are concerned, phytoestrogens are found especially in pulses
(soy, beans, peas, broad beans and lentils), vegetables (cauliflower, broccoli, cabbage, Brussels
sprouts, turnips and turnip tops), wholegrain cereals, nuts, linseeds and sunflower seeds or
derivatives.
Omega-3 fatty acids are, as well known, found especially in blue fish (which is also rich in vitamin
D), but are available today in numerous foods where the concentration of omega-3 fatty acids has
increased through the application of genuine biomagnification, fortification and supplementation
techniques.
In nature vitamin D is found only in some foods, including certain types of fish (salmon, mackerel,
tuna), cod-liver oil and egg yolk. Exposure of arms and legs to sunlight for 10/15 min (equal to
half of the UV-B radiation dose able to cause erythema), determines in any case a synthesis of
vitamin D in the order of 3,000 UI (5 to 10 times the dose found in 100 g of fish and 15 times
greater than that found in egg yolk) (Table 8.3) (Holick, 2007).
As far as dietary supplementation is concerned, evidence justifies vitamin D and calcium

supplementation at doses respectively of 400/800 UI/d and 1,200 mg/d, and less so that of
magnesium (200/250 mg/d) and vitamin B6 (40 mg/d) (De Souza et al., 2009; Fathizadeh et al.,
2010).
8.5 Conclusions
PMS is an extremely frequent disorder in women of fertile age, especially in their third decade
of life, and the most severe cases can transform into PMDD, a truly debilitating disorder able to
periodically limit the work and the social life of affected women.
Among the causes and/or the triggering factors of PMS, today some importance is attributed
to various nutrients and, above all, to a correct intake of vitamin D, calcium, vitamin B and
magnesium. Simple preventive measures such as a correct intake of these nutrients, also through
dietary supplements, not only seems to reduce the severity of the symptomatology in patients
with PMS, but also to have protective effects on women who do not yet suffer from this disorder.
Contemporarily, although awaiting a definite evidence, a comparative analysis of available

literature suggests that the adoption of a correct lifestyle based on a balanced diet, regular
Table 8.3. Food sources of vitamin D2 and D3 (Modified after Holick, 2007).
Sources Vitamin D content1
Natural
Fresh wild-caught salmon (ca. 100 g) ca. 600-1000 UI of vitamin D2
Fresh farm-raised salmon (ca. 100 g) ca. 100-250 UI of vitamin D3 or D2
Canned salmon (ca. 100 g) ca. 300-600 UI of vitamin D3
Canned sardines (ca. 100 g) ca. 300 UI of vitamin D3
Canned mackerel (ca. 100 g) ca. 250 UI of vitamin D3
Canned tuna (ca. 100 g) ca. 230 UI of vitamin D3
Cod-liver oil (1 tablespoon) ca. 400-1000 UI of vitamin D3
Shiitake mushrooms
Fresh (ca. 100 g) ca. 100 UI of vitamin D3
Sun-dried (ca. 100 g) ca. 1,600 UI of vitamin D3
Egg yolk ca. 20 UI of vitamin D2 or D3
Exposure to sunlight, UV-B radiation ca. 3,000 UI of vitamin D
(0.5 of a minimum dose capable of causing erythema)2
Fortified foods
Fortified milk ca. 100 UI/240 ml, in general vitamin D3
Fortified orange juice ca. 100 UI/240 ml, in general vitamin D3
Baby formula ca. 100 UI/240 ml, in general vitamin D3
Fortified yoghurt ca. 100 UI/240 ml, in general vitamin D3
Fortified butter ca. 150 UI/100 g, in general vitamin D3
Fortified margarine ca. 430 UI/100 g, in general vitamin D3
Fortified cheese ca. 100 UI/100 g, in general vitamin D3
Fortified breakfast cereal ca. 100 UI/portion, in general vitamin D3
1 UI stands for International Units equivalent to 25 ng.

2 UV-B radiation minimum dose capable to cause erythema is the dose absorbed after 5-10 min of arms and legs
exposure to sunlight (it varies according to time of day, season, latitude and skin sensitivity).
physical activities and regular outdoor life with a correct exposure to sunlight, represents by itself
a fundamental step in the prevention and in the clinical management of PMS. These interventions
essentially coincide with guidelines for a healthy lifestyle proposed by all the major international
scientific communities and do not exclude the role of dietary supplementation of certain trace
elements and/or the role of pharmacological treatment every time the clinical conditions of the
patient were to require so.
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9. Perimenstrual chocolate craving: from
pharmacology and physiology to cognition and
culture
J.M. Hormes
Department of Psychology, College of Arts and Sciences, University at Albany, State University
of New York, Social Sciences 399, 1400 Washington Avenue, Albany, NY 12222, USA,
jhormes@albany.edu
Abstract
Chocolate craving is a common phenomenon, but only in some cultures. Americans are much
more likely to report chocolate craving than individuals in other cultures that have been surveyed.
Furthermore, in the United States, women are about twice as likely to experience chocolate craving,
compared to men. In about half of female US chocolate cravers, craving fluctuates cyclically
with a well-defined peak in frequency and intensity beginning approximately four days prior to
and lasting until about four days after the onset of menstruation. Though the phenomenon of
cyclically fluctuating chocolate craving has been called premenstrual, it is thus more appropriately
termed perimenstrual craving. The pattern of cyclically occurring chocolate cravings has raised
questions about a possible causal role of the menstrual cycle in eliciting cravings. This chapter
reviews existing accounts of mechanisms underlying perimenstrual chocolate craving, including
pharmacological, physiological, cognitive, and socio-cultural explanations. Evidence generally
does not support a causal role of the active ingredients in chocolate in either the etiology or the
satisfaction of craving. Cyclic fluctuations in hormones do not appear to directly elicit chocolate
craving. The interplay of several cognitive factors, including ambivalent attitudes to chocolate,
dietary restraint, and thought elaboration, on the other hand, may play a role in the etiology of
craving in general, and perimenstrual chocolate craving in particular. A growing body of literature
also suggests that perimenstrual chocolate craving is a culture-bound syndrome, limited to North
America. Based on these findings it is hypothesized that perimenstrual chocolate craving arises
as the result of womens efforts to restrict consumption of a highly ambivalent food, along with
a culturally-driven view of the perimenstrum as a cue signalling permission to engage in an
otherwise prohibited indulgence.
Keywords: perimenstrual, premenstrual, food, sweets, ambivalence, cultural differences
137
J.M. Hormes
Summary points
Perimenstrual chocolate craving, an increase in the frequency and intensity of craving for chocolate beginning
around four days prior to and lasting until about four days after the onset of menstruation, is reported by
around 50% of female chocolate cravers in North America.
In countries outside of North America, chocolate craving is far less prevalent, commonly reported about
equally by men and women, and rarely or not at all linked to the menstrual cycle.
Evidence does not support a role of the pharmacologically active ingredients of chocolate in either the
etiology or satisfaction of cravings, but instead points to a primary role of the sensory properties of chocolate.
Mechanisms associated with the menstrual cycle, such as cyclic fluctuations in levels of hormones, do not
appear to directly cause perimenstrual chocolate craving.
A number of cognitive processes have been implicated in the etiology of perimenstrual chocolate craving,
including conflicting attitudes towards chocolate, cue reactivity, and thought elaboration.
Cultural differences in the nature of craving, including an absence of fully equivalent translations of the word
craving in most languages outside of English, suggest that any causal mechanisms underlying the etiology
of perimenstrual chocolate craving are likely culture-bound.
It appears that culturally defined chocolate-related attitudes and behaviours, along with socially sanctioned
circumstances under which it is acceptable to indulge in otherwise forbidden foods, drive the high prevalence
of perimenstrual chocolate craving specifically in North America.
9. Perimenstrual chocolate craving
9.1 Introduction
As illustrated throughout this volume, food intake is tied to the menstrual cycle in a myriad of
complex ways. This chapter discusses a very specific link, namely the temporal association between
craving for chocolate and the perimenstrual period, which includes the premenstrum and the first
few days of menstruation. Perimenstrual chocolate craving is a well-documented phenomenon,
but its etiology remains poorly understood. This review discusses a number of mechanisms
hypothesized to underlie perimenstrual chocolate craving, roughly in the chronological order in
which they were examined by the field. It closes with a discussion of recent research on cognitive
and cultural factors, integrated into a proposed new model of perimenstrual chocolate craving,
and suggestions for future research.
9.1.1 Food cravings
A food craving is an intense urge that occupies an individuals thoughts and motivates them to
go out of their way to satisfy it (Hormes and Rozin, 2010). Craving differs from the physiological
state of hunger, which can be alleviated by any number of foods, in that a craving is typically only
satisfied by one specific food (Pelchat, 2002) (Table 9.1).
Food cravings are common phenomena in some countries. For example, in a survey of United
States undergraduate students, 94% of women and 75% of men reported ever having experienced
a craving for a specific food (Zellner et al., 1999). Similarly, in Canada, 97% of female and 68% of
male college students endorsed food cravings (Weingarten and Elston, 1990).
Individuals who crave foods experience on average one to two craving episodes per week, and
most result in the consumption of the desired food (Hill and Heaton-Brown, 1994; Weingarten
and Elston, 1991) (Table 9.1). Cravings are most common in younger individuals, and prevalence
decreases with age (Pelchat, 1997).
Table 9.1. Facts about craving.
Definition A craving is an intense urge that motivates an individual to go out of his or her way to satisfy it
(e.g. Hormes and Rozin, 2010)
Specificity Craving differs from the physiological state of hunger, which can be alleviated by any number
of foods, in that it is typically only satisfied by one specific food (Pelchat, 2002)
Frequency Individuals who crave foods experience an average of one or two craving periods per week
(Weingarten and Elston, 1990)
Prevalence 94% of female and 75% of male US undergraduates have experienced a craving for a
specific food (Zellner et al., 1999)
Language The word craving does not lexicalize in most languages other than English (Hormes and
Rozin, 2010)
J.M. Hormes
Research points to a common neuroanatomical substrate for food and drug cravings (Pelchat et
al., 2004). Cravings for cigarettes, alcohol, and drugs of abuse are potent triggers for relapse and
major obstacles to sustained abstinence (Bottlender and Soyka, 2004; Evren et al., 2012; Ferguson
and Shiffman, 2009; Sinha et al., 2006). By comparison, food cravings are mostly harmless;
however, they have been shown to be associated with binge eating episodes in bulimic and
obese women (Bjoervell et al., 1985; Kales, 1990; Mitchell et al., 1985). Even in non-pathological
populations, cravings can elicit strong feelings of guilt (Macdiarmid and Hetherington, 1995). A
better understanding of craving etiology therefore has broad clinical importance.
9.1.2 Chocolate craving and the menstrual cycle
Chocolate is the most commonly and intensely craved food in Western cultures (Rodin et al.,
1991; Rogers and Smit, 2000; Rozin et al., 1991; Weingarten and Elston, 1990), and it is unique
in many ways. Chocolate has a highly attractive aroma and melts at body temperature, producing
extremely pleasant tactual dynamics in the mouth. Cacao beans, the seeds of the Theobroma cacao
tree (Lopez, 2002), contain high levels of lipids, making chocolate a relatively high-fat food. In its
pure form, cacao is bitter and rather unpalatable. A variety of processing techniques, including
roasting, fermentation, and the addition of sugar, bring out the aroma of chocolate not readily
apparent in the raw beans.
Dark chocolate contains the highest concentrations of cacao, while milk chocolate usually has
extra fat and sugar added to it. White chocolate is not a true chocolate, but is simply cocoa
butter mixed with sugar and flavours (Table 9.2). As a result it contains few, if any, of the active
ingredients of pure cacao, with the possible exception of fat-soluble components, such as trace
levels of the cannabinoid anandamide (Bruinsma and Taren, 1999; Parker et al., 2006).
More than half of Americans rate chocolate as their favourite flavour, and a majority prefer milk
to dark or white chocolate. 1 A strong preference for chocolate is experienced as something more
than mere liking by many: in a survey of American undergraduates, 91% of women and 59% of
Table 9.2. Requirements for ingredients in cacao products (FDA, 2011).
Chocolate liquor Milk solids Cocoa butter Sweetener
Semi-/bitter-sweet 30-35%
Milk 10% 12%
White 14% 20% 55%
1 Survey conducted by the National Confectioners Association: http://www.candyusa.com/FunStuff/FunFactsDetail.

cfm?ItemNumber=976.
men had experienced a craving for chocolate (Osman and Sobal, 2006) (Figure 9.1). Chocolate
is the most frequently craved food among sweet cravers in the US (Zellner et al., 1999). Roughly
20% of women, but less than 10% of men rated chocolate as their most intensely craved food in
a survey of undergraduate students and their parents (Rozin et al., 1991).
These figures suggest that chocolate craving is common in the US, and also highlight the
pronounced gender differences consistently observed in studies of craving prevalence, with
women up to twice as likely as men to report strong urges for chocolate (Osman and Sobal, 2006;
Rozin et al., 1991; Weingarten and Elston, 1991; Zellner et al., 1999). Perimenstrual chocolate
craving appears to fully account for these gender differences: across multiple studies, about half
of women surveyed report a well-defined craving peak for chocolate in the perimenstrual period,
beginning from a few days before the onset and extending into the first few days of menses (Hill
and Heaton-Brown, 1994; Rozin et al., 1991; Zellner et al., 2004) (Figure 9.2).
Over the course of several decades, numerous hypotheses have been proposed to explain the
etiology of perimenstrual chocolate craving. Studies examining the pharmacology of chocolate,
the physiological basis of menstruation, cognitive factors, and the cultural context of craving
paint a picture of a complex phenomenon.
9.2 The pharmacology of craving

Cacao contains a number of pharmacologically active ingredients (Bruinsma and Taren, 1999;
Rogers and Smit, 2000), including the sympathomimetic amines tyramine and phenylethylamine
(Michener et al., 1999), the cannabinoid anandamide (Di Tomaso et al., 1996), and methylxanthines
(Smit et al., 2004) (Table 9.3).
100
91.0 89.9
90
81.1
80 75.7
% reporting chocolate craving
70
60
50 45.0
40
30
20
10
0
Undergraduate Undergraduate Age 45 Age 65 Age 80
(any) (regular) (any) (any) (any)
Figure 9.1. Prevalence of chocolate craving (any or regular craving) in US women at different ages (Hormes
and Rozin, 2009; Osman and Sobal, 2006; Zellner et al., 1999).
J.M. Hormes
Plasma concentrations Estrogen
Progesterone
2 4 6 8 10 12 14 16 18 20 22 24 26 28
Follicular Midluteal Luteal Premenstrual

Periovulatory
Days
Figure 9.2. Approximate plasma concentrations of oestrogen and progesterone throughout the menstrual cycle
(day 1 = onset of menstruation) (Hormes, 2010).
Table 9.3. Examples of chocolate ingredients with hypothesized pharmacological effects

(Bruinsma and Taren, 1999; Di Tomaso et al., 1996; Michener and Rozin, 1994; Rogers and Smit, 2000; Smit
et al., 2004).
Type Chocolate ingredient Hypothesized effect
Methylxanthine theobromine hypothesized to exert stimulant effects

caffeine
Cannabinoid anandamide thought to mimic effects of cannabinoid drugs and induce
euphoria, etc.
Sympathomimetic amine tyramine hypothesized to exert stimulant effects
phenylethylamine
Other tryptophan serotonin precursor with wide range of hypothesized effects
casomorphin opoid agonist with wide range of hypothesized effects
Two hypotheses implicate the psychoactive effects of chocolate in perimenstrual craving (Figure
9.3):
1. Physiological changes during the perimenstrum induce a deficit state, which is relieved by
one or more ingredients in chocolate.
2. Ingredients in chocolate exert positive effects, which are most salient during the perimenstrum.
The claim that perimenstrual chocolate craving occurs in response to a nutritional or physiological
deficiency was motivated by observations such as a menstrual drop in levels of magnesium, which
has been linked to a variety of affective and physical premenstrual symptoms (Facchinetti et
Physiological/Pharmacological Cognitive/Psychosocial/Cultural
Nutritional deficits: Ambivalence:

Physiological changes during the Tension between approach
perimenstrum induce a deficit and avoidance tendencies
state, which is relieved by one or toward chocolate cause cravings
more ingredients in chocolate
Pharmacologically Cue reactivity:

active ingredients: Cravings are elicited by
Ingredients in chocolate exert external (and/or
positive effects, which are most internal) cues
salient during the perimenstrum
Hormonal fluctuations: Dietary restraint:

Hormonal fluctuations cause Attempts to restrict intake of
cravings at the time of the chocolate enhance the
perimenstrum, e.g. by salience of chocolate-related
increasing energy needs cues and cause cravings
Figure 9.3. Summary of mechanisms hypothesized to play a role in the etiology of perimenstrual chocolate
craving.
al., 1991; Quaranta et al., 2007; Rosenstein et al., 1994). Research generally does not support
a nutritional or pharmacological deficiency explanation of perimenstrual craving (Pelchat and
Schaeffer, 2000). If the bodys attempts to restore homeostasis in the face of deficits caused
craving it should be rather non-specific and include strong urges for foods of similar nutritional
composition as chocolate, for example, those high in magnesium (e.g. cashews or spinach) or
tryptophan (e.g. peanuts or bananas). Instead, most craving episodes are highly specific, with few
or no apparent substitutes for chocolate to satisfy them (Pelchat, 2002).
It has been speculated that mere caloric depletion increases the frequency of cravings for foods
high in sugar, for example, by enhancing the pleasure of sweet taste. Evidence generally does
not support this assumption. Responsiveness to both sweet and bitter tastes is attenuated when
oestrogen is low (Gong et al., 1989), and consuming a very low-calorie liquid diet tends to
decrease, rather than increase, the frequency of food cravings (Martin et al., 2006). A caloric
depletion hypothesis fails to account both for the unique perimenstrual pattern of many craving
episodes, as well as for the specificity of most cravings: assuming a causal role of a negative energy
balance, perimenstrual craving should be more generally for sweet, fatty, or other calorically
dense foods.
When considering the pharmacology of cacao, it is important to distinguish between potential

physiological triggers of craving and the physiological effects that reduce it. Just like headaches are
not caused by low levels of blood aspirin, but can be treated with aspirin, chocolate craving may
J.M. Hormes
not be triggered by a deficiency in a certain substance, but may be satisfied by the consumption
of that substance. It can thus be hypothesized that instead of alleviating a negative state, chocolate
is craved because its consumption results in positive experiences, such as enhanced mood or
alertness.
Studies examining links between craving and mood states have had mostly mixed results. Mood
states were once reported to be unrelated to food cravings across the menstrual cycle (Bancroft
et al., 1988). Subsequent studies found that depressed individuals consume more chocolate (Rose
et al., 2010), and identified depressed and dysphoric moods as triggers for chocolate craving (Hill
et al., 1991; Willner et al., 1998).
It has been argued that women, who suffer from depression at greater rates than men, use
carbohydrates to self-medicate during times of perimenstrual dysphoria, and a dysphoric
mood induction has been shown to induce a general preference for foods rich in carbohydrates
specifically in women (Corsica and Spring, 2008). Evidence suggests that consumption of
carbohydrates increases the availability of tryptophan and mood-enhancing neurotransmitters
in the brain (Benton, 2002). Tyramine and phenylethylamine, two ingredients in chocolate,
are similar in structure to endogenous neurotransmitters with activating and arousing effects
(Michener et al., 1999). However, significantly higher doses than those found in average sized
servings of chocolate would need to be ingested to achieve these effects (Rogers and Smit, 2000).
Furthermore, recent studies report that consumption of chocolate can lead to significant negative
food-related cognitions (Macht and Dettmer, 2006), guilt, anxiety, and depression (Fletcher et
al., 2007), suggesting that at least in some, chocolate may not only fail to alleviate dysphoria, but
could in fact prolong it (Parker et al., 2006).
The methylxanthines caffeine and theobromine in chocolate increase energetic arousal and well-
being and have been hypothesized to alleviate unpleasant physical symptoms associated with
the perimenstrum (Rossignol et al., 1991; Smit et al., 2004). However, milk chocolate contains
on average only 0.2 mg/g of caffeine and 2 mg/g of the less potent theobromine (Michener and
Rozin, 1994; Shivley and Tarka, 1984). The 120 mg of theobromine found in a 60 g portion
of milk chocolate are far below what is generally considered a reliable placebo-discriminable
dose (Mumford et al., 1994). The same 60 g serving of milk chocolate contains only around
12 mg of caffeine, which is considerably less than the 40-130 mg found in a typical serving of
coffee or tea (James, 1991). There is no evidence for a dose-response difference between dark and
milk chocolate (Smit et al., 2004), and anecdotally, unlike coffee, chocolate is rarely described
as or considered an arousing or energizing food. The effects of any potentially arousing agents
in chocolate are thus small at best and unlikely to play a significant role in causing cravings, in
general, or perimenstrual craving, specifically.
Chocolate contains the lipid anandamide and its analogues, which have been hypothesized to
bind to cannabinoid receptors to heighten sensitivity, induce euphoria, and exert calming and
anxiolytic effects (Di Tomaso et al., 1996). It remains unclear if chocolate contains high enough
concentrations of anandamide to produce these effects to any noticeable degree (Rogers and
Smit, 2000), and if and why effects would be particularly salient during the perimenstrum. Of
note, exogenous administration of the anxiolytic drug alprazolam does not significantly decrease
perimenstrual chocolate craving, suggesting that it is unlikely that chocolate is craved specifically
for its anti-anxiety effects (Michener et al., 1999).
Compelling evidence against a pharmacological basis of chocolate craving in general comes from
a study isolating the orosensory from the pharmacological properties of chocolate (Michener and
Rozin, 1994). Consumption of the equivalent of an average serving of chocolate in the form of
capsulated cocoa powder was largely ineffective at reducing chocolate cravings. White chocolate,
on the other hand, which with the possible exception of the fat-soluble lipid anandamide
contains none of the active ingredient of dark and milk chocolate, but shares its aroma, creamy,
melt-in-your-mouth texture, and high caloric density, reduced craving with about half the
potency of regular chocolate.
9.3 The physiology of perimenstrual craving

The notion of craving has become so closely tied to the perimenstrum that specific food cravings
are included as a criterion in the diagnosis of premenstrual dysphoric disorder (APA, 2000). Of
note, the link between the menstrual cycle and craving appears to extend to a variety of non-
food substances as well. Smokers are more likely to experience craving during the luteal phase
of the menstrual cycle (Carpenter et al., 2006); similarly, alcohol-dependent women identify the
premenstrum as a drinking cue (Epstein et al., 2006) (Figure 9.2).
Based on the pronounced cyclic pattern of chocolate and other cravings in many women, it was
once widely suggested that hormonal fluctuations, or other mechanisms associated specifically
with the menstrual cycle, play a causal role in perimenstrual chocolate craving. An early study
found no significant association between levels of the oestrogen estradiol and the frequency or
types of cravings (Rodin et al., 1991). The hypothesis that a cyclic fall in progesterone (Figure
9.2) induces perimenstrual chocolate craving was not supported in subsequent research that
demonstrated that exogenous administration of progesterone, an inhibitor of estradiol and its
effects, does not effectively reduce craving at the time of the perimenstrum (Michener et al.,
1999).
If cyclic fluctuations in levels of hormones were to play a causal role in approximately half of the
incidence of chocolate craving observed among US women, one would expect craving prevalence
to drop by about 50% in post-menopausal women. However, a survey of three groups of women
ages 45, 65, and 80 found that a significant proportion of women at all ages crave chocolate
regularly (Hormes and Rozin, 2009) (Figure 9.1). Most importantly, there was a striking absence
of a drop in craving prevalence post-menopause that was high enough (i.e. about 50%) to support
a causal role of cyclic fluctuations in hormones in the etiology of perimenstrual chocolate craving
in women pre-menopause (Hormes and Rozin, 2009). In spite of the temporal association
J.M. Hormes
between the menstrual cycle and chocolate craving, the two do not appear to be causally linked
such that perimenstrual changes in levels of hormones directly bring about cravings.
9.4 Craving and cognition

Since the actual onset of menstruation is triggered not by hormonal changes, but rather by localized
events, including increased coiling of spiral arteries, ischemia, and shedding of the endometrium,
it is an occurrence of psychological rather than systemic significance. Furthermore, the typical
pattern of craving reported by many female chocolate cravers in the US is characterized by peaks
in frequency and intensity beginning some days prior to menstruation. As such, perimenstrual
chocolate craving may well be driven by psychological, rather than physiological, factors (Figure
9.3).
9.4.1 Ambivalence to chocolate
Chocolate is widely, though incorrectly, stigmatized as lacking in nutrients and its consumption
is associated with weak willpower. Since it is simultaneously very palatable and high in caloric
density, chocolate can evoke powerful conflicting thoughts and feelings (Cartwright and Stritzke,
2008; Rogers and Smit, 2000). Women in particular hold ambivalent views of sweet snacks as
being both unhealthy and pleasant (Grogan et al., 1997). In a survey of female American college
students, 14% reported feeling embarrassed when buying a chocolate bar at the store (Rozin et
al., 2003). More than a quarter of US women include both positive and negative words when
giving three free associations to chocolate (Rozin, unpublished observation). In some normal-
weight women, consumption of a bar of chocolate elicited both elevated mood and feelings of
guilt (Macht and Dettmer, 2006). Exposing women to calorically dense foods has been shown
to inhibit their salivary response, suggesting that they experience significant anxiety when faced
with palatable foods that are perceived as forbidden (Rogers and Hill, 1989; Rosen, 1981; Wooley
and Wooley, 1981).
Several studies have begun to examine the role of attitudinal and affective ambivalence in the
experience of craving. Data supports a three-factor ambivalence model of chocolate craving,
with three components reflecting underlying approach and avoidance inclinations, along with
guilt (Cartwright and Stritzke, 2008). In US undergraduates surveyed while presented with a bar
of chocolate, the degree of ambivalent affect reported was significantly and positively associated
with their desire to eat the chocolate, but significantly and inversely correlated with the amount of
chocolate subsequently consumed (Hormes and Rozin, 2011). In other words, the more conflicted
they felt, the more respondents wanted to eat the chocolate, but the less likely they were to actually
do so. Giving in to a craving as it emerges tends to enhance positive mood, whereas resisting
before eventually giving in to it triggers increases in negative mood (Hill et al., 1991). Based on
these preliminary findings, the role of ambivalence in craving, and perimenstrual craving, in
particular, should be examined further.
9.4.2 Cue-reactivity and thought elaboration
Eating is elicited by external non-food cues, such as time of day or place (Weingarten, 1984). It
has been argued that cue-induced eating plays a role in chocolate craving, such that certain cues
that are present when eating chocolate become associated with chocolate and trigger cravings
on future occasions (Zellner and Edwards, 2001). Incentive sensitization theory postulates
that repeated activation of mesolimbic dopamine neurotransmission enhances the salience and
motivational value of drug rewards and drug-related cues, thereby transforming mere wanting
(which is thought to be distinct from liking, based on separate underlying neural systems)
into craving (Berridge, 2009; Robinson and Berridge, 1993) The way in which the notion of
incentive sensitization may apply to perimenstrual chocolate craving in particular remains to be
determined.
Chocolate cravers show a strong attentional bias for chocolate-related cues (Kemps and
Tiggemann, 2009; Smeets et al., 2009), and cue-elicited craving appears to interfere with limited
cognitive resources (Kemps and Tiggemann, 2010). Acute chocolate craving specifically impairs
visuo-spatial aspects of working memory performance, suggesting that in terms of underlying
cognitive processes, craving is largely visual in nature (Tiggemann et al., 2010). Indeed, when
people crave, they report experiencing vivid images of the desired substance (May et al., 2004),
and visuo-spatial tasks have been shown to effectively reduce craving for chocolate and cigarettes
(Andrade et al., 2012; May et al., 2010). Recent research demonstrates that mere thoughts about a
craved food can strengthen and maintain cravings over extended periods of time. The Elaborated
intrusion theory postulates that craving is the result of cue-elicited intrusive thoughts about the
craved substance and elaborative processes that increase the salience of thoughts and mental
images related to the craved substance (Kavanagh et al., 2005; May et al., 2012). As is the case
with most theories of craving, more work is needed to apply the construct of elaborated intrusion
specifically to perimenstrual craving.
9.4.3 Dietary restraint
As noted previously, restrained intake of chocolate is valued, especially in women in Western

cultures, and failure to maintain restraint is a sign of weakness and a trigger for feelings of guilt.
Many women attempt to resolve their ambivalence in favour of abstinence from chocolate.
Levels of dietary restraint have been shown to be positively associated with food cravings (Hill
et al., 1991), and deprivation triggers craving and overeating specifically in restrained eaters
(Polivy et al., 2005). Successful maintenance of dietary restraint requires cognitive effort and
attention-demanding tasks, which necessitate allocation of cognitive resources, increase the
likelihood of disinhibited eating in highly restrained eaters (Ward and Mann, 2000). Of note,
women who link chocolate craving to the menstrual cycle reported significantly higher levels of
dietary restraint than those who did not perceive any temporal association (Hormes and Timko,
2011). The possibility that dietary restraint plays a particularly important role in the etiology of
perimenstrual craving should be examined in future research.
J.M. Hormes
9.5 Culture and craving

Food choice is closely tied to cultural identity. For example, most Americans associate the
consumption of chocolate with culture-specific holidays, such as Easter or Valentines Day
(Osman and Sobal, 2006). Somewhat ironically, American culture, which promotes chocolate
as a special treat and antidote for negative mood states, is characterized by the presence of
significant conflict around its consumption. It appears that this ambivalent view of chocolate is
not universal, but rather culturally bound. For example, American women are likely to report
feeling bad after eating chocolate, while Spanish women experience mostly positive or neutral
mood states (Osman and Sobal, 2006).
As noted previously, food cravings are extremely common in the US and Canada. However, in
New Zealand, which is another English-speaking country, a survey of 18 to 45 year-old women
found that only 52% had ever experienced any food cravings unrelated to pregnancy (Gendall et
al., 1997). Sweet cravings are virtually absent in other regions: craving for chocolate represented
a mere 1% and 6% of cravings reported by young Egyptian men and women (Parker et al., 2003).
Rice was the most commonly craved food in a survey of Japanese women (Komatsu, 2008).
Interestingly, in any given country there is an inverse relationship between the amount of raw
cacao produced and the amount of chocolate consumed: Belgian and Luxembourgish per capita
consumption of chocolate is about 5.9 kg a year, but in the worlds two leading cacao producing
countries, the Ivory Coast and Indonesia, annual consumption of chocolate is only about 454
and 36 g per capita, respectively (Richardson, 2003). Furthermore, many non-chocolate sweets
are an important part of local food culture in countries such as Egypt, yet most Egyptians desire
primarily savoury foods (Parker et al., 2003). While access to cacao does not appear to be a
primary factor in determining the nature or prevalence of chocolate cravings, the extent to which
regional availability of processed chocolate products may play a role remains to be determined.
Even in countries that report cravings for specific foods, there is a striking absence of the marked
gender differences that characterize chocolate craving in the US (Zellner et al., 1999). The link
between being female and craving chocolate appears uniquely engrained in American culture:
American men and women believe that chocolate craving occurs more frequently in women
than in men, while Spanish men generally feel that craving is gender neutral (Osman and Sobal,
2006). While half of American women link chocolate craving to the menstrual cycle, only 28%
of Spanish women did the same (Osman and Sobal, 2006).
The notion of chocolate craving has become part of American cultural vocabulary, but marked
cultural differences in the perception and use of chocolate raise questions about the extent to
which craving is an important construct outside of North America. Using a linguistic approach,
a recent study found that the term craving does not lexicalize in a majority of languages other
than English (Hormes and Rozin, 2010). When a rough translation is available, it often refers to
strong desires for food specifically during pregnancy (e.g. antojo in Spanish). Indeed, the cross-
cultural studies described here note the use of descriptive phrases such as what food or drink do
you want very intensely? in place of equivalent translations (Osman and Sobal, 2006; Parker et
al., 2003). Assuming that lexicalization reflects the universality of certain concepts (Wierzbicka,
1999), findings suggests that the notion of craving may be limited in its importance to the US or
North America (Hormes and Rozin, 2010). The role of cultural factors in the high prevalence of
perimenstrual chocolate craving in the US merits further study.
9.6 Conclusions
A number of attempts have been made to explain the phenomenon of perimenstrual chocolate
craving (Figure 9.3). More work remains in to be done, but there is sufficient evidence to rule out
some hypotheses and to begin to integrate others into a new, testable model. Research to date speaks
strongly against a direct role of the pharmacology of chocolate or the physiological mechanisms
involved in regulating menstruation in the etiology of perimenstrual chocolate craving. Though
still preliminary, evidence instead suggests that complex cognitive processes and cultural context
play a key role: in North America, where chocolate is a source of much ambivalence, women in
particular seek to resolve this ambivalence in favour of abstinence, exerting significant cognitive
effort in an attempt to maintain dietary restraint. Under these circumstances, chocolate-related
cues increase in salience, and otherwise fleeting thoughts about chocolate are elaborated upon to
the point at which they are experienced as craving. In this context, the perimenstrum comes to
serve as an easily recognizable, socially acceptable, and culturally sanctioned excuse for women
to break restraint and indulge in otherwise forbidden treats (Rogers and Smit, 2000).
It must be noted that many theories and studies discussed here focus on chocolate craving
in general and more work is needed to examine if and how they can help elucidate the
phenomenon of perimenstrual chocolate craving. In turn, it also remains to be determined if
a better understanding of perimenstrual chocolate craving may shed light on non-menstrual
chocolate craving and craving in men. Similarly, since there is evidence to suggest that cravings
for non-food substances such as tobacco and alcohol may be more prevalent or pronounced
perimenstrually, a better understanding of the etiology of perimenstrual chocolate craving may
potentially generalize to other ingested substances.
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10. The influence of body mass index and
socioeconomic status on pubertal development
Marmara University, School of Medicine, Department of Pediatric Endocrinology, Fevzi
Cakmak Mh, Mimar Sinan Cad. No: 41, Pendik, stanbul, Turkey; abdullahbereket@gmail.com;
atay.zeynep@yahoo.com
Abstract
Puberty is regulated by complex genetic and hormonal mechanisms and also influenced by various
individual and environmental factors. Body mass index (a surrogate marker for nutritional
status) and socioeconomical status are shown in many studies to have significant impact on
pubertal timing. The increase of obesity in children and adolescents has become a major public
health concern worldwide. Studies show that obesity is associated with sexual maturation in
children and adolescents. Although the causal direction of this association remains controversial,
longitudinal studies suggest that increase in body mass index precedes the onset of early puberty
in girls. In developing countries, later age of menarche is attributed to lower socioeconomic
conditions. Effect of socioeconomical status on puberty in developing and developed countries
seem to be opposite and most likely secondary to changes in body mass index.
Keywords: puberty, body weight, leptin
155
Summary points
Worldwide increase in the prevalence of obesity coincided with earlier age of pubertal onset and menarche
According to critical fat mass hypothesis when body fat content reaches to a certain level, that triggers the
signals leading to menarche.
Leptin is the only known signal that transmits the state of nutrition to the reproduction relevant hypothalamus
neurons. Leptin has permissive effect on gonadotropin releasing hormone, luteinizing hormone and follicle
stimulating hormone secretion.
Longitudinal studies suggest that the association between BMI and earlier puberty is causal, i.e. overnutrition
in early childhood can result in an earlier onset of puberty in both girls and boys
In developing countries, inequalities related to socioeconomic status or life setting (urban vs. rural) are still
prominent and might account for important variations in timing of puberty within and among countries
Effect of socioeconomic status (SES) on puberty in developing and developed countries seem to be opposite.
Lower SES is associated with earlier age at menarche in USA which is more likely due to the fact that obesity
is more common in lower SES children in developed countries.
10. BMI and socioeconomic status in pubertal development
Abbreviations
BMI Body mass index
BMI-SDS Body mass index standard deviation score
IUGR Intrauterine growth retardation
NPY Neuropeptide hormone
OR Odds ratio
PROS Pediatric research in office setting
SES Socioeconomic status
10.1 Introduction
Puberty is an important developmental process which is regulated by complex genetic and
hormonal mechanisms, and influenced by various individual and environmental factors.
Nutrition, SES, exposure to endocrine disrupting chemicals, light, altitude, chronic diseases,
social and psychological stress may affect hypothalamic network controlling the pubertal timing
and progression.
Timing and tempo of puberty show ethnic and regional variations and it is subjected to change
also within the same population over the time so called secular trend. As a result of improvement
in socioeconomic conditions and health in the Western societies, the age at menarche had been
declining from 19th to 21st century with a rate of approximately 0.3 years per decade. This decline,
attributed to better nutrition and economic conditions, apparently stopped in the 1970s, probably
as a result of the stabilization of socioeconomic conditions and nutritional status (Atay et al.,
2011). In recent studies it has been demonstrated that the median age at menarche did not change
over the last 20 years in USA and Europe. On the other hand, in developing countries, the age
of both onset of puberty and menarche continues to decline; a study from rural south Mexico
reported that the median age at menarche decreased by almost 2 years between 1978 and 2000
(Malina et al., 2004).
Although these observations suggest an important influence of nutrition and socioeconomical

conditions on the age of puberty and menarche, the exact causal relationship appears to be quite
complex. This paper will review available data on this matter.
10.2 Effect of body mass index on puberty

It has long been known that body weight is important for normal puberty as evidenced by the
observations that being underweight in girls, either due to dieting/malnutrition or intense physical
exercise, is associated with delayed puberty. Secular trend towards earlier age of menarche in the
20th century coincided with the trend towards heavier body weight for children and adolescents
in western societies. Furthermore in developing countries, average age of menarche is delayed
in girls from lower socieconomical class who also have low BMI (a surrogate marker for body
fat) compared to their well off peers who have higher BMI and menarcheal age similar to their
western counterparts (Parent et al., 2003).
The relationship of body weight to menarche was first proposed by Frisch and Revelle (1971)
based on longitudinal anthropometric data from 181 girls collected between 1929 and 1950. In
their critical weight-menarche hypothesis, they claimed that attainment of the critical weight (~48
kg) causes a change in metabolic rate affecting the hypothalamic ovarian feedback by decreasing
the sensitivity of hypothalamus to estrogen. The feedback is then reset at a higher level, increasing
gonadotropins and gonadal hormones, that causes maturation of ovaries and uterus resulting
in menarche (Frisch and Revelle, 1971). This hypothesis argues that the reproductive phase of
female development is triggered at a fixed level of body weight. A close interplay of nutrition and
timing of puberty seems to be meaningful in terms of biological efficiency of reproduction since
the outcome of pregnancy and childbirth will be secured by an adequate level of stored calories
as fat tissue. However, total body weight does not accurately represent the body fat mass since the
lean mass is related to the height of an individual, which is critically relevant in growing children.
The BMI, which includes the ratio of weight to the square of height, represents a more accurate
measurement that reflects at least in part body fat mass compared with the total body weight.
If a threshold exists that reflects the nutrition state as a trigger for the onset of menarche than
the BMI would be a more promising candidate parameter to be similar in girls at the onset of
menarche compared with the body weight. In line with this, later studies demonstrated that the
critical body fat concept would be more suitable for menarcheal timing than the critical weight
hypothesis. The body fat content of shorter lighter girls versus the taller heavier girls at the time of
menarche, although differing in absolute amount, is similar with respect to percent body fat (22%
of their body weight) (Baker, 1985). A loss of 10-15% body weight during adolescence can induce
secondary amenorrhea until the lost amount of fat is reaccumulated (Frisch and Mcarthur, 1974).
The prevalence of childhood obesity (hence total body fat) has increased in the last century and
continues to increase all over the world (both in developed and developing countries) as a result
of sedentary life style and changing nutritional habits. Nearly 43 million children under the age of
5 years are overweight (WHO, 2006). The prevalence of obesity in USA defined as a BMI (weight
in kg/height2 in meters >95th percentile for age and gender) has risen from 5.1% to 11.6% in white
girls and from 5.3% to 22.2% in black girls between the ages 6-11 years within a 30-year-period.
The numbers were similar for boys, namely from 5.6% to 12% for whites and from 2% to 17.1%
blacks (Kaplowitz, 2008).
It has been demonstrated in many studies that obese girls tend to mature earlier. Kaplowitz et al.
(2008) reanalysed the PROS data to determine the relationship between BMI and pubertal status.
They found that the mean BMI z-scores for each age are markedly greater in 6-9 year-old white
girls with versus without breast development. They also demonstrated that the more advanced the
breast development was, the higher the BMI z-score was. They concluded that BMI is a significant
predictor of breast development. Both the white and the black girls who were still premenarcheal
had significantly lower BMI z-scores than postmenarchal girls (Kaplowitz et al., 2001).
Similary, in a recent cross-sectional study of 4,868 healthy schoolgirls (aged 6-18 years) in
Istanbul, we found that in all age groups, the postmenarcheal girls had higher BMI SD scores
than the premenarcheal girls (Table 10.1, Atay et al., 2011).
In the USA, the comparison of results of the National Health Examination Survey cycles II and
III (NHES) with that of the Third National Health and Nutrition Examination Survey (NHANES)
performed 25 years apart revealed that the average age at menarche dropped from 12.75 to 12.54
years. The percentage of overweight girls (BMI>85 percentile) aged 10-15 years increased from
16% to 27% in the same period. The mean BMI z-score increased from 0.05 to 0.32. There was
a positive association between BMI z-score and likelihood of having reached menarche in both
studies. (Anderson et al., 2003).
In a comparative study of Mexican and Egyptian female adolescents, mean age at menarche was
12.1 and 12.9 years respectively. Likelihood of experiencing menarche at an earlier age was 1.45
times greater in Mexican adolescents who were overweight or obese as compared to those with a
normal BMI. For Egyptian adolescents this value was 2.2 (Torres-Mejia et al., 2005).
Although a downward secular trend for age at menarche has recently been slowing down, that of
breast development continues to come down. A Danish study revealed an approximately 1-year
earlier breast development in two cohorts, 15 year apart (Aksglaede et al., 2009). During the
Table 10.1. Body mass index (BMI standard deviation) score of pre- and postmenarcheal girls observed
within age groups (Atay et al., 2011).
Age (year) BMI SD score BMI SD score P-value

premenarchial postmenarchial
10 0.100.05 1.330.39 0.033

11 -0.100.05 0.570.15 <0.001
12 -0.200.08 0.300.09 <0.001
13 -0.500.12 0.060.08 <0.001
14 -1.340.28 -0.240.17 0.002
15 -0.260.64 -0.190.33 0.815
same period mean age at menarche and pubic hair development decreased only by 0.2 years.
The correction of data on puberty timing for BMI did not change the results. Earlier breast
development was not associated with higher levels of gonadotropins. Thus, the authors concluded
that fact (Aksglaede et al., 2009). We also found a similar result with respect to earlier age at
breast development but not at menarche (Atay et al., 2011). The age at menarche has not been
changed in Turkey for almost 4 decades while age at breast development decreased by 0.4 years.
In contrast to the Danish study, we found a considerable association between BMI and the onset
of menarche. Subgroup analysis of girls with premature thelarche (age 4-8 years) also revealed a
strong association between BMI-SDS categories and occurrence of premature telarche (Atay et
al., 2012) (Figure 10.1). 56.2% of girls with premature telarche had BMI-SDS above 1.0 while this
figure was 22.9% in girls without telarche. BMI-SDS of girls who developed breasts were higher
than those without breast development in all age groups.
Davison et al. (2003) reported that early onset of breast development by 9 year could be weakly,
but significantly, predicted by a higher percentage body fat at 5 and 7 years. In this study, up to
14 and 35% of girls reached B2 stage at 7 and 9 years, respectively (Davison et al., 2003). Bau et
al. ( 2009) also found that median age of menarche was similar in German school girls (Berlin)
compared to that in 20 years ago. However, they found that obese/overweight girls reached
menarche significantly earlier (12.5 years), than normal weight (12.9 years), and underweight
girls (13.7 years). The mean total body weight was similar in all girls at menarche irrespective of
age (mean 51.1 kg, standard deviaiton 8.1) and height. BMI-SDS remained the only significant
factor for onset of menarche within a multiple regression model for early menarche (OR 2.1, 95%
CI 1.3-3.3, PZ=0.002). Similarly, in comparison with non-obese girls, the average menarcheal age
of obese girls was 9 months earlier in Japan and 0.9 year earlier in Thailand (Jaruratanasirikul et
al., 1997; Murata and Hibi et al., 1992).
a b
100 100
90 90
80 80
71
70 70
Percentage of girls
60 56.1 60
50 50
42.5
40 40
30 30
22.9
20 20
10 10 6.1
1.4
0 0
BMI-SDS<-1 -1<BMI-SDS<1 BMI-SDS>1 BMI-SDS<-1 -1<BMI-SDS<1 BMI-SDS>1
Figure 10.1. Distribution of body mass index (BMI) and socio-economic status (SES) among girls aged 4-8
years (a) with and (b) without premature thelarche (PT) in Istanbul, Turkey (Atay et al., 2012). The percentage of
girls with BMI-SDS >1 is higher in girls with PT than without PT
10.2.1 Is increased body fat the cause of early puberty or the result of it?
Significant correlation found in many studies between fatness and menarcheal age may indicate
a direct relation between fatness and menarche that can be either causal or consequential.
Alternatively, the link between the two parameters can only be indirect because they share similar
genetic determinants. Wang et al. (2002) examined the influence of early sexual maturation on
fatness in both girls and boys. Early sexual maturation was positively associated with overweight
and obesity in girls while the association was inverse in boys. The prevalence of overweight in
early matured versus the others was 34.4% versus 23.2% in girls and 22,6% versus 31.6% in boys.
The figures for obesity were 15.6% versus 8.1% and 6.7% versus 14.8% respectively. These data is
interesting because early sexual maturation is associated with an increased prevalence of fatness
in girls and leanness in boys. Such a sexual dimorphism could involve genetic and/or endocrine
factors.
In many studies investigating the relationship between menarche and BMI, menarche is
determined by a single time point observational assessment, and status quo method. In such a
study population, a 13 years old girl might have reached menarche more than 2 years before, and
since then her weight and height could have changed significantly. This methodological problem
presented in the available studies suggests that the data are not conclusively informative in terms
of the actual body weight at the time of menarche and it cannot be dismissed that an increase
of the weight might occur as a consequence of menarche rather than as a permissive factor
for menarche. However, there is evidence from longitudinal studies that the increase in obesity
precedes the onset of early puberty in girls. In a large population-based study done in Sweden,
growth data were collected in a sample of children using physician records until age 6 and school
records from ages 7 to 18. From these data, the age at peak height velocity, which is an early
puberty marker in girls but a late puberty marker in boys, could be determined for each child as
a measure of the timing of puberty. There was a negative correlation between the change in BMI
between the ages of 2 and 8 and the age of the peak height velocity. An increase of 1 BMI unit
between ages 2 and 8 was associated with an average of 0.11 years earlier for peak height velocity,
and for children with higher changes in BMI, the effect on the timing of puberty was as great as
0.6 years in boys and 0.7 years in girls. This study suggests that overnutrition in early childhood
can result in an earlier onset of puberty; in this study, the effect seemed to apply to both girls
and boys (He and Karlberg, 2001). To determine the influence of childhood growth and body
composition on the onset of puberty, Boyne et al. (2010) analysed the data from 140 girls and 119
boys from the Vulnerable Windows Cohort Study. The antropometry of children was measured
at birth, at 6 weeks, every 3 months to 2 years, and then every 6 months. Pubertal staging was
performed, starting at 8 years. It was found that birth size in girls had no significant relationship
with any marker of puberty. Gain in height, weight, or BMI during infancy(age 0-6 months)
had inverse associations with the stage of pubarche. Gain in height in childhood (2-8 years) had
a significant correlation with the stage of telarche. Fat mass at 8 year was strongly associated
with the stage and onset of puberty. Overall, faster growth throughout childhood is associated
with advanced puberty apart from menarche. A group of 197 girls in central Pennsylvania were
enrolled in a longitudinal study at age 5 and then re-examined at ages 7 and 9. Fat mass and body
fat percentage were calculated from a formula using height, weight, subscapular and the triceps
skinfold thickness, and bioelectrical impedance. At age 9, girls were classified as earlier (n=44) or
later (n=136) matured on the basis of breast development stage examined by inspection, serum
estradiol levels, and a parent-assessed pubertal development scale. It was found that girls with a
higher percentage of body fat at age 5 and girls with higher body fat or higher BMI percentiles
at age 7 were significantly more likely than their peers to be classified as having earlier pubertal
development at or by age 9 (Davison et al., 2003).
Another recent longitudinal analysis for determination of association between weight status in
early childhood and onset of puberty demonstrated that higher BMI z-score at all ages starting
at 36 months of age was strongly associated with an earlier onset of puberty (Lee et al., 2007).
Kaprio et al. (1995) suggested that the association between relative body weight and menarcheal
age was primarily due to correlated genetic effects, whereas the two parameters were influenced
by separate environmental correlates independent of each other. It can be concluded that the link
between nutritional status and physiological variations in timing of puberty can be significant but
is not particularly strong, suggesting that the relationship is indirect or partial and superseded
by other factors.
In addition to general nutrition, some studies focused on content of diet on puberty. The protein
source of food in early life could also influence the timing of puberty because a high animal vs.
vegetable protein ratio at the ages of 3-5 years is associated with early menarche, after controlling
for body size (Berkey et al., 2000). Similarly, Gunther et al recently have shown that children who
consume more animal protein at 5-6 years have earlier pubertal development than those who
consume vegetable protein. The authors stated that this association was not fully explained by
prepubertal fat mass index SDS (Gnther et al., 2010). More studies are needed to understand by
which mechanism, animal protein might have an influence on pubertal development.
Greater BMI was also associated with an increased likelihood of pubic hair development in
several studies. Rapid weight gain (catch up growth) in intrauterine growth retarded babies is
associated with obesity, hyperinsulinemia and earlier puberty. Children born IUGR with rapid
catch up growth became the heaviest and had the largest waist circumference at 5 years (Ong
et al., 2000). Although larger birth weight babies continue to be overweight as children and
contribute to subsequent risk of obesity based on BMI, it is those babies who are smaller at
birth with rapid growth during infancy who may be most at risk for development of increased
body fat, central adiposity and insulin resistance (Dunger et al., 2006). The development of
hyperinsulinemia may be as early as 1 year of age (Soto et al., 2003). The majority of IUGR babies
attain normal body weight and height by 2 years of age. Then they start to have an adipose body
composition. Increasing body fat and hyperinsulinemia may accelerate adrenal steroid synthesis
causing premature adrenarche that is the development of pubic hair before 8 years of age. The
question whether this earlier adrenarche ends in earlier menarche is still debated. Many studies
demonstrated that these girls present an early to normal onset and progression of puberty and an
adult stature within the target height range (Ibanez et al., 1992). According to Ibanez at al. girls
with premature pubarche who were born IUGR had their pubertal onset, and menarche 0.5 and
0.8 years earlier respectively (Ibanez et al., 2006).
10.3 Leptins role as a link between BMI and puberty

Leptin, a 16 kD 167 amino acid peptide is the only known signal that transmits the state of
nutrition to the reproduction relevant hypothalamus neurons. Leptin is secreted in a pulsatile
manner from the white adipose tissue in direct proportion of the amount of total fat mass.
Leptin receptors have been detected in hypothalamus and the gonadotrope cells of the anterior
pituitary (Jin et al., 1999). Leptin receptors with its 4 alternatively spliced isoforms belong to the
class I cytokine receptor family (Korner et al., 2005). These receptors are highly expressed in the
hypothalamic arcuate and paraventricular nuclei (Sone et al., 2001). Leptin stimulates GnRH,
LH and FSH secretion (Lebrethon et al., 2000; Yu, 1997) (Figure 10.2). Leptins effect on GnRH
secretion may also be mediated through NPY and kisspeptin producing neurons (Smith et al.,
2006). Furthermore leptin has also direct effect on gonads, modulating proliferation, germ cell
differentiation and steroidogenesis (Martos-Moreno et al., 2010).
Mutations in leptin or leptin receptors have been shown to cause hypogonadotropic hypogonadism.
Strobel et al. (1998) reported three patients from a family with codon 105 mutation of leptin gene.
Leptin receptor expressing PMV neuron

+ + +
+ GnRH neuron in +
AVPV Kiss1 neuron Arc Kiss1 neuron
hypothalamus
GnRH
Pituitary
T/E2 T/E2
FSH/LH
Gonads
+
Adipocyte leptin
Figure 10.2. Leptin signaling on gonadotropin releasing hormone (GnRH) release in mouse brain. Leptin
stimulates GnRH release through Kiss1 neurons secreting kisspeptins in anteroventral periventricular (AVPV) and
arcuate nucleus (Arc). It exerts direct action on ventral premammillary (PMV) neurons that stimulates GnRH release
through glutaminergic transmission. Leptin also has direct effect on gonads modulating proliferation, germ cell
differentiation and steroidogenesis (T= testosterone, E2 = estrodiol).
They all had obesity, hyperphagia and hypogonadism. In another family leptin receptor mutation
has been shown to cause early onset obesity, hypogonadism and growth retardation (Clement et
al., 1998). Farooqi et al. (1999) treated a patient with leptin gene mutation by giving recombinant
leptin. They demonstrated the occurrence of nocturnal pulsatile FSH and LH secretion at the
end of a 12-month treatment interval and concluded that leptin played a role in the initiation of
puberty (Farooqi et al., 1999). All these observations strongly suggest that if puberty is triggered
by the nutritional state of an individual, the amount of fat tissue via leptin would be most likely
the critical parameter.
10.4 Effect of socio-economic status on puberty

The timing and the progress of puberty may also be affected by SES. This was mostly noticed as
secular trend of menarcheal age where improvement in the SES, led to improvement in nutrition
and hygiene resulting in a decrease in menarchial age during the late 19th and whole 20th century.
The stabilization of socioeconomic conditions in modern western societies during last 30-40
years resulted in the stabilization of menarcheal age while the secular trend for menarcheal age
is still present in some developing countries (Table 10.2).
In developing countries, inequalities related to SES or life setting (urban vs. rural) are still
prominent and might account for important variations in timing of puberty within and among
countries. A study from rural south Mexico reported that the median age at menarche decreased
by almost 2 years between 1978 and 2000 (Malina et al., 2004). The influence of SES, a likely
marker for better health and nutrition, was illustrated by a recent study from India that reported a
mean age of menarche of 12.1 years for well-off girls and 15.4 years for underprivileged girls (Rao
et al., 1998). Atay et al. (2012) showed that SES had significant influence on the age of menarche
in Turkey, a transitional society. Subjects with lower SES attained B4 and B5 significantly later
than those with high SES (11.7 vs. 11.9 years for B4, and 13.6 vs. 14.4 for B5, P<0.05 and P<0.01
respectively). The average ages of menarche were 12.4, 12.6, 12.8 and 12.8 respectively in subjects
with highest, high, middle and low income families (P<0.05). Thus, girls with highest SES had
menarche 0.4 years earlier in comparison to those with low SES in Istanbul (Table 10.3). In a
Table 10.2. Average (mean or median) menarcheal age in different developing countries (Parent et al., 2003).
Country, year Well-off (years) Underprivileged (years)
India, 1988 12.5 13.7

India, 1998 12.1 15.4
Cameroon, 1999 13.2 14.3
South Africa, 1990 13.2 14.6
Venezuela, 1981 12.3 12.9
Table 10.3. Average age of menarche according to socioeconomical status (SES) in Istanbul, Turkey (Atay et
al., 2011).
SES Age (year)
Highest 12.4
High 12.6
Middle 12.8
Low 12.8
Total 12.7
study done in 1973, also in Istanbul, this difference was more apparent (0.8 years) as the average
ages of menarche were 12.4, 12.9, 13.3 and 13.2 in subjects with highest, high, middle and low
income families (Atay et al., 2011). Thus, it seems that the effect of SES on the age of menarche
and thus secular trend is decreasing in countries where socioeconomical conditions improve.
The effect of SES on puberty may be direct or indirect via BMI, as we have previously shown that
overweight and obesity is more common in children from higher socieconomical conditions
in Turkey (Turan et al., 2007). Alternatively, higher protein intake of children in higher SES
families can be another possible explanation for earlier puberty, as Gnther et al. (2010) recently
have shown that children who consume more animal protein at 5-6 years have earlier pubertal
development than those who consume vegetable protein.
However, results of the studies about the effect of SES on puberty in developed countries are
somehow contradictory. In a study from Eastern Austria, the impact of socioeconomic parameters
on menarcheal age could not be proved (Kirchengast et al., 2007). Although, they used only
educational level of the parents and the kind of school attended to define SES, the authors believe
that this was suitable and the result could be due to the fact that socioeconomic differences in
body build and physique have diminished during the last decades as a result of changes in social
stratification. In contradiction to this, SES had a significant effect on attaining menarche, fourth
and fifth pubertal breast stages in another study (Atay et al., 2011). Defining SES is not so easy and
this may explain contradictory findings. A multi-dimensional measure of SES including income,
education and occupation was used in a study from USA that analysed the impact of SES across
early life on age at menarche (James-Todd et al., 2010). Lower childhood SES and decreasing
SES between birth and age 7 were associated with earlier age at menarche. However, it should
be underlined that in the USA, lower SES is associated with higher BMI whereas in developing
countries lower SES is associated with lower BMI (James-Todd et al., 2010). Thus, effect of SES
on puberty in developing and developed countries seem to be opposite.
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11. Neuroimaging menstrual cycle associated
changes in appetite
Department of Obstetrics and Gynaecology, Department of Biomedical and Molecular Sciences,
Queens University, Kingston K7L 3N6, Canada; vanvugtd@queensu.ca
Abstract
The menstrual cycle provides a unique physiological paradigm in which to study ingestive
behaviour. Caloric intake varies during the menstrual cycle in response to changes in ovarian
steroid levels. Food intake is highest during the luteal phase and early follicular phase before
declining in the second week of the follicular phase, reaching a nadir around the time of ovulation.
This reduction in caloric intake is the result of the periovulatory increase in estrogen which exerts
an anorexigenic effect at the level of the brain. Increased food intake in the early follicular phase
and luteal phase results from reduced estrogen signaling due to low estrogen levels and increased
progesterone levels respectively. In addition to altering expression of hypothalamic neuropeptides
that respond to hormonal signals of energy balance, ovarian steroids influence non-homeostatic
ingestive behavior by affecting corticolimbic brain regions. Neuroimaging studies demonstrate
that activation of the amygdala/hippocampal complex, ventral striatum, and prefrontal cortex
by rewarding stimuli is increased during the late follicular phase compared to the luteal phase or
early follicular phase. This difference may result from ovarian steroid effects on the mesolimbic
dopamine system. Thus, increased dopamine signaling during the periovulatory period may be
the key molecular mechanism responsible for reduced food intake at this time of the menstrual
cycle. It is imperative that future neuroimaging studies involving women and reward incorporate
menstrual cycle phase into the study design.
Keywords: menstrual cycle, neuroimaging, reward, appetite, ingestive behavior, corticolimbic,

dopamine
169
Summary points
Food intake is reduced in the late follicular phase as a result of the periovulatory increase in estrogen which
exerts an anorexigenic effect at the level of the brain.
Ovarian steroids affect homeostatic eating through hypothalamic neuropeptides that respond to hormonal
signals of energy balance.
Ovarian steroids influence non-homeostatic ingestive behavior by acting on corticolimbic brain regions
involved in processing reward, taste, pleasure, emotion, learning and memory.
The ventral striatum, nucleus accumbens, amygdala, and hippocampus were increased during the estrogen
dominant late follicular phase compared to the luteal phase in response to visual food cues.
Activation of the inferior frontal gyrus was increased whereas fusiform activity was decreased in response
to food pictures in the late follicular phase compared to early follicular phase in association with increased
estrogen.
These menstrual cycle phase-dependent differences in activation of reward circuits may involve gonadal
steroid effects on the mesolimbic dopamine system.
11. Neuroimaging menstrual cycle associated changes in appetite
Abbreviations
BOLD Blood oxygen level dependent
DLPFC Dorsolateral prefrontal cortex
fMRI Functional magnetic resonance imaging
HC High calorie
LC Low calorie
mPFC Medioprefrontal cortex
NAc Nucleus accumbens
OFC Orbitofrontal cortex
PET Positron emission tomography
PFC Prefrontal cortex
PMS Premenstrual symptomatology
rCBF Regional cerebral blood flow
STR Sensitivity to reward
VTA Ventral tegmental area
11.1 Introduction
11.1.1 Menstrual cycle related changes in appetite
Food intake varies significantly during the menstrual cycle as documented in both human and
non-human primates (Dye and Blundell, 1997). Caloric intake declines during the second week
of the follicular phase relative to the early follicular phase and luteal phase, reaching a nadir
around the time of ovulation (Figure 11.1). The magnitude of the difference in caloric intake
between late follicular phase and luteal phase ranged from a low of 87 kcal to a high of 500 kcal
(McNeil and Doucet, 2012). Changes in ovarian steroid concentrations offer the most plausible
explanation for this pattern of ingestive behavior. Estrogen concentrations rise exponentially
in parallel with the periovulatory decline in caloric intake. Progesterone increases shortly after
ovulation and is the dominant ovarian steroid during the luteal phase when appetite rebounds.
11.1.2 Effect of ovarian steroids
Ovarian steroid effects on food intake have been convincingly demonstrated in rodent studies.
Increased food consumption and weight gain occurred in ovariectomized rats and was reversed
by estrogen replacement. Co-administration of progesterone blocked these effects of estrogen.
These findings were confirmed in monkeys (Kemnitz et al., 1989). Although it is common for
weight gain to occur in postmenopausal women, gonadal steroid replacement does not prevent
weight gain. A systematic review found no evidence that either estrogen alone or in combination
with a progestin affected weight gain in peri- or post-menopausal women (Kongnyuy et al., 2009).
However, hormone replacement did reduce abdominal fat, and reduced the risk of developing type
II diabetes (Kanaya et al., 2003). This finding of the Heart and Estrogen/progestin Replacement
30
160 * * *
*
*
*
120 20
Plasma LH (mg/ml)
Food intake (g)
80
10
40
0 0
11/9 8/6 5/3 2/0 1/3 4/6 7/9 13/15 13/15
Days relative to LH surge
Figure 11.1. Food intake during the rhesus monkey menstrual cycle. Food intake values are the mean of three
consecutive days during the menstrual cycle (n=7) relative to the day of the luteal hormone (LH) surge. Food intake
progressively decreased during the periovulatory period, before rebounding after ovulation (* P<0.05 compared
with days -2/0) (Van Vugt, 2010; reprinted with permission).
Study (HERS) was confirmed by the Womens Health Initiative Study. A lack of effect of hormone
replacement on weight in the clinical setting does not argue against ovarian steroids mediating
menstrual cycle dependent changes in food intake since hormone replacement regimens do not
mimic ovarian steroid changes typical of the menstrual cycle.
11.1.3 Ovarian steroid site of action
Estrogen and progesterone receptors are concentrated in the hypothalamus, hippocampus,

centromedial amygdala, substantia nigra, dorsal raphe, ventral striatum, caudate, putamen,
NAc, VTA, frontal cortex, bed nucleus of the stria terminalis, and pontine nucleus. The
hypothalamus plays an integral role in appetite regulation, and estrogen and progesterone
affect gene expression of hypothalamic orexigenic and anorexigenic neuropeptides such as
neuropeptide Y, pro-opiomelanocortin, and corticotrophin releasing hormone. Ovarian steroids
also exert extra-hypothalamic effects. Estrogen affects dopamine at multiple sites of the dopamine
transduction pathway including dopamine release, reuptake and receptor expression (McEwen
and Alves 1999). Thus, ovarian steroids may influence homeostatic ingestive behavior by affecting
hypothalamic neuropeptides and non-homeostatic eating or hedonic eating through dopamine
mediated mesolimbic and corticolimbic processing of the cognitive, emotional and rewarding
aspects of food.
11.2 Review
Advances in neuroimaging technologies have made it possible to examine brain activity
associated with hunger, satiety, and adiposity as they relate to appetite regulation. This chapter
will summarize these studies with a focus on functional neuroimaging studies that have examined
how ovarian steroid affect the brains response to appetitive cues.
11.2.1 Neuroimaging modalities
PET and fMRI are the primary functional neuroimaging modalities used to study neural networks
of human ingestive behavior. PET studies indirectly measure neural activity by measuring either
rCBF or cerebral metabolism. In addition, PET is used to quantify receptor density. There has been
a shift towards fMRI because of better temporal and spatial resolution and because it does not
require radioisotopes. Instead, fMRI measures the BOLD signal, specifically deoxyhemoglobin
which is paramagnetic. Deoxyhemoglobin levels decline as a result of increased blood flow to
metabolically active brain regions.
These functional neuroimaging techniques have been used to measure brain responses to food
cues. Food cues used in neuroimaging protocols include taste, smell, sight, and even the thought
of food. Visual food cues consisting of either actual foods or pictures of food are commonly used
because of their relative ease of delivery. Visual food cues are effective because stimuli associated
with primary rewards acquire motivational potency and trigger desire due to their acquired
ability to stimulate reward circuits (classical conditioning).
11.2.2 Brain responses to visual food cues
Functional neuroimaging studies show that brain regions involved in processing reward, emotion,
and cognition are activated when subjects view food pictures. The PFC, anterior cingulate, OFC,
insula, striatum, amygdala, hippocampus, and fusiform are activated by visual food cues in
many studies (see Van Vugt, 2010 for review). A formal examination of this question using a
meta-analytic approach found moderate agreement among the studies. The brain regions most
frequently activated by food cues included the posterior fusiform gyrus, the left lateral OFC, and
the left middle insula. Hunger modulated the response to food pictures in the right amygdala
and left lateral OFC, and energy content modulated the response in the hypothalamus/ventral
striatum (Van der Laan et al., 2011). Experimental and methodological differences between the
various studies likely contribute to only a moderate consensus at this time.
11.2.3 Effect of hunger, adiposity, and gender
Before describing imaging studies that have been conducted in the context of the menstrual cycle,
it is useful to consider neuroimaging studies that have investigated the effects of hunger, adiposity,
and gender on brain responses to appetitive stimuli since these studies help in the interpretation
brain activity changes.
Influence of hunger
Food has greater salience in the fasted state. Food pictures evoked a greater BOLD response
in the OFC, insula, amygdala, parahippocampus, and anterior fusiform of hungry subjects.
Meal-induced satiation reduced blood flow to the striatum, thalamus, insula, hippocampus,
parahippocampus, and cerebellum and increased blood flow to the PFC. Activation in the lateral
and medial OFC, prefrontal cortex, caudate/putamen, fusiform gyrus and the insula was stronger
for low calorie foods in satiated healthy weight females, but stronger for high calorie foods in the
fasted state. Increased responsiveness may result from increased ghrelin levels in the fasted state.
Infusion of ghrelin in subjects who had eaten recently increased hunger and increased activation
of the amygdala, OFC, anterior insula, caudate, VTA/ substantia nigra, and hippocampus in
response to food pictures. Subjects maintained on a eucaloric diet for several days compared
to subjects who ate in excess exhibited greater activity in the inferior temporal visual cortex,
posterior parietal cortex, premotor cortex, hippocampus, and hypothalamus. These finding have
been summarized in recent reviews (Carnell et al., 2012; Van Vugt, 2010).
Influence of adiposity
Food pictures elicit greater activation in obese subjects compared to normal weight subjects.
Regions that exhibited greater activation in obese women include the insula, hippocampus,
anterior cingulate, and dorsal striatum (Rothemund et al., 2007; Stoeckel et al., 2008).
Furthermore, body mass index in the obese group was positively correlated with the degree
of activation in the OFC, mPFC, and putamen in response to high calorie vs. low calorie food
pictures (Stoeckel et al., 2008). In contrast, OFC activity was negatively correlated with BMI in
normal weight women (Killgore and Yurgelun-Todd, 2005). Obese adolescent girls compared
to lean girls showed greater activation in the operculum, insula and anterior cingulate when
anticipating tasting a chocolate milkshake (Stice et al., 2008). Food pictures activated different
brain regions in fasted obese subjects when compared to fasted normal weight subjects, and
activation of limbic and corticolimbic regions by food pictures following a meal was greater in
obese compared to normal weight subjects (Dimitropoulos et al., 2012). Striking differences were
observed when responses in healthy weight controls were compared to individuals with Prader-
Willi Syndrome, a genetic disorder characterized by hyperphagia-induced early onset obesity.
Food pictures elicited greater activation in the amygdala, OFC, mPFC, and frontal operculum in
the pre-prandial compared to the post-prandial state in normal weight controls, whereas Prader-
Willi patients exhibited greater activation after a meal (compared to before) in the amygdala,
OFC, insula, parahippocampus, and fusiform (Holsen et al., 2006). Collectively, these results
suggest that food anticipation elicits greater activation, whereas food consumption produces less
inhibition in obese compared to lean subjects.
Gender differences
Brain responses to food cues differ in men and women. Women had a greater response in the
fusiform, prefrontal cortex, cingulate, and insula. A comparison of brain metabolism measured by
PET reported that food cues stimulated limbic regions in women to a greater extent than in men,
and that attempts to suppress appetite through cognitive inhibition reduced brain metabolism
in the amygdala, hippocampus, insula, OFC, and striatum of men but not women (Wang et
al., 2009). Gender differences also were observed in response to taste. Taste-induced activation
of the insula was greater in women compared to men (Uher et al., 2006), whereas satiation
produced a greater reduction in taste-induced activation in the insula and cerebellum and in
limbic regions including dorsal striatum, amygdala, parahippocampal gyrus, and posterior and
anterior cingulate in males relative to females (Haase et al., 2011). The taste of chocolate following
chocolate satiation stimulated different regions in men (ventral striatum, insula, and OFC) and
women (precentral gyrus, superior temporal gyrus, and putamen). Areas of decreased activation
also were different in men (somatosensory areas) and women (amygdala, hypothalamus) (Smeets
et al., 2006). These gender differences may lead to greater disinhibition in women and a tendency
to overeat in response to food cues. While developmental and endocrine effects of gonadal
steroids, insulin, and leptin may contribute to these gender differences in brain responses, gender
differences in attitudes about food and body image likely play a role as well.
11.2.4 Changes in appetitive brain responses during the menstrual cycle
The menstrual cycle provides a physiological paradigm in which to study ingestive behavior.
While some studies have recognized the need to control for menstrual cycle phase (Frankort
et al., 2011; Gearhardt et al., 2011; Siep et al., 2009; Stoeckel et al., 2008), most imaging studies
involving female subjects do not. To date, there are only two imaging studies that have explicitly
examined the neural substrates of menstrual cycle related changes in eating (Alonso-Alonso et al.,
2011; Frank et al., 2010). Frank et al. (2010) compared corticolimbic brain responses to HC food
pictures, LC food pictures, or control pictures during the late follicular phase and luteal phase,
times during the menstrual cycle when food intake is most discordant. Subjects were imaged
during each phase (using a counterbalanced design) after a 6 h fast. The HC control contrast
activated numerous regions, including the DLPFC, OFC, operculum, NAc, hypothalamus,
pallidum, amygdala, hippocampus, pulvinar, and fusiform during both phases (Table 11.1). Other
regions were selectively activated in the late follicular phase, including right operculum, left NAc,
left putamen, right pallidum, right amygdala, left and right substantia nigra, right hippocampus,
VTA, and right pulvinar (Table 11.1 and Figure 11.2). No regions were selectively activated in the
luteal phase. Differences between follicular and luteal phase responses were even more striking
for LC food pictures as significant activation (with the exception of the fusiform) was restricted to
the late follicular phase. The LC pictures activated the DLPFC, insula, NAc, pallidum, amygdala,
substantia nigra, and hippocampus in the follicular phase, but not in the luteal phase. The HC
LC contrast significantly activated the NAc in the follicular phase and the pulvinar and fusiform
in both phases. The LC HC contrast did not activate any regions in either menstrual cycle
phase. A direct comparison of menstrual cycle phases revealed greater activation during the
follicular phase in the right NAc, right amygdala, and right hippocampus to HC pictures, in the
hippocampus to LC pictures, and the right NAc in response to the HC LC pictures (Table 11.2).
Activation of the right OFC and mid cingulum was greater in the luteal phase to the HC LC
Table 11.1. Comparison of brain responses to food pictures of differing caloric content during the follicular and
luteal phases (Frank et al., 2010; reprinted with permission).
Region2 Follicular phase1 Luteal phase1
Hem Cluster t stat Coordinates Hem Cluster t stat Coordinates
HC control
DLPFC L 188 6.81 -44 32 18 L 34 4.99 -42 32 18
OFC L 90 5.2 -30 36 -12 L 65 5.74 -22 30 -14
Operculum L 23 4.47 -52 8 26 L 7 4.99 -42 32 18
R 74 4.64 60 16 32
NAc L 50 5.62 -14 -2 -10
R 23 4.31 14 0 -10 R 11 4.15 10 -2 -6
Hypothalamus R 2 3.58 8 -4 -4 R 2 3.73 8 -4 -4
Putamen L 12 4.36 -22 2 -8
Pallidum L 34 5.09 -20 -4 -6 L 8 4.23 -18 -6 -6
R 19 4.95 22 -4 -6
Amygdala L 112 5.64 -20 -6 -12 L 21 5.31 -20 -6 -12
R 111 4.86 18 0 -14
Substantia Nigra L 23 5.35 -10 -22 -12
R 27 3.84 10 -18-12
Hippocampus L 99 6.21 -22 -28 -6 L 8 5.4 -18 -6 -12
6 5.23 -18 -6 -12
33 4.98 -32 -16 -12
R 60 5.4 20 -30 -4
27 4.88 34 -8 -16
VTA R 26 3.37 2 -20 -12
Pulvinar L 212 5.92 -16 -32 4 L 78 4.34 -20 -32 2
R 99 5.77 18 -30 2
Fusiform L 766 9.81 -32 -70 -8 L 574 7.88 -32 -54 -16
R 457 8.21 28 -74 -2 R 164 5.87 34 -70 -10
LC control
DLPFC L 42 5.52 -46 34 18
Insula L 8 4.43 -39 6 -10
NAc L 7 3.81 -14 -2 -10
Pallidum L 6 3.82 -20 -2 -6
R 12 4.2 22 -2 -6
Amygdala L 7 3.73 -22 -6 -12
Substantia Nigra L 18 3.79 -10 -22 -12
Hippocampus L 5 3.97 -32 -14 -12
Fusiform L 174 7.21 -34 -52 -12 L 125 7.25 -34 -56 -18
Table 11.1. Continued.
Region2 Follicular phase1 Luteal phase1
HC LC
NAc R 50 5.01 10 -2 -8
Pulvinar L 7 3.59 -22 -28 8
R 12 3.8 20 -30 2 R 30 4.08 22 -26 10
Fusiform L 310 6.82 -26 -64 -16 L 444 9.26 -40 -52 -14
R 308 815 30 -48 -14
1 Activations of the left (L) or right (R) hemisphere (Hem) are indicated. Cluster size refers to the number of
contiguous voxels while the t-score and Montreal Neurological Institute (MNI) coordinates apply to the maximum
voxel within a cluster. Only clusters within regions of interest that were statistically significant (P<0.05 corrected
for multiple comparisons) of at least 5 voxels (except for hypothalamus) are shown.
2 DLPFC = dorsal lateral prefrontal cortex; OFC = orbitofrontal cortex; NAc = nucleus accumbens; HC = high
calorie; LC = low calorie.
Table 11.2. Menstrual cycle phase dependent activation by food pictures of differing caloric content (Frank et
al., 2010; reprinted with permission).
Region2 Follicular > luteal1 Luteal > follicular
HC control
NAc R** 13 3.96 14 0 -10
Amygdala R** 7 3.69 34 0 -22
Hippocampus R* 11 4.01 34 -6 -20
LC control
Hippocampus R* 10 3.73 32 -6 -24
HC LC
NAc R** 18 3.74 12 0 -10
OFC R* 6 3.85 44 52 -6
Mid Cingulum L* 6 3.72 -12 -12 46
1 Activation of the left (L) or right (R) hemisphere (Hem) is indicated. Cluster size refers to the number of contiguous
voxels exceeding the specified threshold using either the Small Volume Correction* or Family Wise Error** utility
within SPM 8 (P<0.05; minimum of 5 voxels). The t-score and Montreal Neurological Institute coordinates apply
to the maximum voxel within a cluster.
2 NAc = nucleus accumbens; HC = high calorie; LC = low calorie.
Figure 11.2. Representative T maps comparing activation in the follicular and luteal phases. Regions of interest,
which include the dorsolateral prefrontal cortex (DLPFC), hippocampus, nucleus accumbens (NAc), orbitofrontal
cortex (OFC), amygdala, substantia nigra (SN), and ventral tegmental area (VTA) are specified in the right column
(along with the Z coordinate) and are designated by an arrow. A t threshold = 3 was applied for presentation
purposes (Frank et al., 2010; reprinted with permission).
contrast. Quantification of the BOLD effect size for the peak voxel within the cluster of activation
confirmed these differences between follicular and luteal phases (Figure 11.3).
Menstrual cycle differences in BOLD responses are likely related to changes in the relative appeal
of HC and LC foods during the menstrual cycle. An online survey found that the appeal of HC
food pictures exceeded LC food pictures at all times of the menstrual cycle, except during the mid
to late follicular phase when the appeal of HC food pictures declined to the same level as LC food
pictures (Frank et al., 2010). This change in food appeal may reflect menstrual cycle-dependent
changes in stimulus salience that lead to different brain responses in the periovulatory phase and
the luteal phase. Activation of the corticolimbic brain by either HC or LC stimuli during the late
1.5
Follicular Luteal
1.0
0.5
Effect size
0.0
0.5
1.0
NAc Amyg Hippo Hippo NAc OFC Mid cing

1.5
HC C LC C HC LC
Figure 11.3. Mean ( SEM) Blood oxygen level dependent effect size for the follicular and luteal phases.
Parameter estimates were extracted for the voxel of maximum activation (identified in the subtraction analysis and
corresponding to the coordinates designated in Table 11.3) for all follicular and luteal phase scans. Responses
that were significantly different between the follicular and luteal phases (P<0.05; paired two-tailed t-test) for
the contrasts high calorie (HC) control (C), low calorie (LC) C, and HC LC are shown. NAc = nucleus
accumbens, amyg = amygdala, hippo = hippocampus, OFC = orbitofrontal cortex, Mid Cing = middle cingulate
(Frank et al., 2010; reprinted with permission).
follicular phase, but only by HC stimuli during the luteal phase may reflect cycle-related food
preferences that affect actual food choices that could lead to a significant reduction in caloric
intake during the late follicular phase compared to the luteal phase.
A subsequent study determined responses to visual food cues in the early follicular phase compared
to the late follicular phase before and after eating (Alonso-Alonso et al., 2011). These phases were
compared because estrogen levels increase significantly over this time period. Activation of the
fusiform gyrus in the fed state exceeded the fasted state in the late follicular phase, but not early
follicular phase (Figure 11.4). Fusiform activity was negatively correlated with the change in
estrogen concentration from early to late follicular phase. This association between estrogen and
BOLD response was restricted to the fasted state (Figure 11.5). Because the fusiform is part of
the ventral visual pathway involved in processing memory, reward, and emotion, it was suggested
that reduced activation of the fusiform may mediate the anorexigenic effect of estrogen, possibly
by reducing the salience of visual food cues. They also observed that food pictures stimulated the
inferior frontal gyrus in the late follicular phase (but not the early follicular phase) of fed subjects
(Figure 11.4). Since the inferior frontal gyrus is involved in executive function and inhibitory
control and increased activation of the inferior frontal gyrus is associated with satiety, these
results may indicate that under the milieu of increased estrogen, greater activation of the inferior
frontal gyrus by visual food cues contributes to the anorexigenic effect of estrogen.
a Inferior frontal gyrus

0.5
0.4
0.3
0.2
Parameter estimates 0.1
0.0
0.1
0.2
0.3
0.4 Early-fasting
0.5 Early-fed
0.6 Late-fasting
Late-fed
b Fusiform gyrus
0.2
0.1
0.0
Parameter estimates
0.1
0.2
0.3
0.4
0.5
0.6
c Insula
0.4
0.3
0.2
Parameter estimates
0.2
0.0
0.1
0.2
0.3
Figure 11.4. Region of interest (ROI) analysis. Brain activity in 3 ROIs [(inferior frontal gyrus (a), fusiform gyrus
(b), and insula (c)] in response to food > nonfood visualization throughout the 4 time points of the study (n=9).
A significant interaction between follicular phase (early/late) and prandial state (fasted/fed) was shown in the
inferior frontal gyrus and fusiform gyrus. The insula showed a similar interaction effect of borderline significance.
Prandial state affected activation (greater in the fed than in the fasted state) only during the late follicular phase.
Columns represent mean parameter estimates (b values) 6 SEMs, which are plotted for each ROI/time point. Brain
maps (depicted in axial sections) highlight activation within each corresponding ROI for the paired comparison
fed minus fasted (fed, fasted) during the late follicular phase (whole-brain analysis: thresholded at P=0.005,
uncorrected; for illustration purposes). L, left; R, right) (Alonso-Alonso et al., 2011; reprinted with permission).
Comparison of the only two studies that explicitly examined the effect of menstrual cycle phase
on BOLD responses to appetitive stimuli is limited by methodological differences. One study
compared the periovulatory period to the luteal phase, whereas the other study compared the
periovulatory period to the early follicular phase. Scans were conducted after a 6 h fast in one
study whereas subjects were scanned sequentially before and after eating in the other. Although
the steroid milieu of the early follicular phase (low estrogen) and luteal phase (elevated estrogen
a b
10 y=-72
Fast
Change in fusiform gyrus activation (rank)
Fed
8 Linear (fast)
Linear (fed) ()
6
0
-50 50 150 250
Change in estradiol (pg/ml) (+)
Figure 11.5. Association of brain activation with estradiol concentration. (a) Change in estradiol concentration
from early to late follicular phase correlated negatively with change in activity in the fusiform gyrus region of
interest only during fasting (P=0.013). (b) Brain coronal sections depict the areas that showed a positive (+) and
negative () significant correlation with estradiol elevation from the early to the late follicular phase in the whole-
brain analysis (Adapted from Alonso-Alonso et al., 2011; reprinted with permission).
and progesterone) are very different, progesterone antagonism of estrogen in the luteal phase may
reduce estrogen-induced anorexia in the luteal phase to a similar degree as that achieved by low
estrogen levels in the early follicular phase. However, similar responses in the two studies were
not observed, even when comparisons were restricted to the fasted state. The finding of reduced
fusiform activation with increasing estrogen in the fasted state prompted us to re-examine
fusiform activity. While the fusiform was significantly activated in both the late follicular phase
and luteal phase, there was not a significant difference between phases. However, this comparison
is tenuous because the effect on the fusiform in the Alonso-Alonso et al. (2011) study was based
on changes in estrogen levels from early to late follicular phase. Estrogen was not measured in
the study by Frank et al. (2010) since the significance of a change in estrogen concentration from
mid-cycle to luteal phase would be uncertain given that there is a large concurrent change in
progesterone level.
11.2.5 Menstrual cycle and reward
Similarities were observed between the Frank et al. (2010) and Alonso-Alonso et al. (2011) study
with a study that examined the effect of menstrual cycle phase on brain responses to monetary
reward (Dreher et al., 2007). Anticipation of an uncertain monetary reward produced greater
activation of the amygdala and OFC in the mid-late follicular phase (Figure 11.6), but greater
activation in the DLPFC and anterior cingulate in the luteal phase. Increased activation of the
amygdala and mid cingulum in the late follicular phase and luteal phase respectively was observed
by Frank et al. (2010). The observation of increased activation of the inferior frontal gyrus in
the late follicular phase by Alonso-Alonso et al. (2011) is consistent with a positive correlation
between estrogen levels in the follicular phase and activation of the DLPFC and fronto-polar
Figure 11.6. Cross-menstrual cycle phase differences in blood oxygen level dependent (BOLD) response during
anticipation of uncertain rewards. Statistical maps overlaid onto structural MRI showing BOLD fMRI responses
greater in follicular phase than in luteal phase in the right amygdala (top) and OFC (bottom) The distributions
of BOLD signal response for each woman is shown on the right (Adapted Dreher et al., 2007; reprinted with
permission).
cortex in response to monetary reward (Figure 11.7). Although money and food represent
different types of reward, increased reactivity of the reward system during the late follicular phase
may explain these similarities in brain responses. Although follicular phase subjects in the Frank
et al. (2010) study were scanned closer to ovulation than subjects in the Dreher et al. (2007) study,
the follicular phase scans in both studies were done during an endocrine milieu of unopposed
estrogen in contrast to the luteal phase scans when both estrogen and progesterone levels are
elevated. Both studies found greater activation of the ventral striatum/NAc in the follicular phase
compared to the luteal phase. In addition, activation of corticolimbic reward regions such as the
putamen and substantia nigra were augmented during the late follicular phase compared to the
luteal phase (Frank et al., 2010). In contrast, another study reported increased activation of the
ventral striatum in the premenstrual phase compared to the late follicular phase, particularly in
women with PMS in anticipation of a monetary reward (Ossewaarde et al., 2011). It is not clear
whether this difference is related to differences in subjects (with vs. without PMS symptoms),
luteal phase timing (mid vs. premenstrual), or reward stimulus (receipt vs. anticipation). Brain
responses to reward anticipation and reward receipt can be different, depending on the region
(Gearhardt et al., 2011; Stice et al., 2008; Uher et al., 2006). Anticipation of food reward or
food receipt activated the insula and somatosensory cortex to a greater extent in obese vs. lean
adolescent girls, whereas activation of the caudate by food receipt was reduced (Stice et al., 2008).
Activation in the anterior cingulate cortex, medial orbitofrontal cortex, and amygdala in response
a b
0.006
0.004
Par. estimates
0.002
0.000
0 50 100 150
-0.002
-0.004
-0.006
Estradiol level (pg/ml) 8
0.015 0.006
0.010 0.004
6
Par. estimates
0.002
0.005
Par. estimates
4 0.000
0.000 0 50 100 150
T-value
0 50 100 150 -0.002
-0.005 2 -0.004
-0.010 -0.006
-0.015 0 -0.008
Estradiol level (pg/ml) Estradiol level (pg/ml)
c Left DLPFC
3
2
Par. estimates
1
0
0 50 100 150
-1
-2
Estradiol level (pg/ml)
d Left fronto-polar cx
1.5
1.0
Par. estimates
0.5
0.0
-0.5 0 50 100 150
--1.0
--1.5
Estradiol level (pg/ml)
Figure 11.7. Statistical maps of regression analyses between brain activity and estradiol level during the
follicular phase of the menstrual cycle. During anticipation of uncertain rewards: estradiol level correlated (a)
positively with activity of the bilateral amygdalo-hippocampal complex and (b) negatively with activity of the
hypothalamus. At the time of rewarded outcome relative to no reward: estradiol level correlated positively with
activity of the (c) bilateral dorsolateral and (d) fronto-polar cortices (Adapted from Dreher et al., 2007; reprinted
with permission).
to anticipation (but not receipt) of food correlated with food addiction scores in young women
scanned in the luteal phase (Gearhardt et al., 2011). Participants with high food addiction scores
compared to low addiction scores showed greater activation in the dorsolateral prefrontal cortex
and the caudate in response to anticipation of food, but less activation in the lateral orbitofrontal
cortex in response to receipt of food.
11.2.6 Sensitivity to reward and the menstrual cycle
STR refers to the ability of one to experience pleasure from natural reinforcers and ranges from
anhedonia (diminished ability) to hedonia (enhanced drive for and an enhanced ability to receive
reward). STR may influence eating behavior. STR was lowest (anhedonic range) in women with
anorexia nervosa (Davis and Woodside, 2002). STR correlated positively with BMI in women in
the normal to overweight range, but negatively with BMI in women in the obese range (Davis et
al., 2004), suggesting that STR may be reduced at both weight extremes. STR in normal weight
subjects correlated with BOLD signal intensity in the ventral striatum, amygdala, orbitofrontal
cortex, midbrain, and ventral pallidum in response to pictures of appetizing foods compared
to bland foods (Beaver et al., 2006). Functional connectivity between the amygdala and ventral
striatum was greater in individuals with increased sensitivity to external food stimuli as measured
by Dutch Eating Behavior Questionnaire (Passamonti et al., 2009). Collectively, these studies
suggest that the neural connection between the ventral striatum and amygdala is an important
component linking STR with the neural network that evaluates the salience of visual food cues.
No studies have specifically examined if STR changes during the menstrual cycle.
11.2.7 Drug versus food reward
Several lines of evidence indicate similarities in reward pathways stimulated by food cues and
those stimulated by nicotine, amphetamine, alcohol, and illicit drugs, such as cocaine and heroin
(Volkow et al., 2008a). Visual cues of drug, alcohol, or nicotine use stimulated craving and
increased activity in the DLPFC, OFC, amygdala, insula, anterior cingulate, and cerebellum of
cocaine abusers, the inferior prefrontal cortex, OFC, insula, and precuneus of heroin addicts, the
amygdala, hippocampus, DLPFC, NAc, putamen, anterior cingulate, OFC, insula, and mPFC of
alcoholics compared to social drinkers, the DLPFC, OFC, anterior cingulate, insula, amygdala,
hippocampus, thalamus, and ventral striatum of smokers compared to non-smokers. There is
considerable overlap in the neural substrates activated by drug cues and those activated by food
cues. Furthermore, a number of neuroimaging studies in humans support the role of dopamine
in both drug- and food-induced reward. Imaging studies in humans have demonstrated that
addictive drug cues activated the mesolimbic dopamine system and that dopamine release was
reduced in chronic users of cocaine, heroin, alcohol, or nicotine (Martinez et al., 2007). Analogous
findings were reported for food cues. Eating chocolate stimulated rCBF in the dorsal striatum
whereas consuming a favorite meal stimulated dopamine release in the dorsal striatum (Small et
al., 2003). Dopamine binding capacity was similarly reduced in the striatum of obese subjects and
chronic users of illicit drugs (Volkow et al., 2008b; Wang et al., 2001). These studies suggest that
the pleasure derived from eating or drug ingestion involves activation of dopamine receptors in
the striatum. These parallels between addictive substances and food, combined with the fact that
a significant proportion of society appears unable to change its eating habits despite being aware
of the health consequences of obesity and suffering the stigma associated with obesity, support
the premise that the Western diet has the potential to be addictive for some people.
11.2.8 Drug reward and the menstrual cycle
Women appear to have greater sensitivity, vulnerability, and health consequences to drugs of
abuse. Preclinical studies indicate a greater effect in females compared to males and that ovarian
steroids are responsible for this gender difference (Becker and Hu, 2008; Roth et al., 2004). A
study conducted in women demonstrated a higher subjective response (ratings of high, euphoria,
energy, liking, wanting more) to amphetamine in the first half of the follicular phase compared to
the luteal phase (Justice and De Wit, 1999). The same authors reported that exogenous estrogen
given to women in the follicular phase in doses that achieved periovulatory estrogen levels
increased the rating of pleasant stimulation, but paradoxically reduced wanting more. Another
study reported that cocaine produced higher ratings of good drug effect, high, stimulated, and
drug quality in the follicular phase compared to the luteal phase of the menstrual cycle (Evans
et al., 2002; Sofuoglu et al., 1999). Interestingly, exogenous progesterone given in the follicular
phase attenuated these subjective ratings (Evans and Foltin, 2005).
11.2.9 Dopamine hypothesis to explain menstrual cycle dependent ingestive behavior
Gonadal steroids likely account for gender differences and menstrual cycle-dependent changes in
subjective responsiveness to psychostimulants and food. An effect of estrogen on dopamine may
provide a unifying mechanism for these effects. Estrogen levels are highest in the periovulatory
period and are unopposed by low progesterone concentrations. Increased dopamine signaling
may occur during this ovarian steroid milieu as a result of estrogen stimulation of dopamine
release and upregulation of dopamine receptors. Dopamine signaling may decline during
the luteal phase as a result of progesterone antagonism of estrogens effect on dopamine and
continue into the early follicular phase due low estrogen levels. Increased dopamine signaling
in the periovulatory period may lead to enhanced activation of the corticolimbic network. Our
observation that the DLPFC, insula, NAc, putamen, amygdala, hippocampus, substantia nigra
and VTA exhibited greater activation in the late follicular phase to food pictures, particularly
LC foods, suggests that the responsiveness of this network is increased by estrogen. Increased
dopamine signaling in the periovulatory period may lead to reward satiation and smaller meals.
In contrast, reduced dopamine signaling may lead to reduced reward and to larger meals and
binge eating during the luteal phase. While it may be counterintuitive that increased dopamine
signaling would terminate a behavior, extracellular dopamine levels that exceed a threshold may
be less rewarding and reduce wanting (Palmiter, 2007). This threshold concept can explain why
drugs like amphetamine and cocaine (which may increase extracellular dopamine levels above
this threshold) inhibit food intake. A link between estrogen, increased extracellular dopamine
levels, and reduced desire is supported by the observation that liking amphetamine was increased
when periovulatory estrogen levels were achieved with exogenous estrogen, but wanting more
was reduced (White et al., 2002).
11.3 Conclusion
Food ingestion is a complex behavior that is influenced by hormones and hypothalamic
neuropeptides that interact to achieve a balance between energy expenditure and energy intake.
However, these homeostatic mechanisms can be superseded by the rewarding nature of highly
palatable foods leading to non-homeostatic or hedonic eating. Ingestive behavior is affected by
incentive motivation as a result of taste, pleasure, emotion, learning and memory which are
fashioned within the corticolimbic brain. Neuroimaging technology can measure activation of
these neural networks in response to appetitive cues such as sight, taste or thoughts of food. Brain
responses to these stimuli are influenced by many factors including hunger, reward potential,
mood, adiposity, gender, and menstrual cycle. Only a few studies have examined the influenced
of menstrual cycle phase and ovarian steroid effects on brain responses to rewarding stimuli.
Activation of several corticolimbic brain regions, including the ventral striatum/NAc, amygdala/
hippocampal complex in response to visual food cues or money were increased during the
estrogen dominant late follicular phase compared to the luteal phase. Activation of the inferior
frontal gyrus was increased whereas fusiform activity was decreased in response to food pictures
in the late follicular phase compared to early follicular phase in association with increased
estrogen concentrations. These menstrual cycle phase dependent differences in activation of
reward circuits may involve gonadal steroid effects on the mesolimbic dopamine system. An
understanding of the mechanisms that affect cyclical changes in food intake during the menstrual
cycle will advance our understanding of the biology of appetite regulation, an important societal
imperative given current obesity rates.
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12. Fluctuations of appetite and food intake
during the menstrual cycle
S.A. Tucci
School of Psychology, University of Liverpool, Eleanor Rathbone Building, Bedford Street South,
Liverpool L37 2LE, United Kingdom; sonia.tucci@liv.ac.uk
Abstract
This chapter aims to review the mechanisms underlying the fluctuations on appetite and food
intake that occur during the reproductive cycle in both animals and humans. Research has shown
that food intake is reduced during the periovulatory period and that this effect is abolished by
ovariectomy. Estradiol has been deemed as the main hormone responsible for this change. It exerts
its effects through activation of nuclear estrogen receptors in brain areas such as the hypothalamus,
hindbrain and reward system. In addition estradiol also seems to increase the satiating potency
of peripheral anorexigenic signals which leads to an early termination of meals. During the luteal
phase there is an increase on intake that has also been well documented. Although the underlying
mechanisms are still not totally clarified, they point toward a progesterone mediated antagonism
of estradiols effects. Food choice is also reviewed and the current literature offers contradictory
results with some reports finding that there is no preference for macronutrients whereas others
found an increase in fat, carbohydrate or protein intake. Studies performed in our laboratory
have found that similarly to others, intake is increased in the luteal phase, but interestingly this
is not accompanied by a variation of subjective measures of appetite supporting the theory of a
weakened post-meal satiety in this phase. The chapter finalizes with a review of neuroimaging
studies which are a relatively recent addition to research and therefore still quite limited in terms
of findings. To date the main variations in brain activity in response to food images seem to occur
in areas related to visual processing. This has led to the preliminary conclusion that an additional
mechanism by which reproductive hormones alter food intake is by change its salience across
the menstrual cycle.
Keywords: estradiol, estrogens, progesterone, cravings, luteal, follicular, cholecystokinin,

hypothalamus, satiety, meals, neuroimaging
189
S.A. Tucci
Summary points
Estradiol, the predominant sex hormone from menarche to menopause affects body composition by
modulating intake and metabolic parameters.
Estrogens exert both tonic and cyclic inhibitory effects on eating.
The phasic or cyclic inhibitory effect of estradiol occurs during the peri-ovulatory phase where estradiol is at
its peak and it is evidenced mainly by a decrease in meal size.
Estradiol exerts its anorexigenic actions by coupling with the nuclear estrogen receptors ER and ER.
Estradiol exerts its anorexigenic effects via actions in central and peripheral signaling mechanisms.
The central effects of estradiol alter mainly neuropeptides involved in feeding behavior and reward areas
associated to hedonic properties of food.
Estradiol increases the satiating potency of cholecystokinin.
The increase in intake that occurs during the luteal phase seems to be a consequence of the antagonism of
estradiols anorexigenic effects by progesterone.
In both animals and humans, intake correlates negatively with estradiol levels.
Neuroimaging studies have found that estradiol seems to modulate activity in brain areas related to processing
of visual images.
12. Fluctuations of appetite and food intake during the menstrual cycle
Abbreviations
AgRP Agouti related peptide
CCK Cholecystokinin
CNS Central nervous system
ER Estrogen receptor
GHSR Growth hormone secretagogue receptor
NPY Neuropeptide Y
OVX Ovariectomized
PPT Propyl-pirazole-triol
PVN Paraventricular nucleus
VMH Ventromedial hypothalamus
12.1 Introduction
In all female mammals embryonic implantation requires an endometrium, however the
mechanisms of embryo implantation differ widely between species. In consequence, endometrial
preparation and remodeling also varies. Female humans and non-human primates shed the
endometrium at the end of the cycle (menstrual cycle), whereas rodents reabsorb it (estrous
cycle) (Salamonsen, 2008).
In most women the menstrual cycle averages 28 days and is divided into two main phases with
the day of onset of menstruation referred to as day 1. The follicular phase starts on day one and
culminates with ovulation (day 14-15) and the luteal phase starts when ovulation takes place and
lasts until the onset of the next menstruation (Owen, 1975).
The menstrual cycle is a consequence of the ovarian cycle which originates the hormonal
fluctuations that alter food intake. The ovarian cycle is controlled by hypothalamic hormones and
although it is present and somehow similar in most mammalian placental females, the immediate
post-ovulatory phase tends to be species specific. For the purpose of this chapter, it is important
to briefly review the ovarian cycle in rats since most animal studies investigating the effects of sex
hormones on appetite and food intake are carried out in this species.
In rats, the estrous cycle lasts 4-5 days. It comprises four phases: proestrus, estrus, metoestrus (or
dioestrus I) and dioestrus (or dioestrus II). Ovulation takes place from the beginning of proestrus
to the end of estrus. In terms of hormonal changes, in the afternoon of the proestrus phase there
is an increase of both LH and FSH. This is followed by an increase of estradiol which reaches peak
levels in proestrus and returns to baseline at estrus. Progesterone secretion peaks twice, during
metoestrus and dioestrus and at the end of proestrus (Freeman, 1988).
S.A. Tucci
Rats do not experience the post ovulatory increase of estrogen and progesterone found in women.
However, both humans and rodents share the decrease of FSH and LH secretion that takes place
in the final phase of the cycle. In terms of food intake, rodents also experience a variation across
the estrous cycle being at its maximum in dioestrus, followed by proestrus, with the smallest
intake observed in estrus (Eckel, 1999). It is important to note that the differences in the post
ovulatory phase of the cycle make rodents poor models to study appetite regulation and food
intake in this phase of the cycle (Asarian and Geary, 2006).
12.2 Estrogen modulation of food intake

Estrogens are the primary female sex hormones in vertebrates. The three major naturally
occurring estrogens are estrone, estradiol, and estriol. Estradiol is the predominant estrogen
in non-pregnant females from menarche to menopause and therefore the focus of this chapter.
Estradiol seems to affect body composition in a dual manner. It modulates appetite and intake
and also alters metabolic parameters such as protein synthesis (Price et al., 1998) and energy
expenditure (Richard, 1986). Nevertheless, the main effect of estradiol is on energy intake since
across the menstrual cycle it varies between 12-38% whereas metabolic rate only fluctuates 2.5%
(Bisdee et al., 1989).
Estrogens exert both tonic and cyclic inhibitory effects on eating (Figure 12.1). The tonic
inhibitory effect on daily intake occurs throughout the cycle and it is evidenced by the increase
on intake and adiposity seen after the menopause in women and in OVX rats. The phasic or cyclic
inhibitory effect occurs during the peri-ovulatory phase where estradiol is at its peak (Asarian
Food intake
LH
Progesterone
Estradiol
Ovulation
Menstruation
Follicular phase Luteal phase
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Figure 12.1. Menstrual cycle depicting hormone levels and appetite fluctuations. When estradiol levels are high
food intake is reduced. Intake increases in the luteal phase.
and Geary, 2006). The analysis of meal patterns shows that the cyclic reduction of intake is due to
a decrease in meal size rather than meal frequency which remains unaffected. Therefore estrogens
seem to alter the physiological mechanisms that control meal size (probably advancing the onset
of satiety) rather than for instance elicit competing behaviors such as an increase in locomotor
activity, which could divert the animals from initiating meals (Eckel, 1999).
Rats and mice show a 25% decrease in food intake during the night that follows ovulation.
Women also experience a similar pattern where food intake is diminished during the 4 days
surrounding the LH surge which corresponds with a peak on plasma estrogen levels (Buffenstein
et al., 1995; Gong et al., 1989). On average food intake is 10% lower during the follicular phase
with no fluctuation seen in anovulatory cycles (Pelkman et al., 2000).
It has been proposed that estrogens regulate feeding by several mechanisms which involve
direct effects through stimulation of estrogen receptors in the CNS and also indirect effects on
peripheral signals.
12.3 Role of estrogen receptors

Estradiol exerts many of its actions by coupling with the nuclear estrogen receptors ER and ER
which are both present in the CNS. ER is mainly found in the hypothalamus, whereas ER has
a broader distribution (Shughrue et al., 1997) (Table 12.1). Both receptors are involved in intake
regulation (Liang et al., 2002). The role of ER has been demonstrated in OVX mice with a null
mutation of this receptor. These animals do not decrease their intake in response to estradiol
administration which suggests that the anorectic actions of estradiol are mediated by this receptor
(Geary et al., 2001). In addition, in OVX rats, the ER agonist PPT induces both a chronic
and acute suppression of intake (Roesch, 2006). ER is also involved in estradiol regulation of
food intake, since its anorexigenic effects can be blocked with anti-sense oligodeoxynucleotides
for ER (Liang et al., 2002). The role of ER is also suggested by the reduction on food intake
observed after administration of estradiol into the PVN, whose estrogen sensitive neurons only
express ER (Shughrue et al., 1997). Furthermore, selective blockade of ER expression in the
VMH does not attenuate the effect of systemic estradiol in OVX rats (Musatov et al., 2007). ER
have been found in the reward system such as the striatum and nucleus accumbens (Creutz and
Kritzer, 2002) and OVX has been shown to reduce dopamine binding in these areas (Le Saux et
al., 2006). This reduction in dopamine reward signaling could lead to a compensatory increase in
food consumption suggesting that estrogen signaling at ER could be relevant to hedonic eating.
12.4 Central effects of estrogen

Although the main effects of estradiol on food intake seem to be mediated through actions on
peripheral signals, some experimental evidence points towards additional direct actions on
central signals. Early studies found that administration of estradiol to the VMH decreased food
S.A. Tucci
Table 12.1. Estrogen receptor involvement on estradiols effect on intake.
Estrogen Localization Effects of estradiol at Receptor antagonist References

receptors receptor level effects
ER Hypothalamus (except Null mutations: no Propyl-pirazole-triol Geary et al., 2001;

paraventricular decrease of intake induces both chronic Roesch, 2006
nucleus) after exogenous and acute suppression
administration of of intake
estradiol
ER Broader distribution Selective blockade Estradiols anorexigenic Liang et al., 2002;
Paraventricular nucleus of ER expression effects can be blocked Musatov et al., 2007
Reward areas in ventromedial with anti-sense
hypothalamus does not oligodeoxynucleotides
attenuate the effect of for ER
systemic estradiol
intake in OVX rats, this leads to an initial suggestion that proposed that one mechanism by which
estradiol decreased food intake was by altering the body weight set point in the VMH (Jankowiak
and Stern, 1974). Additional research reported that in OVX rats estradiol implants in the PVN
decreased intake (Butera and Beikirch, 1989), however this finding has not been consistently
replicated.
Recent studies claim that the central effects of estradiol are a consequence of changes in the
expression of hypothalamic neuropeptides involved in feeding behavior (Olofsson et al., 2009).
In this study NPY/AgRP neurons were identified as essential targets. NPY is one of the most
potent orexigenic agents known whereas AgRP increases food intake by acting as an antagonist
at melanocortin receptors. Stimulation of melanocortin receptors reduces food intake therefore
its antagonism would increase it (Morton and Schwartz, 2001). In mice the abolition of AgRP
neurons eliminated the estrous cycle dependent changes in feeding and body weight (Olofsson
et al., 2009). Moreover, estradiol down-regulates the expression of NPY and AgRP while OVX
increases hypothalamic NPY and AgRP mRNA expression (Titolo et al., 2006). The sensitivity
to NPY effects it is also modulated by estradiol since its administration decreases the orexigenic
effect of exogenous NPY and inhibits the activity of NPY/AgRP neurons in the arcuate nucleus
of the hypothalamus.
12.5 Peripheral effects of estrogen

12.5.1 Ghrelin
Ghrelin is a peptide hormone secreted by the stomach which increases food intake (Kojima et
al., 1999). Some studies indicated that the effects of ghrelin on food intake seem to be modulated
by estrogen levels. For instance, in OVX rats the orexigenic effects of ghrelin are attenuated by
estrogen administration (Table 12.2). This effect is dependent on an intact GHSR, since OVX does
not increase food intake in mice lacking this receptor (Ghsr/ mice). These findings suggest that
OVX increases food intake by releasing ghrelin from the tonic inhibitory effect of estradiol (Clegg
et al., 2007). In terms of estradiols effects on ghrelin secretion, the evidence is not conclusive
with some suggesting that estradiol decreases ghrelin secretion (Clegg et al., 2007) and others
reporting no effects (Dafopoulos et al., 2009).
12.5.2 Cholecystokinin
CCK is a gastrointestinal hormone which plays an important role in digestion and satiation.
CCK signals are transmitted from the periphery to the brain via vagal afferents triggering the
onset of satiation (Smith and Gibbs, 1992). In rats CCK plays an important role in the mediation
of estrogen peri-ovulatory decrease of intake (Table 12.2). These effects are site specific, thus
estradiol implants in the PVN and in the surface of the brainstem but not in other brain areas
potentiate the satiating action of peripheral CCK (Asarian and Geary, 2007). Furthermore, in
OVX rats estradiol increases the satiating potency of exogenous CCK (Butera et al., 1993). Thus,
it seems that at least in rats, estradiol acts on hypothalamic and hindbrain sites increasing the
potency rather than the secretion of CCK. CCK receptor antagonism produces a bigger de-
Table 12.2. Anorexigenic effects of estradiol through interaction with peripheral signals.
Peripheral signals Secretion site Interaction with estradiol References
Ghrelin Stomach Estradiol attenuates ghrelin Clegg et al., 2007

orexigenic actions
Estradiol decreases ghrelin secretion
Cholecystokinin Gut Estradiol increases satiating potency Asarian and Geary, 2007
of cholecystokinin
Leptin Adipose tissue No conclusive findings Pelleymounter et al., 1999;
Clegg et al., 2003, 2007
Progesterone Ovary Progesterone antagonizes Davidsen et al., 2007
anorexigenic effects of estradiol
S.A. Tucci
satiating effect in estrus (when estradiol levels are high) than in dioestrus (when estradiol levels
are low) without having any effects on food intake on its own (Huang et al., 1993).
12.5.3 Leptin
Leptin is a hormone synthesized by the adipose tissue; its circulating levels mirror the total amount
of body fat. In both humans and animals, leptin and estrogen levels increase proportionally
(Caro et al., 1996; Considine et al., 1996). It has been suggested that body weight moderates the
interaction between estrogen, leptin and food intake (Gambacciani et al., 1997). To date, there
is no consensus on the effects of estrogen and OVX on leptin signaling, with some reporting
that estrogens increase leptins anorexigenic actions (Clegg et al., 2003), while others reporting
non-significant findings (Pelleymounter et al., 1999; Chung and Bond et al., 2010) (Table 12.2).
These discrepancies could arise from methodological differences making this a topic that requires
further investigation.
12.5.4 Progesterone
In OVX rats progesterone only affects feeding behavior if administered at high, non-physiological
doses (Butera, 2010). However, in intact rats progesterone increases appetite and consequently
body weight (Wade and Schneider, 1992). This indicates that progesterone might not stimulate
appetite on its own, but could increase energy intake by interacting with estradiol (Davidsen et
al., 2007) (Table 12.2). Furthermore, in OVX rats the anorexigenic effect of estradiol is prevented
by co-administration of progesterone (Blaustein and Wade, 1976; Jankowiak and Stern, 1974;
Wade, 1975). It has been hypothesized thus, that the increase in progesterone during the luteal
phase could somehow antagonize estrogens anorexigenic effects. However, further studies are
required to examine this hypothesis (Davidsen et al., 2007).
12.6 Food intake during menstrual cycle

As mentioned above, in both laboratory animals and menstruating women, food intake is lowest
during the periovulatory phase of the ovarian cycle, when estradiol levels are high (Buffenstein et
al., 1995). Studies using both self-reported (food diaries, interviews, measurement of food weight
at home) (Barr et al., 1995; Dalvit, 1981; Danker-Hopfe et al., 1995; Lyons et al., 1989; Martini
et al., 1994; Pliner and Fleming, 1983) and laboratory measurements (Tucci et al., 2010, 2011;
Figure 12.2) have found that intake increases during the luteal phase of the cycle. The increase in
energy intake reported varies from around 150 kcal (7.5%) per day (Gong et al., 1989; Martini
et al., 1994; Pohle-Krauza et al., 2008) to doubling the amount of intake when compared to the
follicular phase (Lyons et al., 1989). However, it should be noted that not all studies have found
clear effects of phase on total intake.
250 *** **
200
150 Savoury
kcal Sweet
100
50
0
Follicular phase Luteal phase
Figure 12.2. Sweet and savoury items caloric intake in luteal and follicular phases (Tucci et al., 2010). **
P<0.01. *** P<0.001.
12.6.1 Nutrient selection
In terms of estradiols effects on nutrient selection, research has reported controversial results.
While some studies in rats have suggested that macronutrient selection does not vary across the
estrous cycle (Geiselman et al., 1981), others have found fluctuations (Leibowitz et al., 1998).
Research in women has shown similar findings, with some reporting no cyclical effects on food
selection (Cheikh Ismail et al., 2009) whereas other have found some alterations. For instance, a
study by Chung et al. (2010) found that the increased caloric intake observed during the luteal
phase was almost exclusively due to an increase in protein intake. Others have reported that the
increased caloric intake in this phase was due to an increased intake of fat (Johnson et al., 1994;
Li et al., 1999) or carbohydrates (Dalvit-McPhillips, 1983).
Two studies performed in our laboratory found that at least for snack items, participants
consumed more sweet snacks during luteal phase that follicular phase (Tucci et al., 2010, 2011;
Figure 12.3), these findings are consistent with previous reports regarding sweet food (Bowen
and Grunberg, 1990) and chocolate consumption (Hetherington and MacDiarmid, 1993). In
our study, although participants consumed more calories derived from sweet items in the luteal
phase, the ratings of liking for these foods did not increase when compared to the follicular phase
(Figure 12.4). This disconnection between actual intake and a key element of feeding motivation
appears somewhat puzzling. Therefore, these data support the more physiological notion that
the phase based difference in snack food intake are largely consequence of weakened post-meal
satiety rather than driven by altered food cravings and preferences.
Additionally, binge eating is also more likely to occur in the luteal phase (Klump et al., 2008).
Binge eating has been associated with low levels of plasma and brain serotonin (Cross et al.,
2001). Moreover, women with PMS have lower levels of serotonin and more episodes of binge
eating when compared with their non-PMS counterparts (Rapkin et al., 1987). Low levels of
S.A. Tucci
140
120 **
100
80 Follicular
Grams
60 Luteal
40 *
20 *
0
Proteins Carbohydrates Fats
Figure 12.3. Macronutrients intake (grams) of snack items during follicular and luteal phases (Tucci et al.,
2011). * P<0.05, ** P<0.01.
90
80
70
60
50
VAS ratings
Follicular
40 Luteal
30
20
10
0
Enjoyment Palatability Pleasantness
Figure 12.4. Subjective measures of appetite for snack items using a VAT scale of 100 mm (Tucci et al., 2011).
serotonin contribute to the mood changes experienced during the premenstrual period which
are more pronounced in PMS. Carbohydrate consumption can influence brain serotonin levels
by increasing the ratio of tryptophan: large amino acids. Tryptophan, the precursor of serotonin,
competes with large amino acids for entry into the brain. It has been proposed that the increase
in carbohydrate consumption during binge eating is an attempt to elevate brain serotonin levels
in order to improve mood. Both animal and human studies support the hypothesis that low levels
of brain serotonin may play a role in binge eating (Rapkin et al., 1987).
12.6.2 Cravings
Food cravings are common, with up to 100% of women and 75% of men reporting food or
drink cravings (Pelchat, 1997; Rodin et al., 1991). There is a gender difference regarding the
type of food craved with men craving savory food while women crave sweet food, especially
chocolate (Pelchat, 1997; Weingarten and Elston, 1991). Cravings vary across the menstrual
cycle being more intense perimenstrually (Bruinsma and Taren, 1999; Zellner et al., 2004). This
periodicity has led to the idea that cravings for sweets (especially chocolate) are the result of some
physiological adjustment (Bruinsma and Taren, 1999). Two explanations have been proposed,
one suggests that in this period a need state is created which is satisfied by some ingredient in
chocolate (e.g. magnesium or serotonin). The second suggestion is that chocolate contains some
ingredient that causes pleasure, either directly or indirectly through neurotransmitter release (e.g.
endogenous opioids), which for some unknown reason is desired more perimenstrually.
The physiological explanations of perimenstrual cravings are not fully supported. For instance
no significant correlations between levels of estrogen and progesterone and craving (mainly
chocolate cravings) frequency and intensity throughout the cycle have been found (Rodin et
al., 1991). In addition, if chocolate is given in capsules, which avoids identification, cravings are
not satisfied (Michener et al., 1999). The non-physiological causes of chocolate cravings are also
supported by the fact that for instance they are significantly less frequent in Spanish (16.7%) than
in American women (27.8%) (Zellner et al., 1999).
The above findings have led to the suggestion that cravings could be the result of learned
associations between the craving experience and times of the menstrual cycle characterized by
unpleasant physical symptoms (Yonkers et al., 2008). Therefore they could be result of some
learned strategy such as the perceptual properties of the chocolate and/or the fact that chocolate
is viewed as a special treat (Dye, 2001). Others have proposed that cravings are the result of
avoiding certain foods whose intake is restricted during the post ovulatory phase. This food
restriction can lead to craving for the avoided foods, which are usually high fat items (Channon
and Hayward, 1990). This theory is supported by a recent study by McVay et al. (2011) which
found a correlation between fear of fatness and strength of cyclical variation in hunger, food
cravings and amount eaten. Three explanations are offered for these findings, one proposing
that large cyclical variations in appetite and food intake result in concomitant variations in body
weight which in turn increase concerns about body weight. A second explanation proposes
that fear of fatness could trigger cyclical fluctuations in appetite and other eating variables. An
increased fear of fatness will lead to a perception that the fluctuations in hunger that occur as part
of the menstrual cycle are a threat to an idealized body image which in turn would intensify the
focus on hunger, food cravings and food intake. Finally a third explanation is that an underlying
variable is responsible for the association between fear of fatness and cyclical variations in eating
variables.
S.A. Tucci
12.7 Neuroimaging studies

Neuroimaging studies have shown cyclic changes in brain activation during the normal menstrual
cycle with regard to food visualization (Alonso-Alonso et al., 2011; Frank et al., 2010). They
have identified the fusiform gyrus as a potential neural substrate for the anorexigenic effects of
estradiol. This brain area belongs to the ventral visual pathway which is predominantly involved
in object identification (Kanwisher et al., 1997). It has been suggested that interactions between
the fusiform gyrus and the orbitofrontal cortex allow visual cues to elicit the predicted (long-
term) value of food (Murray and Izquierdo, 2007). Estradiol seems to act in this brain area
limiting the salience of food cues (Alonso-Alonso et al., 2011).
A study by Van Vugt (2010) found that visual food cues elicit a stronger response in several brain
regions during the periovulatory period compared with the luteal phase. During the periovulatory
phase images of food activated the lateral orbitofrontal cortex, prefrontal cortex, hippocampus,
amygdala and fusiform gyrus, whereas in the luteal phase only the fusiform gyrus was activated.
Interestingly the insula showed cyclic variation in activation with activation in response to low
calorie foods, but not by high calorie foods in the follicular phase and the opposite in luteal phase.
This finding correlates with ratings of foods during the cycle where high calorie food were rated as
more appealing in all weeks but week two of the menstrual cycle (Kim and Van Vugt, 2008). These
changes could reflect insula activation which might be responsible for the increased preference
for low calorie foods in the second half of the follicular phase which in turn could contribute to
the periovulatory reduction in caloric intake.
12.8 Conclusion
Evidence from animal and human studies has consistently reported that appetite and food
intake fluctuates during the estrous/menstrual cycle. These fluctuations correlate with sexual
hormones variations across the cycle. Estradiol exerts an anorectic effect by acting on central
and peripheral signals that regulate intake. Progesterone on its own does not seem to have
an effect but its combination with estradiol has been deemed responsible of the premenstrual
increase on intake. Both estrogen receptors are responsible of estradiols anorexigenic effects.
ER are located in the hypothalamus whereas ER are more widely distributed and have been
implicated in hedonic modulation of eating. Experimental evidence has shown that estrogens
act directly in the hypothalamus by decreasing the activity of orexigenic signals such as NPY
and AgRP. They have also been shown to alter the brain sensitivity to peripheral signals such
as CCK. The role of sex hormones on ghrelin and leptin signaling needs further investigation.
There is general consensus that food intake is increased in the luteal phase, however, in terms of
nutrient selection there are contradictory findings with some reporting a non-specific increase
in intake and others reporting specific increases of proteins, fats or carbohydrates intake. The
majority thus agrees that carbohydrates tend to be the most craved and over consumed items
and during this phase. Neuroimaging studies have found cyclical variations in brain responses to
food cues, these variations are especially prominent in areas related to visual processing. This is
a relatively unexplored field that will in the future contribute significantly to further clarification
on the effects of female sex hormones in the brain in relation to the regulation of food intake
and appetite.
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13. Dietary strategies contributing to low energy
availability in exercising women: underlying
physiology and impact on the menstrual cycle
Womens Health and Exercise Laboratory and the Department of Kinesiology, Pennsylvania State
University, University Park, PA 16802, USA; niw1@psu.edu
Abstract
Chronic energy deficiency may suppress reproduction to preserve energy for life-sustaining
physiological functions; however, the mechanism is unclear. It is believed that hormones such
as ghrelin and leptin signal a state of low energy availability (EA) to the hypothalamus leading
to the suppression of gonadotropin-releasing hormone (GnRH) secretion. GnRH suppression
inhibits the pulsatile release of luteinizing hormone resulting in inhibition of folliculogenesis
and ovulation. Exercising women may be at an increased risk for the Female Athlete Triad, a
syndrome consisting of three interrelated clinical conditions: low EA, menstrual disturbances
and low bone mass. The Triads etiology has been attributed to chronic energy deficiency induced
by inadequate energy intake to compensate for exercise energy expenditure. Unhealthy dietary
strategies to maintain low EA include clinically diagnosed eating disorders of which amenorrhea
is a diagnostic criterion, disordered eating which has also been linked to menstrual dysfunction
and includes dieting, food avoidance, and vegetarianism, and inadvertent under-eating. Aberrant
eating attitudes may lead to/exacerbate disordered eating and have also been linked to menstrual
disturbances. Exercise may acutely suppress appetite, elicit an anorexigenic gastrointestinal
hormone profile and alter food reward responses; however, more studies are needed to address
whether inadvertent under-eating contributes to menstrual dysfunction.
Keywords: energy balance, disordered eating, female athlete triad, functional hypothalamic
amenorrhea
207
Summary points
Chronic energy deficiency may suppress reproductive function to preserve energy for life-sustaining
physiological functions; however, the mechanism through which this occurs is unclear.
Hormones such as ghrelin and leptin may signal a state of low energy availability to the hypothalamus where
gonadotropin releasing hormone (GnRH) secretion is suppressed. GnRH suppression inhibits the pulsatile
release of luteinizing hormone which results in inhibition of folliculogenesis and ovulation.
Exercising women may be at an increased risk of developing one or more components of the Female Athlete
Triad which consists of three interrelated clinical conditions, i.e. low energy availability, menstrual disturbances
and low bone mass. The etiology of the Triad has been attributed to chronic energy deficiency induced by
inadequate energy intake to compensate for exercise energy expenditure.
Low energy availability may lead to suppressed reproductive function. Both conditions may increase risk of
low bone mass and/or fracture.
Dietary strategies to maintain low energy availability include eating disorders, disordered eating and
inadvertent under eating.
Eating disorders are clinically diagnosed psychiatric conditions that represent the most severe form of energy
restriction where amenorrhea has been considered a diagnostic criterion.
Disordered eating represents many subclinical manifestations, all of which impact the menstrual cycle by way
of the induction of a chronic energy deficit.
High drive for thinness and high dietary restraint may lead to or exacerbate disordered eating and may be
indications of energy deficiency in exercising women with menstrual disturbances.
Exercise may acutely suppress appetite, elicit an anorexigenic gastrointestinal hormone profile and alter
food reward responses leading to inadvertent under eating; however, more studies are needed to address
this effect in exercising women.
Short term gastrointestinal signals that respond to more subtle, daily fluctuations in meal timing and caloric
quantity of food eaten across a single day may be important modulators of menstrual function.
Exercising women may need to plan meals across the day so that caloric and macronutrient content of meals
as well as meal timing are optimal to sustain a healthy energy availability and prevent induction of a chronic
energy deficit that may lead to reproductive suppression.
13. Dietary strategies for low energy availability in exercising women
Abbreviations
AN Anorexia nervosa
BN Bulimia nervosa
DT Drive for thinness
ED-NOS Eating disorder not otherwise specified
FFM Fat free mass
FHA Functional hypothalamic amenorrhea
fMRI functional magnetic resonance imaging
GI Gastrointestinal
HPO Hypothalamic-pituitary-ovarian
LPD Luteal phase defect
NPY Neuropeptide Y
PYY Peptide YY
RDA Recommended dietary allowance
RMR Resting metabolic rate
13.1 Introduction
The HPO axis is sensitive to fluctuations in energy balance. In conditions of energy deficiency
reproductive function may be suppressed to redirect energy away from the energy-costly process
of reproduction to life-sustaining functions such as cellular maintenance and thermoregulation.
Exercising women commonly experience low energy availability by decreasing dietary energy
intake, increasing energy expended through exercise, or both. Chronic energy deficiency and
consequent suppression of reproductive function may manifest as the Female Athlete Triad, a
syndrome of interrelated conditions that exist along a spectrum of severity ranging from optimal
energy balance, regular, ovulatory menstrual cycles and healthy bone mineral density to low
energy availability with or without disordered eating, amenorrhea and osteoporosis (Nattiv et al.,
2007). Exercising women may exhibit chronically low energy intake by way of clinically diagnosed
eating disorders such as AN or BN; however, there is an even greater prevalence of women that
exhibit subclinical indices of disordered eating (Nattiv et al., 2007). It has been suggested that
some women may even present with healthy eating behaviors, but exercise at such a high volume
that they are unable to compensate for exercise energy expenditure by increasing energy intake
and thus, inadvertently under eat (Nattiv et al., 2007). This article will provide an overview of
how particular dietary strategies in which many exercising women engage to maintain low energy
availability impact energy balance and the underlying physiology of menstrual cycle disturbances.
13.2 Energy balance and reproductive function

In a variety of mammalian species, chronic energy deficiency may induce a reversible state of
energy conservation by suppressing reproduction to preserve fuel for life-sustaining processes in
the body (Wade et al., 1996). In animal studies, reducing dietary intake by >30% has continually led
to infertility (Nattiv et al., 2007). Termed FHA, this has been demonstrated in an exercise model
in monkeys in which amenorrhea was induced in response to an exercise-induced energy deficit.
Resumption of menses occurred by increasing caloric intake and body weight while maintaining
daily exercise training (Williams et al., 2001). Several prospective studies in exercising women
have reported menstrual irregularities in women in response to both decreasing energy intake
or increasing exercise energy expenditure. Decreases in LH pulse frequency (Loucks and Heath,
1994) as well as estrogen concentrations (Pirke et al., 1985) have been exhibited in response to
a diet-induced energy deficit. As well, an energy deficit induced by caloric restriction combined
with vigorous exercise has been associated with decreases in estrogen as well as menstrual
disturbances (Bullen et al., 1985). In the latter study, only four of 28 untrained college-aged
women maintained a normal menstrual cycle during training. Those that lost weight experienced
luteal phase defects as well as suppression of the pre-ovulatory LH surge. Within six months of
study termination, subjects regained normal menstrual cyclicity. Notably, one study in young
women showed that an energy deficit induced by diet alone suppressed LH pulse frequency
more so than an equal energy deficit induced by exercise. This study also reported no change in
LH pulse frequency subsequent to the exercise intervention when increased energy intake was
provided to offset the exercise-induced energy deficit. Thus, in agreement with animal studies,
reproductive function is suppressed in exercising women by an energy deficit and not the stress
of exercise per se.
The suppression of reproductive function in response to energy deficiency occurs at the level of
the GnRH pulse generator when metabolic signals in the circulation or via neural pathways relay
information regarding energy status to the hypothalamus (Figure 13.1). Suppression of GnRH
pulsatility inhibits optimal LH pulse frequency signaling to the ovaries to produce estrogen and
the mid-cycle LH surge that causes ovulation. However, the exact mechanism through which this
suppression occurs is unknown. A large amount of evidence supports a key role for leptin in the
modulation of reproductive function, particularly in exercising women (Welt et al., 2004). Leptin
deficiency has been associated with impaired GnRH secretion in mice where ovulation is restored
with exogenous leptin administration (Chehab et al., 1996). In humans, leptin is associated with
both the maintenance of LH pulsatility as well as the LH surge that causes ovulation (Evans and
Anderson, 2012). Interestingly, it has been reported that amenorrheic women as well as those
with disordered eating have lower circulating leptin concentrations as well as a blunted diurnal
rhythm (Laughlin and Yen, 1997). More recent studies suggest that GI peptides like ghrelin may
also play a role in this regulation. GI peptides are secreted from cells along the GI tract in response
to nutrient ingestion, some of which are able to cross the blood brain barrier to send signals to the
brain regarding nutritional status. These GI peptides may signal indirectly by way of stimulation
of neuropeptides that convey GI signals regarding energy balance to GnRH neurons. For example,
ghrelin binds to the growth hormone secretagogue receptor in the hypothalamus and stimulates
Hypothalamus
Gonadotropin
-releasing
hormone +
(GnRH)

Pituitary Ghrelin Leptin

Luteinizing Estrogen Stomach Adipose tissue
hormone
(LH) +
Follicle-
stimulating
hormone
(FSH)

Chronic energy deficit
Ovary
Figure 13.1. Schematic representing metabolic signaling of energy deficiency to hypothalamic gonadotropin
releasing hormone suppression.
NPY release from the hypothalamic arcuate nucleus. NPY is a potent orexigenic neuropeptide
that may inhibit GnRH release when estrogen concentrations are low and stimulate GnRH release
when estrogen concentrations are high. PYY is a GI hormone that binds to the Y2 receptor in the
arcuate nucleus and opposes the actions of ghrelin. PYY is a satiety hormone that has been shown
to be elevated in exercising women with menstrual cycle disturbances; however, the role of PYY in
the suppression of reproductive function is unclear. Neuropeptides that relay hormone signaling
from the GI tract may act directly on GnRH neurons or indirectly by way of a recently discovered
neuropeptide that is believed to be involved in the initiation of puberty and the pre-ovulatory
GnRH surge: kisspeptin. Kisspeptin may act as an intermediary in these pathways transducing
signals from the arcuate nucleus, and other areas that respond to changes in energy balance such
as the paraventricular nucleus, to GnRH neurons (Evans and Anderson, 2012).
13.3 The Female Athlete Triad

The Female Athlete Triad (Figure 13.2) is most prevalent in women training for sports in
which low body weight is emphasized for performance or appearance; however, one or more
Healthy energy status
Intermittent low EA,

Low EA, disordered eating
eating disorders Healthy
bones Healthy
menstrual cycles
ovulatory
Low BMD LPD,
anovulation,
oligomenorrhea
Osteoporosis Amenorrhea
Figure 13.2. Schematic representation of the Female Athlete Triad (Adapted from De Souza et. al., 2004; with
permission). EA: energy availability, BMD: bone mineral density, LPD: luteal phase defect.
components of the Triad have been recognized in non-aesthetic sports as well as high school
athletes and non-competitive exercising women (Nattiv et al., 2007). A high prevalence exists
when considering women diagnosed with one or more components of the Triad, and the
prevalence varies depending on the population of interest. The percentage of exercising women
that have been recognized as having disordered eating has been estimated to be as high as 62%
(Bonci et al., 2008). However, the American College of Sports Medicine emphasizes that women
may also become energy deficient in the absence of disordered eating (Nattiv et al., 2007). Some
exercising women may be unable to compensate for a high volume of exercise energy expenditure
and thus, inadvertently fail to increase energy intake to adequately meet the needs of energy
expended. However, studies have not been performed to determine the existence and prevalence
of inadvertent under eating. The prevalence of menstrual irregularities in exercising women has
been documented as high as 69% (Nattiv et al., 2007). Most studies utilize self-reported menstrual
history to document menstrual disturbances which may underestimate the prevalence as some
women may have a regular cycle length and thus, report normal menses, but may have incidence
of more subtle disturbances. Menstrual cycle disturbances have been documented ranging from
subtle disturbances such as LPD and anovulation to oligomenorrhea (cycle length of 36-90 days)
and amenorrhea. LPD may not be detected as cycle length may be within a normal range (26-35
days); however, may manifest as a short luteal phase of <10 days or inadequate luteal phase where
progesterone does not attain concentrations high enough to sustain the health of the corpus
luteum. One study carefully characterized urinary estrogen, progesterone and LH across 2 to 3
consecutive menstrual cycles in 67 exercising women and demonstrated that 50% of cycles were
abnormal and 33.7% were amenorrheic (Figure 13.3) (De Souza et al., 2010).
13.4 Energy availability

Dietary strategies utilized by exercising women to achieve low body weight and low body
fat promote an energy deficit and consequently, lower energy availability. This may lead to
reproductive suppression and the menstrual cycle disturbances detailed above. Even though
a Luteal phase defect 5% b

1 of 20 women
Anovulatory
Luteal phase 25% 12 of 48
defect 27.1% women***
13 of 48 women*
Ovulatory 95%
19 of 20 women Ovulatory 47.9%
23 of 48 women***
Figure 13.3. Pie chart representing the prevalence of subtle menstrual disturbances among (a) sedentary and
(b) exercising women (Souza et al., 2010; reprinted with permission). * P=0.050; *** P<0.001.
exercising women require an increased energy intake to match energy expenditure, there have
been several reports that they consume the same amount, or even less, than their sedentary
counterparts (Manore, 1999). Energy availability, operationally defined as dietary energy intake
minus exercise energy expenditure, refers to the amount of dietary energy that remains for other
bodily functions after accounting for exercise energy expenditure, and is typically quantified
relative to FFM (Loucks and Thuma, 2003). Optimal energy availability is needed to maintain
reproductive function where 45 kcal/kg FFM is considered to be the energy availability at which
healthy energy balance and menstrual function are sustained. Energy availability can be lowered
by decreasing energy intake, increasing exercise energy expenditure, or both (Nattiv et al., 2007).
Dietary strategies to lower energy availability, explained below, are all avenues through which
energy availability may be lowered to a level at or below which menstrual cycle disturbances
occur. It has been suggested that an energy availability below 30 kcal/kg FFM may suppress
reproductive function. To their detriment, exercising women in a chronic energy deficit may be
weight stable and as a result, presume that they are in a healthy energy balance. However, several
other indications of energy conservation have been demonstrated such as suppressed RMR which
may decrease total energy expenditure and endocrine abnormalities like low triiodothyronine
and elevated circulating concentrations of ghrelin (De Souza et al., 2004). Consequently, body
weight might be stable during a period where energy availability is low.
13.5 D ietary strategies used by exercising women to maintain low body weight
and low body fat
13.5.1 Impact on the menstrual cycle
Several dietary strategies are utilized by exercising women to maintain low body weight and
low body fat ranging from clinically diagnosed eating disorders to subclinical disordered eating
and potentially inadvertent under eating (Table 13.1). Many exercising women may utilize one
or more strategies to maintain low body weight for more aesthetic reasons as opposed to sport
performance. Compared to men, twice as many women perceive themselves to be overweight.
Those that are actively attempting to lose weight are even higher; the proportion of which increase
as body mass index decreases such that nearly nine times the number of lean women as lean men
are engaged in some strategy to lose weight.
Table 13.1. Summary of dietary strategies used to maintain low energy availability and associated menstrual
cycle disturbances.
Dietary strategy Description Associated menstrual

disturbance(s)
Eating disorders1 Clinically diagnosed psychiatric conditions. Most Amenorrhea is a diagnostic

severe form of energy restriction. criterion
Disordered eating Subclinical disorders including energy restriction Subtle (LPD2, anovulation) to
not necessary for health or performance severe (oligo- and amenorrhea)
menstrual disturbances
Dieting Energy restriction to lower caloric intake that is Subtle (LPD, anovulation) to
not necessarily associated with aberrant eating severe (oligo- and amenorrhea)
or body image attitudes menstrual disturbances
Food avoidance Avoidance of particular foods that are high in fat, Subtle to severe menstrual
i.e. red meat, ice cream, dairy products, etc. to disturbances; due to energy
restrict energy intake. Considered a red flag restriction and not lack of a
for potential risk of disordered eating. specific macronutrient
Vegetarianism Avoiding high-fat foods like meat to restrict Anovulation, suppressed
energy intake. Considered a red flag for estrogen and progesterone
potential risk of disordered eating. concentrations
Low energy dense diets Increasing fruit/vegetable intake and decreasing Subtle (LPD, anovulation) to
fat intake to decrease dietary energy intake severe (oligo- and amenorrhea)
Aberrant eating behavior High drive for thinness and/or high cognitive Subtle (LPD, anovulation) to
restraint may lead to or exacerbate severe (oligo- and amenorrhea)
Inadvertent under eating Exercise may inadvertently induce appetite No evidence of menstrual
suppression and lead to accidental energy disturbances, more research is
restriction needed
1 Anorexia nervosa, bulimia nervosa and eating disorder not otherwise specified (ED-NOS).
2 LPD: luteal phase defect.
13.5.2 Eating disorders
Eating disorders are the most severe form of energy restriction to maintain low energy availability
and are clinically diagnosed psychiatric conditions in which individuals may be hospitalized if
health is at risk. AN is characterized by restrictive eating in which individuals have a distorted
body image, viewing themselves as overweight and are afraid of weight gain despite being
<85% expected weight for age and height. Individuals suffering from BN tend to have a normal
body weight and repeat cycles of overeating or binging and then purging or engaging in other
compensatory behaviors such as fasting or excessive exercise. ED-NOS is a category of eating
disorder in which most, but not all criteria for AN or BN are met. Eating disorders are considered
the most severe form of energy restriction and pathogenic weight control and consequently,
are more likely associated with the most severe menstrual disturbance, amenorrhea, which is
currently a diagnostic criterion for AN.
13.5.3 Disordered eating
More recently, disordered eating has been included in describing the arm of the Female Athlete
Triad related to energy availability suggesting that the Triad exists along a spectrum of subclinical
disturbances that may or may not lead to a clinically diagnosed eating disorder. Indices of
disordered eating include, but are not limited to, dieting that is unnecessary for health, sport
performance or appearance, avoiding certain foods or food groups, vegetarianism/veganism,
binge-eating and purging, use of laxatives and/or diuretics and excessive exercise or exercising
despite injury (Bonci et al., 2008).
13.5.4 Dieting
Dieting may lower energy intake and/or lead to insufficient macro- or micronutrient intake
during a period where energy requirements are higher in exercising women than their sedentary
counterparts. It has been suggested that energy intake below 1,800 kcal/day (7,430 kJ/day) is
inadequate to meet the needs of training, especially in adolescent women that are still growing
(Manore, 1999). Additionally, exercising women have increased carbohydrate and protein
requirements. In comparison to the RDA, upwards of 5-10 grams/kg body weight and 1.2-1.4 g/kg
body weight (RDA=0.8 g/kg), respectively, are recommended. Not meeting these requirements
can lead to poor performance, slower recovery and increase risk of injury (Manore, 1999). When
exercising women restrict energy intake and do not adequately meet the needs of exercise energy
expenditure, the incidence of menstrual disturbances increases (Bullen et al., 1985). Subtle to
severe menstrual disturbances have been reported in exercising women that restrict energy intake
(Nattiv et al., 2007). Consequently, larger and more chronic energy deficits may lead to more
severe menstrual disturbances such as oligomenorrhea and amenorrhea. To that end, recovery
of regular, ovulatory menstrual cycles may be a product of increasing caloric intake to match
expenditure and restore a healthy energy availability. As well, time to recovery may depend the
amount of calories supplemented to offset the energy deficit (Williams et al., 2001).
13.5.5 Food avoidance: low-carbohydrate and low-fat dieting
Some exercising women will avoid certain foods (i.e. red meat) or macronutrients like carbohydrate
or fat to maintain low energy intake; however, proper balance of macronutrient intake is essential
in maintaining performance and health (Rodriguez et al., 2009). Avoiding certain foods or
macronutrients, for example avoiding red meat or maintaining a low carbohydrate diet, may
lead to energy restriction in exercising women that have an increased energy requirement. No
particular macronutrient impacts the menstrual cycle more so than another; however, inducing
an energy deficit by way of food avoidance may lead to menstrual cycle disturbances if energy
intake does not adequately meet the needs of exercise energy expenditure. Though, there is no
macronutrient that will impact the menstrual cycle more than another, low carbohydrate diets,
in endurance athletes particularly, may be extremely detrimental with regard to skeletal muscle
metabolism during exercise. Carbohydrates maintain blood glucose concentrations and are a
main substrate for skeletal muscle metabolism during exercise, and they are used to replace muscle
glycogen during post-exercise recovery (Rodriguez et al., 2009). Fat is the only macronutrient
with no recommended value for intake; however, restricting dietary fat intake may be detrimental
because, in addition to energy and use as a main substrate in skeletal muscle metabolism during
prolonged exercise, it provides essential elements of cell membranes and aids in absorption of
fat-soluble vitamins, A, D, E and K (Rodriguez et al., 2009).
13.5.6 Vegetarianism
Vegetarians tend to be leaner and lighter than non-vegetarians and exercising women may
utilize a vegetarian diet to restrict energy intake by avoiding high-fat foods such as meat. These
individuals may report wanting to eat well though the underlying reason of becoming vegetarian
may be energy restriction to induce weight loss (Barr, 1999). Thus, being or becoming a vegetarian
is seen as a red flag or risk factor for disordered eating (Nattiv et al., 2007; Rodriguez et al.,
2009). Adopting a vegetarian diet has been associated with menstrual disturbances. One study
(Pirke et al., 1986) randomized normal-weight women to either a vegetarian or non-vegetarian
diet. In this study, both groups lost an average of 1 kg body weight each week over a six week
intervention period; however, seven of the nine women eating the vegetarian diet were reported
to have anovulatory menstrual cycles characterized with suppressed estrogen, progesterone and
LH concentrations, whereas seven of nine women eating the non-vegetarian diet maintained
regular ovulatory menstrual cycles.
13.5.7 Low energy dense diets
Vegetarian diets tend to be low in dietary energy density (caloric content of food per gram weight;
kcal/gram). Lowering dietary energy density by increasing fruit and vegetable intake and/or
lowering fat intake has been demonstrated to be a key strategy by which successful weight loss
and weight loss maintenance occurs (Ello-Martin et al., 2007). Low energy density diets have
been reported in exercising women with menstrual disturbances as a dietary strategy to maintain
low energy intake (Reed et al., 2011). In this study, consumption of vegetables was greater in
exercising women with menstrual cycle disturbances when compared to exercising, ovulatory
control subjects. Additionally, when compared to exercising ovulatory control subjects, fasting
PYY concentrations, a potential biomarker of satiety, were higher in women with menstrual
cycle disturbances indicating that satiety may be enhanced in exercising women with menstrual
disturbances that consume a diet low in energy density (Figure 13.4). Interestingly, subjects in
this study had high intake of low- or no-calorie condiments which may have been used to make
bland foods taste better without adding caloric value. Additional studies are required to confirm
that lowering dietary energy density is associated with menstrual disturbances.
13.5.8 Aberrant eating behavior phenotypes: dietary restraint and drive for thinness
Though dieting tends to be an initial avenue through which disordered eating manifests,
environmental and social cues such as pressure from coaches or teammates to maintain low
body weight, low self-esteem, and negative eating attitudes like high drive for thinness, dietary
restraint or body dissatisfaction may also contribute to, or exacerbate disordered eating (Nattiv et
al., 2007). Surveys like the Three-Factor Eating Questionnaire and the Eating Disorder Inventory
are utilized to screen for such behaviors. One study (Lluch et al., 2000) demonstrated that
restraint influenced relative energy intake (energy intake after accounting for exercise energy
expenditure) subsequent to acute exercise in normal weight, exercising women. The study
demonstrated a greater decrease in relative energy intake from rest to exercise in restrained
compared to unrestrained women; suggesting that eating behavior may be one factor involved
in the modulation of energy intake subsequent to acute exercise. There is also evidence that
biological factors and genetics may predispose an athlete to disordered eating (Nattiv et al., 2007).
For example, reduced satiety and suppressed secretion of cholecystokinin, a satiety hormone,
has been reported in patients with BN and may be one factor contributing to the uncontrollable
drive of BN patients to binge. It has been suggested that the etiology of eating disorders may be
a b
1.6 100 *
1.4 OV
EAMD 80
1.2
Energy density (kcal/gram)
1.0 * 60
PYY (pg/ml)
0.8
0.6 40
0.4
20
0.2
0.0 0
All food and All food and only
all beverages caloric beverages
Figure 13.4. Bar graphs comparing the (a) dietary energy density and (b) fasting total peptide YY (PYY)
between ovulatory (OV) control and exercise-associated menstrual disturbances (EAMD) subjects (Reed et al.,
2011; reprinted with permission). Data are reported as mean standard deviation; * P<0.05.
genetically linked due to higher prevalence rates of AN and BN in fraternal twins and first degree
relatives of those who have AN. It is interesting to note that aberrant eating behaviors typically
associated with disordered eating and eating disorders could potentially be utilized as surrogate
markers indicating energy deficiency and consequent reproductive dysfunction. High drive for
DT has been associated with suppressed RMR, an indication of energy conservation, as well
as oligomenorrhea and amenorrhea (Gibbs et al., 2011). Additionally, one study demonstrated
that women with high dietary restraint exercised more, were more likely to be vegetarian, have
a history of eating disorders, were more likely to be actively trying to lose weight and reported
irregular menstrual cycles (McLean and Barr, 2003). Thus, indices of disordered eating, like high
dietary restraint or high DT, may be an indication of energy restriction and could potentially
be easily assessed in exercising women to detect or prevent poor dietary habits that may lead to
energy deficiency and consequently reproductive dysfunction.
13.5.9 Inadvertent under eating
Inadvertent under eating may also be an avenue through which exercising women exhibit
low energy availability. It has been suggested that some exercising women may be unable to
compensate for high volumes of exercise energy expenditure and inadvertently fail to increase
energy intake to match energy expended through exercise. Exercising women with menstrual
disturbances exhibit elevated circulating concentrations of ghrelin (De Souza et al., 2004), the
only hormone known to stimulate hunger and increased food intake. Increases in ghrelin are
thought to be part of a homeostatic feedback mechanism involved in increasing body weight
back to a predetermined set point. As well, increases in circulating ghrelin concentrations may
be signaling to the hypothalamus to suppress reproduction by way of suppressed LH secretion
and pulsatility (Evans and Anderson, 2012). However, elevated circulating concentrations of PYY,
a satiety hormone that has been shown to be elevated in exercising women with amenorrhea
(Scheid et al., 2009), may be opposing the impact of increased ghrelin on hunger and food intake
and promoting chronically low energy intake.
In addition to chronic changes in hormone profiles, recent research suggests that changes in
appetite and GI hormone responses induced by acute exercise may contribute to a suppressive
effect of exercise on appetite. Acute bouts of exercise may suppress appetite and lead to a non-
compensatory response in relative energy intake. The mechanism through which exercise
suppresses energy intake is not well understood, but may potentially be one avenue through
which exercising women inadvertently restrict energy intake. Recent literature also suggests that
GI hormones involved in appetite regulation and energy balance exhibit an anorexigenic hormone
profile during and subsequent to exercise. Suppressed concentrations of acylated ghrelin have
been observed during aerobic exercise where after ad libitum energy intake was not different from
a no exercise condition and thus, individuals did not compensate for the energy deficit induced
by exercise (King et al., 2010). Exercise-induced suppression of acylated ghrelin appears to be
transient; however, the increases elicited in the satiety hormones PYY and glucagon-like peptide
I may be sustained through the post-exercise period (Ueda et al., 2009). Thus, the prolonged
participation of women in endurance exercise may promote a cumulative anorexigenic hormone
profile, despite chronic elevations in ghrelin, and aid in the chronic suppression of energy intake
in these women.
Aberrant eating behavior phenotypes and alterations in GI hormone profiles are potential
contributing factors to chronic energy restriction observed in exercising women. However,
integration of these signals occurs through the brain where a complex network of internal (i.e.
GI hormones) and external (i.e. smell, taste, etc.) influences may modulate homeostatic and
hedonic responses to food. The hypothalamus is a central region in the brain involved in the
homeostatic regulation of energy balance. Neuroimaging studies have demonstrated that hunger
stimulates, whereas satiety inhibits, activation of the hypothalamus. The insula, in particular, is
one brain region involved in food reward responses that plays a role in connecting the neural
networks of the hypothalamus to other brain areas signaling hunger and may respond to acute
exercise more so than other food reward brain regions (Cornier et al., 2012). Recent research
has focused on these brain regions to begin to determine how aberrant patterns of food reward,
hunger and satiety signals may differ among obese and lean individuals; however, little is
known about the effect of acute bouts of exercise on food reward and associated brain regions.
Preliminary evidence utilizing fMRI suggests that exercise suppresses the activation of brain
regions associated with food reward. In response to acute exercise, Evero et al. (2012) observed
reduced neuronal responses in the insula and orbitofrontal cortex which is consistent with
reduced pleasure of food, incentive motivation to eat and anticipation and consumption of food.
Further investigation of the suppressive effect of exercise on brain regions associated with food
reward is needed, particularly in exercising women that may be at risk for low energy availability
and thus menstrual disturbances.
13.5.10 Meal timing and meal related gut peptides responses as energy signals
Lastly, it is interesting to note that ghrelin, as well as several other GI hormones involved in
hunger and satiety responses, not only respond to chronic changes in energy status, but also to
decreases in acute energy intake. Higher concentrations of PYY are associated with caloric and
macronutrient content of food as well as meal timing across the day (Hill et al., 2011). Alternatively,
ghrelin has been shown to be negatively associated with similar meal-related parameters (Leidy
and Williams, 2006). As well, ghrelin and PYY oppose the actions of one another on the GI tract
and recent data from our lab shows that they may be reciprocally involved in the modulation
of one another in the circulation (Figure 13.5). It is interesting to posit that these short term GI
signals that respond to more subtle, daily fluctuations in meal timing and caloric quantity of
food eaten across a single day may be important modulators of menstrual function. In support
of this hypothesis, recent data from our lab has shown that in female collegiate soccer players has
demonstrated that energy availability may fluctuate across the day depending on energy content
of specific meals (Figure 13.6). Over time, signaling from day-to-day energy deficits created
by long periods of low caloric intake throughout the day may accumulate and lead to chronic
reproductive suppression. Consequently, future studies could potentially focus on whether meal
timing and caloric content regimens that maintain optimal energy availability on a daily basis
would be associated with menstrual function.
Coefficient = 2.14 PYY Ghrelin

120 2,100
P-value = 0.04
2,000
110
1,900
100 1,800
Peptite YY (pg/ml)
1,700
Ghrelin (pg/ml)
90
1,600
80 1,500
1,400
70
1,300
60 1,200
0 Breakfast Lunch Dinner Snack 0
08:00 10:00 12:00 14:00 16:00 18:00 20:00 22:00 24:00 02:00 04:00 06:00 08:00
Time (hours)
Figure 13.5. 24 h composite profile illustrating an inverse association of total peptide YY and ghrelin. Data are
expressed as mean SEM; P<0.05.
1,200
1000
Energy intake (kcal/d)
800
*
600
* <30 kcal/kg LBM
400 30 kcal/kg LBM
200
n=10 n=8 n=10 n=9 n=10 n=9
0
Breakfast Lunch Dinner
Meal occasions
Figure 13.6. Energy intake at meals during pre-season of Division I female soccer players. Data are expressed
as mean SEM. * P<0.05, LBM: lean body mass
13.6 Conclusions
In conclusion, menstrual cycle disturbances in exercising women have been largely attributed
to a chronic energy deficit and thus, regardless of the avenue through which exercising women
restrict energy intake, it is likely that any avenue that will induce an energy deficit may suppress
reproductive function. Studies suggest that the mechanism through which chronic energy deficits
suppress reproduction may be through altered metabolic hormone signaling. In particular,
ghrelin may be involved in the suppression of LH secretion and pulsatility; however, further
investigation as to this exact mechanism is warranted. It may be advised that exercising women
take care in planning meals across the day so that caloric and macronutrient content of meals as
well as meal timing are optimal to sustain a healthy energy availability and prevent induction of
a chronic energy deficit that may lead to reproductive suppression.
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14. Iodine deficiency: female menstrual cycle to
conception
Department of Clinical and Experimental Medicine and Pharmacology, University of
Messina, University Hospital, Pad. H, A.O.U. Policlinico G. Martino, 98100 Messina, Italy;
francesco.vermiglio@unime.it
Abstract
Iodine is an inorganic substrate essential for thyroid hormone synthesis. Thyroid hormone, in
turn, is essential for a proper gonad maturation and gametogenesis in mammals of both sex.
Two billion people in the general population have been estimated to be exposed to insufficient
iodine intake. When iodine intake is inadequate thyroid hormone production can be deeply
affected, resulting in various degrees of thyroid failure ranging from isolated hypothyroxinemia
to the more severe condition of either subclinical or overt hypothyroidism. Only a few studies
specifically focused on the relationship between iodine deficiency and reproduction. Indeed,
the vast majority of the investigations were aimed at evaluating the relationship between
hypothyroidism (not necessarily iodine-deficiency related) and reproduction. This chapter is
aimed at reviewing the effects of iodine deficiency/hypothyroidism on female reproductive life up
to conception. During the fetal life, the development of male and female reproductive tracts does
not seem to be influenced by thyroid hormones, which conversely seem to play a crucial role later
on, in sexual maturation and gonad development. Hypothyroidism can result in a marked delay
in sexual development in males and, conversely, in precocious puberty, anticipation of pubertal
development set point, menstrual disturbances and reduced fertility in females.
Keywords: iodine deficiency, hypothyroidism, menstrual disturbances, precocious puberty,

thyroid hormones, fertility, sexual maturation
223
Summary points
Iodine is an inorganic substrate essential for thyroid hormone synthesis.
Thyroid hormones are essential for a proper gonad maturation and gametogenesis in mammals of both sex.
When dietary iodine intake is inadequate various degrees of thyroid failure, ranging from isolated
hypothyroxinemia to subclinical or overt hypothyroidism, can occur.
On a worldwide basis, iodine deficiency (ID) is the most important cause of thyroid insufficiency
As a consequence of ID-related thyroid insufficiency, reproduction can be negatively affected.
During the fetal life, the development of male and female reproductive tracts does not seem to be influenced
by thyroid hormones, which conversely seem to play a crucial role later on, in sexual maturation and gonad
development.
Hypothyroidism can result in a marked delay in sexual development in males and, conversely, in precocious
puberty, menstrual disturbances and reduced fertility in females.
Prepubertal hypothyroidism results in disturbed folliculogenesis.
Hypothyroidism-induced infertility, due to the failure of follicular development and pregnancy, can be reverted
by thyroxine treatment.
Severe hypothyroidism can lead to precocious puberty in females, probably due to the spill-over specificity
of increased levels of thyroid stimulating hormone on follicle stimulating hormone receptors.
Pubertal development set point is anticipated in mildly ID-related hypothyroid girls.
Iodine implementation leads to the correction of ID-related mild hypothyroidism, thus delaying the pubertal
set point back to normal.
Reproductive failure in severely ID areas is significantly higher than in mild ID areas.
A dramatic increase in fertility is observed in severely ID areas after iodine prophylaxis was started.
14. Iodine deficiency: female menstrual cycle to conception
Abbreviations
aVWD Acquired type 1 von Willebrands disease
aVWF:Ag Von Willebrand factor antigen
cAMP Cyclic adenosine monophosphate
EPL Early pregnancy loss
FVIII:C Factor VIII activity
hFSH-R Human FSH receptor
ID Iodine deficiency
LT4 Levo-thyroxine
MCRs Metabolic clearance rates
PRL Prolactin
PTU Propylthiouracile
rhTSH Recombinant human TSH
SDS Standard deviation score
T3 Triiodothyronine
T4 Thyroxine
TH Thyroid hormone
TRH Thyrotropin releasing hormone
TSH Thyroid stimulating hormone
UIC Urinary iodine concentration
14.1 Introduction
Iodine is an inorganic substrate essential for TH synthesis. ID is a global public health problem,
and estimates of the extent of the problem were last updated in 2007. Two billion people in the
general population have been estimated to be exposed to insufficient iodine intake, so that this
debilitating health issue affects almost one in three individuals globally. ID is still considered
to be a public health in 47 countries and both children and child-bearing age women represent
subset populations particularly exposed to the most severe consequences of ID. More than 250
million children (30% of school age children population in the world) have insufficient iodine
intake, while, unfortunately, no sufficient data are at present available in both pregnant and fertile
women (De Benoist et al., 2008).
TH is essential for a proper gonad maturation and gametogenesis in mammals of both sex. When
iodine intake is inadequate TH production can be deeply affected, resulting in various degrees
of thyroid failure ranging from isolated hypothyroxinemia to the more severe condition of either
subclinical or overt hypothyroidism. During the fetal life, the development of male and female
reproductive tracts does not seem to be influenced by THs, which conversely seem to play a
crucial role later on, in sexual maturation and gonad development. Hypothyroidism can result in
a marked delay in sexual development in males and, conversely, in precocious puberty, menstrual
disturbances and reduced fertility in females (Jannini et al., 1995, Longcope et al., 1996, Zhang
et al., 1997).
So far, only a few studies specifically focused on the relationship between ID and reproduction.
Indeed, the vast majority of the investigations were aimed at evaluating the relationship between
hypothyroidism (not necessarily ID related) and reproduction. As a consequence this chapter
will be aimed at reviewing the effects of ID/hypothyroidism on female reproductive life up to
conception. The importance of THs in contributing to the stability of the feto-placental unit and
in ensuring normal neurological development in the fetus, once pregnancy has occurred, will not
be discussed, being beyond the scope of this review.
14.2 Effects of hypothyroidism on the female reproductive system

The effects of hypothyroidism on the female reproductive system (Table 14.1) are largely
dependent on both severity and timing of its occurrence. During gestation, ID can result in
various degrees of both maternal and fetal thyroid failure. The most important and investigated
consequences of ID-related maternal and fetal thyroid failure deals with the effects of TH on fetal
brain development.
Concerning the specific effect of hypothyroidism on female reproductive system, the presence of
small ovaries, in which cholesterol and lipid content was reduced, was described many years ago
and attributed to fetal hypothyroidism (Leathem et al., 1959). Apart from this isolated report,
however, the idea that TH is not essential during the fetal development of female reproductive
tract is, today, widely accepted.
Few data are so far available on the effects of hypothyroidism in prepubertal age. The only report
that addressed this specific point demonstrated that prepubertal hypothyroidism resulted in
disturbed folliculogenesis. In this study, rats were rendered hypothyroid by giving them PTU via
the drinking water from birth up to day 40 of extrauterine life. The PTU-induced hypothyroidism
Table 14.1. Effects of hypothyroidism on the female reproductive system at different life periods.
Life period Effect
Fetal life No effect

Prepubertal age Disturbed folliculogenesis
Postpubertal age Reduction in the ovulation rate
hampered the differentiation of granulosa but not its proliferative activity (Dijkstra et al., 1996).
In later stages of development, hypothyroidism has been proved to reduce the ovulation rate in
mature hypothyroid rats (Mattheij et al., 1995). In mature adult female rats, hypothyroidism
resulted in decreased concentrations of plasma LH on the day of diestrus and proestrus, whereas
the plasma concentrations of PRL and progesterone increased as compared with euthyroid rats.
These changes clearly indicate that hypothyroidism causes gonadal disturbances in adult female
rats, and that disruption of the estrous cycle would be the result of the increased concentrations
of plasma progesterone due to the hypersecretion of PRL during the day of proestrus and estrus
(Tohei et al., 1998). Infertility often occurs in mature female hypothyroid mice. These animals
show continuous dioestrus, and ovulate significantly fewer eggs after gonadotropin treatment.
Moreover, they fail to establish pregnancy after mating compared with mature control mice
and have significantly fewer corpora lutea >500 micron in diameter and significantly lower
progesterone concentrations. This hypothyroidism-induced infertility, due to the failure of
follicular development and pregnancy, was reversible by T4 treatment before mating (Jiang et
al., 2011).
So far, only few animal studies addressed the relationship between ID and fertility, even though
damaged reproduction has been recognized as a severe consequence of ID. In 2003, Ferri et al.
studied the effects of iodine supplementation on ovine fertility. The UIC were measured in animals
from 11 breeding farms. UIC in sheep from 8/11 farms analyzed were borderline (100-150 g/l)
or very low (50 g/l). Only in 3 out of 11 farms, sheep UIC was considered adequate (300 g/l).
Both serum TH, total T3 and total T4, were significantly higher in animals with adequate UIC. 32
ewes and 20 rams with a low UIC (78.26.3 g/l) and low TH (T4 39.82.4 ng/ml; T3 0.300.06
ng/ml) were then randomly divided into two groups. One group (16 ewes and 10 rams) received a
sc injection of 1 ml of Lipiodol, containing 480 mg of iodine; the remaining animals were used as
control. The injection of Lipiodol was able to achieve a UIC >300 g/l and a significant (P<0.01)
increase in serum T4 and T3 levels in treated animals. Fertility of both control and treated
animals, ultrasonographically assessed 9 months after iodine supplementation, revealed that
100% of treated ewes mated with treated rams were pregnant, while only 37% of the control ewes
mated with control rams were pregnant. In the lack of histological differences between the ovaries
of the control and treated ewes, the ID-induced hypothyroidism was advocated by the authors
as the main cause of infertility. Indeed, low THs can lead to alterations of endometrial tissue
development and differentiation, which, in turn, may affect gametes survival, fertilization, and
the embryo implantation process. In this view, the presence of TH receptors in the endometrium
further supports this hypothesis (Ferri et al., 2003).
Overall, the detrimental effects of hypothyroidism observed in animal studies seems to affect
more frequently the sexually mature than the immature animals.
14.3 Effects of hypothyroidism on sexual hormones

THs play a role in the modulation of the LH and FSH-mediated control of granulosa cell function.
Experimental data show that THs may exert both a stimulatory and an inhibitory effect on
mammalian granulosa cell gonadotropin-induced steroidogenesis. This ambivalence is probably
due to a variable responsiveness to T3 of granulosa cells isolated from follicles at different stages
of antral development, with small and medium follicles displaying a higher number of T3 binding
sites than large antral follicles. In addition, data from clinical studies demonstrated that TH
replacement therapy increases the success rate of ovulation induced by clomiphene citrate in
women with subclinical hypothyroidism (Raber et al., 2003).
The MCRs of androstenedione and estrone are reduced in hypothyroid women, probably due to
a decrease in hepatic blood flow (Table 14.2). Also, an increase in peripheral aromatization of
androstenedione to estrone has been reported in hypothyroid women. Aromatase catalyzes the
last steps of estrogen biosynthesis from androgens, specifically by transforming androstenedione
to estrone and testosterone to estradiol. The peripheral aromatization of androgens is a major
source of estrogens in men and postmenopausal women but is a less important source of estrogens
in normal reproductive-aged women. The decrease in hepatic blood flow responsible for the
reduced MCRs of androstenedione and estrone in hypothyroid women may also explain the
increase in peripheral aromatization. Indeed, the reduced hepatic blood flow might facilitate the
aromatization from androstenedione to estrone and/or furnish a greater availability of substrate
androstenedione to be aromatized to estrone by liver and other peripheral tissues. Furthermore,
there is evidence that euthyroidism restoration is associated with a decrease in peripheral
aromatization of androgens (Longcope et al., 1990).
The hepatic biosynthetic activity of sexual hormone binding globulin is reduced in hypothyroid
women. As a consequence, both total estradiol and testosterone are reduced and their free fractions
increased. Gonadotropins are usually normal in hypothyroid women but the response of LH
to GnRH stimulation is often delayed or blunted. Hypothyroidism activates the hypothalamic-
Table 14.2. Changes in sexual hormones of hypothyroid women.
Metabolic clearance rates of androstenedione reduced

Metabolic clearance rates of estrone reduced
Peripheral aromatization of androstenedione increased
Peripheral aromatization of estrone increased
Sexual hormone binding protein reduced
Total estradiol and testosterone reduced
Estradiol and testosterone free fractions increased
Gonadotropins unchanged
Luteinizing hormone response to gonadotrophin releasing hormone delayed/blunted
pituitary-thyroid negative feed-back, which results in TRH and TSH increase. As a consequence
of the hypothalamic TRH stimulation on the pituitary lactotroph cells, PRL secretion increases
and galattorrea appears. All these changes are reverted to normal when euthyroidism is restored
(Krassas et al., 2010).
Overall, hypothyroidism may, even at an early stage, have an important impact on conception,
while, once pregnancy has occurred, THs contribute to the stability of the feto-placental unit,
protecting from early loss of the conceptus.
14.4 Changes in pubertal set point in hypothyroid girls

14.4.1 Anticipation of pubertal development set point in ID-related mild hypothyroidism
The long-term effects of iodine repletion on growth and pubertal development in school-age
children were recently reported by Markou et al. (2008). The study was carried out in a large cohort
of 293,000 children, aged 6-17 years from 7 mountainous regions of Caucasus with moderate to
severe ID. All these children received 190 mg of iodized oil by the oral route, the same treatment
being repeated twice over the next 6 months. Two representative groups were then studied: the
iodine negative group (Group I-neg), including 364 children aged 8-14 years, who were examined
1 year before the administration of iodide, and the iodine treated group (Group I-Rx) including
295 children aged 6-16 years, who were examined 4 and 5 years following the last dose of iodine.
Children in the iodine treated group had a significantly greater SDS for height than children
from the iodine negative group, while no differences were found in SDS for weight between the
two groups. The rate of pubertal development in both sexes was evaluated on the basis of the
mean age at which each stage of breast and pubic hair development was achieved. In detail, mean
age of pubic hair development, according to Tanner stages, was significantly shifted to an older
age after an overall dose of 570 mg of iodized oil in males at Tanner stages III, IV and V, and in
females at Tanner stages II, IV and V. Markou et al. (2008) concluded that interventions of iodine
implementation leads to the correction of ID-related mild hypothyroidism, thus delaying the
pubertal set point back to normal.
14.4.2 Precocious puberty in severe hypothyroid females
Hypothyroidism may affect the reproductive system during childhood and adolescence. Severe
hypothyroidism may cause premature puberty in females (Table 14.3).
In 1905, Kendle reported for the first time a case of precocious puberty in a 9 year old girl affected
with endemic myxedematous cretinism. In the meticulous description of this case, menarche was
reported to have started when the girl was 5 years old, and her secondary sexual characters fully
developed. Most importantly, sexual development almost reverted to a prepubertal condition when
thyroid extracts were started (Kendle, 1905). A century later, Anasti et al. (1995) provided us with
a possible explanation on the occurrence of precocious puberty in severely hypothyroid children.
Table 14.3. Changes in pubertal set point in iodine deficiency-related mildly and severely hypothyroid women.
Iodine deficiency-related hypothyroid women Changes in pubertal set point
Mildly hypothyroid women Anticipation of pubertal development set point

Severely hypothyroid women Precocious puberty
The authors hypothesized that elevated levels of TSH could interact directly with the hFSH-R
leading to a specific form of isosexual precocity characterized by breast development, uterine
bleeding, and multicystic ovaries in girls and macroorchidism without excessive virilization in
boys. The possibility that gonadal stimulation recognizes causes other than overproduction of
gonadotropins arises from the observation that girls with severe hypothyroidism and precocious
puberty show prepubertal levels of gonadotropins. At variance with other forms of precocious
puberty, the specific features of precocious puberty in severe hypothyroid girls are reversible upon
treatment with TH. The direct role of TSH in the stimulation of the hFSH-R was very elegantly
proven by Anasti et al. (1995), using rhTSH and transfecting hFSH-R complementary DNA in
COS-7 cells. To assess whether the cAMP response to rhTSH was mediated by hFSH-R, they
examined the extracellular cAMP accumulation in COS-7 cell transfected with hFSH-R, and
could demonstrate that the cAMP response to the stimulation of the combination of recombinant
human FSH and rhTSH was 50% lower than that elicited by FSH alone. Therefore, rhTSH would
act as an inhibitor of hFSH at the hFSH-R, thus suggesting that rhTSH and hFSH would stimulate
the same hFSH-receptor (Anasti et al., 1995).
14.5 Menstrual irregularities in hypothyroid women

Hypothyroidism may be responsible for changes in cycle length and bleeding features. In the 50s
and 60s, the early epidemiological studies indicated that menorrhagia and metrorrhagia (alone
or in combination) were the most common cycle disorders observed in hypothyroid women.
Although less frequently, polymenorrhea and amenorrhea were also reported (Krassas et al.,
2010) (Table 14.4).
In 1989, Wilansky et al. evaluated the functional status of the thyroid gland in 67 apparently
euthyroid menorrhagic women by a TRH-test. 15/67 (23.4%) women had been diagnosed
with mild primary hypothyroidism on the basis of their basal TSH and T4 values, which were
nevertheless within the normal range. All these 15 patients showed an exaggerated response of
serum TSH and T4 to administration of TRH. Menorrhagia disappeared in all of them within
3-6 months after LT4 treatment was started and did not relapse thereafter (Wilansky et al., 1989).
In 1999, menstrual history was investigated in 171 premenopausal women, 6 months before the
diagnosis of hypothyroidism, and in 214 controls. Overall, 23.4% of the hypothyroid vs. 8.4%
Table 14.4. Menstrual disorders in hypothyroid women.
Menstrual disturbances Occurrence
Oligomenorrhea Frequent
Menorrhagia Frequent
Metrorrhagia Frequent
Polymenorrhea Rare
Amenorrhea Rare
of the euthyroid women complained irregular periods. This prevalence was significantly lower
than that (56%) previously reported (Redmond, 2004). In detail, the prevalence of menstrual
irregularities was 19.6%, 23.8% and 25.3% in subclinical, mild and severe hypothyroid women,
respectively. No relationship was found between menstrual disturbances and anti-thyroid
antibody positivity. Oligomenorrhoea and menorrhagia were the most common menstrual
disorders reported (Krassas et al., 1999).
The general trend towards an increased occurrence of menstrual disorders in hypothyroid

women, which resulted in an excessive bleeding (menorrhagia and metrorrhagia), could also
be attributed to a bleeding diathesis due to an acquired type 1 aVWD, mainly characterized by
decreased FVIII:C and aVWF:Ag levels. The pathogenesis of hypothyroidism-associated aVWD
is still unclear. A decrease in aVWF protein synthesis or a decreased response to adrenergic
stimulation (enhancing the aVWF release from endothelial cells) due to TH deficiency is
the most plausible mechanism involved, as also suggested by the finding of a reversal of the
hypothyroidism-associated aVWD, following TH replacement.
Coagulation/fibrinolytic abnormalities have been reported in hypothyroidism and attributed to a

significant reduction in coagulation factors VIII, IX, and XI activities, and lower plasma levels of
coagulation factors VII, X, and XII have been also reported in hypothyroid patients. (Franchini
et al., 2010).
14.6 Iodine deficiency and fertility

In 1996-1997 an epidemiological survey on ID disorders was carried out in Casamance and Senegal
Oriental, two severely iodine deficient areas of Senegal. The study group included a population
of 4,980 women aged 10 to 50, 1,544 of whom were adolescent and 462 were pregnant. All the
women were examined for thyroid size and urinary iodine excretion. Their iodine nutrition was
then related to their fertility rate and reproductive failures. Repeated miscarriages and stillbirth
were associated with low iodine status, with severe ID increasing the risk. Poor nutritional status
and illiteracy had important repercussions in pregnancy outcome, mainly consisting in a four-
fold higher risk in underweight women, and an eight-fold higher risk of failed pregnancy in those
who were illiterate, compared with nutritionally healthy, literate women. In their conclusions the
Authors emphasized the importance of an adequate iodine nutrition, mainly devoted to young
and pregnant women, along with an improved general status of nutrition and education aimed
at preventing reproductive failures (Dillon et al., 2000).
An assessment of the endocrine status, caloric requirement, energy output, fertility, and ecologic
factors was carried out before and during iodine repletion by depot injection in a Central African
population in remote Congo (ex-Zaire) in order to investigate the prevalence, severity, causes,
and potential control of this disorder. Hypothyroidism and endemic cretinism, widely diffuse in
this region before iodine repletion, were eradicated over the years. The social and developmental
consequences observed within the iodine supplemented population were remarkable for an
increase in caloric requirement and a dramatic increase in fertility (Longombe et al., 1997).
A cross-sectional community based study was carried out in Ethiopia from February to May
2005 in 10,998 women aging 15-49 years. The study was aimed at assessing the severity of ID
and its relationship with poor pregnancy outcome. The study protocol included multistage cluster
sampling methods, and WHO/UNICEF/ICCIDD recommended method for goiter classification.
Overall, goiter prevalence was 35.8%. In 4 of the 10 Ethiopia regional states where the study was
carried out this prevalence was greater than 30%, so that they were classified as severely iodine
deficient. In the rest of the Ethiopia regional states, the severity of ID was mild to moderate.
Women with goiter experienced more pregnancy failure than non-goitrous women. Indeed, of
the 3,487 women with goiter, 16.7% had history of one or more reproductive failure in the form
of miscarriage and or stillbirth, whereas reproductive failures were reported in 13.8% of the 7,515
women without goiter. Thus, the presence of goiter was statistically associated with reproductive
failure. Similarly, reproductive failure in severely iodine deficient areas was significantly higher
than in mild iodine deficient areas. Indeed, among 9,489 women from severely iodine deficient
areas, 1,481 (16%) had stillbirth and/or miscarriages, while only 123/1,585 (7.8%) from mild
iodine deficient regions suffered from reproductive failure. In their conclusion the Authors
emphasized the need for a sustainable iodine intervention program, particularly targeted at
reproductive age women, based on Universal Salt Iodization and iodized oil capsule distribution
in some peripheries where ID was more severe (Abuye et al., 2007).
14.7 Hypothyroidism and fertility

The association between hypothyroidism and fertility was addressed in several studies, most of
which are retrospective and uncontrolled. Overt hypothyroidism is usually diagnosed and treated
before gestation. On the contrary, subclinical hypothyroidism can be frequently unrecognized
thus, at least partly, explaining the wide variability of the prevalence of this condition reported in
different studies (0.7-43%). In one of these studies, TRH-tests, along with other thyroid function
parameters, were performed in 185 infertile women with no clinical signs of thyroid dysfunction.
The test, carried out in the early follicular phase, revealed that 80/185 (43.2%) had TSH levels
>20 mU/l following TRH stimulation, and that overall and spontaneous pregnancy rates were
highest in women with normal basal and stimulated TSH, high T4 and low anti-thyroid antibody
titers (Gerhard et al., 1991). In 1994, the association between hypothyroidism and ovulatory
dysfunction was found in all the 3/444 (0.7%) infertile women whose thyroid function had
been retrospectively evaluated (Shalev et al., 1994). The association between hypothyroidism
and ovulatory dysfunction was also studied in 2001 in 30/149 (20.1%) infertile women who
had thyroid abnormalities. The women with functional thyroid abnormalities and ovulatory
dysfunction had a mean duration of infertility significantly longer than that of the control
group, while no relationship was found between infertility and thyroid autoantibodies (Grassi
et al., 2001). Also, the prevalence of increased serum TSH values was highest in the group with
ovulatory dysfunction in a series of 299 women (6.3% compared with 4.8% in the idiopathic and
2.6% in the tubal infertility group and none in the endometriosis group) (Arojoki et al., 2000). In
1993, Joshi et al. found that one of 16 (6.2%) overtly hypothyroid women suffered with primary
and secondary infertility and that this prevalence was similar to that found in normal control
women (Joshi et al., 1993). Similarly, a lower prevalence of hypothyroidism (2.3%) was found
in a large series of 704 infertile women. Also in this case, the prevalence of hypothyroidism was
comparable to that found in the general female population of childbearing age (Lincoln et al.,
1999). A prevalence even lower (0.5%) than that found in 100 control women (1%) was reported
in a series of 438 women with various causes of infertility. Even though median TSH levels
were higher in women with infertility than in controls, only two women, one with ovulatory
dysfunction and one with idiopathic infertility, were affected with subclinical hypothyroidism
(Poppe et al., 2002).
Another series of clinical studies evaluated the effectiveness of the LT4 treatment in restoring
fertility in hypothyroid women. In 1981, low dose of LT4 given to 11/20 hypothyroid women
proved effective in restoring progesterone levels, and two infertile women became pregnant
(Bohnet et al., 1981). In 2003, Raber et al. evaluated the 5 year follow-up of 223 women in whom
absolute causes of infertility were excluded. Infertile women were then divided into 4 groups.
The first group, including women affected with mild subclinical hypothyroidism (increased
basal levels of TSH in the presence of normal T4); the second group, composed of women who
exhibited an exaggerated TRH-stimulated TSH response, but in the presence of normal T4; the
third group, including euthyroid women with normal both basal and TRH-stimulated TSH
response and normal T4; and the forth group, composed of women with normal T4 and basal
TSH in whom TSH response to TRH-test was not evaluated. All the women belonging to the first
and second group were treated with LT4. Overall results indicated that conception rate was higher
(37%) than previously reported and independent of thyroid function prior to LT4 therapy, T4
dose or elevated thyroid autoantibodies. When the women treated with LT4 did not achieve basal
TSH <2.5 IU/l or TRH-stimulated TSH <20 mIU/l, a lower conception rate was observed. Overall
abortion rate was 9% and only first trimester miscarriages occurred. The authors concluded that
high pregnancy and parturition rates found in infertile treated women might be attributed to the
LT4 treatment (Raber et al., 2003). In 2007, Abalovich et al. (2007) investigated the prevalence of
both subclinical hypothyroidism and thyroid autoimmunity in infertile women. For the purposes
of their study, 244 women consulting on infertility and 155 healthy women with confirmed
fertility were recruited. Basal TSH and thyroid peroxidase antibodies were measured in all the
studied women; furthermore, in a subset of 71 women, TSH measurement was also obtained
after TRH stimulation. Women with thyroid autoimmunity were equally distributed in both
fertile and infertile groups. Subclinical hypothyroidism was found in 13.9% infertile and only in
3.9% fertile women. In the former, a pregnancy rate of 44.1% was achieved under levothyroxine
treatment. In their conclusions, the authors emphasized the need for systematic thyroid screening
in infertile women aimed at searching even mild forms of subclinical hypothyroidism (Abalovich
et al., 2007). In 2010, our research group studied retrospectively 216 apparently healthy pregnant
women (whose characteristics are shown in Table 14.5) with no previous history of thyroid
disease and with diagnosis of early miscarriage (before the 12th week of gestation) (Table 14.6).
Miscarriages were classified as very EPL or embryo loss (crown rump length 10 mm) and EPL or
fetal loss (crown rump length>10 mm). The women were subdivided into three groups according
to their thyroid functional status (euthyroid, n=126; subclinically hypothyroid, n=8), and the
presence of thyroid autoimmunity abnormalities but normal thyroid tests (n=24). We found that
both subclinical hypothyroidism and thyroid autoimmunity were independently associated with
very early embryo loss (Table 14.6 and Figure 14.1), but that women suffering from subclinical
hypothyroidism had a lower gestational age at abortion (De Vivo et al., 2010).
Finally, the importance of TSH in predicting poor IVF and as a consequence the importance of
THs in oocyte physiology has been demonstrated by Cramer et al. (2003). They demonstrated,
in a multivariate model, that TSH levels were significantly higher among women who produced
oocytes that failed to be fertilized and that this finding persisted after adjustment for several
covariates, including sperm motility. Also, among women who had at least one oocyte
inseminated, the likelihood that they would have fewer than 50% of their eggs fertilized was
significantly related to higher TSH levels (Cramer et al., 2003).
The bulk of these studies seem to indicate fertility to be deeply affected by hypothyroidism and
promptly restored by LT4 replacement.
Table 14.5. Characteristics of the study population (n=208) (De Vivo et al., 2010; reprinted with permission).
Euthyroidism Thyroid Subclinical P-value1

autoimmunity hypothyroidism
Maternal age (ys) 34.45.4 36.34.9 35.53.7 0.23

Nulliparous (%) 40.9 29.2 50 0.45
Previous miscarriage (%) 25.0 33.3 0 0.17
Gestational age at abortion (wk) 8.21.6 8.22.1 6.50.9 0.02
1 ANOVA.
Table 14.6. Correlation between very early pregnancy loss and considered variables (De Vivo et al., 2010;
reprinted with permission).
Odds ratio 95% confidence interval P-value1
Maternal age 1.05 0.49-2.22 0.9

Nulliparity 0.95 0.43-2.07 0.9
Previous miscarriage 1.0 0.41-2.38 1
Thyroid autoimmunity 3.33 1.28-8.62 0.013
Subclinical hypothyroidism 6.14 1.45-25.4 0.014
1 Logistic regression analysis.
90
ET TAI SH
80
70
60
Miscarriage (%)
50 0.004
40
30 0.002
20
10
0
VEPL EPL
Figure 14.1. Thyroid function in the two kinds of miscarriage (De Vivo et al., 2010; reprinted with permission).
ET = euthyroid; TAI = thyroid autoimmunity; SH = subclinical hypothyroidism; VEPL = very early pregnancy loss;
EPL=early pregnancy loss.
14.8 Conclusions
ID is still a major public health problem, which affects over 1.2 billion individuals worldwide.
On a worldwide basis, ID is the most common (and preventable) cause of thyroid insufficiency.
The extensive research carried out over the years, clearly show that THs play an important
role in regulating normal reproductive behavior and physiology. Until now, only few studies
were specifically aimed at assessing the effects of ID in the female reproductive system. Most
of the available information is indirectly derived from studies designed to evaluate the effects
of hypothyroidism (not necessarily related to ID) on female reproductive function. Even fewer
are the studies aimed at evaluating the potential effectiveness of specific interventions of iodine
prophylaxis in this specific context. Scientific research in this area should be encouraged, thus
contributing to further understanding the role of this trace element in the physiological regulation
of reproduction.
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15. The menstrual cycle and lipid levels
Epidemiology Branch, Eunice Kennedy Shriver National Institute of Child Health and Human
Development, National Institutes of Health, 6100 Executive Blvd., 7B03, Rockville, MD 20852,
USA; mumfords@mail.nih.gov
Abstract
Understanding the complex interplay between lipoprotein cholesterol, endogenous hormones,
and dietary fiber intake is essential for clinical management of women, as well as for the design
and interpretation of studies in reproductive-aged women. Herein, we review the evidence
regarding, first, intra-individual variation in lipoprotein cholesterol levels total, high density
lipoprotein (HDL-C) and low density lipoprotein (LDL-C) across the menstrual cycle; second,
the association between fiber intake and reproductive hormones; and third, the effect of fiber
intake on lipoprotein cholesterol mediated by estrogen. Among women of reproductive age,
the weight of evidence indicates significant fluctuations in lipoprotein cholesterol levels across
the menstrual cycle, with estrogen levels positively associated with HDL-C, and inversely
associated with total and LDL-C. Fiber consumption at or above the recommended intakes was
significantly associated with lower reproductive hormone concentrations and a higher probability
of anovulatory cycles. Moreover, the association between fiber and lipoprotein cholesterol
levels was mediated by estrogen, suggesting that high-fiber diets may have reduced effects in
premenopausal women. Both women and physicians should take menstrual cycle phase into
account when interpreting a womans cholesterol measurement. Cyclic variations in lipoprotein
cholesterol levels have important implications for the design and interpretation of studies among
reproductive-age women.
Keywords: cholesterol, lipoproteins, dietary fiber, estradiol, menstrual cycle
239
Summary points
Lipoprotein cholesterol levels were observed to vary across the menstrual cycle in response to changing
estrogen levels. As such, both women and physicians should take menstrual cycle phase into account when
interpreting a womans cholesterol measurement, or time measurement to specific cycle phases.
Based on the best available evidence, lipoprotein cholesterol varies in response to changing estrogen levels,
with total and low-density lipoprotein cholesterol to be highest during the follicular phase and to decline
during the luteal phase, and high-density lipoprotein cholesterol to be highest around ovulation.
Among premenopausal women, estradiol appears to have a rapid effect on increasing total and high-density
lipoprotein cholesterol and decreasing low-density lipoprotein cholesterol levels, as well as non-acute effects
on decreasing low-density lipoprotein cholesterol levels leading to decreases in total cholesterol levels.
High fiber diets are associated with decreased estradiol concentrations in fertile-aged women and an
increased risk for anovulatory cycles.
Estradiol mediates the association between fiber and lipoprotein cholesterol levels, suggesting that high-fiber
diets may have reduced effects on improving premenopausal womens lipid profiles.
Interplay between hormones, lipids, and dietary intake has important implications for the study and clinical
care of reproductive-aged women.
Abbreviations
BMI Body mass index
DRI Dietary reference intake
FSH Follicle-stimulating hormone
GnRH Gonadotropin-releasing hormone
HDL-C High-density lipoprotein cholesterol
HERS Heart and Estrogen/Progestin Replacement Study
HT Hormone therapy
IOM Institute of Medicine
LDL-C Low-density lipoprotein cholesterol
NCEP National Cholesterol Education Program
TC Total cholesterol
VLDL-C Very low density lipoprotein cholesterol
WHI Womens Health Initiative
15.1 Introduction
According to the NCEP guidelines, a LDL-C level <100 mg/dl is optimal, as levels 100 mg/dl put
an average adult at increased risk for CVD (Expert Panel, 2001). While CVD has been reported
to be the leading cause of death among women, the prevalence of CVD among women aged 20-
39 years is half that for men in the same age group (7.8% versus 15.9%) (Roger et al., 2012). The
CVD sex disparity narrows with increasing age, with women having a higher CVD prevalence
than men after age 80 (86.7% versus 80.1%), which has led researchers to consider estrogen as a
potential modifying factor in CVD risk. While current NCEP blood cholesterol guidelines are not
sex-specific, if the weight of evidence supports estrogen levels impacting cholesterol metabolism
in the body, future guidelines should be tailored separately for men and women.
During the early 1990s, based upon studies revealing an increased risk for CVD among
postmenopausal women compared to premenopausal women, HT was recommended to
postmenopausal women as a means of heart disease prevention (Stampfer and Colditz, 1991).
While the HERS (Hulley et al., 1998) and WHI (Rossouw et al., 2002) observed significantly
improved lipid profiles among women using exogenous estrogens, HERS found no decreased risk
while the WHI actually found an increased risk for total cardiovascular disease among women
taking estrogen plus progestin compared to women taking a placebo. Neither HERS nor WHI
demonstrate a direct relationship between lipid levels and CVD due to potential confounding by
age, and duration and indication of treatment. However, they do support the cardioprotective
properties of HT, and especially estrogen-only HT. Furthermore, while estrogen-only HT showed
the largest improvements in lipid levels among postmenopausal women (Rossouw et al., 2002),
there is also evidence to suggest that among premenopausal women, oral contraceptives may
improve the lipid profile (Burkman, 1993). While the effects of endogenous estrogens among
premenopausal women on the lipid profile have not been well studied, there are strong biological
mechanisms to support a beneficial role of estrogen in lipoprotein metabolism (Knopp et al.,
2006; LaRosa, 1990). Dietary fiber intake has also been consistently associated with decreased
levels of lipoprotein cholesterol, though some evidence suggests that pre- and postmenopausal
women respond differently to fiber intake (Ganji and Kuo, 2008; Vega-Lopez et al., 2001), and
that estradiol, the predominant estrogen form among non-pregnant premenopausal women,
may mediate fibers effect on lipoprotein cholesterol in premenopausal women (Mumford et al.,
2011b).
In this chapter, based on a review of the epidemiologic evidence and the results from a recent
large prospective study, we will explore the interplay between lipoprotein cholesterol, fiber intake,
and endogenous hormones including estradiol, LH, FSH, and progesterone (Figure 15.1). First,
we will describe variation in lipoprotein cholesterol levels across the menstrual cycle and evaluate
the association between endogenous estrogens and lipoprotein cholesterol levels. Second, we
will examine the association between fiber intake and reproductive hormone levels. Third, we
will examine whether the effect of fiber intake on lipoprotein cholesterol levels is mediated by
estrogen. These findings have important implications for not only the design and interpretation
of studies among women of reproductive age, but for clinical practice as well.
15.2 Estrogen and lipoprotein cholesterol

Exogenous estrogen has been shown to affect the lipid profile, leading to the hypothesis that
endogenous estrogen may have similar effects (Mumford et al., 2010). Understanding the impact
of both exogenous and endogenous estrogen on lipid levels is critical in the proper screening
and management of atherosclerosis, CVDs primary antecedent. Furthermore, the effects of
endogenous and exogenous estrogen may vary, and an evaluation of the role of endogenous
estrogen on lipoprotein metabolism may help to elucidate the role of estrogen in protecting
premenopausal women against CVD. A clear understanding of the intra-individual variability
of lipoprotein cholesterol across the menstrual cycle and the association between estradiol and
lipoprotein cholesterol has important clinical implications regarding the appropriate standard of
care for women undergoing cholesterol testing.
Fiber Estrogen LH/FSH/P Cholesterol
Figure 15.1. Interplay between fiber intake, reproductive hormones and lipoprotein cholesterol levels. FSH =
follicle-stimulating hormone; LH = luteinizing hormone, P = progesterone.
15.2.1 Epidemiologic evidence
Over the past several decades, numerous epidemiological studies have evaluated the role
of endogenous estrogens in lipoprotein metabolism, yielding conflicting results, with some
observing fluctuating plasma lipid levels throughout the menstrual cycle, while others finding a
lack of variation in lipid levels over the menstrual cycle (Mumford et al., 2011a). Limitations of
previous studies that contribute to these inconsistencies include small sample sizes, short follow-
up (i.e. usually for only a single menstrual cycle), heterogeneity in markers of ovulation, and/or
lack of a standardized method to time lipoprotein and hormone measurements. Most studies only
compared lipoprotein cholesterol levels between the follicular and luteal phases of the cycle, and
did not estimate associations between hormone levels and lipoproteins at multiple points during
the cycle. Many studies found that only certain component measures of lipoprotein cholesterol
(TC, HDL-C, LDL-C, or triglycerides) differ between cycle phases.
While studies have shown inconsistent findings, the weight of evidence does support the
hypothesis that lipoprotein cholesterol levels vary across the menstrual cycle. The most rigorous
studies show that TC and LDL-C are lower during the luteal phase, corresponding to the time of
the menstrual cycle when estrogen and progesterone levels are high compared with the follicular
phase. Furthermore, as demonstrated in studies with multiple measurements over the menstrual
cycle (Lamon-Fava et al., 1989; Mumford et al., 2010), HDL-C levels have been shown to be
typically highest during the late follicular and periovulatory phases, a finding that tended to not
be observed in studies that only compared measurements during the follicular and luteal phases
(De Leon et al., 1992; Jones et al., 1988; Kim and Kalkhoff, 1979; Schijf et al., 1993). The increases
in TC and LDL-C immediately prior to ovulation, and peak levels of HDL-C at ovulation, are
of great physiological importance as cholesterol, and VLDL-C in particular, is the precursor
for steroid synthesis. Among the studies reviewed, the mean changes in TC levels across the
menstrual cycle varied between 4 and 10%, LDL-C between 4 and 12.5% and HDL-C by 11%.
The mean within individual variability reported for TC ranged from 8 to 19% while triglyceride
levels did not cyclically vary.
Not only do lipoprotein cholesterol levels differ between the follicular and luteal phases, but they
have been shown to vary on a day-to-day basis throughout the cycle. In the largest study to date,
hormones and lipoprotein cholesterol were measured at up to eight visits per cycle, for up to
two cycles in a cohort of 259 healthy, regularly menstruating women (Mumford et al., 2010).
Collection of these fasting blood samples was timed to specific phases of the menstrual cycle using
fertility monitors. These multiple measurements enabled evaluation of the patterns of means and
the variability of lipoprotein cholesterol levels across the cycle. As shown in Figure 15.2, TC and
LDL-C follow a similar pattern across the menstrual cycle, with levels increasing rapidly after
menses, peaking during the follicular phase and then declining throughout the luteal phase (Figure
15.2a and 15.2b). The peak levels of TC and LDL-C were observed during the follicular phase
prior to the rise and peak of estrogen, with TC and LDL-C levels declining during the luteal phase,
corresponding to rising and peak concentrations of estrogen and progesterone. HDL-C levels
were highest around ovulation, corresponding to high levels of estrogen (Figure 15.2c), whereas
a b
168 140 102 140
166 120 120
Total cholesterol (mg/dl)
100
LDL cholesterol (mg/dl)

100 100
Estradiol (pg/ml)
164
Estradiol (pg/ml)
80 98 80
162
60 60
160 40 96 40
158 20 20
Total cholesterol Estrogen 94
LDL-C Estrogen
156 0 0
Menses Follicular Ovulation Luteal Menses Follicular Ovulation Luteal
c d
140 140
52 56 120
120
HDL cholesterol (mg/dl)
Trigylcerides (mg/dl)
100 55 100
Estradiol (pg/ml)
Estradiol (pg/ml)
51 80 80
54
60 60
50
40 53 40
49 20 52 20
HDL-C Estrogen TRY Estrogen
0 0
Menses Follicular Ovulation Luteal Menses Follicular Ovulation Luteal
Figure 15.2. Mean levels of (a) total cholesterol, (b) low-density lipoprotein cholesterol (LDL-C), (c) high-density
lipoprotein cholesterol (HDL-C), and (d) triglycerides (TRY) and estrogen levels across the menstrual cycle among
259 women enrolled in the BioCycle Study (adapted from Mumford et al., 2011a).
triglyceride levels varied without a consistent pattern across the cycle (Figure 15.2d). Minimum
variation in TC, LDL-C and triglycerides was observed during menses and around ovulation, with
HDL-C showing the most variability around ovulation (Mumford et al., 2011a).
Owing to the observed day-to-day changes in both the mean and variability of lipoprotein
cholesterol levels, studies have analyzed the association between estradiol and lipoprotein
cholesterol levels across the cycle (Mumford et al., 2011a). Positive correlations and associations
between HDL-C and estradiol levels have been observed. TC and LDL-C levels have been found
to be inversely associated with estradiol levels, although findings have not been statistically
significant in all studies. In the most recent study, endogenous estradiol was also positively
associated with TC and HDL-C, and inversely associated with LDL-C in acute effects models
(which consider hormones and lipoprotein cholesterol measured on the same day), and
significantly and inversely associated with TC and LDL-C levels during the cycle in persistent
effects models (which consider hormones measured the visit immediately prior to measurement
of lipoprotein cholesterol levels) (Mumford et al., 2010). These models took into account repeated
measurements across the cycle, levels of other circulating reproductive hormones and other
factors known to impact these associations, such as age and BMI. Further adjustment for physical
activity and dietary intake did not alter the results. These findings suggest that estradiol has a
rapid effect on increasing TC and HDL-C levels and decreasing LDL-C levels, as well as non-acute
effects on decreasing LDL-C, which lead to decreases in TC.
15.2.2 Biological rationale
There is strong biological evidence for fluctuations in lipoprotein cholesterol levels during the
menstrual cycle in response to fluctuating estrogen levels (Mumford et al., 2011a). Formation
rates of all lipoprotein fractions increase to some degree under the influence of estrogen, but
removal rates are variably increased or decreased (Knopp et al., 2006). Previous research supports
the hypothesis that improvements in the atherogenic nature of the plasma lipid profile in response
to endogenous or exogenous estrogen consist of increasing VLDL synthesis, which subsequently
increases HDL-C and decreases LDL-C. These changes depend upon a sophisticated interaction
of many components. The upregulation of the LDL receptors increases clearance of LDL-C, while
upregulation of the ATP-binding cassette transporter and apoA-I increases HDL synthesis, and
the suppression of hepatic class B scavenger receptors expression leads to decreased hepatic
selective cholesterol uptake from HDL and further effects on LDL (Zannis et al., 2006). The
evidence reviewed supports these findings in that endogenous estrogen improves the lipid
profile by elevating HDL levels and lowering LDL levels. An alternative hypothesis is that fluid
retention and hemodilution during the luteal phase of the cycle induced by rising progesterone
levels accounts for a portion of the reduction in lipoprotein cholesterol levels during the luteal
phase. However, previous studies show that the cyclic changes in lipoprotein cholesterol levels
are greater than the accompanying changes in plasma volume (Adlercreutz and Tallqvist, 1959;
Cullinane et al., 1995; Pahwa et al., 1998).
15.2.3 Clinical implications
Taken together, the menstrual cycle phase should be taken into account when evaluating
lipoprotein cholesterol levels among reproductive-aged women. Although on average, Mumford
et al. (2010) found only a 5-8% change in mean levels by cycle phase, these differences have
potential clinical implications for women of reproductive age. Women crossed clinical boundaries
of acceptable lipoprotein cholesterol levels when tested at different phases of the menstrual cycle.
Specifically, fewer women were classified as having high cholesterol when measured during the
luteal phase compared with the follicular phase (TC: 7.9 vs. 14.3%; LDL-C: 10.5 vs. 17.8%). Based
on these results, the mid-follicular phase may be the best phase for measurement to reduce false
negatives, if we assume that management of a womans cholesterol should be based on a level
outside the NCEP guidelines at any point during the cycle. While treatment decisions regarding
the lipid profile may still require repeated samples above the recommended level, using a standard
method to time lipid measurements may improve the interpretability of results and consequently
reduce the overall number of tests. Notably, the observed changes found by Mumford et al. (2010)
occurred among healthy women. It is possible that variability in lipoprotein cholesterol levels over
the course of the menstrual cycle could be even greater among other groups of women. Testing
during menses is recommended to facilitate consistent comparisons due to reduced variability
during this time and because this menstrual cycle phase can be more reliably identified than
others. Implementation of uniform timing of cholesterol testing in reproductive-aged women
would improve interpretation in clinical settings as well as future studies. These findings are
important in that they show that the standard of care based on men are not necessarily appropriate
for women, and that women need to be studied directly. Thus, considering menstrual cycle phase
in the development of clinical guidelines for reproductive-aged women could improve the current
standard of care.
15.3 Fiber, reproductive hormones and ovulatory function

Increased intake of fiber has been promoted due to fibers favorable association with certain
health outcomes (Anderson et al., 2009). High-fiber diets have been associated with reduced risks
of cardiovascular disease, stroke, diabetes, colon cancer, and breast cancer. In particular, evidence
from randomized controlled trials, observational studies, and animal models demonstrates that
dietary fiber lowers levels of TC and LDL-C (Anderson et al., 2009; Brown et al., 1999), which are
common risk factors for cardiovascular disease. Current recommendations from the American
Heart Association, the US Department of Agriculture, and the Institute of Medicine (IOM)
suggest that individuals should consume 20-35 g fiber/d depending on caloric intake (Krauss et
al., 2000; US DHHS and USDA, 2005). This is in contrast to the average fiber intake in the United
States, which is substantially below these recommendations (13.8 g fiber/d for reproductive-aged
women) (Trumbo et al., 2002). Several studies have reported inverse associations between fiber
intake and estrogen concentrations in older women (Gaskins et al., 2009), presumably because
of a decrease of b-glucuronidase activity in feces that result from high fiber consumption and
leads to a decreased reabsorption of estrogen in the colon. Although certain beneficial effects
of fiber on chronic diseases have been observed, the effect of intake on endogenous hormones
and anovulation in younger women has had limited study. The influence of fiber intake on
reproductive hormone concentrations and anovulation is of particular interest in reproductive-
aged women, given the effect of these hormones on conception and pregnancy maintenance.
Similar to inconsistent findings in the variability of lipoprotein cholesterol across the menstrual
cycle, the association between fiber intake and reproductive hormone concentrations has had
inconsistent findings. In regards to estradiol, some have found a statistically significant inverse
association while others have observed no significant effect (Gaskins et al., 2009). Only three
studies have evaluated fibers association with LH (Gaskins et al., 2012), with only one observing
a significant inverse association (Gaskins et al., 2009). Past research has been limited by small
sample sizes and few measurements, using non-standard methods to time serum collection, and
low ranges of fiber consumption.
The most rigorous prospective study, having multiple measurements of hormones and diet
over 2 menstrual cycles (using a standardized method to time menstrual cycle phase), found
that fiber consumption at or above the recommended intakes was significantly associated with
decreased estradiol, LH, FSH, and progesterone concentrations (Figure 15.3) and a substantially
elevated probability of anovulatory cycles (Figure 15.4) in women of reproductive age (Gaskins
et al., 2009). The significant association between fiber and reproductive hormone concentrations
persisted and remained strong, whether fiber intake was considered as a continuous variable or
categorized according to groupings on the basis of the DRI. Although this is a single study, these
180
160
140
120
Estradiol (pg/ml)
100
80
60
40
20
22 g fiber/day <22 g fiber/day
0
Menses Follicular Ovulation Luteal
Figure 15.3. Fiber consumption 22 grams/day associated with lower estradiol concentrations across the
menstrual cycle (based on data from Gaskins et al., 2009).
5
Log odds ratio (95% confidence intervals)
1
<10 g/day 10-16 g/day 16-22 g/day 22 g/day
Figure 15.4. Fiber consumption 22 grams/day associated with increased odds of anovulation (based on
data from Gaskins et al., 2009).
findings call into question whether current DRIs are applicable to women of reproductive age
who are trying to conceive. High fiber diets cause a decrease of b-glucuronidase activity in feces
that leads to decreased reabsorption of estrogen in the colon. In addition, fiber binds to estrogen
in the intestine, increasing its fecal excretion.
In women with normal reproductive function, one might anticipate that in response to lower
concentrations of estradiol, an intact hypothalamic-pituitary-ovarian axis would respond by
increasing FSH and estradiol production from the follicles followed by ovulation. Although this
response could explain why some women who consume higher intakes of dietary fiber have
been shown to have normal ovulatory function, at least in one study, anovulatory women have
not exhibited this response; rather, they had consistently lower concentrations of reproductive
hormones (Gaskins et al., 2009). Thus, the decreased hormone concentrations associated with
higher fiber intakes could result in anovulatory cycles due to the close relation between diet
and the hypothalamic-pituitary axis. However, more research is needed to understand the exact
biological mechanisms. It is also possible that dietary fiber intake is associated with other lifestyle
factors related to increased menstrual irregularity, such as intense physical activity, low or high
BMI, or low-fat/low-calorie diets. However, in research to date, the association between fiber
intake and ovulatory function are not altered by adjustment for a wide variety of demographic,
lifestyle, and dietary characteristics (Gaskins et al., 2009).
Understanding the effects fiber has on other reproductive hormones, in addition to estradiol,
is key given the complex positive and negative feedback mechanisms at play between these
hormones under control of the hypothalamic-pituitary-ovarian axis (Gaskins et al., 2012).
However, there is little biological rationale to support an independent association between fiber
intake and LH levels. Some epidemiological studies have linked high fiber diets with decreased
leptin concentrations that in turn have been shown to suppress GnRH; however, no experimental
studies have confirmed a direct association. Conversely, experimental studies provide strong
biological rationale to support the inverse association between fiber intake and estrogen via
a decrease of beta-glucuronidase activity in feces. The one methodologically rigorous study
evaluating the independent effect of fiber on LH levels found no association after estradiol levels
were taken into account (Gaskins et al., 2012). This research supports the hypothesis that through
fibers influence on estrogen, fiber subsequently influences other menstrual hormones due to the
strict feedback mechanisms, which dictate hormonal fluctuations in the menstrual cycle. These
findings put into perspective the direct role of fiber on estradiol as it appears to have cascading
effects on additional pathways including other reproductive hormones.
15.4 Fiber and lipoprotein cholesterol mediated by estrogen

Because leading dietary and public health associations continue to endorse high-fiber diets, a
better understanding of the direct and indirect effects of fiber intake on lipoprotein cholesterol
levels is essential (Mumford et al., 2011b). This knowledge could provide further insight regarding
possible mechanisms, as well as valuable knowledge for interpreting studies of fiber intake among
women of reproductive age.
The major mechanism of reduced serum cholesterol levels to increased fiber intake is thought
to work through bile acid metabolism (Jenkins et al., 2000). However, increased fiber intake
does not always lead to an increased fecal output of bile acids, suggesting that the reduction
in cholesterol may work through another mechanism. Alternatively, as discussed dietary fiber
may alter serum sex hormone concentrations, which could in turn affect lipid metabolism. As
noted previously, high fiber intake in women has been associated with lower levels of estradiol
(Gaskins et al., 2009). There is also evidence that pre- and postmenopausal women respond
differently to fiber intake (Ganji and Kuo, 2008), since premenopausal women have been found
to have smaller reductions in lipoprotein cholesterol levels in response to fiber intake than
postmenopausal women. The difference in response to fiber between pre-menopausal and post-
menopausal women could be because estradiol mediates fibers effect on lipoprotein cholesterol
in premenopausal women.
In a study of healthy premenopausal women, fiber consumption at or above 22 g/day was

associated with lower TC and LDL-C levels, independent of measured endogenous estradiol
level, when estradiol was set at certain levels (Mumford et al., 2011b) (Figure 15.5). The controlled
direct effects of high fiber intake were in fact larger than the total effects, since the effect of fiber on
cholesterol through estradiol has been shown to increase cholesterol levels (Figure 15.6). The fact
that direct effects were larger than total effects suggests that estradiol mediates the effect of fiber
on lipoproteins; high-fiber diets may also have reduced effects among premenopausal women.
The observed direct effects of fiber on TC and LDL-C provide further insights regarding possible
biologic mechanisms of fiber on lipoprotein metabolism, suggesting that fiber has a direct effect
on lowering lipoprotein cholesterol levels, in addition to its effect that operates through estradiol.
15.5 Conclusion
Understanding the complex interplay between lipoprotein cholesterol, endogenous hormones,
and dietary fiber intake, is essential for clinical management of women, as well as for the design
and interpretation of studies in women of reproductive age.
0.15
0.10
(95% confidence intervals)
0.05
0.00
0.05
0.10
0.15
Total cholesterol HDL-C LDL-C TRY
Figure 15.5. Total effects of 22 grams/day fiber consumption associated with decreased total cholesterol
and low-density lipoprotein cholesterol levels (based on data from Mumford et al., 2011b). LDL-C = low-density
lipoprotein cholesterol, HDL-C = high-density lipoprotein cholesterol, TRY = triglycerides.
0.15
0.10
(95% confidence intervals)
0.05
0.00
0.05
0.10
0.15
Total cholesterol HDL-C LDL-C TRY
Figure 15.6. Direct effects of 22 grams/day fiber consumption associated with decreased total cholesterol
and low-density lipoprotein cholesterol levels independent of estradiol when estradiol was set to 45 pg/ml
(based on data from Mumford et al., 2011b). LDL-C = low-density lipoprotein cholesterol, HDL-C = high-density
lipoprotein cholesterol, TRY = triglycerides.
We observed that lipoprotein cholesterol levels vary during the menstrual cycle and are
significantly associated with endogenous reproductive hormone levels. TC and LDL-C tend
to be highest during the follicular phase and decline during the luteal phase. HDL-C tends to
be highest around ovulation. Both women and physicians should take menstrual cycle phase
into account when interpreting a womans cholesterol measurement. Cyclic variations also have
important implications on research among women of reproductive age. Measuring cholesterol
levels during menses is recommended for consistent comparisons due to reduced variability in
cholesterol levels during this phase and because this phase can be more reliably identified and
scheduled than others.
Fiber consumption at or above the recommended intakes has been shown to be significantly
associated with decreased reproductive hormone concentrations and a substantially elevated
probability of anovulatory cycles in women of reproductive age (Gaskins et al., 2009). Although
the observed association between fiber intake and estradiol is consistent with several observational
studies and randomized trials, further studies are needed to confirm these findings with respect
to ovulation, and elucidate the role of fiber intake on reproductive health to inform current
recommendations for adequate fiber intake in young women. Evidence supports the notion that
through fibers influence on estrogen, fiber subsequently influences other menstrual hormones
due to the strict feedback mechanisms, which dictate hormonal fluctuations in the menstrual
cycle (Gaskins et al., 2012).
Not only does estrogen mediate the association between fiber and other reproductive hormones,
but it has also been shown to mediate the association between fiber and lipoprotein cholesterol
levels (Mumford et al., 2011b), suggesting that high-fiber diets may have reduced effects among
premenopausal women. The observed direct effects of fiber on TC and LDL-C provide further
insights regarding possible biologic mechanisms of fiber on lipoprotein metabolism, suggesting
that fiber has a direct effect on lowering lipoprotein cholesterol levels, in addition to its effect that
operates through estradiol. These findings not only highlight the inter-related nature of hormones,
lipids, and dietary intake, but have important implications for the study of reproductive-age
women.
Acknowledgements
This work was supported by the Intramural Research Program of the Eunice Kennedy Shriver
National Institute of Child Health and Human Development.
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16. Menstrual patterns in adolescents with eating
disorders
A. Deltsidou
Department of Nursing, School of Health Sciences, Technological Educational Institution of Sterea
Ellada, 3rd km Old National Road of Lamia-Athens, 35100 Lamia, Greece; deltsidou@teilam.gr
Abstract
Menstrual irregularities are a commonly found manifestation of all eating disorders and lead to
noteworthy adverse medical effects. The causality of menstrual disorders is multifactorial and the
outcome of a multidimensional interaction of numerous components, including body weight loss,
reduced body fat and leptin levels, unusual eating behaviors, physical activity, and psychosomatic
stressors. Eating disorders arise with a high occurrence of sports, such as physical activity and
combat sports, and activities requiring weight control. Nutritional behaviors may have a great
influence on the gynecological health of adolescents. Adolescents with anorexia nervosa might
demonstrate amenorrhea due to hypothalamic suppression and they are prone to osteoporosis
and fractures. Moreover, about 50% of adolescents suffering from bulimia nervosa may exhibit
oligomenorrhea or menstrual irregularities because of the hypothalamic disruption. The rate is
greater in young people, who usually demonstrate low self-esteem, an unclear body shape in which
body weight is deemed to be too high, ineffectiveness, meticulousness and an impression of loss
of control, with compensatory behavior including food management and the use of inadequate
approaches to weight management. Among the significant results in female athletes are the
menstrual irregularities, the decline of the bone mineral absorption and osteoporosis, resulting in
rise to suspected female athlete triad. Treatment requires multidisciplinary management, with the
support of physicians, psychologists/psychiatrists, nutritionists, trainers and family. This chapter
of the book will review the occurrence of menstrual irregularities in young girls suffering from
eating disorders, its multifaceted causality, the pathophysiology, and the consequent adverse
outcomes for linear and pubertal growth, bone mineral deposit, and mental function. Current
investigations and trends on handling of menstrual abnormalities in young girls suffering from
eating disorders are discussed.
Keywords: eating disorders, adolescents, menstrual dysfunction, amenorrhea, oligomenorrhoea
255
A. Deltsidou
Summary points
Eating disorders arise with a high occurrence of sports, such as physical activity and combat sports, and
activities requiring weight control.
Adolescents with anorexia nervosa might demonstrate amenorrhea due to hypothalamic suppression and
they are prone to osteoporosis and fractures.
Adolescents suffering from bulimia nervosa may exhibit oligomenorrhoea or menstrual irregularities because
of the hypothalamic disruption.
The causality of menstrual disorders is multifactorial and the outcome of a multidimensional interaction
of numerous components, including body weight loss, reduced body fat and leptin levels, unusual eating
behaviors, physical activity, and psychosomatic stressors.
Hypoleptinemia is correlated with decreased levels of luteinizing hormone and estradiol. Hypoleptinemia
might also work directly at the level of the pituitary gland and ovary where leptin receptors are present.
16. Menstrual patterns in adolescents with eating disorders
Abbreviations
AN Anorexia nervosa
BN Bulimia nervosa
CNS Central nervous system
DSM-IV Diagnostic and statistical manual of mental disorders, fourth edition
DSM-IV-TR Statistical manual of mental disorders, fourth edition, text revision
EDNOS Eating disorder not otherwise specified
FSH Follicular stimulating hormone
GnRH Gonadotropin-releasing hormone
HPG Hypothalamic pituitary gonadal
IGF-I Insulin growth factor I
16.1 Introduction
Eating disorders are multifaceted diseases (Figure 16.1) producing noteworthy morbidity and
mortality (Katzman, 2005). Among young girls, they are deemed to be the third most predominant
chronic disease, after obesity and asthma (Chamay-Weber et al., 2005). Most studies have been
supported by data gathered from non-representative samples designated at a regional level, or
have examined the dissemination of indications by using questionnaires (Hoek and Van Hoeken,
2003). The findings of American and European studies suggest that the percentage of anorexia
nervosa varies between 0.3% and 6% (Hoek and Van Hoeken, 2003). For bulimia nervosa the
occurrence throughout the lifespan has been projected to vary between 0.6% and 3% (Preti et
al., 2009).
Eating disorders among adolescents and the habit of detrimental weight restraint activities have
reached epidemic proportions in developed countries. These behaviors vary from disproportionate
dieting to a full eating disorder. The American Psychiatric Association (2000) describes three
major categories of eating disorders: AN, BN and EDNOS. At times diagnosis in adolescents is
confounding given that they do not usually meet the exact diagnostic criteria delineated in the
DSM-IV-TR (Bravender et al., 2007).
The onset of these illnesses in early childhood or adolescence will potentially also result in
postponement of menarche, disruption of pubertal growth, and menstrual irregularities.
Menstrual dysfunction constitutes a common clinical characteristic of all types of eating disorders
(Poyastro Pinheiro et al., 2007). The etiology of menstrual disorders is multidimensional, counting
numerous contributors such as weight loss, reduced body fat, hypoleptinemia, abnormal eating
attitudes, intense physical activity, and psychological stressors (Table 16.1).
A. Deltsidou
Genetic factors
Physical risk
Impulsivity factors
Sociocultural Adverse life events

factors and difficulties
Family factors Perfectionism
Figure 16.1. The multidimensional linkage and complexity of eating disorders (Adapted from National
Collaborating Centre for Mental Health, 2004).
Table 16.1. Causes of menstrual irregularity (American Academy of Pediatrics et al., 2006, with permission).
Endocrine causes
Poorly controlled diabetes mellitus
Polycystic ovary syndrome
Cushings disease
Thyroid dysfunction
Premature ovarian failure
Late-onset congenital adrenal hyperplasia
Acquired conditions
Stress-related hypothalamic dysfunction
Medications
Exercise-induced amenorrhea
Eating disorders (both anorexia and bulimia)
Tumors
Ovarian tumors
Adrenal tumors
Prolactinomas
16.2 Eating disorders

16.2.1 Anorexia nervosa
AN is deemed to be a long-lasting disorder that affects between 0.3 and 1.2% of adolescents (Hoek
and Van Hoeken, 2003). Patients with AN refuse to maintain an ideal body weight in relation to
their somatometric features. In young people, this will possibly be interpreted as failure to reach
the projected weight increase. Individuals also have a profound concern about weight increase or,
worse, about becoming obese and experience difficulty in appreciating their body shape.
Mortality owing to AN is about 5.6% per decade, the highest mortality rate for psychiatric
illnesses. Medical complications occur frequently and AN can affect every organ system in the
body (Katzman, 2005). Quality of life might also be seriously damaged, with higher rates of social
separation, co-morbid psychiatric illnesses, failure to accomplish educational objectives, and
unemployment (Wentz et al., 2001). Early intervention is essential for a positive result.
The deep fear of weight increase and the lack of self-esteem cannot be underestimated and they
are components that cause distress to the adolescent with anorexia. Moreover, these patients may
have a variety of personality disorders: for example, perfectionism, obsession, social withdrawal,
high-achievement (with rare satisfaction) and depression are often noted in these patients. The
adolescent with anorexia might completely impede nutritional consumption (restrictive subtype)
or can have periods of eating too much and vomiting (bulimic subtype) (American Psychiatric
Association, 2000).
Although the adolescent girl with anorexia often demonstrates signs such as faintness, vertigo or
exhaustion, it is because her weight loss has caused amenorrhea that she frequently seeks help
(or an upset parent makes her seek help). The exact mechanism of amenorrhea in the young girl
suffering from anorexia is not recognized. Nevertheless, the simple caloric restraint suppresses
the hypothalamic-pituitary axis (Golden et al., 1997).
We define amenorrhea as the absence of menstruation for three normal menstrual cycles (Table
16.2) in females who have previously menstruated (Schillings and McClamrock, 2002). This is
used to help confirm the diagnosis of AN. Moreover, an adolescent is thought to be amenorrheic
when her menses happen only after hormonal therapy.
Amenorrhea due to malnutrition-induced deficiency in gonadotropin, predominantly LH

secretion, is identified to be one of the most common consequences of AN. Women with normal
weight may demonstrate amenorrhea if they have a low body fat ratio. In girls with AN this
phenomenon can be observed before their considerable weight loss, and may continue after
weight gain. On the contrary, menstruation might continue in certain underweight adolescents
(Poyastro Pinheiro et al., 2007).
A. Deltsidou
Table 16.2. Normal menstrual cycles in young females (American Academy of Pediatrics et al., 2006, with
permission).
Menarche (median age) 12.43 years

Mean cycle interval 32.2 days in first gynecological year
Menstrual cycle interval Typically 21-45 days
Menstrual flow length 7 days or less
Menstrual product use Three to six pads or tampons per day
Moreover the adolescent with anorexia has an increased risk of acquiring osteopenia and full-
blown osteoporosis (Brooks et al., 1998). While the pathophysiology of osteoporosis is not well
recognized, it is accepted that adolescence is an essential time for bone mineralization. Estrogen
seems to play a primary role (Hergenroeder, 1995), although dietary elements are also critical
(Rock et al., 1996).
The main aims of care provided to adolescents with anorexia are particularly body weight
increase and the opportunity to manage eating habits. Thus, although oral contraceptives have
effectively re-established menstruation in such adolescents in clinical trials, there is no proof that
they significantly improve osteoporosis. One study (Hergenroeder et al., 1997) that investigated
women presenting with amenorrhea for various reasons recommended that long-lasting therapy
with oral contraceptives and calcium supplementation (duration of more than 12 months) will
possibly have a favorable effect, although other studies (Mnster et al., 1992) do not confirm this
finding.
16.2.2 Bulimia nervosa
BN has a prevalence of 1-5% (Hoek and Van Hoeken, 2003). The prominent characteristic of
BN is the exhibition of frequent events of binge eating associated with incongruous behavior to
avoid weight gain, characteristically self-induced vomiting. Moreover, the contemporary DSM-
IV diagnostic criteria in the recent (fourth) edition of the Diagnostic and Statistical Manual
of Mental Disorders of the American Psychiatric Association (DSM-IV) require that both the
binge eating and the improper compensatory behavior happen at least twice a week for 3 months
(American Psychiatric Association, 2000). Patients suffering from BN are usually adolescent girls
of regular weight who are very upset about their silhouette and weight.
The key feature of a binge eating disorder is the demonstration of frequent binge eating events of
individuals with BN. In contrast to those with BN, patients with a binge eating disorder are liable
to be overweight or fat and middle-aged (Walsh, 2011).
Even though adolescents with BN may keep their body weight within normal limits, they may
demonstrate amenorrhea between 7 and 40% due to hypothalamic-pituitary dysfunction.
However, abnormal cycles (oligomenorrhoea) are more frequent in women with BN, occurring
at a prevalence between 37 and 64%. Investigations of biochemical studies demonstrated that
abnormal menstruation in women with BN is related to decreased LH levels and a lower LH
pulse rate, and reduced levels of estradiol and noradrenalin. The incidence of polycystic ovaries
in adolescents with BN is high (76-100%), and in these girls, oligomenorrhoea is considered to
be associated with an insulin-induced increase in circulating androgen levels. This may occur
because of considerable variability in food produced by constricting diets and binge eating
(Poyastro Pinheiro et al., 2007). Parameters that have been related to menstrual dysfunction in
normal weight BN consist of vomiting on a frequent basis, low circulating thyroxine levels and
low nutritional fat consumption (Gendall et al., 2000), a background of AN, and weight loss in
the past resulting in less than 92% of the appropriate body weight (Poyastro Pinheiro et al., 2007).
Oligomenorrhoea in women with bulimia does not seem to influence their bone mineral
concentration. One study (Sundgot-Borgen et al., 1998) fascinatingly revealed that physical
activity focusing on weight loss had a protective result in women with bulimia in contrast to
those with anorexia. Thus, osteoporosis does not constitute a problem for women with bulimia,
especially for those who engage in physical activity on a regular basis.
However, a young girl with bulimia demonstrating menstrual abnormalities needs to have a
limited evaluation including a thorough history, physical assessment, and laboratory studies.
If the menstruation does not occur in more than three months, a progesterone challenge test
(administration of medroxyprogesterone acetate in a dosage of 10 mg daily for seven days)
is recommended. Withdrawal bleeding occurring two to seven days after treatment suggests
adequate levels of estrogen. An adolescent with long-lasting anovulation who is not underfed is
treated by administering progesterone every three months or by stimulating menstruation with
combined oral contraceptive pills (Seidenfeld and Rickert 2001).
16.2.3 Eating disorder not otherwise specified
EDNOS occurs to a less obvious extent than AN or BN due to the heterogeneity of the major
clinical eating disorders that are included in this category. EDNOS encompasses a variety of
clinically disordered eating attitudes, incorporating those that are used as criteria for AN or BN,
those that have uncommon clinical manifestations of AN and BN, and those with a binge-eating
disorder. Owing to the inconsistency among individuals diagnosed with EDNOS, up to 50-60%
of children, adolescents, and young individuals with eating disturbances are encompassed in this
diagnostic category (Fairburn et al., 2007).
Many studies have investigated other components related to the presence or absence of
amenorrhea in adolescents with eating disorders. These studies concentrated on samples including
individuals with broader definitions of AN, incorporating women fulfilling all the diagnostic
criteria of AN excluding amenorrhea (partial syndrome AN). Adolescents with amenorrhea had
a considerably lower body mass index than those without amenorrhea (Gendall et al., 2006).
On the contrary, Garfinkel et al. (1996) detected that the decrease in weight was equivalent in
A. Deltsidou
adolescents with and without amenorrhea. There were no noteworthy discrepancies in eating
disorder symptomatology, personality, psychiatric comorbidity, or family history between those
who presented with amenorrhea and those who did not. The degree to which the presence of
menstrual disturbances is associated with psychological characteristics, personality features, and
coexistence of other psychiatric diseases needs to be clarified.
Given that eating disorder manifestations are identical in girls with AN with or without
amenorrhea, it is necessary to think about the strength of amenorrhea as a diagnostic criterion.
Amenorrhea happens in women with normal-weight BN, in other psychiatric patients, and in
female athletes. Unexpectedly, the role of menstrual dysfunction as diagnostically pertinent to
BN or EDNOS has not been discussed, regardless of the frequency of the symptom (Poyastro
Pinheiro et al., 2007).
16.3 Eating disorders and menstrual irregularities

Amenorrhea is considered to be one of the four diagnostic criteria of AN (Pinheiro et al.,
2007). Nevertheless, numerous adolescents with BN and EDNOS also demonstrate amenorrhea
or oligomenorrhoea (American Psychiatric Association, 2000). Whether or not amenorrhea
should be included as a diagnostic criterion for children and adolescents is a matter of great
debate (Garfinkel et al., 1996). The absence of menses in early teenage years and the physiologic
abnormal menstrual cycles that typically happen after menarche, limit the use of this criterion
in adolescents (Golden et al., 2003). Additionally, there are a percentage of girls who exhibit all
the signs and indications of AN, but maintain their menstruation regardless of being seriously
underfed and of low body weight (Garfinkel et al., 1996).
Despite this ongoing debate, amenorrhea is currently one of the diagnostic criteria. Of significance
is the fact that the onset of an eating disorder before, during, or after puberty can result in primary
or secondary amenorrhea or oligomenorrhoea (Poyastro Pinheiro et al., 2007).
Menstrual irregularities are very usual among young people (Slap, 2003). The occurrence
of secondary amenorrhea in adolescents has been stated to be between 2.6 and 8.5% and of
menstrual disturbances between 11.3 and 26.7%. Menstrual disturbances in adolescents during
the first years after the onset of menses are usually justified by immaturity of the HPG axis
and are thought to return to normal with increasing steadiness of the axis. Nevertheless, there
is insufficient information about the etiology of menstrual irregularities among young girls.
Most of the studies are cross-sectional, and only a few have taken into account the primary
endocrine mechanisms of menstrual dysfunction in young girls. Weight decrease and physical
activity have been found to be the main causes of menstrual irregularities in college students.
It has also been suggested that oligomenorrhoea or menstrual disturbances in young girls will
possibly be a primary indication of PCOS rather than a phase in the biological evolution of the
HPG axis. On the other hand, there are not sufficient data about endocrinological characteristics
of oligomenorrhoea and amenorrhea in adolescents, which are correlated with environmental

causes (Wilksten-Almstrmer et al., 2007).
16.3.1 Hypothalamic-pituitary-ovarian axis
Young girls with AN have hypogonadotropic hypogonadism, also commonly called hypothalamic
amenorrhea. This condition indicates a state of gonadal hypofunction (hypo-oestrogenemia and
anovulation/amenorrhea) owing to anatomic and/or functional disturbances of the hypothalamic/
pituitary parts (Figure 16.2). The endocrine basis for hypogonadotropic hypogonadism is
disturbed due to inadequate secretion of hypothalamic GnRH and/or pituitary gonadotropins
(FSH and LH) (Davis, 2004).
Among the hypothalamic causes of hypogonadotropic hypogonadism are post-radiation

hypothalamic dysfunction, tumor, Kallmanns syndrome, isolated GnRH insufficiency, physiologic
postponement of puberty, eating disturbances and weight decrease, stress, and extreme physical
activity. The pituitary causes of hypogonadotropic hypogonadism consist of prolactinomas, other
CNS tumors, inflammatory or infiltrative processes, and pituitary infarction.
A careful history and physical assessment might indicate iatrogenic causes, stress, eating
disturbances/weight loss, or intensive physical activity and they will possibly detect
galactorrhoea or anosmia (one indication of GnRH insufficiency owing to Kallmanns syndrome).

Hypothalamus
Negative
feedback
Gonadotropin-releasing
hormone (GnRH)
Anterior pituitary
Negative feedback
Inhibits secretion
of GnRH
Gonadotropins
(LH and FSH)
Inhibits responsiveness
to GnRH
Gonads

Sex steroid hormones
(oestrogens and progesterone)
Figure 16.2. The negative feedback of the hypothalamic-pituitary-ovarian axis. The increased levels of steroid
hormones inhibit the secretion of GnRH from hypothalamus (modified from Homburg, 2008).
A. Deltsidou
Hyperprolactinemia may be documented by determining the prolactin serum level. Generally,

it is recommended to use imaging studies of the hypothalamus and pituitary (MRI or CT) to
diagnose or exclude a mass lesion, mainly in the case of an increased prolactin level, which might
be owing to a prolactinoma or other tumor applying pressure to the pituitary gland (Vyver et
al., 2008).
Studies have revealed that young girls with AN demonstrate a prepubertal or early pubertal
pulsatile secretion pattern of LH with age-inappropriate 24-h LH secretion patterns arising in
young women aged 17 to 23 years old who have AN and primary or secondary amenorrhea.
These findings recommend that females with AN have a reversion of the hypothalamic-pituitary-
ovarian axis, with a related obstruction to the regular menstrual function (Vyver et al., 2008).
16.3.2 Anorexia nervosa
Amenorrhea related to AN is considered to be a subsequent event of hypothalamic dysfunction.

The reason is multifactorial, and includes weight loss, reduced body fat, hypoleptinemia, unusual
eating patterns and behaviors, physical activity, and psychological stressors.
Menstrual function and fat mass
Some researchers recommend that the lowest or threshold weight for menarche is about 17%
fat as a percentage of body weight. Correspondingly, the authors stated that more or less 26-
28% fat as a percentage of body weight is reached at the achievement of typical growth and that
a lowest weight equivalent of 22% body fat is essential for the maintenance or continuation of
menstruation in young girls older than 16 years of age (Vyver et al., 2008).
The reasons for maintenance of menses in a few very skinny adolescents are indistinct. The
findings of the study conducted by Miller et al. (2004) indicated that a relative deficiency in fat
mass in the face of strict starvation can be unfavorable to the maintenance of menstruation.
Moreover, menstruating adolescents were also found to have greater levels of leptin and IGF-I.
Menstrual function and leptin
A factor which might contribute to the hypogonadism of starvation is the low level of the
hormone leptin. Leptin is a protein hormone produced by adipose tissue that is implicated in
energy homeostasis (Figure 16.3). Leptin levels have been detected to be inferior in individuals
with AN compared with controls. Leptin has also been revealed to estimate fat mass and eating-
disordered behavior in both underfed individuals with AN and individuals throughout the
process of recovery. Moreover, there is increasing evidence that leptin significantly contributes to
reproduction. Hypoleptinemia is correlated with decreased levels of LH and estradiol and might
also work directly at the level of the pituitary gland and ovary where leptin receptors are present.
A leptin level lower than 2 g/l has been suggested as the essential level value for amenorrhea
(Hebebrand et al., 2007). Fasting causes reduced leptin levels earlier than the beginning of weight
Adipose Weight loss Adipose Weight gain Adipose

tissue tissue tissue
Decreased Increased
leptin leptin
Increased food intake Decreased food intake

decreased energy increased energy
expenditure expenditure
Figure 16.3. The Leptin hormone produced by adipose tissue is implicated in energy homeostasis (Modified
from Friedman, 2002).
loss and will possibly provide an explanation as to why, in a few individuals, amenorrhea occurs
before the onset of weight loss (Vyver et al., 2008).
Menstrual function and eating disorders
In about two thirds of young people with AN, lack of menstruation occurs before noteworthy
weight loss. This indicates that there are causes other than considerable weight loss leading to
amenorrhea in AN. The influence of eating disorders on menstruation as well as the impacts
of physical activity and eating disorders on serum hormone levels have been examined. The
findings demonstrate that women undertaking a physical activity have a low serum LH mainly
owing to low caloric intake rather than to exaggerated physical activity (Vyver et al., 2008).
Correspondingly, Warren and Perlroth (2001) stated that the prominent reason for GnRH
deficiency in athletes is low energy availability.
Menstrual function and exercise
Intense physical activity is usually observed in adolescents with AN. Extreme physical activity in
athletes with no eating disorder has been associated with menstrual dysfunction. Amenorrhea
as a result of extreme exercise occurs between 5 and 25%, depending on the type and level of
activity, and is caused by hypothalamic dysfunction related to a reduction in pulse rate of GnRH,
with resultant reduced levels of LH, FSH, and estradiol. When accompanied by malnourishment,
weight decrease and extreme physical activity, the odds of amenorrhea emerging earlier and for
a prolonged time rise (Vyver et al., 2008).
Extreme physical activity, as practiced by competitive female athletes and dancers, may lead to
amenorrhea. In adolescents, it might also cause a delay in menarche (Davis, 2004).
A. Deltsidou
There are three components that contribute to the female athlete triad, which include eating
disorders, amenorrhea, and osteoporosis (Figure 16.4). The female athlete triad is produced by
a lack of equilibrium between caloric availability and caloric consumption. This consecutively
activates compensatory mechanisms, such as weight loss or energy maintenance, followed by a
reproductive dysfunction and concurrent hypo-estrogenism (Vyver et al., 2008).
16.4 Complications of menstrual dysfunction

Menstrual irregularities in young people with eating disturbances may have extensive influences
and exert a significant effect on growth and pubertal development, peak bone mass gaining, and
cognitive function. These complications may not be entirely reversible.
Eating disorders usually appear at the beginning of sexual maturity, when typical variations in
body composition might come together with the growing pressures of puberty to result in body
appearance disruption. The subsequent undernourishment may lead to pubertal postponement
or interruption (Vyver et al., 2008).
In some girls with eating disturbances a deficiency of linear growth and a steady short stature
have been observed (Katzman, 2005). This is due to increasing levels of estrogen and IGF-I bone-
tropic hormones during normal puberty that trigger longitudinal bone growth. The decreased
levels of estrogen and IGF-I in girls with AN might play a role in variations in linear growth
(Vyver et al., 2008).
Moreover, peak bone mass attainment happens during the teenage years (Katzman, 2005). The
presence of an eating disorder during puberty may lead to a failure to obtain peak bone mass and
lead to a decreased bone mineral mass with a probable higher risk of fractures (Vyver et al., 2008).
Low energy
availability with
or without an eating
disorder
Functional
hypothalamic Osteoporosis
amenorrhoea
Figure 16.4. The low energy availability may cause osteoporosis in female athletes due to functional
hypothalamic amenorrhea (modified from American College of Sports Medicine, 2007).
Women with persistent amenorrhea have been found to have deficiencies in cognitive function,
counting recall, verbal and working memory, visual reproduction, reading, and oral language
(Chui et al., 2007).
16.5 Conclusions
Menstrual irregularities are one of the frequent characteristics of all types of eating disturbances
(Poyastro Pinheiro et al., 2007). Factors contributing to menstrual dysfunction in young girls
with eating disorders include body weight, body fat composition, leptin and other hormones,
eating behaviors, physical activity, and psychological stressors. The fact that eating disturbances
appear more frequently in puberty, a period of intense physical and mental development, makes
the management of this condition more difficult. The implication of menstrual irregularities in
girls with eating disorders is predominantly significant when taking into account its effect on
linear growth, pubertal growth, bone mineral mass, and mental functioning.
There are numerous studies focusing on the menstrual irregularities in adults with eating
disturbances, but their findings may not apply to adolescents. Thus, further investigation into
menstrual irregularities in adolescents with eating disturbances, including factors influencing
menarche, is necessary. The recognition of biological indicators for the anticipation of menarche
and the reappearance of menstruation is significant in the management and prevention of
considerable morbidity in these individuals. Menstrual irregularities in young girls with
eating disorders have long-lasting consequences. Future investigation of women with AN will
probably help us evaluate the influence of estrogen on cognitive function and this might provide
information that helps us to understand the management and prognosis of AN.
Finally, research on the etiology of menstrual irregularities in girls with eating disorders will
offer researchers and professionals a clear understanding of the possible consequences of these
disturbances on these girls general health.
Consequently, better recognition of the psychological and biological elements that lie beneath
these behavioral attitudes might offer a valuable way of improving both our appreciation of, and
our aptitude to manage, this disorder.
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Annals of the New York Academy of Sciences 1135, 253-264.
Walsh, B.T., 2011. The importance of eating behavior in eating disorders. Physiology & Behavior 104, 525-529.
Wilksten-Almstrmer, M., Hirschberg, A.L. and Hagenfeldt, K., 2007. Menstrual disorders and associated factors
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Conception
17. Role of micronutrients in the periconceptional
period
Unit of Obstetrics and Gynecology, Department of Clinical Sciences, Hospital L. Sacco, University
of Milan, Via G.B. Grassi, 74, 20157 Milan, Italy; irene.cetin@unimi.it
Abstract
Unhealthy preconceptional diet and lifestyle significantly contribute to impaired reproduction. In
particular, micronutrient deficiencies have been associated with significantly high reproductive
risks, ranging from infertility to fetal structural defects and long-term diseases. On the other
side, healthy dietary patterns and micronutrient supplementation during the periconceptional
period are related to improved birth outcome, probably through alterations in maternal and
fetal metabolism due to micronutrients role/involvement in enzymes, signal transduction,
transcription pathways, oxidative stress and epigenetic modifications. The periconceptional
period is a wide concept that encompasses preconception, conception, implantation, placentation
and embryogenesis, all of which may be affected by maternal nutrition and, specifically, by
micronutrient imbalances. However, up to now conclusive evidence has been provided solely for
periconceptional folate supplementation for the prevention of neural tube defects.
Keywords: nutrition, pregnancy, periconceptional period, micronutrients
273
Summary points
Micronutrient deficiencies have been associated with significantly high reproductive risks, ranging from
infertility to fetal structural defects and long-term diseases.
Healthy diet and micronutrient supplementation prior to and after conception are related to improved fertility
and birth outcomes.
It may be hypothesized that micronutrients affect pregnancy outcomes through alterations in maternal and
fetal metabolism due to their role/involvement in enzymes, signal transduction and transcription pathways
and oxidative stress during the different stages from the preconceptional to the post-partum period.
Prospective studies related to the association between periconceptional maternal nutrition and pregnancy
outcomes are still scarce.
Conclusive evidence is currently available solely for periconceptional folate supplementation and prevention
of NTDs.
17. Role of micronutrients in the periconceptional period
Abbreviations
CpG Cytidine-guanosine
DHA Docosahexaenoic acid
EVT Extravillous trophoblast
HHCY Hyperhomocysteinemia
IUGR Intrauterine growth restriction
LBW Low birth weight
MTHFR 5,10 methylenetetrahydrofolatereductase
NTDs Neural tube defects
OS Oxidative stress
PUFA Polyunsatured fatty acid
RA Retinoic acid
ROS Reactive oxygen species
SODs Superoxide dismutases
17.1 Introduction
Unhealthy preconceptional diet and lifestyle of both women and men significantly contribute
to impaired reproduction. Preconception care has been defined as a set of interventions that
aim to identify and modify risks to a womans health or pregnancy outcome through prevention
and management. In this context, the importance of proper nutrition prior to and throughout
pregnancy has long been known to optimize health and well-being of both mother and
baby (Hammiche et al., 2011). Although many of required nutrients are present in food, the
physiological demands during preconception, pregnancy and nursing may require additional
dietary supplementation. Moreover, most women of reproductive age, and above all pregnant
women, do not get enough trace elements in their diet, representing an important topic of
public health not only in developing countries but also in industrialized countries where dietary
patterns, typified by snacking, breakfast skipping, fast foods, soft drinks and convenience foods,
are nutritionally unbalanced and fail to meet recommended daily allowance for micronutrients.
Micronutrient deficiencies have been associated with significantly high reproductive risks,
ranging from infertility to fetal structural defects and long-term diseases. At the time of
conception, usually when pregnancy has not yet been confirmed, maternal nutritional status
is an important determinant of embryonic and fetal growth (Figure 17.1). In fact, the onset of
several malformations and pregnancy related disorders (i.e. congenital abnormalities, fetal loss,
miscarriage, insufficient fetal growth, premature birth and pre-eclampsia) may occur during the
periconceptional period. Deficiencies of specific antioxidant activities associated with selenium,
copper, zinc and manganese in the periconceptional period can result in poor pregnancy
outcomes, including intrauterine growth restriction and preeclampsia, probably related to altered
placentation mechanisms. Moreover, mothers diet and lifestyle are known to influence the long-
term health of her children: in fact, inadequate levels of maternal nutrients during the crucial
Maternal factors
(i.e. nutrition and Placental
lifestyle, diseases) function
Fetal growth and Changes in

Genetic factors metabolism
development
and organ function
Fetal metabolic and

endocrine milieu Fetal programming
(i.e. insulin,
glucocorticoids)
Figure 17.1. Factors involved in fetal growth and development.
period of fetal development may lead to reprogramming within the fetal tissues that predisposes
the infant to chronic illnesses in adulthood (De Boo et al, 2006).
On the other side, healthy diet and micronutrient supplementation prior to and after conception
are related to improved birth outcomes. Among these, increasing calcium and magnesium intake
can reduce the risk of pregnancy-induced hypertensive disorders; ensuring adequate intake of iron,
zinc, iodine, calcium and folic acid during pregnancy can improve birth outcomes; improving the
intake of folic acid before pregnancy can reduce birth defects and maternal megaloblastic anaemia;
providing zinc supplements during pregnancy can improve birth weight and reduce prematurity.
In addition, micronutrients involved in the correct function of the immune system, such as
vitamin A and iron, may also be implied in the careful balance between immune surveillance
to protect the mother and immune tolerance in regard to the semiallogenic conceptus. Regular
periconceptional multivitamin use has been found to be associated with reduced risk of small
for gestational age births and preterm births in non-overweight women (Catov et al., 2011).
However, the biological mechanisms responsible for these association are not completely clear.
It may be hypothesized that micronutrients affect pregnancy outcomes through alterations in
maternal and fetal metabolism due to their role/involvement in enzymes, signal transduction and
transcription pathways and OS. Since different pregnancy stages represent a continuum, from
the preconceptional to the post-partum period, an injury acting before conception or in early
pregnancy may have long-term consequences.
17.2 Role of specific dietary micronutrients

17.2.1 Antioxidants
Cells are constantly exposed to oxidants from both physiological processes, such as mitochondrial
respiration, and pathophysiological conditions such as inflammation, foreign compound
metabolism and radiation. The body defenses against oxidants include antioxidant enzymes and
radical scavengers of which many are nutritionally dependent. Failure of enzymatic, endogenous
and nutritional antioxidants may lead to oxidative damage to biomolecules such as membrane
lipids, proteins and DNA, as well as deregulation of cell cycle control. OS constitutes a unifying
mechanism of injury of many different disease processes that occurs when there is an imbalance
between the generation of ROS and the antioxidant defense systems in the body so that the latter
become overwhelmed. OS is generated during normal placental development; however, when
supply of antioxidant micronutrients is limited, exaggerated OS within both the placenta and
maternal circulation occurs, possibly resulting in adverse pregnancy outcomes. The placenta is
armed with antioxidant defenses, including the selenium-dependent glutathione peroxidases,
thioredoxin reductases, selenoprotein-P and copper/zinc and manganese superoxide dismutases
(Cu/Zn and Mn SODs). The pathogenesis of adverse pregnancy outcomes including preeclampsia
and IUGR and a number of neonatal outcomes has been shown to be associated with OS.
Many antioxidants are obtained from the diet, such as vitamin E and other tocopherols,
vitamin C, -carotene, whereas proteins and peptides, such as glutathione, ceruloplasmin and
metallothionein, are synthesized endogenously. Vitamin E (-tocopherol) is a chain-breaking
antioxidant that prevents the propagation of free radical damage in biologic membranes, thus
defending PUFA from auto-oxidation. Vitamin C or l-ascorbic acid, present mainly in vegetables
and fruit, exhibits a protective effect against free-radical-induced oxidative damage by acting as
a reducing agent. It is also important in the synthesis of collagen, carnitine and catecholamines.
Vitamin C is commonly included in low doses (<200mg/day) within multivitamin preparations
for pregnancy but has also been given in higher doses (up to 1000 mg/day) as a supplement,
alone or in combination with vitamin E. Although the concentrations of these vitamins remain
significantly reduced in women with preeclampsia, supplementation with higher doses of vitamin
C and E have not been shown to prevent development of preeclampsia in either high-risk or
lower-risk women (Conde-Agudelo et al., 2011).
Selenium
Plant foods are the major dietary sources of selenium in most countries. The growing fetus requires
selenium, which is transported across the placenta by passive diffusion down a concentration
gradient. Recurrent early pregnancy loss has been associated with reduced serum selenium
concentrations compared to healthy controls, probably linked to reduced glutathione peroxidase
activity culminating in reduced antioxidant protection of biological membranes and DNA
during the early stages of embryonic development. Selenium deficiency has been confirmed in
women suffering from preeclampsia and this continues to be linked with glutathione peroxidase
inadequacy. Results on the effect of optimization of selenium status in women at risk of adverse
pregnancy outcomes are still controversial (Mistry et al., 2012).
Zinc
Zinc is abundantly present in meat, seafood, pulses, legumes and whole-grain cereals. Zinc is
an essential constituent of over 200 metalloenzymes participating in carbohydrate and protein
metabolism, nucleic acid synthesis, antioxidant functions (through Cu/Zn SOD) and other
vital functions such as cellular division and differentiation, making it essential for successful
embryogenesis. The World Health Organization estimated that suboptimal zinc nutrition affected
nearly half the worlds population. Benefits of zinc supplementation in suboptimal diet seem to
include reduced incidence of pregnancy-induced hypertension and of low birth weight (Uriu-
Adams et al., 2010).
Copper
The richest dietary sources of copper include shellfish, nuts, seeds, legumes, grains bran and
germ, liver and organ meats. Copper is an essential cofactor for a number of enzymes involved
in metabolic reactions, angiogenesis, oxygen transport and antioxidant protection, including
catalase, SOD and cytochrome oxidase. This nutrient exhibits several biological roles being
involved in connective tissue formation, iron metabolism, cardiac function, immune function
and central nervous system development. Interestingly, new insights are emerging into the role of
iron and copper in neurocognitive and neurobehavioral development during the last two thirds
of gestation. Copper is essential for embryonic development. Maternal dietary deficiency can
result in both short-term consequences, including early embryonic death and gross structural
abnormalities, and long-term consequences such as increased risk of cardiovascular disease and
reduced fertilization rates.
17.2.2 Folate
Folic acid, a water-soluble B-complex vitamin required for DNA synthesis and cell division,
is a nutrient currently recognized as important prior to and during pregnancy because of its
proven preventive properties against NTDs. 70% of NTDs could be prevented if the embryo is
exposed to protective amounts of folic acid during the critical window of organogenesis. Folate
is widely distributed in foods (green-leafy vegetables, fruits, liver, bread, etc.). Inadequate dietary
folate intake results in a reduction of DNA biosynthesis and thereby of cell division, leading
to anemia, leucopenia and thrombocytopenia. A decrease in the methylation cycle results in
HHCY, implicated in the etiology of endothelial disfunction. Moreover, as an effective scavenger
of oxidizing free radicals, folic acid acts as antioxidant and can protect bio-constituents such
as cellular membranes or DNA from free radical damage. Supplementing dietary intake with
folic acid has been recommended because of the difficulty for woman to obtain the extra folate
required periconceptionally through the diet alone. The current recommended daily intake for
folic acid is 400 g for women of preconception age. The recommended dose is higher (4,000 g)
for women who have had an infant with a NTD (Lamers, 2011).
17.2.3 Vitamin B12 or cobalamin
Vitamin B12, or cobalamin, is found primarily in animal products. B12 functions as an enzyme
to catalyze mitochondrial conversion of methylmalonic acid to succinyl-CoA, essential for
synthesis of hemoglobin as well as metabolism of fat and protein. Vitamin B12 also functions as
a cofactor, with folic acid, for generation of methionine from homocysteine in the cytosol. Low
vitamin B12 levels are related to HHCY and high methylmalonic acid. Anaemia, myelopathy and
neuropathy are the main clinical manifestations of vitamin B12 deficiency. Vitamin B12 has also
been identified as a crucial nutrient for fetal development. Low serum levels of B12 have been
linked to negative impacts in cognitive, motor and growth outcomes. Low cobalamin levels also
may be related to depression in adults. On the whole, the defects in the one-carbon metabolism,
implying folate, vitamin B6 and B12, play a crucial role in intrauterine programming of adult
diseases (Ronnenberg et al., 2002).
17.2.4 Vitamin A
Vitamin A can be obtained from food either as pre-formed vitamin A, in the form of retinol
or retinyl-esters which come from animal sources, or as provitamin A form from plants, i.e.
provitamin A-carotenoids such as -carotene. The highest concentration of vitamin A is found
in liver and fish liver oil. Yellow and green leafy vegetables provide provitamin A-carotenoids.
Adequate vitamin A is essential for proper visual functioning, fetal growth, reproduction,
immunity and epithelial tissue integrity. Because vitamin A is lipid soluble, it crosses the placenta
easily and has a long half-life. Carotenoids exert antioxidant properties. Vitamin A deficiency is
linked to xerophthalmia and vitamin A-deficiency anemia whereas hypervitaminosis seems to be
involved in teratogenesis, liver abnormalies and bone mineral loss. It has been supposed that even
the brain may be susceptible to an imbalance of RA, particularly the hippocampus (McCaffery
et al., 2003).
17.2.5 Calcium
Calcium is essential for bone development. The dynamic balance between skeletal calcium
storage and fetal nutritional needs can affect the maternal calcium equilibrium adversely.
When completing a diet history during preconception counseling, it is important to ask about
dietary calcium consumption (milk, fortified orange juice, etc.), calcium supplementation and
use of antacids to assess the womans overall calcium intake. Vitamin D intake is necessary to
facilitate calcium absorption. Higher birth-weight babies, a reduced risk of preterm delivery and
preeclampsia, and lower infant blood pressure have all been linked with a high calcium intake
during pregnancy. Current recommendations indicate an intake of 1000 mg/day of calcium for
pregnant and lactating women who are 19-50 years old and 1,300 mg/day for pregnant and
lactating women who are younger than 19 years old. Moreover, calcium supplementation should
be provided to women at increased risk of preeclampsia (WHO, 2012).
17.2.6 Iron
Worldwide, iron deficiency represents the most common nutritional deficit that can exist with or
without anemia. Prior to conception and during pregnancy, women should eat iron-rich foods (lean
meat, poultry and iron fortified cereals). Foods that inhibit iron absorption, such as whole-grain
cereals, unleavened whole-grain breads, legumes, tea and coffee, should be consumed separately
from iron-fortified foods. The Centers for Disease Control and Prevention recommends 18 mg/
day for women and 27 mg/day for all pregnant women. A relationship has been observed between
low ferritin (<12 g/l), a marker of depleted iron stores, and reduced birth weight, as well as
between elevated ferritin (70 g/l), a biomarker of acute or chronic inflammation, and increased
risk of low birth weight and IUGR (Ronnenberg et al., 2004). Despite an iron accumulation in
the developing fetus against a concentration gradient and the consequent protection of the fetus
in case of maternal iron deficiency (Cetin et al., 2011), iron-deficient anemia in pregnancy is
linked to spontaneous prematurity, IUGR and altered behavioral and neural development (Beard,
2003). Data on human infants are consistent with altered myelination of white matter, changes in
monoamine metabolism in striatum and functioning of the hippocampus. Iron supplementation
in low risk pregnancy raises or maintains serum ferritin level above 30 g/l and reduces incidence
of anemia, but seems to have no detectable effect on any substantive measures of either maternal
or fetal outcomes (Milman, 2011).
17.3 C ritical stages and mechanisms potentially affected by nutrition in the

periconceptional period
The periconceptional period consists of preconception, conception, implantation, placentation
and embryo- or organogenesis stages and specific cellular events that occur during the distinct
stages of embryogenesis. Besides genetics, each of these steps may be affected by maternal nutrition
and, specifically, by micronutrient imbalances (Table 17.1; Figure 17.2; Berti et al., 2011).
17.3.1 The preconceptional period
Despite the paucity of studies dealing with preconceptional dietary patterns, it is clear that there
is a strong relation between imbalance of micronutrients before conception and the successful
onset, as well as healthy development of pregnancy (Cetin et al., 2010).
Optimizing the health of the mother before conception is important for improving pregnancy
outcome. Studies have consistently shown that planned pregnancies generally have better
outcomes than unplanned ones. However, one-third to one-half of live born infants in the
United States are the result of unintended pregnancy. These findings suggest that preconception
counseling should be provided to all women of childbearing age, regardless of their immediate
pregnancy plans.
Table 17.1. Micronutrients intake recommendation in the periconceptional period.
Micronutrient Strength of Quality of recommendation

recommendation1
Vitamin A B The recommended dietary allowance of preformed vitamin A for women

is 700 retinal activity equivalents per day, with a tolerable upper
intake level for pregnancy of 3,000 retinal activity equivalents /day or
10,000 IU/day).
Folic acid A All women of reproductive age should be advised to ingest 0.4 mg of
synthetic folic acid daily.
Vitamin D B There is insufficient evidence to recommend for or against routine
screening or vitamin D supplementation during preconception
counseling. Currently, no data are available for the optimal dose
before and during pregnancy.
Calcium A Calcium supplements should be recommended if dietary sources are
inadequate.
Iron A At a preconception visit, screening should be conducted for all women
with risk factors for iron deficiency in order to identify and treat anemia
and improve perinatal outcome.
Iodine A Women should be informed about the importance of maintaining
adequate daily dietary iodine intake of 150 g during preconception
and at least 200 g when pregnant or lactating. Public health efforts
to implement salt iodization programs should be encouraged for all
women who reside in regions with endemic iodine deficiency.
1 Based on GRADE (grading of recommendations, assessment, development and evaluation) system (Gardiner et
al., 2008). A: recommendation based on consistent and good-quality patient-oriented evidence; B: recommendation
based on inconsistent or limited-quality patient-oriented evidence.
Moreover, assessing hematologic indices before conception could be of great utility, as they are
likely to reflect status in the periconceptional period. On the contrary, biomarker concentrations
assessed at various times throughout pregnancy are affected by plasma volume expansion,
consequently the interpretation of the relation between these measures and birth outcomes can
be challenging.
Some observational studies show an association between short interpregnancy interval and
increased risks of adverse outcomes probably due to maternal depletion of nutrients. Depletion
of maternal folate seems to be mainly involved, with particular regard to the risk of IUGR.
Antioxidants, folate,
Antioxidants Calcium, antioxidants B12, calcium
Chromatin
aberrations Pregnancy diseases:
Epigenetics preeclampsia diabetes
Methylation
ROS Fetal
Alterations of fetal
Miscarriage growth, IUGR, macrosomia programming
Infertility
Effects in
later life
Ist trimester
Fetal period
Preconceptional Implantation Post partum
period period
Embryogenesis
2 1 placentation 12 40
LMP
Folate, B12
Years
Conception Delivery
Weeks of gestation
Figure 17.2. Pregnancy stages from preconception to post-partum period (Cetin et al., 2010).
Preconceptional folic acid supplementation is a goal of public health to prevent NTDs. All
women planning pregnancy or capable of becoming pregnant should be counseled to take a daily
multivitamin with folic acid (400 to 800 g) to reduce the risk of neural tube defects.
Another potential problem is that women of reproductive age and pregnant women enter pregnancy
without adequate iron reserves or are already iron deficient (Milman, 2011). Pre-conceptional
anemia, particularly iron-deficiency anemia, was found to be associated with reduced infant
growth and increased risk of adverse pregnancy outcomes (Ronnenberg et al., 2004). Plausibly,
it may be supposed that either anemia in the periconceptional period has an independent effect
on infant growth by influencing hormone synthesis or the moderate preconceptional anemia
turns to a more severe anemia during pregnancy leading to the observed growth deficits. In a
prospective observational study in China, it was observed that the risk of preterm delivery and
LBW was increased more than 2-fold in moderately anemic women and more than 3-fold in
those with severe anemia during early pregnancy (4-8 weeks) (Zhou et al., 1998).
An open question is whether iron supplementation, which is generally started in the second half
of pregnancy, could reduce the rates of preterm LBW, or whether anemia plays a role in placental
development solely during early pregnancy and therefore must be prevented prior to conception.
Interestingly, prophylactic iron supplementation given from about 12 weeks of gestation to the
third trimester among low income women in a randomized controlled trial evidenced that birth
weight was significantly higher in the supplemented groups than in controls (Siega-Riz et al.,
2006).
In childbearing women, insufficient energy stores may negatively affect ovulation, menses
and challenge the beginning of pregnancy. On the other hand, excessive fat stores may inhibit
conception by affecting ovulation because of insensitivity to insulin, excess of male sex hormones
and over-production of leptin. Maternal obesity has been linked to subfertility, having a child
with a congenital anomaly, and several other pregnancy complications (gestational diabetes,
preeclampsia, cesarean delivery, macrosomia, difficult delivery, and stillbirth/early neonatal death).
The relationship between diet and fertility is not well-defined, except at dietary extremes. All of
the data in humans are from observational studies.
Analysis of data from the Nurses Health Study II, a prospective cohort study of more than
116,000 women aged 24-42 years, evidenced that the consumption of iron supplements and
non-heme iron from foods may decrease the risk of ovulatory infertility (Chavarro et al., 2006).
Folate seems to be important for oocyte quality and maturation. Zinc plays a role in ovulation and
the menstrual cycle. Oocyte maturation, ovulation, luteolysis and follicle atresia are also affected
by ROS unbalance. For this reason, a positive role of the antioxidant status may be explained by
counteracting ROS effects (Ruder et al., 2008). Similarly, folate, zinc and thiols affect apoptosis,
which is important for regulation of follicle atresia, degeneration of the corpus luteum and
endometrial shedding. Poor folate status and HHCY are mainly crucial due to their involvement
in cell division (e.g. of oogonia or of granulose cells), inflammatory cytokine production, OS,
apoptosis and defective methylation reactions.
In this context, DNA synthesis is important for the development of oocytes, and several enzymes
involved in DNA synthesis are zinc- or vitamin B-dependent. Deficiencies of vitamins A, C
and D have been associated with diminished fertility in rats and rainbow trout (Ebisch et al.,
2007). However, though addition of vitamins C and E has been shown to strengthen antioxidant
defenses in in vitro media, oral antioxidant supplementation in human studies does not provide
definite, conclusive evidence.
Micronutrients are also essential for male fertility, in fact human seminal plasma contains several
trace elements that play an important role in the normal function of sperm. Selenium is essential
for testosterone biosynthesis and spermatozogenesis (Mistry et al. 2012). Low concentrations
of folate in seminal plasma may be associated with DNA damage in spermatozoa. Poor Zn diet
may be an important risk factor for low quality of sperm and idiopathic male infertility. However,
there is a considerable lack of information on the exact role of micronutrient supplementation in
normal reproductive functions as well as in the treatment of male infertility.
17.3.2 Conception and implantation
The initial stages of preimplantation development, from fertilized ovum (zygote) to a solid mass
of cells (morula, blastocyst), occur as the embryo transits the Fallopian tube (Figure 17.3). The
blastocyst reaches the uterine cavity 2-3 days after fertilization. Implantation occurs around days
6-7 postconception. Normal implantation is critical for a successful pregnancy. Implantation is
Figure 17.3. Events from ovulation to the implantation of blastocyst.
characterized by invasion of the maternal tissues of the uterus by fetal trophoblast, and the degree
to which trophoblast invades the decidualized endometrium (decidua) and inner third of the
myometrium appears to be a major determinant of pregnancy outcome.
Maternal vitamin B6 status seems to influence reproductive events from the start of pregnancy.
Conceivably, the effects of poor pre-pregnancy maternal vitamin B6 status on the early gestational
events could be explained firstly through the involvement of vitamin B6 dependent-co-enzymes
in the metabolism of amino acids, lipids, nucleic acids and glycogen. Then, by considering
the association between vitamin B6 deficiency and impairment of enzymes involved in the
structural integrity of arterial walls, it could be supposed to affect implantation and early
placental development. Moreover, a nutritionally unbalanced diet characterized by low intakes
of B-vitamins, folate, vitamin B6 and vitamin B12, implicated in the homocysteine pathway,
may cause the increase of homocysteine concentration, which can ultimately lead to HHCY.
This biochemical derangement seems to be detrimental for reproductive outcome as elevated
total homocysteine concentrations in follicular fluid have been inversely associated with altered
number of preantral follicles, retrieved oocytes, and embryo quality. Moreover, early interaction
of nutrients with the epigenetic system may lead to variations associated with chromatin
remodeling and regulation of gene expression that underlie the developmental programming of
pathological consequences in adulthood. After fertilization, the genome of the zygote undergoes
rapid demethylation at coding sequences and at repetitive sequences. After implantation, the
embryonic ectoderm and mesoderm genome is hypermethylated, through de novo methylation,
whereas the extra-embryonic cell genome, such as the primary endoderm and trophoblast,
remains hypomethylated. These processes might be enhanced by methyl donors provided by the
folate-methionine pathway (Figure 17.4). In this context, methyl-supplements and zinc adequate
diets during pregnancy may affect phenotypic modifications in offspring. A methyl-deficient
diet around the time of conception results in significant changes in the methionine cycles within
ovarian follicles, leading to adult offspring being heavier and fatter, insulin-resistant, eliciting
altered immune responses and with elevated blood pressure. These clinical outcomes seem to be
associated with modification in methylation status of 4% of 1,400 gene-associated CpG islands,
mainly in male fetuses, to indicate that periconceptional specific dietary inputs to the methionine
cycles may affect a significant part of genome in offspring with long-terms implications for adult
health. Furthermore, polymorphisms of folate-related genes are reported to be associated with
the prevalence of placental abruption, mainly when folate status is low, suggesting a genetic-
nutrient interactive effect in disease risk (Guant et al., 2003).
17.3.3 Placentation
The placenta is an organ for exchange of oxygen, nutrients, antibodies, hormones, and waste
products between the mother and fetus. Placental vascular development represents a crucial
process for adequate fetal development.
The progenitor villous trophoblast cell proliferates throughout gestation, differentiating along
two pathways to form either invasive EVT or syncytiotrophoblast. Invasive EVT invades decidua
and remodels the spiral arteries. The syncytiotrophoblast is a specialized epithelium covering the
villous tree and has several functions, such as transport of gases, nutrients, and waste products
and synthesis of peptide and steroid hormones that regulate placental, fetal, and maternal systems.
Folic acid
Methylation S -Adenosyl- (unmetabolized)
reactions methionine
S -Adenosyl- Methionine Dihydrofolate

homocysteine
B12 Tetrahydrofolate
Homocysteine
5, 10 -methylen-
tetrahydrofolate
Purine
MTHFR biosynthesis
5, 10 -methyl-
tetrahydrofolate Pyrimidine
biosynthesis
Figure 17.4. Folate pathway. MTHFR = 5,10 methylenetetrahydrofolatereductase.
The surface area of capillaries involved in gas exchange in the mature intermediate and terminal
villi was strongly and inversely related to serum ferritin suggesting that anaemia influences the
pattern of placental vascularization.
Inadequate trophoblast invasion has been implicated in the pathophysiology of such conditions
as preeclampsia, preterm premature rupture of membranes, preterm labor and IUGR. In
preeclampsia, the cytotrophoblast infiltrates the decidual portion of the spiral arteries, but fails to
penetrate the myometrial portion. Thus, the large, tortuous vascular channels characteristic of the
normal placenta do not develop; instead, the vessels remain narrow, resulting in hypoperfusion.
Environmental, immunological, and genetic factors all appear to play a role in this process. OS
and inflammatory mediators are also involved in the abnormal implantation associated with
pre-eclampsia and IUGR. Abnormal placentation leads in fact to placental ischemia, resulting in
generation of placental OS and increased levels of lipid peroxidation (Figure 17.5).
The use of antioxidants seems to be useful to reduce the damage on placenta, thereby the risk
of pre-eclampsia because of its limited antioxidant enzyme capacity in the first trimester. Serum
lipid peroxide levels are significantly higher and serum vitamin E levels significantly lower in
women with preeclampsia than in women with normal pregnancies.
At present, nutrition has not been assessed during the periconceptional period or during
early pregnancy in women that later develop preeclampsia. However, we may hypothesize that
nutrients can affect OS by increasing or decreasing free radicals or antioxidants or by providing
substrates for the formation of ROS. Moreover, they could modify inflammatory response in
Environmental,
immunological,
genetic factors
Abnormal
trophoblast invasion
Placental ischemia/hypoxia
Preclinical stage
Antioxidant
defences
Increased release of
pro-infiammatory factors
in the maternal circulation
Clinical signs of
Endothelial dysfunction pre-eclampsia
& Hypertension
systemic inflammation Proteinuria
Figure 17.5. Pathogenesis of preeclampsia.
the period when placentation occurs. In particular, adequate intake of vitamin C and E during
the second trimester of pregnancy is recognized to improve the biochemical incidence of OS.
The postulated involvement of deficiencies of trace elements in preeclampsia relates to the
fact that they are present in many enzymes and proteins involved in redox regulation such as
metallothionein (zinc), ceruloplasmin (copper), SOD (copper, selenium, zinc) and glutathione
peroxides (selenium).
Some placenta events are postulated to arise from deficiencies of either folate and/or vitamin
B12 or defects within the methionine-homocysteine metabolic pathways.
As an example, HHCY has been shown to provoke vascular inflammation, to decrease the
bioavailability of NO that is an important endothelium vasodilatator, and seems to be associated
with the production of ROS. This means that folate-deficiency or HHCY may underlie endothelial
dysfunction, and therefore placental endovasculature, a theory supported by the observation
that elevated serum homocysteine concentrations have been associated with an increased risk of
diseases, such as atherosclerotic, thromboembolic and neuro-degenerative disorders (Forges et
al., 2007). HHCY has also been implicated in other adverse pregnancy outcomes such as placental
abruption or infarction. As already mentioned, folate is potentially important in a number of
crucial early stages of placental development, including extravillous trophoblast invasion,
angiogenesis, and secretion of metalloproteinases, and this may address to the benefit of longer-
term folic acid supplementation throughout pregnancy to prevent pregnancy disorders associated
with deficient placental development, including preeclampsia (Williams et al., 2011).
Maternal vitamin B6 status was observed to influence reproductive events throughout the
entire course of pregnancy. Significantly lower plasma concentrations of vitamin B6 have been
demonstrated in women with previous recurrent spontaneous miscarriage compared with
control women. Furthermore, significantly lower plasma vitamin B6 was detected among women
with placental abruption or infarction compared with control women. In addition, some studies
have documented associations between low vitamin B6 status and inflammatory responses, and
inflammation has been linked to early pregnancy loss.
17.3.4 Embryogenesis
Because developing organ systems directly respond with permanent adaptations to the availability
of nutrients during critical periods of rapid growth, timing of adequate maternal nutrition is
important to both fetus and child development. Moreover, fetal growth is vulnerable to maternal
nutrition, especially during the preconception period and first weeks of gestation, since it has the
potential to affect epigenetic mechanisms in the placenta and fetus.
The association between maternal folate status and fetal NTDs is well recognized. Neural tube
develops into the spine and NTDs occur when the brain and skull and/or the spinal cord and
the protective spinal column do not develop properly within the first 4 weeks after conception.
In general, women are advised to take 0.4 mg/day when planning a pregnancy whereas they are
recommended to take 4.0 mg/day if they experienced a previous pregnancy affected by NTD.
Folate functions as a co-enzyme in single-carbon transfers in the metabolism of amino acids and
nucleic acids. Moreover, folate is the substrate donor in the remethylation of homocysteine into
methionine, catalyzed by methionine synthase and MTHFR. Altered homocysteine metabolism
leading to HHCY has been proposed as the mechanism involved in NTDs given that higher
homocysteine levels were found in plasma or amniotic fluid of NTD infants and their mothers
with respect to non-NTD individuals (Tamura and Picciano, 2006). Also maternal dietary pattern
is associated with the risk of having NTDs offspring independent of periconception folic acid
supplementation: low maternal intake of vegetable oil, vegetables, fruits, fish and whole grains is
associated with a more than two-fold increased NTDs risk (Vujkovic et al., 2009). Furthermore,
low-dose periconception folic acid supplementation is associated with increased fetal growth
resulting in higher placental and birth weight, and decreased risks of having a child with low
birth weight or being small for gestational age. This effect is probably mediated by epigenetic
modifications in the preimplantation embryo which may result in increased placental and fetal
growth patterns (Timmermans et al., 2009).
Neural crest cells are involved not only in the embryogenesis of the neural tube, lip and palate,
but also in cardiovascular development. The migration and differentiation of neural crest cells is
influenced by homocysteine, and vitamin B12 is an important determinant in the homocysteine
pathway, thereby contributing to the embryogenesis of the heart in the first weeks after conception.
Periconceptional intake of thiamine, niacin and vitamin B6 seems to contribute to the prevention
of orofacial cleft defects. This result may be explained by considering vitamin B6 involvement
into the homocysteine pathway.
Retinoids are also thought to be involved in the development of several embryonic systems. RA
plays a crucial role in central nervous system development, i.e. neural crest survival, neuritis
outgrowth and hindbrain patterning. Vitamin A and RA are part of the required normal
regulatory system. Nevertheless, excessive intake (>10,000 IU/day) has also been shown to be
teratogenic in animals. Excessive vitamin A may disturb all three stages of palatogenesis: during
shelf outgrowth, it may decrease cell proliferation and thus prevent tissue development; it may
prevent shelf elevation by affecting extracellular matrix composition and hydration; during shelf
fusion, it may affect epithelial differentiation and apoptosis, which precludes the formation of a
continuous palate. In general, high doses of vitamin A affect palatogenesis through interference
with cell proliferation and growth factors such as transforming growth factor and platelet-
derived growth factor. The overall recommendation is that vitamin A or retinol intake below
3,000 g (10,000 IU)/day is safe.
Severe maternal zinc deficiency in early pregnancy results in impaired implantation, abortions
and fetal malformations, including cleft lip and palate, brain and eye malformations, numerous
abnormalities of the heart, lung and urogenital systems. Biochemical and functional abnormalities
can occur as a result of a zinc deficit. The consideration that zinc deficiency represents a teratogenic
risk in humans may be supported by the correlation of low plasma zinc concentrations in the
first and third trimesters of pregnancy with an increased risk for malformations and low birth
weight, respectively. Zinc deficiency is thought to influence embryonic and fetal development
through reduced cell proliferation, or reduced protein synthesis or reductions in rates of tubulin
polymerization. Increased rates of cellular oxidative damage or increased rates of apoptosis and
reduced binding of hormones and transcription factors dependent on zinc-finger regions may
also be involved.
17.4 Conclusions
Preconceptional nutrition is crucial for an optimal onset and development of pregnancy. Two
major studies (Chavarro et al., 2007; Timmermans et al., 2012) have proven an important role
for maternal and paternal diet in improving fertility and pregnancy outcome. Unfortunately,
nutritional intake of childbearing-age women appears to be inadequate during the preconceptional
period mainly in terms of micronutrients, also considering the global shift towards the Western
diet. Thus, efforts to increase awareness of a healthy diet and lifestyle should be strengthened not
only throughout pregnancy but also before, given that pregnancies are often unplanned.
However, conclusive evidence is currently available solely for periconceptional folate

supplementation and prevention of NTDs, and prospective studies related to the association
between periconceptional maternal nutrition and pregnancy outcomes are still scarce.
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18. Prevalence and predictors of periconceptional
folic acid use
Department of Obstetrics and Gynaecology, Trinity College, University of Dublin, Coombe
Women and Infants University Hospital, Dublin 8, Ireland; deirdre.j.murphy@tcd.ie
Abstract
The benefits of periconceptional folic acid supplementation in prevention of neural tube defects
are well established. However, use of periconceptional folic acid remains disappointingly
low despite clear evidence of benefit typically less than one-third of women actually take
periconceptional folic acid supplements. This chapter considers the many factors influencing
the prevalence of periconceptional folic acid use demographic, socioeconomic, lifestyle,
gynaecological, previous pregnancies, current pregnancies, other medical conditions and access
to folic acid supplements. The importance of underlying awareness and knowledge is discussed,
including the various sources of information. The role that these factors may have in shaping
future strategies to promote folic acid supplementation is also considered.
Keywords: neural tube defects, pregnancy, supplements.
293
Summary points
The use of folic acid supplements periconceptionally reduces the risk of the fetus developing neural tube
defects (NTDs). Preconceptional use is particularly important given that the neural tube closes by the sixth
week of gestation.
The use of periconceptional folic acid supplements remains disappointingly low typically less than one-third
of women actually take supplements. Poor uptake applies to both preconceptional and postconceptional
time periods.
Many factors influence periconceptional folic acid supplement use, particularly demographic factors,
socioeconomic factors and whether the pregnancy was planned. Often these factors are interlinked. In
addition, there is a correlation between the factors influencing periconceptional folic acid supplement use
and the risk of adverse perinatal outcomes including NTDs.
Even women at further increased risk of NTDs (e.g. epileptics, previous baby with a NTD) have relatively
low rates of use of periconceptional folic acid supplements (and often at the standard dose rather than the
recommended high dose).
An appreciation of these factors is critical in influencing health promotion strategies particularly targeting
women who are less likely to take periconceptional folic acid supplements.
18. Prevalence and predictors of periconceptional folic acid use
Abbreviations
CDC Centre for Disease Control
FA Folic acid
NTD Neural tube defect
18.1 Introduction
NTDs are a collection of malformations of the brain and spinal cord, including spina bifida and
anencephaly, arising from failure in closure of the neural tube. The formation of the neural tube
occurs early in the embryonic period and closure is completed by the sixth week of gestation.
The worldwide prevalence of NTDs range from approximately 0.05 to 6 per 1,000 births with
significant geographic and population specific variations (Beaudin and Stover, 2009).
The benefit of periconceptional FA in reducing NTDs was identified from studies in the 1970s
and 1980s. This resulted in the Medical Research Council Vitamin Study of FA, concluding that
supplementary FA can significantly reduce the risk of a NTD (MRC, 1991). The significance
of these results was recognized internationally and various national bodies recommended that
women of childbearing age should take FA supplements (400 g/day) before conception and
up to 12 weeks of gestation to reduce the risk of a NTD (Table 18.1). Some countries have also
adopted food fortification programmes with FA, notably the USA. Nevertheless, inadequate levels
of FA intake persist among many women of childbearing age in these countries and individual
FA supplementation is still advised.
Because the neural tube forms and closes early in the embryonic period, often before the woman
realises she is pregnant, taking FA supplements preconceptionally is particularly important to
achieve maximum benefit.
Table 18.1. Historical background to the use of folic acid supplements.
1970s and 1980s The relationship between folic acid deficiency and neural tube defects suggested.
1991 British MRC RCT published: neural tube defects could be reduced by about 75%.
1992 UK Department of Health recommends that all women planning a pregnancy take 400 g
of folic acid daily through supplementation.
1992 US Public Health Service recommends that all women planning a pregnancy consume
400g of folic acid daily through fortification, supplementation and diet.
1998 US FDA requires that grain-based foods, cereals, and dietary supplements be enriched with
folate.
Despite its clear benefit in preventing NTDs, periconceptional FA supplement use remains low
and a cause for concern. The purpose of this article is to review the extent of periconceptional
FA supplement use and the many factors influencing it (Table 18.2). In addition, the significance
for health promotion strategies is considered.
Table 18.2. Factors influencing periconceptional folic acid supplement use.
Awareness and knowledge of periconceptional folic acid

Demographic factors
Maternal age
Maternal ethnicity and race
Geographical location
Relationship and marital status
Socioeconomic factors
Education level
Occupation
Income
Social work involvement
Lifestyle factors
Alcohol consumption
Cigarette smoking
Recreational drug use
Diets and exercise
Gynaecological factors
Previous fertility treatments
Previous contraception use
Factors relating to previous pregnancies
Parity
Previous early pregnancy loss
Previous neural tube defect affected pregnancy
Previous stillbirth
Factors related to a current pregnancy
Pregnancy planning
Gestational age booking for antenatal care
Type of antenatal care
Other medical conditions
Factors related to access to folic acid
Factors related to health promotion strategies
18.2 Literature review

Articles relating to periconceptional FA supplement use were identified using Medline (United
States National Library of Medicine). A large number of articles from a wide variety of geographical
locations were identified. Emphasis was placed on more recent articles and articles with large
numbers of participants.
This article considers each factor separately. It should be acknowledged, however, that there is
significant overlap and correlation between many of these factors. There is, in general, a correlation
between the many factors influencing healthy lifestyle behaviours (including periconceptional FA
supplement use) and the risk of adverse perinatal outcomes including NTDs.
18.2.1 Terminology
The terms preconception, postconception and periconception are often used in relation to the
timing of FA supplement use.
Preconception refers to the time period before conception occurs.
Postconception refers to the time period after conception occurs. A woman usually recognizes
this phase by the absence of her menstrual period and/or pregnancy symptoms (and
confirmed with a positive pregnancy test). However, these features usually only become
apparent sometime after conception (at least 2 weeks).
Periconception refers to the time period from just before conception through to early
pregnancy, though there is no universal definition as to when this phase begins or ends. In
this article, periconception refers to both pre and postconception.
18.2.2 Rates of folic acid supplement use
Taking FA before and during early pregnancy is critical if FA is to reduce NTDs. Therefore, rates
of preconceptional FA supplement use are particularly significant. Though acknowledging that
individual rates vary somewhat, there is no doubt that recommended use of periconceptional FA
supplements remains disappointingly very low. Virtually all reviews in the literature report rates
of periconceptional FA supplement use of 30% or less (Figure 18.1). Rate of use increases when
women discover they are pregnant (i.e. postconception).
30%
Used
70%
Not used
Figure 18.1. Typical rates of periconceptional folic acid supplement use.
A large prospective cohort study in an Irish urban obstetric population of 61,252 women over
seven years from 2000 to 2007 reported that only 28% of women used FA supplements as
recommended (i.e. pre- and post-conception), though 85% used FA supplements at some stage in
the pregnancy (McGuire et al., 2009). Similar results were reported in a large prospective cohort
study in a Norwegian obstetric population of 22,500 women over three years from 2000 to 2003.
They found that only 28% of women used FA supplements pre- and post-conceptionally, though
72% used FA supplements at some stage in the pregnancy (Nilsen et al., 2006).
Some studies have examined the use of FA supplements among the wider population of women of
childbearing age (rather than an obstetric population). The CDC in the USA carried out annual
national surveys on the awareness, knowledge and use of FA supplements among women of
childbearing age (18 to 45 years) from 2003 to 2007. The most recent survey (2007) of 2,003
women reported a rate of FA supplement use of 40% (CDC, 2008).
In an early audit following the recommendation of FA supplement use in the UK, a large London
teaching hospital reported on the rate of use in an obstetric population one year after the UK
Chief Medical Officers recommendation. They found that only 3% used preconceptional FA
supplements and 19% used postconceptional FA supplements (Clark and Fisk, 1994).
Though it appears that FA supplement use amongst women who are planning pregnancy or in
early pregnancy has marginally increased since then, it is also clear that there is considerable
scope for improvement.
18.2.3 Awareness and knowledge
A distinction is often made between awareness of FA (i.e. having simply heard of FA) and
knowledge of FA (i.e. having a greater degree of understanding about the role of FA in pregnancy).
Knowledge may refer to many different aspects of FA use the benefits of FA in NTD prevention,
the need to take FA preconceptionally for maximal benefit, the need to take FA in the form of a
supplement (and the appropriate daily dose) and possible good food sources of FA).
An awareness and knowledge of the importance of FA in the prevention of NTDs is critical in

determining the likelihood of a woman actually using periconceptional FA (Figure 18.2). The
majority of women appear to be aware of FA, but a significantly smaller number of women have
a clear understanding of periconceptional FA and its benefits.
A systematic review (of 26 systematic reviews and/or meta-analyses from 1989 to 2006) on
periconceptional FA use found that the majority of studies reported rates of awareness of 60
to 75%, although different methodologies meant direct comparison between many of these
studies was difficult (Stockley and Lund, 2008). The CDC survey reported that 81% of women
of childbearing age were aware of the existence of FA, though only 12% reported knowing that it
should be taken before pregnancy (CDC, 2008). It should be borne in mind that many of these
Awareness Knowledge
of folic acid of folic acid
Periconceptional folic
acid supplement use
Figure 18.2. The relationship between awareness and knowledge and periconceptional folic acid supplement use.
studies were conducted following major health promotion campaigns in the 1990s, though more
recent studies have not shown any significant difference in awareness and knowledge.
Information sources
Women obtain their information from a variety of sources. Stockley and Lund (2008) identified
common sources of information: mass-media advertising, magazines, newspaper articles, friends
and family. Although health professionals were cited less frequently, they were considered more
credible. Family doctors were a more frequent source of advice than other healthcare professionals
(including midwives).
Older women appear to be more likely to hear about FA from a healthcare professional. The CDC
Survey found that older women were more likely to hear about FA from a health care provider
(37% of women aged 25 to 34 years and 36% of women aged 35 to 45 years) compared with
younger women (17% of women aged 18 to 24 years) (CDC, 2008).
Translating awareness and knowledge into use
In theory, a high level of awareness and knowledge of the benefits of FA should translate into a
high degree of periconceptional FA supplement use. However, the evidence suggests that this is
not the case. This trend is understandable in the context of an unplanned pregnancy. However,
evidence suggests that some women do not start using postconceptional FA supplements even
after the pregnancy is confirmed. A recent study from Scotland explored the reasons behind this
phenomenon. They held group discussions among 211 postpartum women to discuss the reasons
for not taking periconceptional FA. Despite awareness and knowledge of its importance, reasons
highlighted were busy lives, forgetting to take the supplements, linking FA to morning sickness
and doubts about the benefit of FA supplements following a previous healthy pregnancy outcome
without the use of supplements (Barbour et al., 2011) (Table 18.3).
The CDC survey reported that women of child-bearing age who did not take any vitamin or
mineral supplements on a daily basis cited the following reasons forgetting, no need, no reason
and already get balanced nutrition. Pregnant women are more aware and knowledgeable about FA
compared with non-pregnant women of childbearing age. Food fortification was not mentioned
specifically as a reason why these women did not take supplements (CDC, 2008).
Table 18.3. Reasons why women do not use folic acid supplements.
Lack of awareness or knowledge of folic acid supplements

Feel that they already have a balanced diet
Busy lifestyle
Forgetting to take the supplements
Associating folic acid supplements with morning sickness
Previous healthy pregnancy outcome without using folic acid supplements
Awareness and knowledge of among doctors
The evidence suggests that the majority of doctors routinely recommend periconceptional FA
supplements, though more specific knowledge (in relation to dosage and timing) is sometimes
lacking. A questionnaire survey of 87 primary care doctors from Southern Israel found that,
while 94% routinely recommended periconceptional FA supplements, only 12% knew the correct
timing and 47% the correct dosage (Abu-Hammad et al., 2008).
However, recommendations on the optimal duration of FA intake before conception lack clarity.
The UK Department of Health Expert Advisory Group (1992) advises FA supplements prior to
conception and the NICE Antenatal Care Guidelines advises FA supplements before conception
(NICE, 2008a). Therefore, some vagueness over timing of preconceptional FA supplements is
understandable.
18.2.4 Demographic factors
Maternal age
Younger women are less likely to use periconceptional FA supplements with a gradual increase
in use with increasing maternal age (Table 18.4). In particular, use declines considerably among
women aged less than 25 years. McGuire et al. (2010) reported rates of periconceptional FA
supplement use of less than 5% among women aged less than 25 years, 20% among women
aged 25 to 29 years and 40% among women aged greater than 30 years. Nilsen et al. (2006) also
reported a similar trend with rates of 6% among women aged less than 25 years and 11% among
women aged greater than 25 years. The CDC survey reported a similar trend among a general
population of women of child-bearing age with rates of FA supplement use of 30% among women
aged 18 to 24 years compared with 47% among women aged 25 to 34 years and 40% among
women aged 35 to 45 years (CDC, 2008).
However, while the literature generally supports the view that rates of FA supplement use are
significantly higher among older women, this is not universal. There may be specific factors
among certain populations that alter this general trend, such as cultural influences. A study of
Table 18.4. Demographic factors affecting use of periconceptional folic acid supplementation.
Decreased supplement use amongst
Maternal age young women

Maternal ethnicity and race immigrant women
Geographical location women from rural communities
Relationship and marital status single women and unmarried women
1,083 Turkish women reported that women aged greater than 35 years were less likely to be aware
of the need to use FA supplements (Baykan et al., 2011).
In addition to actual periconceptional FA supplement use, awareness and knowledge of

periconceptional FA supplement use is less in women aged 18 to 24 years. The CDC reported
that only 61% of women aged 18 to 24 years were aware of FA compared with 87% of women
aged 25 to 34 years (CDC, 2008).
Maternal age, however, should not be considered in isolation. Younger women are more likely to
have other exacerbating factors particularly unplanned pregnancy and socioeconomic factors.
Maternal ethnicity and race
Women with a different ethnic origin to the native population (defined as women who have a
different nationality or a different country of birth) often have different rates of periconceptional
FA supplement use (Table 18.4). In particular, ethnic differences that cause a barrier to
communication are associated with lower rates of periconceptional FA supplement use, i.e.
if they speak a language different to that country or their health-care provider. A number of
studies have identified that immigrants to some Western countries appear to be less likely to use
periconceptional FA supplements after controlling for other factors.
In Europe, Brough et al. (2009) reported significant differences in the rate of preconception
FA supplement use among 402 pregnant women in the first trimester in East London 19% of
Caucasian women compared with 5% of African women, 8% of West Indian women and 12% of
Asian women. Van Eijsden et al. (2006) reported lower rates of periconceptional FA supplement
use among non-Dutch speaking women and non-Western women (based on country of birth)
with lack of the ability to speak the native language identified as a key factor.
In North America, Kingston et al. (2011) reported lower rates of FA supplement use before
and during pregnancy among immigrant women to Canada compared with Canadian-born
women. Yang et al. (2001) reported lower rates of FA intake among Hispanic and non-Hispanic
black women compared with non-Hispanic white women of childbearing age. The CDC survey
reported very little difference in rates of FA use between Hispanic and non-Hispanic women
(38%) of childbearing age (CDC, 2008).
There is evidence from the USA that non-white women are less likely to use periconceptional FA
than white women. However, the gap appears to be narrowing. The CDC survey reported rates of
FA supplement use of 40% among white women compared with 36% among non-white women.
However, this gap narrowed considerably from the previous CDC survey in 2005 (only 23% of
non-white women used FA supplements) (CDC, 2008).
However, different ethnic origins may also exert a positive influence on FA use depending on
the population. McGuire et al. (2010) reported that, while native Irish women had higher rates
of periconceptional FA supplement use compared with non-Irish women (approximately 30%
compared with 15%), women from North America, Australia and New Zealand had higher rates
of periconceptional FA supplement use than Irish women (approximately 40%).
Geographical location
Understandably, there is considerable variation in periconceptional FA supplement use between

different continents and countries and also within individual countries. Women who live in rural
areas appear to have a lower rate of periconceptional FA supplement use (Table 18.4). Watson
et al. (2006) reported that women living in rural areas of New South Wales in Australia were
less likely to use periconceptional FA supplements compared with women living in urban areas.
Tamim et al. (2009) also reported a significant difference in periconceptional FA supplement
use between women attending urban hospitals (19%) compared with women attending rural
hospitals (3%) in Lebanon. Zeng et al. (2011) reported that poor levels of FA awareness among
Chinese women of childbearing age living in rural areas.
Relationship and marital status
Unmarried women appear to have a lower rate of periconceptional FA supplement use compared
with married women, though the evidence is somewhat limited (Table 18.4). McGuire et al.
(2010) reported that periconceptional FA supplement use was 7% among unmarried women
compared with 39% among married women. Nilsen et al. (2006) reported periconceptional FA
supplement use of 4% among single women, 8% among women co-habiting with their partner
and 12% among married women.
18.2.5 Socioeconomic factors
An individuals socioeconomic status refers to her level of education, occupation and

income (Figure 18.3). These factors play a critical role in determining periconceptional FA
supplement use (Table 18.5). The evidence has consistently and clearly shown that women of
a higher socioeconomic status (in terms of educational level and income) are more likely to
Educational
level
SE status
Income Occupation
Figure 18.3. The important elements of socioeconomic status.
Table 18.5. Socioeconomic factors affecting use of periconceptional folic acid supplementation.
Educational level women with lower educational levels

Occupation women not in employment
Income women with lower incomes
Social work involvement women requiring social work involvement
use periconceptional FA supplements. In addition, socioeconomic status appears to be a more

significant factor compared with other factors.
Educational level
Women with higher educational levels have higher rates of periconceptional FA supplement
use (Table 18.5). Studies usually categorise educational level as primary level, secondary level
(including high school) or third level (including college or university). Nilsen et al. (2006)
reported rates of periconceptional FA supplement use of 2% among women with primary level
education compared with 7% among women with secondary level education and 13% among
women with third level (university or college) education. The CDC survey reported rates of FA
supplement use among women of child-bearing age of 29% among women with primary level
education (less than high school education) compared with 36% among women with second
level education (high school education) and 48% among women with third level (college
education) (CDC, 2008).
There is some evidence suggesting a widening gap between women of lower educational levels
compared with women of higher educational levels in terms of awareness, knowledge and use
of periconceptional FA supplements. De Walle and De Jong-Van den Berg (2007) reported a
2-yearly cross-sectional study from 1995 to 2003 in the Netherlands. Correct FA supplement use
among women of lower educational levels had decreased over that time-period (to 22% in 2003)
compared with increased FA use among women of higher educational levels over that same time
period (to 59% in 2003). De Walle and De Jong-Van den Berg (2008) also subsequently reported
a further cross-sectional study indicating a similar trend with periconceptional FA supplement
use of 31% among women of lower educational levels compared with 61% among women of
higher educational levels.
Paternal (as well as maternal) educational levels also play a role and, indeed, follow the same trend
between lower maternal educational levels and lower periconceptional FA supplement use. Nilsen
et al. (2006) reported rates of periconceptional FA supplement use of 5% among women whose
partners had primary educational level compared with 8% among women whose partners had
secondary educational level and 13% among women whose partners had university or college
educational levels.
Occupation
Women not in employment have lower rates of periconceptional FA supplement use compared
with employed women. In addition, women with higher professional occupations are more
likely to use periconceptional FA supplements compared with women employed in other
occupations (Table 18.5). McGuire et al. (2010) reported that women not in employment had a
rate of periconceptional FA supplement use of approximately 5% compared with 31% of women
in employment. Women who were classified as professionals had rates of 50% compared with
25% of women who were classified as having other employment (McGuire et al., 2010).
Income
Women with higher incomes have higher rates of periconceptional FA supplement use
compared with women with lower incomes (Table 18.5). Nilsen et al. (2006) reported rates of
periconceptional FA supplement use of 8% among women with income of less than 200,000 NOK
compared with 11% among women with an income of 200,000 to 300,000 NOK and 15% among
women with incomes greater than 300,000 NOK (Nilsen et al., 2006). The CDC reported rates of
FA supplement use of 32% among women earning less than $25,000 compared with 43% among
women earning greater than $50,000 (CDC, 2008).
Social work involvement
There is evidence that women requiring social work interventions are less likely to use
periconceptional FA supplements compared with women who do not need such involvement
(Table 18.5). McGuire et al. (2010) demonstrated that women requiring social worker intervention
were less likely to use periconceptional FA supplements (14% compared with 65%).
18.2.6 Lifestyle factors
Alcohol consumption
There is limited evidence on the effect of alcohol consumption on periconceptional FA supplement

use. McGuire et al. (2010) reported that women who consumed low to moderate amounts of
alcohol (<15 units/week) prior to pregnancy appeared to have better rates of periconceptional
FA supplement use than women who never consumed alcohol or women who consumed excess
amounts of alcohol (>15 units/week). They found rates of 26% among occasional alcohol
consumers, 37% among women who consumed 1 to 5 units/week, 36% among women who
consumed 6 to 9 units/week and 27% among women who consumed 10 to 14 units/week
compared with 15% among women who never consumed alcohol, 12% among women who
consumed 15 to 20 units/week and 4% among women who consumed >20 units/week (McGuire
et al., 2010) (Figure 18.4 and Table 18.6).
Cigarette smoking
Women who smoke are less likely to use periconceptional FA supplements than women who
do not smoke. Nilsen et al. (2006) reported that 10% of women who did not smoke used
periconceptional FA supplements compared with 5% of women who smoked. McGuire et al.
(2010) reported that 33% of women who did not smoke used periconceptional FA supplements
compared with 8% of women who smoked (Table 18.6).
40%
Alcohol consumption
35%
30%
25%
20%
15%
10%
5%
0%
Never consumed 19 units/week 1015 units/week >20 units/week
Figure 18.4. The influence of alcohol consumption of periconceptional folic acid supplement use (McGuire et
al., 2010).
Table 18.6. Lifestyle factors affecting use of periconceptional folic acid supplementation.
Alcohol consumption women who never consume and consume >20 units/week
Cigarette smoking women who smoke cigarettes
Recreational drug use women who use recreational drugs
Recreational drug use
Women who use recreational drugs are less likely to use periconceptional FA supplements.
McGuire et al. (2010) reported that 25% of women who never used recreational drugs took
periconceptional FA supplements compared with only 10% of women who used recreational
drugs (at any stage of their life) (Table 18.6).
Diet and exercise
There is little evidence on the impact of diet and exercise programmes on periconceptional
FA supplement use. Clark and Fisk (1994) reported that some women modified their diets to
improve their intake of FA. However, it is now recognised that such dietary measures alone are
often insufficient to achieve adequate FA levels and the use of FA supplements is preferred.
18.2.7 Gynaecological factors
Previous fertility treatments
Women attending gynaecology services for fertility assessments or treatments have a higher rate
of periconceptional FA supplement use (Table 18.7). However, while the rates are increased, they
are still surprising low among this highly motivated population (who are in close contact with
health care professionals including gynaecologists).
Table 18.7. Gynaecological factors affecting use of periconceptional folic acid supplementation.
Increased supplement use amongst
Previous fertility treatments previous fertility assessment or treatment

Previous contraception use previous use of combined oral contraceptive pills
McGuire et al. (2010) reported that 79% of women who received fertility treatments took
recommended doses of periconceptional FA supplements compared with only 27% of women
who did not receive any fertility treatment. It is remarkable, however, that 21% of women who had
received fertility treatment did not take recommended periconceptional FA supplements. Nilsen
et al. (2006) also found a similar trend in their Norwegian population with women who underwent
ovarian hyperstimulation and in vitro fertilisation being more likely to use periconceptional FA
supplements (16% and 25% respectively compared with 10%). However, again, the overall low
figures in this group are notable (Nilsen et al., 2006).
A high level of awareness and knowledge about the benefits of periconceptional FA

supplementation in NTD prevention does not necessarily result in increased use among women
attending for fertility assessments or treatment.
Frishman et al. (2001) reported on the awareness, knowledge and use of preconceptional FA
among 86 subfertile women attending a reproductive medicine practice in Rhode Island, USA.
Subfertile women were more aware and knowledgeable about FA supplements (65% compared
with 13% in the control group) and used preconceptional FA supplements more often (52%
compared with 29% in the control group). However, 30% of women who were aware of the
benefits of preconceptional FA supplements did not take it. They concluded that, even in this
highly motivated population, only 50% were taking preconceptional FA supplements and
knowledge alone did not ensure use (Frishman et al., 2001).
A study of 400 subfertile women attending a reproductive medicine clinic in Ottawa, Canada
found that, although the general awareness regarding periconceptional FA supplementation was
good, there were still significant gaps in knowledge (Vause et al., 2009).
Previous contraception use
There is some evidence to suggest that women who have used the oral contraceptive pill in the
past are more likely to use FA supplements in the periconceptional period (Table 18.7). This
is understandable given that women who plan their pregnancies are much more likely to use
periconceptional FA supplements (and some of the women stopping their combined oral
contraception are likely to be planning a pregnancy). De Walle and De Jong-Van den Berg
(2008) in a previously referred to cross-sectional study from the Netherlands, found that previous
use of the combined oral contraceptive pill was associated with increased periconceptional FA
supplement use.
18.2.8 Factors relating to previous pregnancies
Parity
The evidence regarding the influence of parity on periconceptional FA supplement use

is conflicting. Larger studies have found little influence of parity on periconceptional FA
supplement use. Some smaller studies have found that multiparous women are less likely to use
periconceptional FA supplements (Table 18.8).
McGuire et al. (2010) reported similar rates of periconceptional FA supplement use among
nulliparous women (27%) compared with multiparous women (28%). Nilsen et al. (2006) found
similar rates of periconceptional FA supplement use among nulliparous women and women with
a single previous delivery (11%). However, this rate dropped to 8% among women with 2 previous
deliveries and 7% among women with 3 or more previous deliveries, although the difference was
marginal. Women who used periconceptional FA supplements in a previous pregnancy were more
likely to use supplements on a subsequent pregnancy (De Walle and De Jong-Van den Berg, 2008).
Previous neural tube defects affected pregnancy
Higher doses of periconceptional FA supplements are recommended in women who have had a
previous pregnancy affected by a NTD or women with a strong family history of a NTD (Table
18.8). A number of studies have examined the impact of having a previous pregnancy affected by
a NTD on subsequent periconceptional FA supplement use (although these studies are somewhat
dated ranging from 1999 to 2002).
While there appears to be an increased awareness and use of periconceptional FA supplements

in subsequent pregnancies, the overall rate appears to be disappointingly low. In addition, those
women who take periconceptional FA supplements appear to be doing so at the standard doses
rather than the recommended higher doses.
A study from Texas, USA reported that only 33% of 195 women who previously had a baby with a
NTD took regular FA supplements (compared with 25% of women without history of a baby with
a NTD). Interestingly, this study also found that only 56% of these high-risk women could recall
discharge advice regarding future FA supplementation suggesting greater vigilance by healthcare
professionals may be required (Canfield et al., 2002).
Table 18.8. Factors related to previous pregnancies affecting use of periconceptional folic acid supplementation.
Parity nulliparous women (in some studies)

Previous NTD affected pregnancy women with a history of a neural tube defect affected pregnancy
Previous early pregnancy loss women with a history of early pregnancy loss
Previous early pregnancy loss
Women who have suffered a miscarriage are more likely to use periconceptional FA supplements
on a subsequent pregnancy (Table 18.8). In separate studies, Tamim et al. (2009) (Lebanon) and
Timmermans et al. (2008) (the Netherlands) reported that women with a previous spontaneous
miscarriage were more likely to use recommended periconceptional FA supplements.
Previous stillbirth
History of a previous stillbirth does not influence periconceptional FA supplement use, although
the evidence is limited (Table 18.8). Nilsen et al. (2006) reported similar rates of periconceptional
FA supplement use among women with no history of stillbirth (but who had a previous delivery)
compared with women with a single previous stillbirth and women with more than 1 previous
stillbirth (10%).
18.2.9 Factors relating to a current pregnancy
Pregnancy planning
While the rates of reported unplanned pregnancies varies considerably from country to country,
the evidence is universally emphatic women with planned pregnancies are far more likely to
use periconceptional FA supplements (Table 18.9).
A recently published large study from the USA assessed the relationship between pregnancy
intention and preconception health behaviours including FA supplement use among 35,351
non-pregnant women of reproductive age (18 to 45 years). They found that women intending
pregnancy in the following 12 months were more likely to take FA supplements compared
with women not intending future pregnancy. Women intending pregnancy after 12 months or
ambivalent about future pregnancy were no more likely to be engaging in healthy preconception
behaviours than women not intending future pregnancy (Chuang et al., 2011).
This trend is consistently repeated throughout the literature. McGuire et al. (2010) reported that
only 3% of women with unplanned pregnancies used periconceptional FA supplements compared
Table 18.9. Factors related to a current pregnancy affecting use of periconceptional folic acid supplementation.
Pregnancy planning women who plan the pregnancy

Gestational age at booking women who book at earlier gestations
Type of antenatal care women using private health insurance
with 41% of women with planned pregnancies. Nilsen et al. (2006) also reported that only 4% of
women with unplanned pregnancies took periconceptional FA supplements compared with 12%
of women with planned pregnancies.
Equally, it is worth reflecting on the fact the majority of women with planned pregnancies do not
take recommended FA supplements. A Swedish study on 270 women attending antenatal clinics
assessed pregnancy planning found that, although 75% were very well or rather well planned,
only 20% took recommended periconceptional FA supplements (Tyden et al., 2011).
Gestation booking for antenatal care
Women booking at earlier gestations are more likely to use periconceptional FA supplements (Table
18.9). In addition, the later the booking visit, the less likely they are to use supplements. McGuire
et al. (2010) reported that 45% of women who booked before 12 weeks took periconceptional FA
supplements compared with only 29% at 12 to 20 weeks and 11% after 20 weeks.
Type of antenatal care
Women who use private health care antenatal services are more likely to use periconceptional
FA supplements, though the evidence is limited (Table 18.9). McGuire et al. (2010) found that
private patients were more likely to use FA supplements (65% of private patients compared with
14% of public patients) in an Irish context.
18.2.10 Other medical conditions
Certain medical conditions are associated with an increased need for FA use, particularly obesity,
diabetes mellitus and epilepsy (especially if on anti-epileptics). There is some evidence to suggest
that periconceptional FA supplement use is slightly better in women with certain chronic diseases.
Nilsen et al. (2006) reported that, in general, women who had more than one chronic disease
used periconceptional FA supplements more often (increasing rates from 10% in healthy women
to 11 to 18% among women with more than one chronic disease). Interestingly, epilepsy was not
associated with a significantly increased use of periconceptional FA supplement use in this study.
NICE guidance (2008) recommends the higher dose of FA (5 mg/day) for women with diabetes
because of the increased risk of congenital abnormalities including NTDs. This recommendation
was based on the Guideline Development Groups opinion.
18.2.11 Factors related to access to folic acid supplements
Poor rates of periconceptional FA use may result from difficulties in accessing or obtaining FA
supplements. These difficulties may arise from poor socioeconomic circumstances (i.e. unable to
purchase FA supplements) or, particularly in the developing world, may not be available locally.
Studies from developing countries have consistently shown poor rates of periconceptional
FA supplement use. Making FA supplements easily available (e.g. free of charge) improves
periconceptional FA supplement use (Stockley and Lund, 2008).
18.2.12 Factors related to health promotion strategies
Individual health promotion campaigns within various countries have been used with varying
success. Stockley and Lund (2008) found that integrated health promotion campaigns could
improve the use of FA supplements. However, printed resources and the mass media (in isolation)
are not effective in the long-term. Health-care based initiatives can be effective and more likely
to be successful if they include making supplements easily available.
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19. Lower fertility associated with
periconceptional obesity and underweight
E. Derbyshire
School of Healthcare Science, Manchester Metropolitan University, John Dalton East Building,
Oxford Road, Manchester M1 5GD, United Kingdom; e.derbyshire@mmu.ac.uk
Abstract
It is well documented that obesity rates are rising worldwide, but equally, we must not overlook
the fact that thinness can also impact on a womans fertility. There has been an emergence
of information linking raised body mass index to reduced oocyte and endometrium quality,
impaired ovulation, implantation, insulin resistance, hyperandrogenism and altered levels of
insulin growth factors, cytokines and leptin; all of which may reduce fertility. With regard to
lower body weight, energy balance is reciprocally linked to reproduction, through the actions of
the hypothalamic-pituitary-gonadotrophin axis and reduced levels of fertility hormones. There
is also evidence linking high and low body weights (generally following a U-shaped curve) to
increased time-to-pregnancy. Certain dietary and lifestyle practices, including smoking, coffee
and soda drinking may also feed in here. In terms of the success of reproductive technologies,
there is some information suggesting that obesity leads to the worst outcomes following treatment
using assisted reproductive technologies, including lower pregnancy and live birth rates and a
higher miscarriage rate (although conflicting meta-analyses have been published). Comparatively
fewer studies, however, have studied how underweight can affect the successful of reproductive
technologies, although the research that has been published to date will be outlined in this review.
Keywords: body weight, time-to-pregnancy, conception, oocyte quality, miscarriage
313
E. Derbyshire
Summary points
Both rising body weights and social pressures to attain lower weight thresholds can lead to women feeling
anxious about their body size.
Obesity and underweight can impact on fertility, health in pregnancy and pregnancy outcomes.
Underweight is thought to impact on fertility by reducing levels of oxidisable fuel, which in turn leads to
changes in the hypothalamic-pituitary-gonadotrophin axis, including the secretion of key fertility hormones
(also known as the metabolic fuel hypothesis).
High intensity and frequencies of physical activity could also lead to menstrual dysfunction, impacting on a
womans fertility.
Time-to-pregnancy may be longer at the extremes of body mass index (following a U-shaped curve),
particularly when coupled with a multitude of unfavourable lifestyle factors (smoking, coffee drinking, high
body mass index, socially deprived).
There is some (weak) evidence that underweight can affect ooctye quality and retrieval when women are
undergoing fertility treatments.
Overall, there appears to be inconsistent evidence that over and underweight can impact on the success of
fertility treatments.
19. Lower fertility associated with periconceptional obesity and underweight
Abbreviations
ART Assisted reproductive technologies
BMI Body mass index
FR Fecundability ratio
FET Frozen-thawed embryo
GnRH Gonadotrophin-releasing hormone
HPG Hypothalamic-pituitary-gonadal axis
ISCI Intra cytoplasmic sperm injection
IVF In vitro fertilisation
LH Luteinising hormone
TTP Time-to-pregnancy
WHO World Health Organisation
WHR Waist-to-hip ratio
19.1 Introduction
Unfortunately, not all couples fall pregnant quickly and there is now an emergence of new
research suggesting that body weight may have a role to play. This problem does not only appear
to be confined to developed regions as evidence from newly industrialised countries indicates
that similar problems are arising. Take China, for example. Here rising rates of overweight are
increasingly being linked to reduced success of fertility treatments (both IVF and ICSI) and at
lower BMI cut-offs than seen in other study populations (Li et al., 2010). Equally the effects of
Americanisation in these regions may also be contributing to eating disturbances, disordered
attitudes and behaviours in this population group; a result of increased desire for thinness. In
Japan, there has been as increased incidence of low bodyweight amongst women of childbearing
age, and more women are reporting consumption of a low energy diet (Hayashi et al., 2006).
We can also see how rates of obesity and underweight amongst females vary globally using data
from the World Health Organisation and International Body Weight Classification Scale (Table
19.1). To determine this scale the weight-for-height BMI index is applied. This is defined as body
weight in kilograms divided by the square of the height in metres (kg/m2). For example, a female
who weighs 54 kg and whose height is 1.7 m will have a BMI of 1.74: 54 kg / (1.75 m2) = 54 /3.1
= 17.4 (categorised as underweight). This index serves as a useful guide, although there has been
some dispute about its accuracy, particularly if self-reported (Stommel and Schoenborn, 2009).
In terms of obesity (BMI30), it can be seen that around 43.8% Saudi women are obese, compared
with 30% in the UK and just 12% in Sweden. The number of women classified as underweight
(BMI<18.5) also varies globally with around 35.6% of women living in India falling into this
E. Derbyshire
Table 19.1. Adult international classification of underweight, overweight and obesity according to body mass
index (World Health Organisation, 2012).
Classification Body mass index (kg/m2)
Principal cut-off points Additional cut-off points
Underweight <18.50 <18.50

Severe thinness <16.00 <16.00
Moderate thinness 16.00-16.99 16.00-16.99
Mild thinness 17.00-18.49 17.00-18.49
Normal range 18.50-24.99 18.50-22.99
23.00-24.99
Overweight 25.00 25.00
Pre-obese 25.00-29.99 25.00-27.49
27.50-29.99
Obese 30.00 30.00
Obese class I 30.00-34.99 30.00-32.49
32.50-34.99
Obese class II 35.00-39.99 35.00-37.49
37.50-39.99
Obese class III 40.00 40.00
category, compared with just 5.9% in the United Kingdom (WHO, 2012). It is important to
consider how body weight varies globally, which is largely determined by the environment in
which that person is living, but equally, an individuals genetic makeup must also be considered.
The health risks of obesity and underweight in pregnancy are well documented, in terms of the
risks this can pose to the baby (Derbyshire, 2011). However, the importance of getting body
weight right before becoming pregnant seems to be a key health recommendation that has been
overlooked until recently. In the UK, the National Institute for Health and Clinical Excellence
(NICE) published a report recommending that women should aim to achieve a body weight
within healthy ranges before conceiving. In particular, health professionals are advised to help and
encourage women with a BMI of 30 or more to reduce their weight before becoming pregnant.
In their report it was recommended that health professionals should explain that losing 5-10%
of excess weight could have significant health benefits for both mother and child. It was also
recommended that further weight loss, to achieve a BMI within the healthy range (between 24.9
and 18.5 kg/m2) should also be encouraged, using evidence-based behaviour change techniques
(NICE, 2010). Unfortunately, this report let to a lot of debate, with many women not fully
understanding the benefits of achieving a healthy weight before falling pregnant, or how this
may impact on their fertility.
This chapter first sets out to explain how obesity and underweight can both impact on fertility
from a physiological perspective. The second part of the chapter outlines how a womens body
weight may impact on her TTP and, finally, the third section of the chapter goes on to describe
how obesity and underweight can both influence the success of fertility treatments.
19.2 Lower fertility and body weight

19.2.1 Overweight
Firstly, with regard to obesity the causes of reduced fertility are very different to when a
woman is underweight. For example, obesity increases features of PCOS and impairs insulin
resistance, further exacerbating the metabolic and reproductive features of PCOS which include
hyperandrogenism and subfertility (Motta, 2012). Obesity may also affect a womans oocyte (egg)
quality. Research indicates that raised levels of insulin, glucose, free fatty acids and changes in
adipokine levels may all impact on oocyte maturation, both independently and in combination
(Purcell and Moley, 2011). Analysis of ovarian cells also shows that lipid accumulation, higher
rates of oxidative stress and systemic inflammation can also be activated in the ovary, lowering
fertility (Robker et al., 2011).
Obesity can also lead to changes in a womans hormone milieu. The combined effects of
underpinning genetics, increased food intake and a sedentary lifestyle leads increased levels
of adipocytes (fat cells). Adipose tissue, often thought to be a redundant fat mass is actually a
complex and highly active endocrine organ, secreting hormones including leptin, in addition
to hormones and cytokines as outlined in Figure 19.1. Leptin, in particular, has been linked to
reduced fecundity, namely by acting on the HPG axis, which is a common cause of oligoovulation
and anovulation (Brewer and Balen, 2010). There is also emerging evidence that normal levels
of adipose tissue adipokines are needed for HPG function, regular ovulation and embryo
implantation, but more research is needed to understand how these may exert their effects
(Tersigni et al., 2011). Interestingly, studies using animal models have identified that even
paternal obesity could impair the likelihood of implantation, reducing the chances of womans
pregnancy (Mitchell et al., 2011).
19.2.2 Underweight
From an energy and physiological perspective pregnancy and lactation are regarded as being
the most demanding phases of the female lifecycle. Pregnancy requires around 50,000 calories
over and above normal metabolic requirements whilst lactation requires around an extra 500
to 1000 calories a day (Hytten and Chamberlain, 1991). The nature of the relationship between
bodyweight and the capacity to conceive appear to follow a U shape curve, with women in
the healthy weight ranges (Table 19.1) having the best chances of conception (Davies, 2006).
As we will see in this section, the physiological mechanisms that regulate a womans fertility
(particularly in the case of underweight) are closely linked to those that control energy balance.
E. Derbyshire
Hypothalamus
Genetic Food intake
factors Exercise Altered
gonadotrophin
secretion
Adipocytes Leptin Pituitary
Insulin LH
IR
Pancreas
Ovary
SHBG
Liver
Testosterone
Oocyte Oestrogen Endometrium
Progesterone
Hormones and chemokines effected
obesity:
Oestrogens
Androgens Fertilisation Embryo Receptivity
SHBG
Insulin
LH Clinical pregnancy
Leptin rate
Adiponectin Pregnancy Miscarriage
IL6 Live birth rate
PAI1
TNF
Figure 19.1. Effects of obesity on the hypothalamic-pituitary-gonadal axis and reproductive potential (Brewer
and Balen, 2010, reprinted with permission).
IL6 = interleukin-6; IR = insulin resistance; PAI1 = plasminogen activator inhibitor type-1; SHBG = sex hormone-
binding globulin; TNF = tumour necrosis factor-.
Rose Frisch from the Harvard Centre of Population Studies was one of the first scientists to link
excessive dieting and exercise to amenorrhoea. It was proposed that a certain level of body fat
was needed to convert androgens to oestrogen, as well as acting as a storage site for hormones. It
was also though that body fatness may influence the direction of oestrogen metabolism to more
potent or less potent forms; leaner women make more catechol oestrogens. Taking these points
into consideration, it was concluded that a minimum ratio of fat to lean mass was needed for
menarche (around 17% body fat) and about 22% needed for maintenance of womens reproductive
function (Frisch, 1987). Unfortunately, this theory has now recently been overruled with the
metabolic fuel hypothesis.
19.2.3 Metabolic fuel hypothesis
Scientists have now linked lower fertility to reduced levels of oxidisable fuel (body fat, along
the lines of Rose Frisch), but more specifically relate this to changes in the HPG axis (Mircea et
al., 2007). Limited supplies of body fat leads to the altered secretion of key hormones which are
needed to maintain reproductive function (Mircea, 2007). For example, when levels of oxidisable
fuel are low this is thought to be detected by fuel receptors found at the peripheral (leptin, central,
ghrelin) and central (neuropeptide Y, melanocortin, orexin) levels. These detectors then inhibit
the release of GnRH and LH; key hormones involved in steroidogenesis, reproductive cyclicity
and subsequent fertility (Mircea et al., 2007).
This is clearly shown in Figures 19.2a and 19.2b. When there are appropriate levels of metabolic
fuels (Figure 19.2a), GnRH is released from neurosecretory cells in hypothalamus which, in
turn, stimulates the secretion of LH and then follicle development begins (also stimulated by
the presence of FSH) helping to regulate ovulation. During follicle development low levels of
estrogens have a negative feedback effect on the secretion of GnRH from the hypothalamus
(estrogen exerts stimulatory effects on its own synthesis and secretion). The inhibition of GnRH
then slows and LH production is again stimulated causing ovulation to return.
a b
Abundant The primary effect
availablility of low availability
of oxidizable of oxidizable
metabolic fuels metabolic fuels
Hypothalamic GnRH Hypothalamic GnRH
1. Pulse 4B. 1. Inhibited 4B. No
generator Surge pulses surge
(Posative (No positive

Pituitary gonadotropins feedback) Pituitary gonadotropins feedback)
(E negative 2. LH, FSH 5. LH, (Enchanced E 2. No LH, 5. No
feedback) pulses FSH surge negative FSH release surge
feedback)
Gonadal steroid Gonadal steroid

secretion secretion
3. Estradiol (E) 3. Little estradiol (E)
progesterone and progesterone
synthesis, secretion
Normal bone mass, 6. Ovulation
cognitive function Loss of bone mass, 6. No ovulation
impaired cognitive
4A. Steroid-dependent function (chronic)
reproductive behaviors
4A. No steroid-dependent
reproductive behaviors
Figure 19.2. The effects of (a) abundant and (b) low oxidizable fuels on fertility. Low levels of metabolic fuel
leads, ultimately, through inhibition of the gonadotrophin-releasing hormone (GnRH) pulse generator which in
turn leads to reduced follicle development, low levels of estrogen and/or progesterone secretion and failure to
generate a luteinizing hormone (LH) surge thus impacting on fertility (Schneider, 2004, reprinted with permission).
E. Derbyshire
When levels of metabolic fuel are in deficit, for example, in food-deprived individuals (Figure
19.2b) the GnRH pulse generator is inhibited. This leads to multiple effects including reduced
levels of GnRH secretion, inhibition of follicle development and therefore reduced secretion
of estrogen and progesterone. This ultimately means that there will not be an LH surge and
subsequently fertility is likely to be impaired. The GnRH pulse generator plays a central role in
this pathway and when exogenous treatments are given, subsequent endcrinological pathways
are often restored (Schneider, 2004).
19.3 Physical activity

The effects on energy restriction on fertility are well documented. However, equally increased
energy expenditure can also affect womens reproductive function. In one recent systematic
review, evidence looking at menstrual dysfunction amongst females athletes published over
the last two decades was collated. Scientists identified that that the endocrine role of adipose
tissue appeared to play a key role in the regulation of metabolism and reproduction. It was
found that this could manifest in the form of primary and secondary amenorrhea, luteal phase
deficiency, oligomenorrhea and anovulation, all of which could impact on fertility (Roupas and
Georgopoulos, 2011).
The Norwegian North-Trndelag Health Study has looked at the effect that energy balance and
physical activity could have on the reproductive system. Patterns of physical activity and fertility
levels were assessed using questionnaires completed by a sample of 3,887 women aged <45 years.
Study findings showed that increased frequency, duration and intensity of physical activity were
significantly linked to subfertility and childlessness. After adjusting for confounders (parity,
smoking, marital status) scientists concluded that women who were active on most days were
3.2 times more likely to have fertility problems than inactive women. In terms of level of activity,
exercising to exhaustion was also 2.3 times more likely to increase the odds of women having
fertility problems, compared to women taking part in low intensity activities (Gudmundsdottir
et al., 2009). There are interesting findings and demonstrate that high intensity and frequency
of physical activity could also impact on a womens fertility. More work is needed to establish
whether this is attributed to the level of activity per se or combined effects of low body weight.
These health ramifications may need to be communicated to health professionals who, in turn,
can offer advice to active females experiencing fertility problems.
19.4 Body weight and time-to-pregnancy

There are a host of factors that can affect couples TTP which include diet quality (caffeine, alcohol
and nutrient intakes, exercise levels and smoking (Derbyshire, 2011). However, increasingly
bodyweight is also thought to play a role in the timing and chances of conception.
Epidemiological research shows that a womans fecundability seems to be lower at the extremes
of BMI. Danish scientists have conducted an internet-based cohort study, known as the Snart-
Gravid (Soon Pregnant) study. Scientists recruited 1,651 Danish women (18-40 years) who were
in a stable relationship and planning for pregnancy. Their baseline weight was self-reported in
addition to their retrospective weight at 17 years age. Participants, once recruited, were contacted
by e-mail every 2 months until pregnancy occurred.
Results showed that women who had gained 15 kg since the age of 17 years had longer TTPs
compared with those who maintained a stable weight (-5 to +4 kg). Equally, after adjustment for
BMI the FR (probability of conception) was 1.27 (95% CI: 0.98-1.64) for women with a WHR of
87 compared to 1.44 (95% CI: 0.98-1.64) for those with a WHR <74 cm. Underweight (BMI<20)
was associated with reduced fertility among nulliparous women (FR=0.82, 95% CI: 0.63-1.06)
and this increased amongst parous women (FR=1.61, 95% CI: 1.08-2.39). As shown in Figure
19.3, these findings demonstrate that fecundability was reduced in overweight and obese women
(BMI >30). For women who were underweight, nulliparity rather than multiparity was linked to
a reduced FR and increased TTP (Wise et al., 2010).
Two US studies have also studied links between obesity and TTP/fertility. Using data from the
Collaborative Perinatal Project (based at 12 study centres across the United States) fecundability
was found to be reduced overweight (OR=0.92, 95% CI: 0.84-1.01) and obese (OR=0.82, 95%CI:
0.72-0.95) compared to healthy weight primiparious women (OR 0.66 95% CI: 0.49-0.89) (Gesink
Law et al., 2007). Similarly, in the US National Longitudinal Survey of Youth, obese women were
2.5
2.0
1.5
1.0
Fecundability ratio
Parous
Nulliparous
0.5
0.2
16 18 20 22 24 26 28 30 32 34 36 38
Body mass index (kg/m2)
Figure 19.3. Relationship between womens body mass index and fertility. It can be seen, in particular, how
underweight (body mass index less than 19) links to a reduced fecundability ratio, leading to a longer time-to-
pregnancy. Curves are adjusted for age, partners age, cycle regularity, cycle length, partners body mass index,
physical activity, smoking, alcohol intake, intercourse frequency and waist circumference (Wise et al., 2010,
reprinted with permission).
E. Derbyshire
less likely to have their first child by 47 years and the probability of having more than one child
was lower (Jokela et al., 2008).
Interesting a UK observational study of 2,112 pregnant women retrospectively asked them about
their TTP. TTP was significantly longer if the womans body mass index was >25 kg/m2 (P<0.001).
Couples who had >4 negative lifestyle variables (smoking, coffee drinkers, high BMI, socially
deprived) had a sevenfold longer TTP. The same authors also reported that underweight women
(defined as a BMI<19) had a fourfold longer TTP than women with a normal BMI. Specifically,
women with a low BMI required an average of 29 months to conceive compared to an average
6.8months for women with a normal weight profile (Hassan and Killick, 2004).
19.5 Bodyweight and reproductive technologies

There is a wealth of literature investigating the effect of BMI on the success of reproductive
technologies. A recent systematic review and meta-analysis pooled together the findings from 33
different studies, the equivalent to 47,967 treatment cycles. Results showed that women who were
overweight (defined as a BMI 25) were significantly less likely to fall pregnant clinically or have
a live birth, in addition to having a higher miscarriage rate (Rittenberg et al., 2011).
The British Fertility Society on the basis of such evidence and to promote health in pregnancy
recommends that women attain a healthy body weight before undergoing reproductive
treatments. More specifically, it is advised that obese females lose 5-10% of their body weight
before undergoing fertility treatments, or obtain a BMI <35 kg/m2, or in the case of younger
mothers a BMI <30 kg/m2 (Balen and Anderson, 2007).
However, another recent publication contested links between obesity and unfavourable ART
outcomes. Authors reported that none of the included studies found a positive links between
obesity/overweight and ART complications, e.g. ectopic or multiple pregnancy and that this may
only marginally reduce success rates (Koning et al., 2012). However, the broader long-term effects
of obesity on pregnancy and birth outcomes were not considered in this article.
With regard to underweight and the success of reproductive treatments, seven studies appear to
have investigated this to date (Table 19.2). The findings from these studies are inconsistent with
four reporting that underweight has no significant effects on the success of fertility treatments
and three reporting some implications. Of the studies documenting some effects, a large Danish
study reported that underweight may lead to problems retrieving womens embryos (Pinborg et
al., 2011) and Wittemer et al. (2008) found that underweight could impact on embryo quality. Li
et al. (2010) found that the clinical pregnancy rate was slightly lower for underweight compared
to overweight women, although no other significant findings were identified and Veleva et al.
(2008) found that underweight women had a higher risk of miscarriage after fertility treatments,
compared with healthy weight women. On the whole, further research is needed before it can
be firmly concluded that underweight affects the success of fertility treatments. Taking this on
Table 19.2. Impact of underweight on in vitro fertilisation (IVF) / intra cytoplasmic sperm injection (ICSI)
outcome.
Study Sample population BMI1 Study findings

(kg/m2)
Denmark n=1,417 cycles; <18.5 There was a negative association between

(Pinborg et al., 2011) IVF/ICSI treatment BMI and the number of oocytes retrieved
(P<0.01). Less embryos were available from
underweight women (P=0.03).
Canada n=116 cycles; <18.5 There was no difference in the endometrial
(Shalom-Paz et al., 2011) IVF treatment thickness, rates of implantation or
pregnancy between BMI. Pregnancy rate for
underweight women was 50% compared
with 47.9% for normal weight and 27.2%
for obese women.
China n=1,107 women; <18.5 Underweight women showed no differences
(Li et al., 2010) IVF/ICSI treatment in ovarian stimulation and IVF outcome;
although their clinical pregnancy rate was
lower (31% vs. 37% in overweight women).
Finland n=3,330 cycles; <20 or The relationship between BMI and
(Veleva et al., 2008) IVF/ICSI/frozen <18.5 miscarriage was U-shaped (P=0.01).
thawed embryo Underweight women were at a higher risk
treatment of miscarriage than women with normal
weight.
Norway n=5,019; <18.5 Underweight was not related to any impaired
(Fedorcsak et al., 2004) IVF/ICSI treatments IVF or ICSI outcomes.
France n=398 couples; <20 Certain IVF parameters were affected but
(Wittemer et al., 2000) IVF/ICSI treatments bodyweight did seem to impact on oocyte
quality. When BMI value was <20 kg/m2,
64.2% of the retrieved oocytes were of
good quality whilst for a BMI 20-25 kg/m2,
66.7% of the retrieved oocytes were of
good quality.
UK n=333, IVF patients <19 The dose of gonatotrophin, number of
(Lashen et al., 1999) eggs, fertilisation rate, serum oestradiol
concentration, human chorionic
gonadotrophin concentration, implantation
or miscarriage rate was not significantly
different between underweight and normal
controls.
1 BMI = body mass index.
E. Derbyshire
board, as with overweight one can question whether it is morally, ethically and scientifically fair to
exclude women from access to fertility treatments given that the evidence-base is not consistently
conclusive.
19.6 The way forward

On the whole, there is good evidence to suggest that high and low body weights can impact on
fertility and time-to-pregnancy, as well as womens health in pregnancy and the health of the
offspring. It seems that the public could benefit from health strategies to promote the importance
of attaining a healthy weight before falling pregnant, as these seem to be areas that are overlooked.
When it comes to the success of fertility treatments, it seems somewhat unjust that these are
restricted for women with a high or low BMI (as they are currently in the UK) as the scientific
evidence in relation to the success of these is inconsistent and somewhat lacking. However, it
makes sense that women should set out to achieve a healthy body weight in order to maintain
good health should they fall pregnant. Taking the evidence on board, greater emphasis should be
placed on these messages, rather than portraying that fertility treatments are less likely to work
in these population groups.
References
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Brewer, C.J. and Balen, A.H., 2010. The adverse effects of obesity on conception and implantation. Reproduction
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Gudmundsdottir, S.L., Flanders, W.D. and Augestad, L.B., 2009. Physical activity and fertility in women: the North-
Trondelag Health Study. Human Reproduction 24, 3196-3204.
Hassan, M.A. and Killick, S.R., 2004. Negative lifestyle is associated with a significant reduction in fecundity.
Hayashi, F., Takimoto, H., Yoshita, K. and Yoshiike, N., 2006. Perceived body size and desire for thinness of young
Japanese women: a population-based survey. British Journal of Nutrition 96, 1154-1162.
Hytten, F. and Chamberlain, G., 1991. Clinical Physiology in Obstetrics. Blackwell Scientific Publications, Oxford,
UK.
Jokela, M., Elovainio, M. and Kivimaki, M., 2008. Lower fertility associated with obesity and underweight: the US
National Longitudinal Survey of Youth. American Journal of Clinical Nutrition 88, 886-893.
Koning, A.M., Mutsaerts, M.A., Kuchenbecher, W.K., Broekmans, F.J., Land, J.A., Mol, B.W. and Hoek, A., 2012.
Complications and outcome of assisted reproduction technologies in overweight and obese women. Human
Lashen, H., Ledger, W., Bernal, A.L. and Barlow, D., 1999. Extremes of body mass do not adversely affect the
outcome of superovulation and in-vitro fertilization. Human Reproduction 14, 712-715.
Li, Y., Yang, D. and Zhang, Q., 2010. Impact of overweight and underweight on IVF treatment in Chinese women.
Gynecological Endocrinology 26, 416-422.
Mircea, C.N., Lujan, M.E. and Pierson, R.A., 2007. Metabolic fuel and clinical implications for female reproduction.
Journal of Obstetrics and Gynaecology Canada 29, 887-902.
Mitchell, M., Bakos, H.W. and Lane, M., 2011. Paternal diet-induced obesity impairs embryo development and
implantation in the mouse. Fertility and Sterility 95, 1349-1353.
Motta, A.B., 2012. The role of obesity in the development of polycystic ovary syndrome. Current Pharmaceutical
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Pinborg, A., Gaarslev, C., Hougaard, C.O., Nyboe Andersen, A., Andersen, P.K., Boivin, J. and Schmidt, L., 2011.
Influence of female bodyweight on IVF outcome: a longitudinal multicentre cohort study of 487 infertile
couples. Reproductive BioMedicine Online 23, 490-499.
Purcell, S.H. and Moley, K.H., 2011. The impact of obesity on egg quality. Journal of Assisted Reproduction and
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Schneider, J.E., 2004. Energy balance and reproduction. Physiology and Behavior 81, 289-317.
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bmi/index.jsp?introPage=intro_3.html.
20. Preconception care and barriers to addressing
overweight and obesity: a focus on weight
loss advice and weight loss strategies
M. Dekker Nitert1, H.L. Barrett1, S. de Jersey2, K. Matusiak1, H.D. McIntyre3 and L.K. Callaway1
1University of Queensland, School of Medicine, Royal Brisbane and Womens Hospital, Butterfield
Street, Herston Qld 4029, Australia; 2Department of Nutrition and Dietetics, Royal Brisbane
and Womens Hospital, James Mayne Building, Butterfield Street, Herston Qld 4029, Australia;
3Mater Health Services, MMH Annexe, Raymond Terrace, South Brisbane Qld 4101, Australia;
m.dekker@uq.edu.au
Abstract
Obesity in women of childbearing age is on the rise. Obesity in pregnancy is associated with
an increased risk of pregnancy complications for the mother, and macrosomia for the baby.
Reducing maternal weight prior to pregnancy is difficult to achieve, but reduces the risk for
these complications. Diet and lifestyle interventions are usually successful only in the short
term. Bariatric surgery has proved to be successful in weight loss prior to pregnancy although
it is recommended that maternal weight is stable for more than six months prior to conception.
Preconception care visits are opportunities to make women aware of the risks of their body
weight on complications of pregnancy, to provide them with accurate information about their
body weight and successful weight loss strategies. Current preconception care varies widely in
content and health care provider and is currently attracting women higher education levels and
a higher disposable income than the general obstetric population. Future preconception care
strategies should focus on alternative ways of providing all women with information about the
risks of obesity on pregnancy complications but ensure that women with lower levels of education
are especially catered for.
Keywords: obesity, pregnancy, preconception care, weight loss
327
M. Dekker Nitert et al.
Summary points
Maternal obesity is increasingly common and associated with difficulties with conception, and maternal and
neonatal complications in pregnancy.
Weight loss interventions have proven difficult to implement.
Preconception weight loss reduces pregnancy complications but little is known about the longer term impact
on the infant.
Further studies are required:
To examine the comparative benefits and risks of maternal weight loss at different stages: preconception,
periconception and during pregnancy.
To examine the impact of preconception weight loss on pregnancy weight gain and implications of this.
To assess longer term infant outcomes following preconceptual weight loss.
20. Preconception care and barriers to addressing overweight and obesity
Abbreviations
BMI Body mass index
GDM Gestational diabetes mellitus
LAGB Laparoscopic adjustable gastric band
20.1 Introduction
In this chapter, we will provide an overview of the importance of obesity prior to pregnancy and
the evidence relating to the maternal and neonatal benefits of weight loss prior to pregnancy. We
will then examine the barriers to addressing this prior to pregnancy. We will identify areas that
require further research, and potential opportunities that could be maximized.
20.2 Obesity in women of childbearing age

Obesity and overweight are a major modifiable risk factor for adverse pregnancy outcomes
(Johnson et al., 2006) and are regarded as the most common clinical risk factor for the development
of high risk and complicated pregnancies (Callaway et al., 2006; Wolfe, 1998).
In line with the age and gender adjusted general population prevalence, around one third women
of childbearing age in the USA and Australia are overweight and obese (Callaway et al., 2006).
The rates in Europe vary from 6.2 to 36% with women in Eastern Europe and the Mediterranean
countries having higher rates of obesity (Berghofer et al., 2008).
20.2.1 Obesity is associated with reduced fertility
Obesity is associated with reduced fertility, which appears to be due to an increased risk of ovulatory
infertility (Maheshwari et al., 2007). Obesity is also associated with PCOS, which is a common
cause of anovulatory infertility. Whether obesity is a cause or a consequence of PCOS remains
contentious. However, compared with normal weight women with PCOS, those with obesity have
less regular menses, poorer ovulatory rates and lower pregnancy rates. Maternal obesity has also
been reported to be associated with a longer time between onset of unprotected intercourse and
conception with overweight women taking a median 1 month longer to become pregnant and
obese women a median of 2 months longer than normal weight women. In a prospective study
of 500 women with subfertile or infertile partners requiring donor sperm insemination, only 4
of 22 obese women (18%) conceived within one year of treatment, in comparison to 45% of the
overall group (Zaadstra et al., 1993).
20.2.2 Maternal and neonatal outcomes and weight loss prior to pregnancy
Weight loss in non-pregnant overweight and obese adults is associated with positive health
outcomes, and has been widely endorsed. Although there is no randomized controlled trial of
weight loss prior to pregnancy, accumulated data is highly supportive of the idea that weight loss
prior to pregnancy can improve pregnancy outcomes (Figure 20.1). In a population based cohort
study, modest amounts of weight loss prior to pregnancy reduced the risk of gestational diabetes
(Glazer et al., 2004). Also, morbidly obese women, who have LAGB surgery and lose weight
prior to pregnancy improve their pregnancy outcomes (Dixon et al., 2005). However, metabolic
stability after the LAGB procedure should be achieved prior to conception.
In a case control study, 79 women who underwent LAGB placement (mean BMI prior to
procedure 45.9 kg/m2, with a mean weight loss prior to pregnancy of 28.3 kg) were compared to
matched obese controls (mean BMI 43.7 kg/m2). The obese controls had a 19% rate of gestational
diabetes and 38% experienced hypertensive disorders of pregnancy. In contrast the women post
LAGB had a markedly decreased gestational diabetes rate (6.3%) and experienced hypertensive
disorders of pregnancy (10%). These rates were very similar to the complication rates found in
the normal population where this study was conducted (Dixon et al., 2005).
20.2.3 Adverse outcomes of pre-pregnancy weight loss
Are there any potential adverse outcomes associated with pre-pregnancy weight loss? Pre-
pregnancy and early pregnancy dieting might be associated with folic acid deficiency or ketone
body formation, which could potentially contribute to fetal abnormalities. However, it appears
that the risk of neural tube defects is actually lower in obese women who diet prior to or in early
pregnancy (Shaw et al., 1996). At present, there is no clear evidence of harm from weight loss
through standard lifestyle measures prior to pregnancy, although this needs to be confirmed by
well-designed interventional trials (Table 20.1).
Preconception
weight loss
Subsequent
pregnancy
GDM Preterm birth

Pregnancy-induced Large for gestational age
hypertension Small for gestational age
Mother Baby
Figure 20.1. Positive effects of preconception weight loss. Preconception weight loss has positive effects on
reducing complications of pregnancy for mother and baby.
Table 20.1. Adverse outcomes of pre-pregnancy weight loss.
Publication Species Weight loss period1 Adverse outcome
Zhang et al., 2010 Sheep Periconception Increased adrenal mass in newborn lambs
Increased cortisol stress response in females at
4months of age
Heijmans et al., 2008 Human Early/late gestation Increased obesity
Increased metabolic and cardiovascular disease
Increased mental health disorders
Edwards and McMillen, Sheep Periconception Increased arterial blood pressure and rate
2002 pressure product
Joshi et al., 2003 Rat Preconception Reduced vital organ growth
Zncreased blood glucose and cholesterol in
adult offspring
Watkins et al., 2008 Mouse Preconception Increased systolic blood pressure
Blunted vascular responsiveness
1 Weight loss period refers to the period during which the mothers was exposed to reduced food intake:
periconception includes period prior to conception, conception and implantation; early gestation includes first
trimester and late gestation includes third trimester of pregnancy.
From animal studies, there is a growing body of evidence that undernutrition in the periconceptional
period implicates changes to the hypothalamic pituitary adrenal axis, adult glucose control and
cardiovascular changes. In particular, dietary restriction of normal and overweight ewes in the
periconceptional period results in increased adrenal mass in both male and female lambs as well
as an increased cortisol stress response in females at 4 months of age. These changes appear to
be caused by epigenetic modifications of the maternally imprinted gene IGF2 in the adrenal and
suggest that it is the maternal metabolic response to dietary restriction in the periconceptional
period and not maternal body weight per se at conception that significantly determines offspring
hypothalamic pituitary axis outcomes (Zhang et al., 2010). Epigenetic modifications in IGF2 have
also been observed in humans who were prenatally exposed to famine during the Dutch Hunger
Winter, with the effects persisting 6 decades later (Heijmans et al., 2008). Exposed individuals
have higher rates of obesity, metabolic and cardiovascular disorders, but also of mental health
problems than their sex-matched siblings. Periconceptional weight loss has been shown to have
both positive and negative effects on the glucose tolerance in offspring in well-controlled animal
studies. Female offspring of obese ewes who were dietary restricted in the month prior to and one
week post-conception have a lower total fat mass and this may result in improved adult glucose
tolerance (Rattanatray et al., 2010). However, research in normal weight ewes has demonstrated
that moderate periconceptional undernutrition impairs glucose tolerance in the offspring (Todd
et al., 2009), as well as causes an increased arterial blood pressure and rate pressure product in
twin fetal sheep in late gestation independent of food quantity during gestation (Edwards and
McMillen, 2002). Similarly, undernutrition of rat dams for 8 weeks prior to conception affects
the growth of vital organs and results in increased blood glucose and cholesterol concentrations
in the adult offspring (Joshi et al., 2003). Further, low protein diet during the oocyte maturation
stage in mice resulted in elevated systolic blood pressure and attenuated vascular responsiveness
compared to controls (Watkins et al., 2008).
In neither humans nor animals, have there been detailed studies of the relationship between
pre-pregnancy weight loss and subsequent weight gain during pregnancy. Pregnancy outcomes
from this scenario have not been investigated. In contrast, there is evidence that modest amounts
of weight loss between pregnancies can reduce the risk of gestational diabetes in the subsequent
pregnancy (Glazer et al., 2004). Modest increases in BMI, even in the normal weight range
can increase the risk of maternal, intrapartum, fetal and neonatal complications. Therefore,
obese women should be advised about the need to continue to lose weight prior to any future
pregnancies, and also for long term good health. Ideally, for preconception and interpregnancy
weight loss to succeed, a combination of nutrition counseling, exercise programs and weight
management consultation would be available prior to attempting another pregnancy.
Recent recommendations to improve preconception care have emphasized the importance of

addressing overweight and obesity in women of childbearing age, and particularly at the pre-
pregnancy health check (Johnson et al., 2006).
20.3 Preconception care

Preconception care is not a new concept; the importance of a healthy mother for good pregnancy
outcomes has been described for hundreds of years (Atrash et al., 2008). Since organogenesis
occurs very early in pregnancy, prenatal interventions usually materialize too late to forestall
preventable malformations. Therefore, preconception care can be beneficial and should perhaps
not be limited to a specific consultation with a health care professional but rather be part of
continuing care for women in the reproductive age-span (Atrash et al., 2008). The Centre for
Disease Control convened a Select Panel on Preconception Care which defined preconception
care as interventions that aim to identify and modify biomedical, behavioral, and social risks
to a womans health or pregnancy outcome through prevention and management, emphasizing
those factors which must be acted on before conception or early in pregnancy to have maximal
impact (Atrash et al., 2008).
20.3.1 Preconception care, behavior change and weight loss
In contrast to the general population, there is some evidence that access to preconception
counseling in women with pre-gestational diabetes is associated with behavior change and better
pregnancy outcomes (Willhoite et al., 1993). In addition, in non-pregnant adults weight loss
advice by a primary health care provider is associated with weight loss attempts. A randomized
trial of an obesity and weight gain prevention program targeting women 18-28 years of age found
the intervention reduced their body weight, BMI, waist circumference and waist-hip ratio (Eiben
and Lissner, 2006). This intervention provided an individualized program targeting physical
activity, diet and weight control with an emphasis on empowering participants to develop
strategies for overcoming barriers (Eiben and Lissner, 2006). The use of appropriate behavior
modification techniques rather than simply focusing on education may have contributed to the
success of this one year program. Although long term weight loss and weight maintenance are
very difficult to achieve, short term weight loss is achievable. It could be argued that the goal of
a healthy pregnancy and healthy baby is highly motivating and that weight loss attempts prior to
pregnancy therefore are more likely to succeed than at other times, although at present, there is
no data to support this.
20.3.2 Success of weight loss strategies prior to pregnancy
Many overweight and obese women attempt to lose weight prior to pregnancy according to
their self-reports (Callaway et al., 2009b). In fact, a survey study performed in Brisbane showed
that over half (55.6%) of women with a BMI>25 had tried to lose weight and that 45% actually
had lost weight prior to pregnancy. Indeed, there was a positive correlation between weight
loss attempts and BMI, e.g. there were more weight loss attempts with increasing BMI. There is
evidence that in subfertile women, small amounts of weight loss (5-10%) result in improvement
in conception rates achieved either naturally or with assisted reproductive techniques (Clark et
al., 1995). Therefore, a key strategy is to ensure that women who are presenting with difficulty
with conception are supported in weight loss efforts.
Current guidelines (NICE, 2010) recommend weight loss in overweight and obese women
preceding natural or assisted reproduction. However, high quality evidence regarding strategies
and timing of weight loss interventions are lacking.
20.3.3 General advice about preconception weight loss
The literature is filled with controversy around the type of strategies that are most effective for
weight loss. In essence, most strategies are characterized by short term weight loss, followed by
longer term weight regain (Figure 20.2). The difficulty with this in the preconception period is
that the period of weight regain may well coincide with pregnancy. At present, there is no good
data assessing weight regain in pregnancy following preconception weight loss.
Lifestyle intervention
In general, most approaches with short term success combine diet and exercise. Some include
psychological approaches which may assist in obtaining a successful outcome. In overweight
and obese women presenting with subfertility, a 6-month program of weekly sessions resulted
in an average weight loss of 10.2 kg and improved pregnancy rates. Each session consisted of 1
h of customized exercise and 1 h of group counseling seminar on weight loss issues such as diet,
physical activity and problem-solving as well as encouraging a further two sessions of exercise
Exercise
Diet: quantity Bariatric
surgery
Preconception
weight loss
Diet: composition Pharmacology

changes
Figure 20.2. Strategies for preconception weight loss. Multiple strategies are effective at reducing weight prior
to pregnancy.
per week (Clark et al., 1998). Changing the behavior of health care professionals, or changing
the organization of care has not been shown to make a difference (Flodgren et al., 2010). Further,
there is evidence that brief interventions in primary care can assist people to lose small amounts
of weight (Yanovski, 2011).
Diet composition and quantity
Dietary treatment of obesity aims to shift the balance from caloric intake to caloric expenditure.
In the context of preconception, there are no clear answers to the questions regarding the relative
strengths of any particular dietary prescription, such as low carbohydrate vs. Mediterranean vs.
low glycemic index vs. low fat. Dietary guidelines for women prior to conception are based on
general adult dietary guidelines. It is probably wise to tailor the dietary approach according to
patient preference.
Very low energy diets (typically 450-600 kCal) result in rapid weight loss, and are superior to other
dietary and drug strategies. These strategies are most suitable for obese individuals, and should
be undertaken with medical supervision and appropriate dietary counseling. These approaches
are contraindicated during pregnancy and lactation. Therefore, effective contraception is required
while undertaking these types of diets. Further, we believe it is prudent to undertake radical
approaches such as this well in advance of pregnancy, to allow a period of weight stabilization.
Pharmacological therapy
Pharmacological therapy is considered an adjunctive therapy to improve weight loss and long-
term maintenance. Sibutramine, rimonobant, and orlistat are approved drugs for the treatment
of obesity that result in greater weight loss than diet alone. However these drugs are not
recommended for women trying to conceive. There is some evidence that insulin-sensitizing
drugs (such as metformin), which are considered safe to use during pregnancy, may be effective
for weight loss in overweight and obese women at similar success rates to conventional weight
loss drugs, but further study is needed.
Bariatric surgery prior to pregnancy
Bariatric surgery prior to conception is associated with improved maternal and neonatal outcomes
(Maggard et al., 2008), and should be considered for women who have had unsuccessful weight
loss attempts, who have complications of obesity (such as hypertension or type 2 diabetes), or
who have large amounts of weight to lose.
It is recommended that women who undertake bariatric surgery should leave a two year window
following surgery before conception, which highlights the importance of detailed contraceptive
advice. It is also important to ensure adequate replacement of vitamins and minerals, e.g. vitamins
B12 and A, as depending on the type of bariatric surgery, deficiencies are common, exacerbated
by pregnancy and of great importance for maternal and fetal health.
20.3.4 Many weight loss attempts prior to pregnancy are unsuccessful
Many people who often report weight loss attempts do not succeed. However, behavior changes
associated with these weight loss attempts are often short in duration (Jeffery et al., 2000).
Attempts at losing weight through increased physical activity have not been as successful as
hoped, most likely due to the fact that increases in physical activity are modest and therefore
not associated with a major change in energy use. Diet as the sole method to lose weight is
more successful in the short term, but is not associated with longer term weight maintenance.
Combining dietary and exercise methods are generally more successful. Successful weight losers
and maintainers are more likely to participate in daily physical activity, less likely to use over the
counter diet products and are more likely to use self-monitoring activities such as weighing and
food diaries. There is an apparent gap between people stating that they try to lose weight and
their behavior to achieve weight loss, e.g. when questioned in detail, less than half of the women
stating that they wanted to lose weight actually reported using a combination of fewer calories
and increased physical activity (Bish et al., 2005).
20.3.5 Evidence for durable weight loss through lifestyle modification is limited
Current regimes for maintenance of weight loss have been known to be unsuccessful for at least 30
years (Stubbs et al., 2011). A majority of people who manage to lose weight in studies reported in
the literature have regained that weight after five years. Examining the predictors of longer-term
weight loss success, a systematic review found that weight loss maintenance is indeed difficult to
achieve (median success of 15%). However the effects of diet are positively mediated by group
therapy, behavior modification and active follow-up (Ayyad and Andersen, 2000). However, most
people trying to lose weight are not enrolled in specific programs, but do so on their own. There
are no reliable data about the rates of successful weight loss and weight maintenance in the
preconception period in the literature.
20.3.6 Global perspective issues in low and middle income countries
The majority of the authors personal experience in dealing with maternal obesity in the context of
pre conception care relates to higher income countries, in particular Australia. However, similar
problems are emerging in low and middle income countries, where their potential importance
as a side effect of urbanization, dietary changes and reduced physical activity frequently receives
scant attention in the face of other major health challenges (McIntyre et al., 2011).
Both obesity and undernutrition pose health risks in low-income countries, on a societal level
and even within individual households. Diabetes and obesity are more prevalent in urban areas
of low-income countries owing to rapid nutritional, lifestyle, and socioeconomic transitions.
Poverty also clusters with metabolic syndrome and cardiovascular risk factors in urban slums.
20.4 Barriers to preconception care

20.4.1 Lack of pregnancy planning
There are several barriers to adequate preconception care that need to be considered (Figure
20.3). Firstly, many pregnancies are not planned, which limits the potential for preconception
intervention (Green et al., 2002). Marital status, age and socioeconomic status are positively
correlated with planned pregnancy and therefore pre-pregnancy counseling. Given that
obesity is related to lower socioeconomic status, obese women may be less likely to present for
preconception care. This is especially the case in health care systems where preconception care
is not reimbursable. Secondly, women comply poorly with even relatively simple and easily
implemented periconceptual health recommendations such as folic acid supplementation which
are followed by 50% of the population in most countries (Callaway et al., 2009b; Ray et al., 2004).
The complex lifestyle changes required for weight loss prior to pregnancy are even more difficult
to establish and adhere to.
Unplanned
pregnancy Obesity is not
I am not seen as a
overweight problem by
or obese the community
Lack of advice
Figure 20.3. Barriers to preconception care. Barriers within the individual and within the community that prevent
women from obtaining preconception advice and care.
Both preconception care and pregnancy planning are not pervasive in the Australian context. In
a survey of women in Brisbane, only 220/412 women (53.4%) reported having seen a health care
professional for a preconception health care check whereas 266/412 (64.6%) reported that they
had planned the current pregnancy (Callaway et al., 2009a). These numbers indicate that better
outcomes could be obtained if more women planned their pregnancy and consulted a health care
professional prior to falling pregnant.
20.4.2 Women do not identify themselves as obese
Additionally, both overweight and obese women tended to underestimate their fatness with 36%
of overweight women and 84% of obese women inaccurately categorizing themselves in a lower
BMI category (Callaway et al., 2009b). This misrepresentation could imply that these women
underestimate the risks that their weight has for themselves and their infants in a pregnancy.
Since obesity is prevalent, it may also be perceived as normal.
Our findings highlight the importance of calculating and advising women of their actual BMI and
BMI category and therefore risk group when they present for preconception care. In a study of
obese women early in pregnancy found that when women were informed of the risks associated
with obesity they desired further information about these risks and interest in weight loss prior
to becoming pregnant (Kominiarek et al., 2010).
20.4.3 Lack of advice or support to lose weight prior to conception
In the aforementioned survey study in Brisbane, 26.1% of overweight and obese women were
offered advice to lose weight from a variety of sources, including friends and family. Of the
overweight and obese women undertaking a preconception health check with their doctor, only
20% recalled that they were recommended to lose weight. This suggests that there is a need for
accurate information regarding the risks of overweight and obesity prior to pregnancy to be widely
available, so that consistent messages are received. Alternatively, women may not accurately recall
being provided with weight loss advice if they did not act on the advice. However, there was
no difference in self-reported weight loss success between women who did receive weight loss
advice from their doctor and those who did not. Health care professionals should perhaps receive
additional information about the risks of overweight and obese women for complications in
pregnancy. They can then pass this information on to their patient in targeted lifestyle advice.
Prospective studies on the success of targeted lifestyle advice at the preconception health check
for overweight and obese women should be conducted to indicate if this is a successful approach
for increasing awareness and promoting weight loss. While diet and physical activity are effective
in achieving weight loss individual responses and adherence are highly variable. Individualized
advice and behavior change strategies are likely to be important, matching these to womens
needs to enhance weight management success (Stubbs et al., 2011). This may be especially true
for women looking for a healthy pregnancy and a healthy baby. However, education levels are
associated with the level of knowledge and preconception visits to health care professionals,
and efforts to increase knowledge about the risks associated with obesity during pregnancy in
women with lower education levels should include additional measures besides information
during preconception visits.
20.4.4 Cultural aspects
Population-based strategies to reduce obesity and diabetes in low-income countries appear

desirable, but implementation poses many problems. Apart from resource issues, there are
cultural barriers to weight reduction in low income countries. In some traditional societies,
overweight may carry esteem as a visible sign of strength, wealth, and affluence. This in turn may
lead to a cultural reluctance to lose weight, especially among women.
20.5 Aims for preconception care

20.5.1 Increasing awareness of preconception issues
There are two randomized controlled studies showing that engagement in preconception care
programs can improve aspects of preconception health behavior (compliance with folic acid,
reduction in alcohol intake, increased awareness of lifestyle factors), but there is no evidence
that these programs resulted in women losing weight prior to pregnancy (Elsinga et al., 2008;
Hillemeier et al., 2008). It would seem sensible that women should be educated regarding the
short and long-term risks of obesity to themselves and their offspring. However, there is no
evidence that this type of education translates into the complex behavioral change that is required.
We assessed knowledge regarding the risks of overweight and obesity in pregnancy (Nitert et
al., 2011). Most women correctly identified that obese women have an increased risk of overall
complications, including gestational diabetes and hypertensive disorders of pregnancy compared
to normal weight women. Women were less aware of neonatal complications. Knowledge was
similar amongst women independent of pre-pregnancy BMI and independent of public or private
care. Higher educational status was associated with more knowledge of the risks of overweight
and obesity in pregnancy. Educational status is an important predictor of birth outcomes, and
is associated with better knowledge of other preconception health issues such as periconceptual
folate supplementation (Johnson et al., 2006). In order to improve knowledge regarding the risks
of obesity, targeting public health messages at those with lower levels of education would be
important.
20.5.2 Individualized approaches to preconception care
Given that a healthy baby is a highly valued outcome of pregnancy, increasing womens knowledge
about the impact on overweight and obesity on neonatal problems such as birth defects might
encourage women to actively attempt to lose weight prior to pregnancy. A meta-analysis of
Leventhals common-sense models as a theoretical basis for intervention programs identified
moderate to strong relationships between knowledge of disease, coping behaviors and outcomes
(Hagger and Orbell, 2003). Tailored diet and exercise interventions for at-risk individuals have
been shown to be effective in improving outcomes in type 2 diabetes (Table 20.2). It would also
seem sensible that women who seek pre-pregnancy advice should have their height and weight
measured, and should be offered referral to appropriate services to support achieving a healthy
weight prior to conception (Table 20.2). Therefore a program that will encompass an increase in
knowledge of the risks of obesity for maternal and neonatal pregnancy outcomes in conjunction
with tailored lifestyle changes may improve pregnancy outcome for obese women.
20.5.3 Infertile women
As outlined earlier, overweight and obese women are more likely to have difficulty with
conception, and require assistance with fertility. Any overweight or obese woman presenting for
assistance with fertility, is by definition, seeking preconception care. Issues outline in this chapter
should be carefully assessed and addressed in this group of women.
20.5.4 Public health approaches
Beside individual preconception care, public health interventions could be used to improve
knowledge and awareness of preconception and pregnancy behaviors that increase the risk for
adverse pregnancy outcomes. We are not aware of any comprehensive public health campaigns that
have been undertaken to improve preconception care. A recent study of providing an educational
video to increase preconception knowledge and awareness in women with pre-existing diabetes
proved to be a relatively easy and cost-effective successful intervention (Holmes et al., 2012).
As suggested above, public health interventions perhaps should be targeted specifically at those
segments of the population with the highest barriers to preconception care, e.g. those least likely
to attend individual preconception care visits with health care professionals.
20.6 Conclusions
The increasing levels of overweight and obesity in women of childbearing age are associated with
increased risk for pregnancy complications for mother and baby. Preventive strategies focus on
Table 20.2. Summary points for an individualized approach to preconception care.
Service provider1 Goal
General Practitioner Accurate weight/height measurements and frank discussion

about risks with actual body mass index1
Dietician, physiotherapist or General Practitioner Tailored diet and exercise advice with respect for patient
preferences and capabilities
1 Other service providers could be involved in this process.
the preconception period to achieve weight loss. Weight loss is difficult to achieve but appears
beneficial in preventing in maternal complications of pregnancy. Individualized programs are the
most effective in producing behavioral change. It is not yet clear if preconception weight loss has
adverse effects for the infant. The interactions between preconception weight loss and gestational
weight gain have not been investigated either.
Preconception care visits with a health care profession offer excellent opportunities to make
women aware of their body weight, the risks that they have for developing complications of
pregnancy and directing to supported weight management services. However, there are many
barriers for preconception care including underestimation of BMI and lack of specific advice
on body weight and the need for weight loss. This is especially true in women with lower levels
of education and in health systems where these visits are not reimbursed. Other ways of raising
awareness and knowledge levels in women about the risks for complications of pregnancy in
overweight and obese women include public health measures. These measures should be tailored
to reach all women of reproductive age.
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and female fecundity: prospective study of effect of body fat distribution on conception rates. BMJ 306, 484-487.
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21. Herbal medicine and fertilisation
Department of obstetrics and gynaecology, Princess Anne Hospital, University Hospital
Southampton NHS foundation trust, Coxford Road, Southampton SO16 5YA, United Kingdom;
linden.stocker@uhs.nhs.uk
Abstract
Fecundity has been long used by many cultures as a maker of health and wellbeing. Herbal
remedies have been developed over centuries to try to prevent or aid fertilisation by improving
levels of male and female sub-fertility. Medical herbalism is now thriving in Europe and in
the United States, however the methodology and patient beliefs are complex and diverse. In
most countries, herbal medicine and practitioners are poorly regulated, herbal products are
often neither registered nor controlled and research in to this area is lacking. Phytomedical
medicine needs further well designed clinical trials to augment the existing evidence before clear
recommendations can be made about their safety and effectiveness.
Keywords: assisted reproductive techniques, herbalism, fertility, infertility, phytochemistry,

phytomedicine
343
Summary points
Herbal medicine is the application of botanical remedies with therapeutic or other human health benefits.
Some practices such as Traditional Chinese Medicine have remained timeless throughout successive
generations, whilst over recent years herbal medicine has become more commonly used in the Western world.
Generally the emphasis of herbal medicine is on the augmentation and preservation of health, rather than
solely as a treatment for an ailment or disease.
The Western world uses herbal medicine most commonly to aid or improve fertility, as opposed to preventing
fertilisation. The application of herbal medicine can be used for female and male factors, unexplained
infertility and as an adjunct to assisted reproductive techniques.
Phytochemistry is still under developed or utilised and few well-designed trials into the efficacy and safety
of herbal medicine are in existence. This is in part because it is inherently difficult to design or execute these
studies.
Further well conducted randomised controlled trials are needed to assess the safety and efficacy of herbal
medicine.
Abbreviations
ART Assisted reproductive techniques
COX Cyclo oxygenase
GABA Gamma aminobutyric acid
GMC General medical council
HFEA Human and embryology fertilisation act
IL Interleukin
IUI Intrauterine insemination
IVF In vitro fertilisation
MHRA Medicines and healthcare products regulatory agency
TCM Traditional Chinese medicine
TNF Tissue necrosis factor
WHO World Health Organization
21.1 Introduction
The therapeutic action of plants has been used to enhance health for centuries, and herbal
remedies may be considered the oldest form of medicine. Traditional herbal treatments have been
used by all cultures, religions and races for ailments ranging from insomnia to impotence, and
minor colds to malignancies. Some practices such as TCM have remained timeless throughout
successive generations, whilst over recent years herbal medicine has become more commonly
used in the Western world.
Fecundity has long been used as a maker of health and wellbeing and many cultures have
developed herbal remedies to try to aid fertilisation by improve levels of male and female sub-
fertility. Conversely, plant material has been used to prevent fertilisation and impede conception
since Ancient Egyptian times, as described by the ancient Kahun gynaecological papyrus
(Haimov-Kochman et al., 2005).
21.1.1 Herbal medicine in the developed world
The modern-day use of herbal medicine has been influenced by the historic context within which
these substances were first used. Many of these herbs have been found to have a use which has
been developed within a particular culture or country. However, the common dogma of all types
of herbal medicine is a holistic approach to life, and equilibrium between the mind and body and
environment (Figure 21.1). Generally the emphasis is on the augmentation and preservation of
health, rather than solely as a treatment for an ailment or disease (WHO, 2005).
Todays patients are far more involved in their own healthcare than their ancestors. There is less
didactic instruction from healthcare professionals and patients are encouraged to be involved
Improved self-esteem
Decreased anxiety
Relaxation
Feeling of welbeing
Herbal medecine
Figure 21.1. Diagram of the holistic benefits of herbal medicine.
in their own management and treatment regimes. This, in conjunction with a greater wealth of
relevant and accessible information that has become available with the widespread use of the
internet, means that patients are increasingly interested in herbal healing. The subset of people
who seek fertility treatment, or attend contraception clinics, are unusual amongst patients so
far as that the majority are young, fit and well. They are often highly motivated to pursue their
treatment goal and as a result are more likely to explore the unconventional option of herbal
medicine than their physically or mentally unwell counterparts.
Medical herbalism is thriving in Europe and in the United States and the sale of these medications
is an expanding industry. The availability of herbal products, as demonstrated by the plentiful
array of high-street shops and online companies, means that consumers can readily purchase a
wide range of herbs. Research conducted for the MHRA in 2009 showed that 26% of adults in the
UK had taken a herbal medicine in the last two years, mostly bought over the counter in health
food shops, pharmacies and even supermarkets. Advertising for these products is ubiquitous and
the practise of herbal medicines can be lucrative.
21.1.2 Definition of herbal medicine
Phytotherapy is the practical application of botanical remedies. These plant derived materials
or preparations have therapeutic or other health benefits. Herbs, herbal materials and herbal
preparations and finished herbal products are all included under the umbrella title of herbal
medicines and are used in various forms (Table 21.1 and 21.2). These contain either raw or
processed ingredients from one or more plants.
Table 21.1. Explanation of the terminology used in the preparation of herbal medicine (WHO, 2000).
Description Component parts Production method
Herbs Leaves, flowers, fruit, seeds, roots, bark or Crude plant material
other plant parts (entire or fragmented)
Herbal materials Juices, gums, fixed oils, essential oils, Steaming, roasting, baking
resins, or dry powders of herbs
Herbal preparations Comminuted, or extracts, tinctures and Extraction, fractionation, purification,
fatty oils of herbal materials concentration, or other physical or
biological processes including steeping/
heating herbal materials in alcoholic
beverages or other materials
Finished herbal products Preparations of one or more herbs. May Mixture of the above
contain excipients in addition to the
active ingredients
Table 21.2. Explanation of the terminology used on some herbal products.
Terminology Explanation
Tea/infusion Adding boiling water to fresh or dried botanicals and steeping them
Decoction Simmered in boiling water
Tincture Soaking in a solution of alcohol
Extract Purifying by using a substance that removes specific chemicals
Capsules or tablets Drying an extract into a compressed pellet
21.2 Why do patients use herbal medicine?

The Western world commonly uses herbal medicine to aid or improve fertility, as opposed to using
herbs as a contraceptive and to prevent fertilisation. Patients are keen to avoid the adverse effects
of conventional treatment. There is a perception amongst some patients that herbal medicine has
fewer side-effects and is inherently safer than orthodox medication. Patients are often pragmatic
when they perceive their existing treatment to be incomplete or ineffective and the majority of
herbal medicines are used simultaneously with prescribed treatment.
Patients often seek the advice of a herbalist before making contact with primary care services.
They may wish to experiment with over-the-counter medication before embarking on a
cocktail of prescription drugs, or they may already be taking herbs for another condition and
consider adapting these to their current needs. Herbalism may be cheaper to the consumer than
prescription medication.
Once a couple seek the advice of a fertility expert, they will invariably be faced with laboratory
tests and invasive investigations. This may be followed by fertility drugs, terminology and medical
intervention which means that there is a tendency to medicalize sub-fertility. The patient may
have previously viewed their reproductive potential as a variation of normal, rather than a disease
state. Herbs may be used by this population to circumvent the medicalized chain of events.
Infertility rates range from 3.5% to 16.7% in more developed nations (Boivin et al., 2007). The
mainstay of treatment for these couples is ART which are costly, both financially and emotionally.
Their modest success rates means that couples are willing to seek help from herbal medicine as
a replacement for or adjunct to ART.
21.3 Specific medicinal herbs

The enormity of herbal products which are used for fertilisation means that it would be
impossible to include them all in this chapter. Table 21.3 presents a selection of plants in wide
usage, including in preparations used TCM and dietary supplements. These specific examples are
by no means exhaustive, but are exclusively from the WHO series of monographs of commonly-
used medicinal plants (WHO, 2009). This is a rare authoritative source of information created to
regulate herbal medicines and to ensure safety, efficacy and quality. Table 21.3 presents details on
the medicinal uses of these herbs, none of these uses are supported by clinical evidence and there
is insufficient data to support these applications in the literature. The majority of the therapeutic
claims are well documented in folk medicine, whilst others have a long history of use described
in pharmacopoeias and traditional systems of medicine.
The descriptions included in this chapter do not imply the authors endorsement or approval
of the use of these herbs. We merely aim to present details of these plants for the purposes of
information.
21.4 The evidence behind the practise of herbal medicine

Phytochemistry, the field of research into isolating the active compounds within botanical
materials, has been in existence since the 1960s with the aim of validating the use of herbs.
However few well-designed trials into efficacy are in existence. This is in part because it is
inherently difficult to design or execute these studies.
Plants are a collection of complex pharmacologically active chemicals, often used in combination
and in non-standardised quantities. Quality of manufacture is a difficult problem; plants differ in
their chemical content. There are many variables, for example the growing conditions, the plant
Table 21.3. Selected plants for use in fertilisation (Adapted from WHO, 2009).
Latin name Common name(s) Documented role in Safety profile in pregnancy

fertilisation
Bulbus Allii sativi Garlic Aphrodisiac/emmenagogue Non-teratogenic effects known

Flos calendulae Calendula, Chinese Treatment for amenorrhoea No information
safower, marigold (systemic) and inflammation of
vulva and vagina (topical)
Fructus foeniculi Fennel, ladys Aphrodisiac/emmenagogue, Should not be used in
chewing tobacco thought to have oestrogenic pregnancy
and anti-androgenic affects
Radix glycyrrhizae Chinese liquorice An emmenagogue and as a Should not be used
contraceptive
Fructus hippophas Buckthorn, armarillo Ammenorrhoea No information
recens
Herba hyperici St Johns Wort, devils Emmenagogue No information
scourge
Herba leonuri Bladderwort, lions A remedy for female Avoid
tail reproductive disorders;
stimulates the muscles of the
uterus and to treat delayed
menstruation
Folium melissae Balm mint, lemon To treat amenorrhoea No information
balm
Folium menthae American mint, An emmenagogue No information
piperitae peppermint
Herba origani Oregano, common An emmenagogue, for treating Avoid during pregnancy
marjoram oligomenorrhoea and
impotence
Herba pegani African rue An aphrodisiac and Not recommended during
harmalae abortifacient and possible pregnancy
positive effect on male fertility
Herba thymi Common thyme An emmenagogue Not established but widespread
use has not resulted in any
safety concerns
variety and farming methods, which will dictate the unique qualities of a particular crop (similar
to the concept of terroir in wine). These botanical variations are generally accepted, but mean
that the concentrations of compounds in standardised herbal products can vary significantly.
There are few studies which look at individual herbs or specific compounds derived from
herbs alone. For this reason it is rare to find satisfactory preclinical evidence to satisfy an ethics
committee. In addition, phytomedical research lacks the funding which supports drug companies
because it is difficult to patent herbs. When there is supportive data, this is often of poor quality as
a result of badly designed and executed trials. These (often incomparable) results are then quoted
as evidence, which may conflict with the findings of other studies. This lack of robust data and
standardisation is an unfamiliar concept in Western medicine.
Conventional medicine aims to assess new treatments through controlled clinical trials and the
most reliable evidence comes from systematic reviews. These ask a specific question and then a
comprehensive literature search is performed to make clinical recommendations based on the
best quality papers. A meta-analysis is a systematic review that combines the results of all the
studies into a single statistical analysis of results. The systematic reviews of herbal medicine
have to disregard many studies which do not provide robust data (Flower et al., 2009, 2012). For
example, the Cochrane review of Chinese herbal medicine for endometriosis considered only 2
papers from a total of 110 trials (Flower et al., 2009). The heterogenicity of the trials involved in
herbal medicine mean it is a topic which does not lend itself well to meta-analysis and a common
conclusion is that further well designed studies are needed.
The information on herbal treatment that follows draws on the available data. Many of the
studies focus on TCM as it is well established and increasingly popular. TCM consists of complex
prescription based on Chinese diagnostic patterns and follows a very different approach to
Western medicine. TCM focuses on restoring homeostasis of whole body systems in which
imbalance is perceived, using meridians of treatment such as stagnation of the blood. A typical
example which demonstrates this is shown in Table 21.4. It is a treatment which is dependent
on the practitioner and their individualised approach to their patients. In TCM the herbs are
prescribed in non-standardised quantities and the patients are often required to prepare the
herbal tonics themselves. Data which quotes the use of TCM is further confounded because TCM
combines medicinal herbs with food therapy, acupuncture, massage and exercise and is often
used in conjunction with western traditional medicine.
The fundamental principles and the approach used in Chinese herbalism means that evidence is
more likely to be based on individual case experiences and expert opinion than to have evidence
derived from systematic reviews. The TCM practitioner designs a treatment which is individually
tailored and may encompass various treatment modalities to simultaneously treat a myriad of
symptoms as opposed to a specific pathology.
TCM papers are only usually accessible in literature searches if they have been translated into
English and this will decrease their accessibility to the Western world. Often reviews of the
evidence pool the results of different herbal medicines and neglect to search Chinese databases in
the same way that extensive searching of British and American databases occurs. The systematic
reviews often do not have the participation of TCM practitioners who are key in understanding
Table 21.4. An example of a Chinese herbal remedy used in subfertility and irregular menses.1
Ingredients Effects
Evodia root Warming the channel, dispelling cold and promoting blood circulation
Cassia twig
Chinese angelica root Nourishing the blood and activating blood flow, regulating menses
Chaunxiong rhizome
Red peony root
Ass hide glue Nourishing blood/ stopping bleeding
Moutan bark Purging the latent fire of blood
Ginseng Supplementing qi and regulating the stomach and enlivening the spleen
Liquorice root
Fresh ginger
Pinellia tuber
1 Information from Meridian-warming decoction in Gongwang et al. (2002).
TCM, in the same way that Western clinicians would be asked for their expert opinion on new
drugs and clinical trials.
Further well conducted randomised controlled trials are needed to assess the safety and efficacy
of herbal medicine. In China where widespread use of TCM is practised as a matter of routine,
it follows that there would be a good opportunity for such trials to be executed. However, the
Chinese believe that it is unethical to withhold potentially beneficial treatment and therefore few
double-blind placebo controlled trials exist.
The Western doctor needs to remain cautious when advising patients about phytomedicine
because of the lack of evidence surrounding such practice. It is good practice to provide the
patient with the available information (including negative findings where available) in a format
that they can understand so that they can make an informed decision about the use of herbal
medicine.
21.5 The practise of herbal medicine to affect fertilisation

21.5.1 Increasing fertilisation rates in subfertile couples
The major causes of subfertility and difficulty achieving fertilisation are anovulation and ovulatory
disorders (25%), tubal factors (20%), sperm defects (25%) and unexplained infertility (25%). In
about 30% of couples, multiple factors will be found (Stabile et al., 2000). Herbal remedies may
be used for any of these conditions and lend themselves to conditions which have a number of
components, such as multiple confounders and unexplained infertility (Figure 21.2).
21.5.2 Female factors
Anovulation/polycystic ovarian syndrome
PCOS as the most common cause of secondary amenorrhoea and anovulation, is the most
common endocrinopathy to affect women of reproductive age (Anonymous, 2004). The Cochrane
library has conducted a systematic review to evaluate the efficacy and safety of Chinese medicinal
herbs in sub-fertile women with PCOS. It reported on 4 studies with 344 participants who used
TCM in combination with Western medicine. It concluded that the efficacy of clomiphene
citrate may be augmented by TCM but those women who were resistant to orthodox methods of
ovulation induction, may also be resistant to TCM methods. Whilst clinical pregnancy rates may
be increased by the addition of TCM, live birth rates were not reported by these studies (Zhang
et al., 2010).
There are studies which suggest that herbal products can also improve female sub-fertility by
increasing the likelihood of ovulation. Unfortunately these are often under-powered, not controlled
or otherwise flawed. An example of this is the use of Vitex agnus-castus (chasteberry) which has
been used to treat ovulatory dysfunction. Unsubstantiated claims exist that other herbs including
Cimicifugae rhizoma (black cohosh), Radix Angelicae Sinensis (dong quai), Chamaelirium luteum
Female factors
Annovulation
Tubal patency
Male factors
Adjunct to Herbal Semen parameter
ART medicine Impotence
Unexplained factors
Relaxation anxiety
Insomnia
Figure 21.2. Diagram demonstrating the different applications of herbal medicine in subfertility.
(false unicorn root), Radix urticae2 (nettle), Oenothera sp (evening primrose), Rubus idaeus
(raspberry leaves), Viburnum opulus (cramp bark), Caulophyllum thalictroides (blue cohosh),
Triticum aestivum (wheat grass) and bromelain (protease enzymes extracted from the pineapple
plant, family Bromeliaceae) increase female fertility (Covington and Burns, 2006).
Tubal factors
In China the treatment of endometriosis with TCM to promote fertility is common practice.
There is limited data available to compare with conventional treatments, but a Cochrane review
evaluated the treatment of endometriosis and did not demonstrate a difference in pregnancy
rates between treatment with gestrinone (an anti-progesterone) and TCM (Flower et al., 2009).
It has been hypothesised that TCM can improve tubal factor infertility when used following tubal
surgery (Jiang et al., 2006). Herba hyperici (St Johns Wort) has been used in an attempt to reduce
the formation of adhesions by external application over the abdomen. This is a novel concept for
the Western doctor, as surgery is their treatment of choice for cases of mechanical obstruction.
However, in other areas of medicine the external application of medicine leads to absorption
and systemic distribution of the active substance. There are descriptions of the effects of TCM
on pelvic inflammatory disease when administered orally or via a herbal enema. These report
symptom relief, although these trials are of poor quality. Research is increasingly being conducted
that describes possible immunological and anti-inflammatory effects of Chinese herbal medicine,
such as cytokines (TNF-a, IL-6, IL-8) suppression, COX-2 inhibition, antioxidant activity, and
pain relief via opioid, dopaminergic, and GABAergic mechanisms, that would verify clinical
benefit in pelvic inflammation (RCOG, 2012).
21.5.2 Male factors
Semen parameters
In up to half of couples who experience subfertility, a male factor is contributory (Fisher and
Hammarberg, 2012). Reports have been made which suggest that TCM can cause improvements
in sperm parameters and a reduction in serum anti-sperm antibody titres. These studies although
not controlled, showed results in vitro and in vivo, with demonstrable effects on subsequent
pregnancy rates (Crimmel et al., 2001). Herbs used in Chinese herbalism such as Astragalus
membranaceus and Acanthopanacis senticosi have also been reported to have positive effects on
sperm motility and viability (Liu et al., 2004). In an uncontrolled study, Pinus pinaster bark
(French maritime pine tree) improved sperm morphology and mannose receptor binding (Roseff,
2002). Although there is no supportive data, there are claims that Serenoae repentis fructus (saw
palmetto) and Ginko biloba (ginkgo) strengthen the male reproductive system.
2 Radix urticae is described by the WHO (2009) monographs, but not for this purpose.
Impotence
Herbal remedies for impotence, for example Panax ginseng (red ginseng) (Jang et al., 2002), have
been suggested to be more effective than placebo in treating erectile dysfunction.
21.5.3 As an adjunct to reproductive techniques
Sub-fertile couples are increasingly utilising ART including ovarian stimulation with or without
IUI or IVF. There are no randomised controlled trials comparing the use of TCM and ART
(Cheong et al., 2010). There are reports of women using other herbal remedies, for example
the phytoestrogen Trifolium pratense (red clover) at the time of ART in order to optimise their
success rates. Luteal phase support with high dose phytoestrogens, has been used in successful
IVF pregnancies but there are also concerns that excessive phytoestrogens found in diets rich in
soy may impede fertilisation.
21.5.4 Unexplained sub-fertility
Couples should be informed that stress can affect the couples relationship and is likely to reduce
libido and frequency of intercourse which can contribute to fertility problems. The herbalist
will believe that the body has a capacity to self-heal and herbs should be chosen to support
wellness and vitality in order to break down barriers to fertilisation. Agents should support the
homeostatic balance of the whole person and counteract inhibitory environmental pressures.
Herbs such as Herba hyperici (St Johns Wort) have long been used to treat anxiety and depression,
and Valeriana officinalis extract (valerian) is widely used in insomnia, a common complaint of
the anxious fertility patient.
21.5.5 Decreasing fertilisation rates in fertile couples
In the developed world traditional contraception is widely available and highly effective, and in
the UK supplied free of charge. This excellent provision means that herbal medicine is little used
as a contraceptive. For couples who do use herbal methods, most probably prevent implantation
rather than directly affecting fertilisation.
As is often the case with herbal medicine there are discrepancies between studies. A non-
randomised study on a single sperm donor suggested that Serenoae repentis fructus (saw palmetto)
and Ginko biloba (ginkgo) may have caused a reduction in his sperm motility in vitro (level III
evidence) (Ondrizek et al., 1999a). In a similar small study, Herba hyperici (St Johns Wort) and
Echinacea sp resulted in decreased oocyte penetration in the zona free hamster test and denatured
the DNA within the sperm (Ondrizek et al., 1999b).
21.6 Safety and regulation of herbal medicine

Opinions dominate information in herbal medicine. Few medicinal herbs have been adequately
scientifically evaluated for their proposed application. Preparations containing botanical
components and their extracts lack safety and efficacy data. Even less information is available about
the active ingredients. Furthermore, in most countries herbal medicine and herbal practitioners
are poorly regulated, and herbal products are often neither registered nor controlled. Assurance
of the safety, quality, and efficacy of medicinal plants and herbal products has now become a
prime concern. In contrast, most countries have regulatory bodies to govern the registration of
qualified medical practitioners, for example the GMC. Strict regulation also exists to monitor
the drugs that these professionals can prescribe, as monitored by the MHRA in the UK. There
is also legislation to control fertility treatments including IVF techniques which is provided by
the HFEA.
In 2011 the European Union enforced a new directive which dictates that the sale of herbal
medicines must be registered. These products must meet safety, quality and manufacturing
standards, and come with information outlining possible side-effects (EC, 2004). In the UK
there are also plans to provide regulation of herbal and traditional medicine practitioners. It is
proposed that herbalists will become members of the health professions council and entered onto
a statutory register, which is proposed to open in the autumn of 2013.
China is the only country in the world where orthodox medicine and herbal medicine are practised
alongside each other. In the 1950s there was a fear that TCM would be superseded by modern
Western medicine and so clinical trials were conducted to promote and justify the tradition. This
is still maintained and TCM has its own department at the Ministry of Public Health, is taught
in standalone medical schools, practised in hospitals and has dedicated research institutions. In
95% of the hospitals practising Western medicine there are departments of traditional Chinese
medicine, most with inpatient beds and patients can opt for Chinese or Western treatment
(Hesketh and Zhu, 1997).
21.7 The power of the placebo effect

The herbal placebo effects are those that are attributable to the administration and consumption
of the herbs, but are not due to the inherent biological activity contained within the substance.
This effect could be due to the conditioned response of patients that any medication must cause
an effect, ideally meeting expectations. Those patients who engage in herbal medicine must have
a belief that it could provide them with benefit, or they would not pursue this methodology. In
addition fertility patients may perceive their herbal practitioner as emotionally sympathetic to
their needs, and this may be a factor in the positive action of herbs.
21.8 Pitfalls of herbal medicine and fertilisation

Couples trying to conceive are often vulnerable and have profound psychological difficulties in
dealing with their childlessness (Schmidt, 2009). These couples are extremely susceptible to the
virtuous claims of herbal medicines, and have a greater uptake of complementary medicines than
the general population (Coulsen and Jenkins, 2005). The desperation for fertilisation may render
them less thorough in their analysis of the treatment options on offer. Whilst some of the herbs
available may be of benefit, there are unsubstantiated claims made about herbal medicine without
sufficient safety profiles to support their use.
In a review it was found that infertility is potentially humiliating and emasculating to men and has
a profound adverse impact on masculinity (Fisher and Hammarberg, 2012). Herbal medicines
can often be purchased without the need to see a healthcare professional, often online as opposed
to in person, and this could be seen as appealing to this patient group.
Patients who use phytomedicine do not always disclose this to medical professionals. Herbal
medicines can adversely interact with other drugs, have problematic anticoagulant effects during
surgery or have undesired actions of which the patient may be unaware. For example, some
phytomedicines used to promote fertilisation may cause uterine contractions and act as an
abortifacient or pose a risk of fetal abnormality.
Women will have concerns about disclosing their use of herbal remedies to clinicians if they
feel that they will not be taken seriously, especially if previous responses had been dismissive.
Clinicians have a duty of care which includes being accepting of patient choice, and being
informed as to the herbal medicines that are available and taken by their patients. Encouraging
open discussion about these treatments and providing opportunities for discussion will promote
the desired environment for optimal care.
21.9 Future scope

Despite the increasing use of herbal medicines, there is still a significant lack of research data in
this field. Both patients and health-care professionals need up-to-date, authoritative information
on the safety and efficacy of medicinal plants. A wide range of herbal treatments are used by
sub-fertile couples, and to a lesser extent in fertile couples wanting contraceptive effects. Few
randomized trials have been performed to evaluate herbal treatments when used to promote
or reduce fertilisation. High-quality studies need to be conducted to demonstrate the safety
and effectiveness of these therapies before clear recommendations can be made by medical
professionals about their safety and effectiveness. It is anticipated that programs such as the Good
Practice in Traditional Chinese Medicine Research in the Post-Genomic Era funded under the 7th
framework programme will help facilitate these in the future (Anonymous, 2012).
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22. Diet containing endocrine-disruptors and
reproductive health
D. Balabani1 and A. Krivograd Klemeni2
1Pulp and Paper Institute, Bogiieva 8, 1000 Ljubljana, Slovenia; 2Faculty of Health
Sciences, University of Ljubljana, Zdravstvena pot 5, 1000 Ljubljana, Slovenia;

damjan.balabanic@gmail.com
Abstract
There is increasing concern about chemical pollutants that are able to mimic hormones, the
so-called endocrine disrupting compounds (EDCs), because of their structural similarity to
endogenous hormones, their ability to interact with hormone transport proteins or because
of their potential to disrupt hormone metabolic pathways. Thus, the effects of endogenous
hormones can be mimicked or, in some cases, completely blocked. A substantial number of
environmental pollutants, such as polychlorinated biphenyls, dioxins, polycyclic aromatic
hydrocarbons, phthalates, bisphenol A, pesticides, alkylphenols and heavy metals (arsenic,
cadmium, lead, mercury), have been shown to disrupt endocrine function. These compounds can
cause reproductive problems by decreasing sperm count and quality, increasing the number of
testicular germ cells and causing male breast cancer, cryptorchidism, hypospadias, miscarriages,
endometriosis, impaired fertility, irregularities of the menstrual cycle, and infertility. However, in
most cases, human exposure to EDCs is through the ingestion of contaminated food.
Keywords: contaminated food, environmental pollutant, hormone
359
Summary points
In recent decades, there has been increasing concern regarding disruption of the endocrine system in living
organisms by synthetic organic chemicals, so called endocrine disrupting compounds (EDCs).
EDCs such as bisphenol A, alkylphenols, phthalates, pesticides, dioxins, polychlorinated biphenyls, polycyclic
aromatic hydrocarbons and heavy metals are environmental pollutants that have been shown to significantly
alter the endocrine balance in living organisms.
Humans and most wildlife do not have biochemical pathways to detoxify or excrete these chemicals, so they
tend to accumulate them in the body.
EDCs can cause reproductive problems in humans by decreasing sperm count and quality, increasing the
number of testicular germ cells and causing male breast cancer, cryptorchidism, hypospadias, miscarriages,
endometriosis, impaired fertility, irregularities of the menstrual cycle, and infertility.
Human exposure to EDCs may result from the ingestion of contaminated food and water, inhalation of air
and absorption of EDCs through the skin, of which the ingestion of contaminated food is the most common.
EDCs like bisphenol A, phthalates, pesticides, dioxins, polychlorinated biphenyls, arsenic, methylmercury,
cadmium and lead are typical food contaminants.
Food packaging can interact with the packaged foodstuff by diffusion-controlled processes which mainly
depend on chemical properties of the food packaging materials and the foodstuff, temperatures at packaging,
during heat treatment and storage, exposure to UV light and storage time of the product. This interaction
can lead to food packaging material compounds including EDCs leaching from the packaging to the food.
Preventing the discharge of EDCs is an important factor in the prevention of endocrine reproductive disorders
in humans and animals. Chemical policies and legislations at local and national levels, as well as globally,
need to be formulated, financed and implemented to ensure the best public health.
22. Diet containing endocrine-disruptors and reproductive health
Abbreviations
As Arsenium
BPA Bisphenol A
Cd Cadmium
E2 Estradiol
EDCs Endocrine disrupting compounds
HDPE High density polyethylene
Hg Mercury
PAHs Polycyclic aromatic hydrocarbons
Pb Lead
PCBs Polychlorinated biphenyls
PET Polyethylene terephthalate
PVC Polyvinyl chloride
UHT Ultra-high temperatures
22.1 Introduction
In recent decades, there has been increasing concern regarding disruption of the endocrine system
in living organisms by synthetic organic chemicals or EDCs. Many authors reported that the
environment is contaminated with numerous EDCs that exert hormonal activity (e.g. Balabani
et al., 2011). Environmental pollutants such as BPA, alkylphenols, phthalates, pesticides, dioxins,
PCBs, PAHs and heavy metals (e.g. Cd, Pb, As and Hg), have been shown to significantly alter
endocrine balance in living organisms. Human exposure to EDCs may result from the ingestion
of contaminated food and water, inhalation of air and absorption of EDCs through the skin,
of which the ingestion of contaminated food is the most common. Typical food contaminants,
such as BPA, phthalates, pesticides, dioxins, PCBs, arsenic, methylmercury, Cd and Pb are well
characterized in food (Balabani et al., 2011; Muncke, 2009).
Most of the above mentioned EDCs are organochlorine substances, which means that they
contain chemically bound carbon and chlorine. This binding is strong and resistant to
biochemical and physical degradation. Humans and most wildlife do not have biochemical
pathways to detoxify or excrete these chemicals, so they tend to accumulate them in the body.
The log [octanol/water partition coefficient] (Kow) values for most EDCs indicate a high degree
of lipophilicity (Birkett, 2003).
The current knowledge of the way in which EDCs affect humans is patchy, although there is some
evidence that these compounds have the potential to induce deleterious changes in the human
reproductive system (Balabani et al., 2011). In summary, there is evidence from many countries
that exposure to EDCs can decrease sperm count and quality, increase in the number of testicular
germ cells and the incidence of male breast cancer, cryptorchidism and hypospadias, disturb
menstrual cycle and result in intrauterine growth restriction and polycystic ovarian syndrome
(Carlsen et al., 1992, 1995; Muoz de Toro et al., 2005) (Table 22.1). The evidence supporting
the effect of EDCs on wildlife is stronger, although the validity of the results is widely disputed
(Balabani et al., 2011).
22.2 Food packaging as a source of endocrine disrupting compounds

The purpose of food packaging, apart from marketing purposes, is to preserve food by protecting
it from mechanical inuences, air (oxygen as a oxidizer), microbial contamination, temperature
instability, light (and UV radiation), loss of gas (carbonated beverages) and foreign aroma
compounds. Different materials are used to package foodstuffs: cardboard, paper, regenerated
cellulose, plastics, metals, glass, ceramics, rubbers and elastomers, wood, cork and textiles
(Muncke, 2009). Most metal cans have polymeric coatings, and paper or carton packaging often is
coated or laminated with plastic as the effective food contact material, essentially making plastics
the main food contact material in todays packaging landscape (Castle, 2007). Food packaging
Table 22.1 Impact of endocrine-disruptors on human reproductive health.
Endocrine Impact on human reproductive health References

disrupting
compounds
Bisphenol A hypospadias, cryptorchidism, decrease in the quantity and Balabani et al., 2011; Bergeron
quality of sperm production, decreased fertility in males, et al., 1999; Carlsen et al.,
endometrial hyperplasia 1992
Alkylphenols hypospadias, cryptorchidism, decrease in the quantity and Carlsen et al., 1992, 2005
quality of sperm production, testicular cancer
Phthalates hypospadias, cryptorchidism, reduced anogenital distance, Balabani et al., 2011; Fisher,
nipple and areola retention, abnormal seminiferous cord 2003
formation, reduction in fetal testosterone levels, abnormally
located leydig cells
Pesticides hypospadias, cryptorchidism, decrease in the quantity and Balabani et al., 2011; Garry,
quality of sperm production, various cancer, miscarriage 2004
Cadmium infertility, prostate cancer, testicular injury in the fetus Nampoothiri and Gupta, 2006;
Xu et al., 1996
Lead infertility, miscarriage, decreased birth weight Bellinger, 2005; Nampoothiri
and Gupta, 2006
Arsenic miscarriage, decreased birthweight Balabani et al., 2011; Tapio
and Grosche, 2006
Mercury infertlity, miscarriage, congenital malformations, Balabani et al., 2011; Choy et
disturbances in the menstrual cycle al., 2003
can interact with the packaged foodstuff by a diffusion-controlled processes which mainly
depend on chemical properties of the food packaging materials and the foodstuff, temperatures
at packaging, during heat treatment and storage, exposure to UV light and storage time of
the product (Arvanitoyannis and Bosnea, 2004). This interaction can lead to food packaging
material compounds leaching from the packaging to the food, a process also known as migration.
Compounds that can leach from plastic food packaging materials are starting substances used
for the initial polymerization step, like monomers or catalysts, and additives that are included
during the manufacturing process to achieve special material properties (phthalates for material
softening). Starting substances can leach either because of incomplete polymerization during the
formation of the material, or because of material degradation over time. Furthermore, starting
substances or additives can contain impurities, which again might leach from the packaging.
Leaching of EDCs also occurs from the other types of packaging materials such as metal, paper
and carton (Table 22.2).
Table 22.2. Endocrine-disruptors in food and food-packaging materials.
Endocrine Source References

disrupting
compounds
Bisphenol A food contact surface lacquer coatings for cans, plastic Balabani and Krivograd
bottles, contaminated fruits and vegetables, recycled Klemeni, 2011; Fasano et
paperboard packaging materials al., 2012
Alkylphenols contaminated fruits and vegetables, paper packaging Balabani and Krivograd
materials, cans, plastic bottles Klemeni, 2011; Fasano et
al., 2012; Fernandez et al.,
2008; McNeal et al., 2000
Phthalates contaminated fruits and vegetables, meat, fish and milk, Balabani and Krivograd
paper and plastic packaging materials, plastic bottles Klemeni, 2011; Balabani et
al., 2011; Fasano et al., 2012;
Fierens et al., 2012
Pesticides contaminated drinking water, fruits, compotes and cooked Nougadre et al., 2012
fruit, eggs and egg products, meat and vegetables
Cadmium contaminated fruits, vegetables, tea, rice, milk, fish and meat Oymak et al., 2009
Lead contaminated fruits, vegetables, milk, fish and meat Nasreddine and Parent-Massin,
2002
Arsenic contaminated drinking water fruits, vegetables, milk, fish, Alam et al., 2003; Mukherjee et
seafood and meat al., 2006; Sirot et al., 2009
Mercury contaminated drinking water, fruits, vegetables, milk, meat Bjrnberg et al., 2005; Choy et
and fish al., 2003; Clarkson, 2002
22.3 I mpact of diet containing endocrine disrupting compounds on human

reproductive health
22.3.1 Bisphenol A
BPA (2,2-bis(4-hydroxyphenyl)propane) is an organic compound composed of two phenol rings

connected by a methyl bridge, with two methyl functional groups attached to the bridge. BPA is
used as a source material for the production of phenol resins, polyacrylates and polyesters, but
mainly for the production of epoxy resins and polycarbonate plastics. Epoxy resins are used as
food contact surface lacquer coatings for cans, protective coatings and finishes, adhesives and
as coatings for PVC pipes. Polycarbonate plastics are used in the manufacture of household
appliances, food packaging and plastic bottles because they have high impact strength, hardness,
toughness, transparency, resistance to temperatures, many acids and oils (Fasano et al., 2012).
BPA is also used in resin-based dental sealants and bonding agents (Pulgar et al., 2000). As a
result of an increase in the use of products based on epoxy resins and polycarbonate plastics,
human exposure to BPA (both environmental and through food) has increased.
BPA is an endocrine disruptor with estrogenic activity and an estrogenic potency that is
approximately 1104 less than that of E2 (Bergeron et al. 1999). Several studies have suggested
adverse endocrine disruptive effects of low doses BPA on adults, such as decreased quantity
and quality of sperm production, decreased fertility in males, recurrent miscarriages, persistent
alterations in peripubertal mammary gland development and polycystic ovarian syndrome
(Balabani et al., 2011; Carlsen et al., 1992, 1995; Muoz de Toro et al., 2005), as well as effects
on the fetus, such as an increased incidence of hypospadias and cryptorchidism, stimulation of
mammary gland development and endometrial hyperplasia (Balabani et al., 2011; Carlsen et
al., 1992, 1995).
22.3.2 Alkylphenols
Alkylphenols are the final products in the biodegradation of alkylphenol polyethoxylates, which
are non-ionic surfactants widely used in detergents, paints, cans, paper packaging materials,
plastic bottles, pesticides, cosmetics and other formulated products (Balabani and Krivograd
Klemeni, 2011; Fasano et al., 2012).
Based on a meta-analysis of 61 studies, it has been suggested that the human sperm count and
quality have decreased in the past 50 years (Carlsen et al., 1992). There are also indications of
an increased incidence of testicular cancer in human adults, as well as an increased occurrence
of cryptorchidism and hypospadias in human fetuses (Carlsen et al., 1995). Alkylphenols have
the potential to interfere with the sexual development and reproduction of vertebrate organisms.
Because a balance of steroid hormones is required to maintain normal gonadal differentiation
and morphological development of male and female vertebrates, it is possible that exposure
to EDCs may result in altered development of primary and secondary sexual characteristics
Nonylphenol is a well-studied model EDC with estrogenic properties that is widely present in
foods (Guenther et al. 2002). In food packaging, nonylphenol originates from oxidation of the
antioxidant additive trisnonylphenyl phosphite (McNeal et al., 2000). Levels of nonylphenol in
different packaging materials were recently assessed and were found to range from below 0.03
g/g in a PET water bottle to 287 g/g in PVC cling lm (Fernandes et al., 2008). Nonylphenol
was also detected in different types of retail-purchased foods: up to 78 ng/l were found in PET-
bottled mineral water (ToyoOka and Oshige, 2000), up to 40 g/kg in beverage cartons of UHT
whole milk and up to 32.3 g/kg in HDPE-bottled milk (in bottle sterilization) (Casajuana and
Lacorte, 2004).
22.3.3 Phthalates
Phthalates are synthetic chemicals that are widely used in the production of plastics, additives
in paper production, as solvents in inks used in food packaging and in certain cosmetics
(Balabani and Krivograd Klemeni 2011; Balabani et al. 2011; Fasano et al. 2012). Phthalates
may also be found in different type of food groups (Table 22.3). When certain phthalate esters
are administered during the period when the male reproductive tract is developing in utero,
certain abnormalities can occur (e.g. reduced anogenital distance, nipple and areola retention,
cleft phallus, hypospadias and undescended testes) (Balabani et al., 2011; Fisher et al., 2003).
The effects observed in rodents have been associated with a reduction in testosterone synthesis
by the fetal testis (Balabani et al., 2011; Fisher et al., 2003). The wide spectrum of abnormalities
that are induced following in utero exposure of rodents to certain phthalate esters is similar to the
spectrum of reproductive disorders believed to occur within the human population. Testosterone
production is essential for the normal masculinisation of the male reproductive tract, but dibutyl
phthalate and diethylhexyl phthalate induce a 60-85% reduction in fetal testosterone levels during
the critical period of development. Unlike other anti-androgens, phthalates do not interact with
the androgen receptor. Epidemiologically, testicular cancer and poor semen quality have been
linked to birth cohort effects and, by definition, hypospadias and cryptorchidism are male
congenital abnormalities suggesting that all these disorders have their root in fetal development
22.3.4 Pesticides
Recently, the potential of certain pesticides to act as EDCs has been reported (Tables 22.4 and
22.5). Conventional toxicological testing of pesticides may miss the potential of a substance
to disrupt the endocrine system, especially at the low concentrations likely to be found in the
environment (Balabani et al., 2011). EDCs in pesticides are active in vivo at extremely low
doses and it has been suggested that the permitted levels in food may be too high. Links between
pesticide exposure and endocrine disruption has been implicated in the aetiology of various
cancers, miscarriage and other reproductive disorders (Garry, 2004). Recent studies suggest
adverse endocrine-disrupting effects such as hypospadias, cryptorchidism and other birth
defects, on the fetus exposed to pesticides (Balabani et al., 2011; Garry, 2004).
Table 22.3. Phthalates determined in different food groups.
Food group Phthalates References
Fruits and dimethyl phthalate, diethyl phthalate, diisobutyl phthalate, di-n- Fierens et al., 2002;
vegetables butyl phthalate, benzylbutyl phthalate, di(2-ethylhexyl) phthalate, Self and Wu, 2012
dicyclohexyl phthalate, di-n-octyl phthalate, diisononyl phthalate,
diisodecyl phthalate
Sport drinks benzylbutyl phthalate, di-n-butyl phthalate, di(2-ethylhexyl) phthalate, Self and Wu, 2012
di-n-octyl phthalate, diisononyl phthalate, diisodecyl phthalate
Artificial juice benzylbutyl phthalate, di-n-butyl phthalate, di(2-ethylhexyl) phthalate, Self and Wu, 2012
drink di-n-octyl phthalate, diisononyl phthalate, diisodecyl phthalate
Meat and meat dimethyl phthalate, diethyl phthalate, diisobutyl phthalate, di-n- Fierens et al., 2002
products butyl phthalate, benzylbutyl phthalate, di(2-ethylhexyl) phthalate,
dicyclohexyl phthalate, di-n-octyl phthalate
Fish and fish dimethyl phthalate, diethyl phthalate, diisobutyl phthalate, di-n- Fierens et al., 2002
products butyl phthalate, benzylbutyl phthalate, di(2-ethylhexyl) phthalate,
dicyclohexyl phthalate, di-n-octyl phthalate
Milk dimethyl phthalate, diethyl phthalate, diisobutyl phthalate, di-n- Fierens et al., 2002;
butyl phthalate, benzylbutyl phthalate, di(2-ethylhexyl) phthalate, Self and Wu, 2012
dicyclohexyl phthalate, di-n-octyl phthalate, diisononyl phthalate,
diisodecyl phthalate
Nutraceutical benzylbutyl phthalate, di-n-butyl phthalate, di(2-ethylhexyl) phthalate, Self and Wu, 2012
products di-n-octyl phthalate, diisononyl phthalate, diisodecyl phthalate
22.3.5 Heavy metals
The general human population is exposed to heavy metals at trace concentrations either voluntarily
through supplementation or involuntarily through the intake of contaminated food (Table 22.6)
and water or following contact with contaminated soil, dust or air. Heavy metal pollution in
water bodies is a serious environmental issue, threatening not only aquatic ecosystems, but also
human health through the contamination of drinking water (Balabani et al., 2011; Mukherjee et
al., 2006; Tapio and Grosche, 2006). Some metals, such as Cd, Pb, As and Hg, are non-essential
xenobiotics that are known to be harmful to human health (Balabani et al., 2011; Bellinger,
2005; Nampoothiri and Gupta, 2006; Tapio and Grosche, 2006; Xu et al., 1996). Heavy metal
contamination can take place during the handling and processing of foods, from the farm to the
point of consumption. Thus, besides growth of plants in contaminated soils and the feeding of
animals on feeds containing heavy metals, other factors may contribute to food contamination
such as food contact contaminations. Exposure to toxic levels of any of these environmental
contaminants may result in impaired health in adults, but the toxicological effects of heavy metals
Table 22.4. Detected pesticides in different food groups
Food group Pesticides References
Fruits and vegetables 2-phenylphenol, acephate, acrinathrin, azoxystrobin, Nesreddine and

azinphos-methyl, bupirimate, carbofuran, chlorothalonil, Parent-Massin, 2002;
chlorpyrifos, chlorpyrifos-ethyl, chlorpyrifos-methyl, Nougadre et al., 2012
cyfluthrin, cyprodilin, dichlorvos, diflubenzuron,
diphenylamine, endosulfan, etofenprox, fenbuconazole,
fenhexamid, fludioxonil, imidacloprid, iprodione,
methamidophos, methidathion, penconazole, phosalone,
pirimiphos-methyl, procymidone, propargite, spiroxamine,
tetraconazole, trifloxystrobin, vinclozolin
Bread and dried bread 2-phenylphenol, chlorpyrifos-methyl, pirimiphos-methyl Nougadre et al., 2012
products
Eggs and egg products lindane Nougadre et al., 2012
Meat lindane Nougadre et al., 2012
Soft drinks 2-phenylphenol, imazalil, pyrimethanil, thiabendazole Nougadre et al., 2012
Table 22.5. Common endocrine-disrupting pesticide groups: their effects and modes of action.
Pesticide group Hormones Mechanisms References

affected
Carbamates androgens, androgen receptor dependent; estrogen Goad et al., 2004;

estrogens, receptor interference with cellular microtubule Lu et al., 2004;
steroids formation in estrogen-sensitive cells Morinaga et al., 2004
Organochlorines androgens, competitive inhibition of androgen receptor, Daxemberger, 2002;
estrogens, inhibition at estrogen-sensitive reporter, Lemaire et al., 2004;
prolactin binding to androgen receptors, interference in Scippo, 2004; Storrs
induction of aromatase and Kiesecker, 2004
Organophosphates estrogens induction of estrogen-related genomic activity Gwinn et al., 2005;
Jeong et al., 2006;
Kang et al., 2004;
Pyrethrins estrogens, antagonism or potentiation of estrogen action Kim et al., 2004
progesterone by inhibition of progesterone action
Triazines androgens competitive inhibition of androgen receptors, Ishihara et al., 2003;
binding to androgen-binding proteins; Meulenberg, 2002
induction or inhibition of aromatase
Table 22.6. Heavy metals determined in different food groups.
Food group Heavy metal References
Fruits and vegetables cadmium, lead, arsenic, mercury Hamurcu et al., 2010; Muoz et al., 2005;
Radwan and Salama, 2006;
Meat and meat products cadmium, lead, arsenic, mercury Muoz et al., 2005
Fish and fish products cadmium, lead, arsenic, mercury Choy et al., 2003; Muoz et al., 2005
Milk cadmium, lead, arsenic, mercury Muoz et al., 2005
are often more devastating in the developing reproductive system of children (Balabani et al.,
2011; Bellinger, 2005).
Cadmium
Cd is a highly toxic heavy metal and it is one of the most important environmental pollutants in
industrialized countries. For the general human population the main sources of Cd exposure are
food, drinking water and tobacco smoke (Satarug and Moore, 2004). Cd has pronounced toxic
effects on most organisms and produces significant testicular injury in the fetus (Xu et al., 1996).
This injury may include a progressive reduction of testis weight, accompanied by irreversible
damage to the seminiferous tubules and decreased viability of testicular cells. Furthermore, Cd
can cause a significant reduction in gonadotropin binding, which alters the steroidogenic enzyme
activity of granulosa cells and thus a dysfunction in the production of hormones, leading to
infertility (Nampoothiri and Gupta, 2006).
Lead
Pb is the most abundant of all the heavy metals on earth (Tong and McMichael, 1999). The
main sources of Pb exposure are paints, dust, soil, kitchen utensils and leaded gasoline (Jrup,
2003). Pb in food originates mainly from atmospheric deposition and adherence of Pb-rich soil
particles to fruits and vegetables (Nesreddine and Parent-Massin, 2002). It is estimated that
approximately half of human Pb intake is through food, with half originating from contaminated
fruits and vegetables. The content of Pb in plant foodstuffs is found to be higher than in foodstuffs
originating from animals (Nasreddine and Parent-Massin, 2002).
Pb toxicity can result in a wide range of biological effects on various organ systems depending on
the level and duration of exposure. Pb exposure during the early stages of human life poses a risk
for the health and functional ability of vulnerable fetuses and infants. High levels of paternal Pb
exposure appear to reduce the male fertility and to increase the risk of miscarriage and reduced
fetal growth (preterm delivery and low birth weight; Bellinger, 2005). Exposure to Pb during
fetal development and breast feeding depends on the maternal burden and factors that modulate
Pb transfer through the placenta and the mammary gland. A recent study by Nampoothiri and
Gupta (2006) has shown that Pb can cause a significant reduction in gonadotropin binding, which
alters the steroidogenic enzyme activity of granulosa cells and thus interferes with the production
of hormones, leading to infertility.
Arsenic
Exposure to excess As, principally from contaminated drinking water, is considered one of the top
environmental health threats worldwide (Mukherjee et al., 2006). Most of this exposure is from
natural geological sources of As that contaminate groundwater (Mukherjee et al., 2006). It has
been established that fish and seafood can accumulate organic As from their environment (Alam
et al., 2003; Sirot et al., 2009). The concentrations of As in fruits and vegetables depends on the
soil content, water contamination, air pollution and the usage of fertilizers (Alam et al., 2003).
Epidemiological studies have linked chronic exposure to As in drinking water with increased risks
of developmental and reproductive problems (Balabani et al., 2011; Tapio and Grosche, 2006).
Mercury
Hg is a naturally occurring metal that has several forms: (1) metallic or elemental Hg, which is
commonly used in dental fillings and thermometers; (2) inorganic compounds that are used in
skin care and medicinal products; and (3) organic compounds that are used in fungicides and
paints (Bjrnberg et al., 2005). Hg is one of the most toxic heavy metals commonly found in
the global environment, including in the lithosphere, hydrosphere, atmosphere and biosphere
(Clarkson, 2002). It persists in the environment and accumulates in the food chain (Bjrnberg
et al., 2005; Choy et al., 2003; Clarkson, 2002). The general human population is exposed to Hg
(usually in an inorganic form and at very low concentrations) primarily through the diet with
contaminated fish, dental amalgam, water consumption and as a result of inhalation (Balabani
et al., 2011; Clarkson, 2002). The level of Hg in foods is inconsistent and reflects the level of
pollution of the local environment. Plants absorb only limited amounts of Hg through their
roots, even from highly contaminated soils (Nasreddine and Parent-Massin 2002). Hg and its
compounds are a significant threat to human health, particularly to pregnant women, women
of child-bearing age, developing fetuses and breast-fed infants. In vitro studies have shown that
Hg is capable of inducing sperm abnormalities (Choy et al., 2003). According to Balabani et
al. (2011), Hg can cause spontaneous abortion, stillbirths, congenital malformations, infertility,
disturbances in the menstrual cycle and inhibition of ovulation.
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23. Anemia, Roux-en-Y gastric bypass and
conception
Department of Obstetrics and Gynecology, Faculty of Medicine, University of Sao Paulo, Av. Dr.
Enas de Carvalho Aguiar 255, 10th floor, suite 10037, So Paulo, SP CEP 05403-000, Brazil;
clinicanomura@terra.com.br
Abstract
Bariatric surgery is a weight loss intervention that provides significant decrease of obesity-related
mortality. However, anemia after bariatric surgery may affect as many as two thirds of the patients
and is attributed to iron deficiency. Roux-en-Y gastric bypass (RYGB) is a malabsorptive procedure
that may lead to malnutrition and progressive iron depletion. Food intolerance to certain foods,
particularly red meat, is due to reduction in intake capacity after bariatric surgery with RYGB.
As a consequence, the total iron intake is very low, leading to a substantial decrease in haem iron
bioavailability. Conception after bariatric surgery may help prevent obesity-related gestational
complications. Maternal malnutrition is not without potential risks during pregnancy. Women
undergoing gastroplasty with RYGB for morbid obesity and who subsequently wish to become
pregnant should be referred for counseling with a nutritionist before and during pregnancy.
Long-term nutritional and hematological monitoring should be a priority in patients becoming
pregnant after bariatric surgery. Women who undergo RYGB need appropriate counseling before
becoming pregnant in order to minimize the risk of anemia in pregnancy.
Keywords: anemia, hemoglobin, Roux-en-Y gastric bypass, bariatric surgery, conception.
373
Summary points
Iron is an essential nutrient for life and iron deficiency anemia is particularly associated with Roux-en-Y gastric
bypass (RYGB) surgery.
Anemia following RYGB may be multifactorial, resulting from impaired iron absorption in the proximal
gastrointestinal tract, inadequate oral intake because of food intolerance or occult blood loss.
RYGB is a malabsorptive procedure that may lead to malnutrition in pregnant women, and deficits during
pregnancy are not completely eliminated despite dietary guidance and micronutrient supplementation.
Women who undergo RYGB need appropriate counseling before conception in order to minimize the risk
of anemia in pregnancy.
Long-term nutritional and hematological monitoring should be a priority in patients becoming pregnant after
bariatric surgery.
23. Anemia, Roux-en-Y gastric bypass and conception
Abbreviations
RYGB Roux-en-Y gastric bypass
BMI Body mass index
23.1 Introduction
Bariatric surgery is a weight loss intervention that provides significant decrease of obesity-
related mortality (Adams et al., 2007; Sjstrm et al., 2007). However, despite this benefit,
the postoperative development of anemia is a concern, especially if the woman intends to get
pregnant after the procedure.
Anemia after bariatric surgery may affect as many as two-thirds of the patients and is attributed
to iron deficiency (Brolin et al., 2002). Intestinal iron absorption is regulated according to body
iron reserves and the intensity of erythropoiesis. However, RYGB is a malabsorptive procedure
that may lead to malnutrition and progressive store depletion (Bal et al., 2011).
RYGB can be performed either by laparoscopy or by open laparotomy. The RYGB configuration
is characterized by the creation of a vertical gastric pouch of approximately 30 ml, a Roux-en-Y
jejunal limb of 100 cm and a biliopancreatic limb of 60-80 cm. The gastric reservoir has a length
of 8-10 cm and a volume of 30-50 ml, and it disconnects from the food stream during secretion
of gastric acid, pepsin, and intrinsic factor. The small intestine is divided at a distance of 30-
50 cm distal to the ligament of Treitz. By dividing the bowel, the surgeon creates a proximal
biliopancreatic limb that transports the secretions from the gastric remnant, liver and pancreas.
The Roux limb is anastomosed to the new gastric pouch and its function is to drain consumed
food. A silastic ring (6.3 cm in diameter) is placed around the pouch 3 cm proximal to the end-
to-side gastrojejunal anastomosis in order to prevent future dilatation (Faintuch et al., 2007).
Iron deficiency anemia is particularly associated with RYGB surgery, far more so than with purely
restrictive procedures. Iron bioavailability is compromised after gastroplasty with RYGB due to
decreased food intake, a lack of gastric acidity and the bypass of the duodenum (Ruz et al., 2009).
Kalfarentzos et al. (2001) diagnosed anemia in 46% of the patients 4 years after gastroplasty.
Furthermore, anemia may persist despite oral iron replacement therapy (Mizn et al., 2007).
Varma et al. (2008) identified the need for long-term parenteral iron replacement therapy after
malabsorptive bariatric procedures, especially in premenopausal women.
23.2 Iron homeostasis

Iron is an essential nutrient for life, as it is vital for several biological reactions, such as oxygen
transport, cell proliferation and cellular-immune responses (Andrews, 2008). Most body iron
circulates as hemoglobin, which is recycled during red cell senescence. Other stocks include the
liver and myoglobin, and a small amount is bound to plasma transferrin. Menstruating women
may lose 2 mg of body iron daily on average.
Dietary iron comprises haem (animal sources 10%) and non-haem (cereal and vegetable sources
90%) forms. Daily, 1-2 mg of iron is absorbed mostly at the duodenum. Haem iron is better
absorbed than non-haem iron; absorption occurs at the apical surface of duodenal enterocytes
via different mechanisms. Non-haem iron primarily exists in a ferric form (Fe3+), which must
first be reduced to the ferrous form (Fe2+) for better absorption. The non-bioavailable Fe3+ is
reduced to the Fe2+ form by stomach acid, dietary ascorbic acid and luminal reductases. The
non-haem iron is absorbed by intestinal luminal cells through a specific transporter (divalent
metal transporter) and released into the circulation where it binds to transferring (Muoz et al.,
2009). The absorption of non-haem iron is inhibited by simultaneous consumption of phytates
(in cereals and legumes), tannins (in coffee and tea) and calcium. The co-administration of
tetracyclines, proton pump inhibitor and antiacid medication can diminish the absorption of
non-haem iron (Table 23.1).
Absorption of haem iron into the enterocyte occurs by an as yet incompletely identified haem
carrier protein 1. Once in the enterocyte, haem iron is probably metabolized by haem oxygenase
to release Fe2+, which enters a common pathway with non-haem iron. It is not clear whether
haem carrier protein 1 has physiological roles in other tissues, since the protein is also expressed
in the kidneys and liver (Andrews, 2008).
Within the enterocytes, most Fe2+ is transported through the basolateral membrane, via
ferroportin channels, to the bloodstream, where it is bound to serum transferrin, which carries
the bound iron to target cells. Intracellular iron may remain in the cell for use or storage (ferritin),
and this iron is never absorbed into the body, except when lost due to enterocyte senescence
(Muoz et al., 2009).
Table 23.1. Factors that affect the absorption of iron by the human body.
Positive factors
Deficiency state (increases absorption)
Bioavailability of haem iron in the meat
Haem iron increases the absorption of non-haem iron
Ascorbic acid
Negative factors
Phytate (rice, beans, soybeans, vegetables)
Polyphenols and tannins (black and mate tea, coffee)
Dietary calcium
Soy protein
The absorption of iron is dependent on the bodys iron stores, hypoxia and rate of erythropoiesis.
It is upregulated by iron deficiency and increased erythropoiesis, downregulated in the presence
of inflammation and iron repletion and mediated by the regulator of iron homeostasis, hepcidin,
which blocks iron release from enterocytes and macrophages. The main systemic regulator
of iron absorption and macrophage release is hepcidin, which negatively interacts with and
degrades ferroportin in response to iron overload and inflammation (Nemeth et al., 2004).
Hepcidin reduces circulating iron by blocking its absorption in the intestine and its release from
macrophages. Transferrin, ferritin and hepcidin are produced by the liver. The latter two are
acute phase reactants, and their levels may be elevated during infection, inflammation, or stress
(Andrews, 2008).
Iron distribution via the circulation occurs by the binding of iron to transferrin, which then
transports the iron to the sites of use and storage. Transferrin-bound iron enters target cells
(erythroid, immune and hepatic cells) through a highly specific process of receptor mediated
endocytosis. The release of iron from transferrin is promoted in the interior of the cell; Fe3+ is
reduced to Fe2+ by a ferrireductase and transported to the cytoplasm. To form haem, iron must
cross an ion-impermeable membrane to enter the mitochondria by an importer (mitoferrin)
(Andrews, 2008).
Hemoglobin iron has substantial turnover. Macrophages and the liver are the main storage sites
for iron. Macrophages from the reticuloendothelial system promote phagocytosis of senescent
erythrocytes, and haem is metabolized, releasing iron into the cytoplasm. Iron can be stored
in the cytosol as ferritin or hemosiderin, when ferritin is broken down within the lysosomes.
Hemosiderin represents a very small fraction of stored body iron and is mostly found in
macrophages. In the liver, iron is sequestrated in hepatocytes predominantly in the form of
ferritin or hemosiderin (Camaschella and Strati, 2010).
23.3 RYGB and anaemia

Anemia following RYGB may be multifactorial, resulting from impaired iron absorption in the
proximal gastrointestinal tract, inadequate oral intake because of food intolerance or occult blood
loss. Determining the true prevalence of anemia after RYGB is difficult. Nevertheless, it has been
reported that 5-64% of this postbariatric population suffers from it (Cable et al., 2011). A high
incidence of 38% of anemia has been reported in the late postoperative setting with a 3.5% long-
term transfusion requirement (Amaral et al., 1985). Some studies, however, have reported that
the incidence of anemia after RYGB is about 10% (Avgerinos et al., 2010). In a large population
of 720 patients followed up for 1 year, Cable et al. (2007) found a prevalence of 36%. Anemia
typically occurs with deficiencies of iron, folate and vitamin B12 anywhere from 8 weeks to 2
years following surgery (Vargas-Ruiz et al., 2008). The main sites of absorption of vitamins in the
intestinal tract are included in the Table 23.2.
Table 23.2. The main sites of absorption of vitamins in the intestinal tract.
Nutrient Site
Thiamine (B1) jejunum; duodenum

Riboflavin (B2) jejunum; duodenum
Niacin (B3) jejunum; duodenum
Vitamin B6 jejunum; duodenum
Folate (B9) jejunum; duodenum; ileum
Vitamin B12 (requires intrinsic factor) terminal ileum
Vitamin C jejunum; duodenum; ileum
Iron duodenum
Food intolerance to certain foods, particularly red meat, is due to reduction in intake capacity
after bariatric surgery with RYGB. As a consequence, the total iron intake is very low, leading to
a substantial decrease in haem iron bioavailability (Love and Billet, 2008). Moreover, the limited
gastric capacity interferes with the amount of gastric acid produced. As a result, the oxidation of
non-haem iron is impaired, thus reducing the absorption of iron into enterocytes. Furthermore,
a low serum iron concentration after bariatric surgery was found to be more common among
women (51%) than men (20%), and this was attributed to menstrual blood losses (Amaral et
al., 1985).
Meat is a major source of haem iron. Diminished intake of red meat after RYGB contributes
to anemia in post-bariatric patients. As iron supplementation is required to reduce the risk of
iron deficiency anemia following RYGB, prophylactic multivitamin supplements are routinely
prescribed for these patients (Von Drygalski et al., 2011). However, despite their being on
iron supplementation regimens, some patients develop iron deficiency anemia. Avgerinos et
al. (2010) demonstrated that, in patients receiving iron supplementation, the risk factors for
anemia following RYGB are menstruation and peptic ulcer disease. Brolin et al. (1998) found that
iron deficiency anemia is very resistant to oral iron supplementation, especially in menstruating
women, leading them to recommend prophylactic oral iron in high doses of 320 mg of ferrous
sulfate daily (providing 100 mg of elemental iron).
Symptoms of iron deficiency may be nonspecific and they include fatigue and muscle weakness,
dyspnea, pica, chest and oral discomfort. The laboratory findings include low serum ferritin,
elevated total iron binding capacity, low mean corpuscular volume and low intracellular
hemoglobin concentration. Iron deficiency anemia is typically hypochromic and microcytic.
The risk of iron deficiency increases over time. Some studies have reported that over half of the
patients had low ferritin levels 4 years after the RYGB (Nomura et al., 2011; Skroubis et al., 2002)
and that such low levels can persist for up to 7 postoperative years (Avinoah et al., 1992). Because
oral iron supplementation is associated with poor absorption and adverse gastrointestinal effects,
parenteral intravenous iron may be needed by a significant number of patients (Cable et al., 2011;
Heber et al., 2010).
Patients who have undergone RYGB are at risk for vitamin B12 deficiency, which may occur
within several months of the surgery, depending on tissue stores and intake of B12-rich foods
(red meat). The decreased intake of B12 should not be overlooked because of the exclusion of
the source of the gastric intrinsic factor, since the formation of the B12-intrinsic factor complex
is essential to absorption in the distal ileum. Moreover, gastric acid is necessary for adequate
vitamin B12 release and absorption. Prevention of vitamin B12 deficiency following RYGB is vital
(Fujioka et al., 2011). Supplementation can be given via injections of cyanocobalamin. Treatment
of severe B12 deficiency with parenteral B12 may result in hypokalemia due to the increased
potassium needs of the responding cellular elements, and RYGB patients may be at a higher risk
if vomiting or poor oral intake has occurred.
Folate deficiency is common after RYGB and it occurs in 38% of patients. Inadequate intake and
prolonged vomiting are the main reasons in most cases. Folic acid is the composite of a larger
group of compounds known as folates, which are necessary for DNA synthesis and are vital for
normal cellular proliferation, especially for hematopoietic cells. Folate deficiency following RYGB
may lead to megaloblastic anemia, which may be more prevalent in women than in men, and
has been reported to cause fetal neural tube defects. Multivitamin supplements containing 800
microgram of folate have been found to prevent folate deficiency.
23.4 Conception after bariatric surgery with RYGB

Conception after bariatric surgery may help prevent obesity-related gestational complications.
However, maternal malnutrition is not without potential risks during pregnancy, and the woman
must be aware of some aspects (Table 23.3). Obese women present an elevated risk for infertility,
macrosomia, gestational diabetes, preeclampsia, caesarean delivery and infectious morbidity (Chu
et al., 2007). The rates of many adverse maternal outcomes may be lower after bariatric surgery
when compared to pregnancy before surgery (Wax et al., 2008; Weintraub et al., 2008). Strategy to
improve the maternal nutritional status during pregnancy is presented in the Table 23.4.
Despite improvement in maternal outcome, maternal anemia due to iron deficiency is a matter
of concern. A favorable iron status at the beginning of pregnancy is a prerequisite for a good
course and normal development of the fetus. Since long-term sequelae of bariatric surgery
include vitamin and mineral deficiencies such as iron, vitamin B12 and folate, iron deficiency is
expected to be relatively frequent during pregnancy after bariatric surgery with RYGB. Therefore,
appropriate treatment should be instituted and iron status should be monitored carefully.
Maternal anemia can have a strong impact on maternal and perinatal outcomes. Low hemoglobin
concentrations at delivery and poor pregnancy outcome have been reported in several studies.
Table 23.3 Things to consider before becoming pregnant after Roux-en-Y gastric bypass.
To be achieving the nutritional needs in the diary intake

Do not have mineral and micronutrient deficiencies (Fe, vitamins, calcium, etc.)
Regular use of multivitamin supplementation
To be controlled medical conditions (e.g. hypertension, diabetes, etc.)
Table 23.4. Strategy to improve the maternal nutritional status during pregnancy after Roux-en-Y gastric bypass.
Consultation with the surgeon to know which bariatric surgery was performed and the occurrence of any
complications
Evaluation of micronutrient deficiencies and refer to the nutritionist to ensure adequate dietary intake
Monitoring blood count, mineral, and vitamin levels
Supplementation with oral vitamins and if necessary by parenteral forms
Surveillance for postpartum anemia, iron or vitamins deficiency
Iron deficiency anemia is associated with preterm birth, low birth weight and small-for-
gestational-age newborns (Ren et al., 2007). However, despite the presence of maternal anemia,
the assessment of fetal wellbeing during pregnancy, by cardiotocography and fetal biophysical
profile, is not compromised in pregnancies after gastroplasty with RYGB (Nomura et al., 2010).
In addition, this condition predisposes to postpartum anemia and may have a negative influence
on the womans physical and emotional state (Beard et al., 2005).
RYGB is a malabsorptive procedure that may lead to malnutrition in pregnant women, limiting
the helpfulness of iron absorption improvement in the 2nd half of gestation. Anemia and other
nutritional deficits during pregnancy are not completely eliminated despite dietary guidance and
micronutrient supplementation (Faintuch et al., 2009).
A broad evaluation of micronutrient deficiencies at the beginning of pregnancy is strongly

recommended. Less than 60% of postoperative bariatric surgery patients continue taking
multivitamin supplements for a prolonged period of time. Therefore, maternal iron stores,
vitamins and hemoglobin levels (Table 23.5) should be investigated at the beginning of prenatal
care and iron supplementation should be implemented during pregnancy considering the time
interval between gastroplasty and conception.
Women undergoing gastroplasty with RYGB for morbid obesity and who subsequently wish
to become pregnant should be referred for counseling with a nutritionist before and during
pregnancy. The recommended diet (15-25 kcal/kg/day) targets a pregnancy weight gain of 7-11
kg as prescribed for overweight women (BMI 26.1-29.0 kg/m2) by the Institute of Medicine.
Table 23.5. Routine monitoring during pregnancy after Roux-en-Y gastric bypass.
Complete blood count each trimester

Iron studies (ferritin, serum Fe, transferrin)
Vitamin B12
Folate
Prothrombin time
25-OH vitamin D
The women are instructed to consume small meals and to cut all foods in tiny pieces, especially
meat and sausages, as well as uncooked fruits and vegetables. The women are also asked to
permanently maintain consumption of low-fat foods (<30% total energy), especially of saturated
fats, and to refrain from consuming energy-dense or fatty snacks and sweets, such as fried foods,
chocolate, ice-cream, cookies and sugared beverages (Beard et al., 2008). In our experience,
dietary compliance during pregnancy is not optimal, mean weight gain is high and patients
display various comorbidities. Nutritional monitoring and dietary guidelines should be a priority
(Dias et al., 2009).
There are no established guidelines for dietary control. Beard et al. (2008) have made some
valuable suggestions, including a daily minimum of 60 g of protein, 400 g of folate, 50-100
mg of elemental iron and at least 1000 mg of calcium (maximum of 1,500 mg). In respect to
this, routine nutrition screening, use of appropriate supplements and compliance monitoring
are imperative for these women.
The average total iron requirement in normal pregnancy has been estimated to be 1,190 mg,
taking into account maternal adaptations, fetus, placenta, and blood loss during delivery. An
adequate maternal iron status is essential to provide normal development of the fetus and
maturity of the newborn.
Oral iron supplementation is appropriate at the beginning of pregnancy. Multivitamin and

iron supplements containing 60-100 mg/day of elemental iron and 5 mg/day of folic acid are
prescribed for all patients. In addition, the patients also receive 1000 mg/day of calcium and
vitamin B12 injections (1000 g) at 3-month intervals (Table 23.6). Diagnosed prepartum iron
deficiency anemia is treated first by oral administration of elemental iron (120-180 mg/day);
however, intravenous iron should be administered if hemoglobin levels do not improve (Heber
et al., 2010). In the case of severe maternal anemia after 32 weeks of gestation, intravenous iron
therapy is an option. A dose of 200 mg of iron III-hydroxide saccharate diluted in 250 ml of
0.9% sodium chloride solution is recommended to be administered intravenously over a period
of 40-60 min, twice a week for 4 weeks (total dose of 1,600 mg). In our experience, the need for
intravenous iron therapy or packed red cell transfusion is significantly more frequent among
women who become pregnant 4 years after RYGB compared to <4 years (30.8% vs. 0%, P=0.026)
Table 23.6. Recommended diet and supplementation during pregnancy after Roux-en-Y gastric bypass.
Energy: 15-25 kcal/kg/d

Protein: 60 g/d
Calcium: 1.0-1.5 g/d, oral supplementation
Iron (elemental): 60-100 mg/d, oral supplementation
Vitamin B12: 1000 g; intramuscular, each trimester
Folate: 5.0 mg/d, oral supplementation
(Nomura et al., 2011). Pregnancy after four years of RYGB is associated with low hemoglobin
levels and the need for stricter iron supplementation.
23.5 Conclusions
Long-term nutritional and hematological monitoring should be a priority in patients
becoming pregnant after bariatric surgery. Dietary guidelines are clearly needed since current
recommendations are insufficiently tested within this context. Women who undergo RYGB need
appropriate counseling before becoming pregnant in order to minimize the risk of anemia in
pregnancy.
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Fertility-infertility
24. V itamin D and assisted reproductive
technology
A. Mahdavi1 and H. Hosseini Jadda2
1Tehran University of Medical Sciences (TUMS), Shariati Hospital, Department of Obstetrics,
Gynecology and Infertility, North Karegar Street, 1411713135 Tehran, Iran; 2Milad Hospital,
Department of Gastroentrology, Hemmat Highway, before Chamran Bridge, 1449614531 Tehran,
Iran; at-mahdavi@tums.ac.ir
Abstract
Vitamin D is well known for its role in calcium and phosphorus homeostasis and bone
mineralization, but this vitamin has significant effects on the growth and differentiation of a
variety of tissues. The beneficial roles of this steroid in decreasing the risk of many chronic
illnesses including common cancers, insulin resistance, autoimmunity, infectious diseases and
cardiovascular problems via recognized pathways are well recognized. Most tissues and cells in
the body have a vitamin D receptor and due to global pandemic of this vitamin new insights has
been attracted into the reproductive and fertility functions of this vitamin. In fact, this steroidal
hormone affects most systems in the body and its receptor has been demonstrated in reproductive
tissues both in male and female. This chapter reviews literature relating to vitamin D status and
reproduction in animals and human beings, then focuses on research in women undergoing
assisted reproductive technology.
Keywords: vitamin D, assisted reproductive technology, fertility, follicular fluid, in vitro

fertilization, intracytoplasmic sperm injection
387
Summary points
Vitamin D affects ovary (sex hormone production, folliculogenesis) and endometrium (implantation).
Vitamin D has positive effects on androgen synthesis, spermatogenesis and sperm maturation.
Significant linear correlation exists between the levels of vitamin D in follicular fluid and serum.
Powerful data about the role of vitamin D in assisted reproduction is scarce and there exists some inconsistent
data.
The optimal concentration of 25-hydroxy vitamin D to improve human fertility is not realized and randomized
clinical trials of vitamin D supplementation to find the best level are warranted.
24. Vitamin D and assisted reproductive technology
Abbreviations
ART Assisted reproductive technology
BMI Body mass index
CES Cumulative embryos score
ET Embryo transfer
ICSI Intracytoplasmic sperm injection
1,25(OH)2D 1,25-dihydroxy vitamin D
25(OH)D 25-hydroxy vitamin D
24,25(OH)2D 24,25-dihydroxy vitamin D
MSEQ Mean score of embryo quality
VDR Vitamin D receptor
VDRE Vitamin D response element
24.1 Introduction
Since the first successful in vitro fertilization resulting in the birth of Louise Brown in 1978
(Steptoe and Edwards, 1978), gradual improvements have occurred in the field of ART. These
include improvements in gamete collection, preparation, selection and micromanipulation,
introduction of new drugs and culture systems plus different stimulation protocols, clinical
applications of genetics in ART, strategies for reducing complications, in addition to gamete and
embryo donation and surrogacy motherhood.
Despite these dramatic developments, the failure of the many ART cycles still challenges the
infertility specialist to find new strategies to enhance ART outcomes. One of the possible fields
of focus may be the effect of vitamins and micronutrients on the success of ART. Vitamin D is
well known for its role in calcium and phosphorus homeostasis and bone mineralization, but
this vitamin has significant effects on the growth and differentiation of a variety of tissues. The
beneficial roles of this steroid in decreasing the risk of many chronic illnesses including common
cancers, insulin resistance, autoimmunity, infectious diseases and cardiovascular problems via
recognized pathways are well recognized (Grundmann and Versen-Hynck, 2011; Heaney, 2008;
Holick, 2007; Perez-Lopez et al., 2011). The fact that most tissues and cells in the body have a
vitamin D receptor (Holick, 2007) and due to global pandemic of this vitamin (Heaney, 2008;
Holick, 2007; Perez-Lopez et al., 2011) new insight has been attracted into the reproductive and
fertility functions of this vitamin. In fact, this steroidal hormone affects most systems in the body
and its receptor has been demonstrated in reproductive tissues both in male and female (Barrett
and McElduff, 2010; Blomberg Jensen et al., 2010; Kinuta et al., 2000).
This chapter reviews literature relating to vitamin D status and reproduction in animals and
human beings, then focuses on research in women undergoing ART.
24.2 Vitamin D
Vitamin D is a steroid hormone which can regulate up to 3% of the human genome and its
receptor (VDR) is a member of the nuclear steroid receptor super family; regulation of its
expression is very important (Barrett and McElduff, 2010; Jones et al., 1998). There are two
forms of this vitamin: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). This latter
is the one discussed in this article. In the liver it is 25-hydroxylated to form 25(OH)D and by
1-hydroxylase in the kidney is converted to the active metabolite1,25(OH)2D. The enzyme
24-hydroxylase catabolizes the active metabolites to inactive metabolites. Vitamin D status is
assessed by measuring serum 25(OH)D which has a variable half-life (Barrett and McElduff, 2010;
Holick, 2007; Lerchbaum and Obermayer-Pietsch, 2012). This vitamin acts through genomic (via
changes in gene transcription, taking hours to days) and non-genomic (via interaction with a cell
surface receptor and second messengers, taking seconds to minutes) pathways.
The classical function of vitamin D is to maintain calcium and phosphorous homeostasis through
bone, kidney and intestinal organs. Its non-classical function is suggested by the presence of
vitamin D receptor and the 1-hydroxylase enzyme in a variety of tissues. Skeletal muscle, skin,
cardiovascular system, insulin resistance and the immune system are affected by non-classical
functions of this steroid hormone (Figure 24.1; Barrett and McElduff, 2010; Holick, 2007;
Lerchbaum and Obermayer-Pietsch, 2012).
Vitamin D
Classic eects Non classic eects
Intestine Endometrium (implantation, endometriosis)

Calcium absorption
Synthesis of calcium binding proteins Immune system (chronic inflammation, endometriosis)
Phosphorus absorption
Ovary (steroidogenesis of sex hormones, folliculogenesis)
Kidney
Synthesis of 1,25(OH)2D3 Pancreas (insulin secretion, insulin resistance)
Phosphate reabsorption
Calcium reabsorption Placenta
Bone Testis (androgen synthesis, spematogenesis, spem maturation)

Promotes bone formation
Promotes bone resorption by
increasing the number and
activity of osteoclasts
Figure 24.1. Vitamin D effects (classical and non-classical).
24.3 Vitamin D expression and function in reproduction

24.3.1 Animal
The majority of research on vitamin D and fertility is conducted in animal models, especially
in rats. VDR has been found in the genital tract of rodents and most studies focused on the
direct effects of vitamin D or its calcium-mediated effects (Lerchbaum and Obermayer-Pietsch,
2012). Lines of evidence were drawn from vitamin D deficient and knockout animals. Since the
interest in vitamin D and animal fertility, vitamin D deficient animal models have shown reduced
fertility rates and fertility capacity, diminished mating success, impaired neonatal growth,
increased pregnancy complications, gonadal insufficiency, reduced aromatase gene expression,
low aromatase activity, hypogonadism, bone malformations, uterine hypoplasia, impaired
folliculogenesis, spermatogenesis and infertility (Halloran and DeLuca, 1980; Lerchbaum and
Obermayer-Pietsch, 2012; Yoshizawa et al., 1997).
24.3.2 Human
Human reproduction is affected by many factors. There is a seasonal variation in human fertility
because the ovulation rates and endometrial receptivity and resulting conception rate is decreased
during the winter, whereas the conception rate peaks during the summer (Rojansky et al., 1992,
2000). Other epidemiological studies have revealed that vitamin D has a seasonal variation and
nearly one-third of healthy adults are vitamin D deficient at the end of winter. Well-designed
studies will show whether seasonal variation in human reproduction is related to seasonal
variation of this vitamin concentration.
The biologically active form of vitamin D is 1,25(OH)2D and the vitamin D status is mainly
assessed by the measurement of serum 25(OH)D concentration. The normal range of vitamin
D is difficult to determine, but most articles refer to a serum level of 25(OH)D >30 ng/ml as
a sufficient vitamin D status. Levels of 20-30 ng/ml and <20 ng/ml are recognized to reflect
vitamin D insufficiency and deficiency, respectively (Holick, 2007). However, the best level of
vitamin concentration to improve fertility has yet to be defined.
24.3.3 Women
VDR mRNA is expressed in the ovaries and endometrium (Agic et al., 2007) with a role in
steroidogenesis of sex hormones (Parikh et al., 2010). In the ovaries, 1,25(OH)2D stimulates
progesterone, estradiol and estrone production (Parikh et al., 2010). Insulin and vitamin D
synergistically stimulate estradiol production, and vitamin D also enhances the inhibitory effect
of insulin on IGFBP-1 production (Parikh et al., 2010).
Also, the active form of the 1-hydroxylase gene is expressed in human endometrial stromal cells
especially in early pregnant decidua (Vigano et al., 2006). It has been shown that 1,25(OH)2D
regulates human chorionic gonadotropin expression and secretion in human placenta (Barrera
et al., 2008) and increases placental sex steroid production (Barrera et al., 2007).
HOXA10 is necessary for embryo implantation and fertility. It has been documented that
HOXA10 expression is up-regulated by 1,25(OH)2D by binding to VDR and interacting with
vitamin D response elements (VDRE) in the HOXA10 regulatory region (Daftary and Taylor,
2006; Du et al., 2005).
24.3.4 Men
Spermatids, vesicles within the caput epididymis, and glandular epithelium of cauda epididymis,
seminal vesicles and prostate are the sites of VDR and Vitamin D metabolizing enzymes
(Blomberg Jensen et al., 2010). So this steroid is considered important for spermatogenesis and
maturation of human spermatozoa.
In a recent study evaluating the actions of vitamin D at a molecular level in sperm survival and
capacitation, lower doses of vitamin D induced cholesterol efflux, protein phosphorylation and
sperm survival (Aquila et al., 2008). In addition, 1,25(OH)2D increases the fertilizing ability of
human sperm by increasing intracellular calcium, motility and acrosin activity (Aquila et al., 2009).
Moreover, 1,25(OH)2D reduces triglycerides to increase the lipase activity. Lipid metabolism
probably increases to meet the energetic demands during capacitation by reducing energy storage
and increasing energy expenditure (Aquila et al., 2009). In a very recent study it was suggested
that expression of CYP24A1 at the annulus of human spermatozoa may serve as a novel marker of
semen quality and an objective proxy for sperm function (Blomberg Jensen et al., 2012).
24.4 Vitamin D and ART

When reviewing the effects of vitamin D in reproduction and especially ART, its effects on male
gonad and gametes, oocytes, embryos, uterus and implantation should be considered. This section
reviews available and relevant English language publications in Pubmed until April 2012. At the
time of this review, specific data on the effects of vitamin D metabolites in women undergoing
assisted reproductive techniques are sparse, insufficient and inconsistent.
The presence of vitamin D metabolites (25(OH)D, 1,25(OH)2D and 24,25(OH)2D) in follicular

fluid was documented for the first time in ten healthy women undergoing IVF-ET (Potashnik et
al., 1992). The results showed that elevated estradiol levels during gonadotropin-induced ovarian
stimulation was associated with a significant increase in serum 1,25(OH)2D, but not 25(OH)D
and 24,25(OH)2D levels.
Studies investigating the relationship or rather association of vitamin D metabolites with ART
results are sparse and sometimes contradictory. Nearly two decades later, Ozkan et al. (2010)
for the first time found significantly higher levels of follicular vitamin D in women becoming
pregnant after in vitro fertilization (Table 24.1). 84 infertile women undergoing IVF were enrolled
and in the case of pregnancy were followed till the intrauterine gestational sac was visible on the
transvaginal ultrasound scan. They showed a high correlation between serum and follicular fluid
levels of 25(OH)D and higher implantation rate and clinical pregnancy across tertiles of vitamin
D in follicular fluid of women candidates for ART. Also, significant inverse correlations were
noted between follicular fluid 25(OH)D and BMI. The number of embryos transferred differed
between pregnant and non-pregnant women, but multivariable logistic regression analysis
confirmed follicular 25(OH)D level as an independent predictor to achieve a successful IVF
cycle. They showed an increased likelihood for achieving clinical pregnancy by 6% for each ng/
ml increase in follicular 25(OH)D. Despite the relatively small sample size, and in the absence of
any significant relationship with ovarian response, they proposed that higher circulating vitamin
D levels improved endometrial receptivity.
Table 24.1. Participant and in vitro fertilization cycle characteristics by outcome of the cycle (Ozkan et al.,
2011, reprinted with permission).
Clinical pregnancy Not pregnant P-value2

(n=26) (30.95%)1 (n=58) (69.04%)1
Follicular fluid 25(OH)D

ng/ml 34.4215.58 25.6210.53 0.013
mmol/l 86.0538.96 64.0426.32
Age (years) 33.884.57 34.865.08 0.565
Body mass index (kg/m2) 23.774.00 26.376.60 0.164
Race3 0.801
Black (%) 4/23 (17) 8/53 (15)
White (%) 17/23 (74) 33/53 (62)
Other race (%) 2/23 (9) 12/53 (23)
Baseline FSH4 (mIU/ml) 7.763.08 8.162.09 0.360
Gonadotropin dose (Amps)5 28.1014.27 44.9627.19 0.001*
Days of COH4 10.731.43 11.931.70 0.002*
Estradiol on hCG4 day (pg/ml) 2,2971,171.86 2,266.311,101.07 0.961
Oocytes retrieved (n) 12.886.33 12.026.72 0.525
Overall fertilization rate (%) 53.0021.00 45.0024.00 0.154
Embryos transferred (n) 2.560.66 1.981.16 0.011*
1 Continuous data are presented as mean standard deviation.

2 Statistically significant: * P<0.05.
3 Information on race was not available for the entire cohort.
4 FSH = follicle stimulating hormone; COH = controlled ovarian hyperstimulation; hCG = human chorionic
gonadotropin.
5 Gonadotrophin dose per ampoule = 75 IU.
In another prospective study we recruited eighty-two infertile women undergoing ART for a cohort
study and 25(OH)D levels in follicular fluid and serum were measured (Aleyasin et al., 2011). A
significant correlation was found between the levels of vitamin D in follicular fluid and serum
(r=0.767, P=0.001), so it was concluded that the level of follicular 25(OH)D reflects body stores of
this vitamin (Figure 24.2). There was no linear correlation between metaphase II (and/or embryo
quality) and levels of 25(OH)D in follicular fluid and serum. Also, no significant difference was
found in pregnancy rates between the tertiles of 25(OH)D level in follicular fluid. The fertilization
rate decreased significantly and the implantation rate increased (not significantly) with increasing
tertiles of 25(OH)D level in follicular fluid. In contrast to the previous study, the logistic regression
model including the variables related to clinical pregnancy did not show that the follicular vitamin
D level could be an independent predictor of clinical pregnancy (Table 24.2).
30
25
Vitamin D in follicular fluid (ng/ml)
20
15
10
r = 0.77
p = 0.001
5
5 10 15 20 25 30
Serum vitamin D (ng/ml)
Figure 24.2. Correlation between serum and follicular fluid vitamin D (with permission from Aleyasin et al.,
2011).
Table 24.2. Participant and assisted reproductive technology cycle characteristics by outcome of the cycle
(Aleyasin et al., 2011, reprinted with permission).
Clinical pregnancy (n=24)1 Not pregnant (n=53)1 P-value
Female age (year) 28.673.84 30.284.35 0.119

Male age (year) 32 (27-50) 34 (24-51) 0.312
Infertility duration (year) 5.5 (1-20) 7 (1-19) 0.396
Table 24.2. Continued.
Clinical pregnancy (n=24)1 Not pregnant (n=53)1 P-value
Type of infertility2 0.161

Primary (n, %) 15 (62.5%) 45 (77.6%)
Secondary (n, %) 9 (37.5%) 13 (22.4%)
Gravidity 0 (0-2) 0 (0-3) 0.290
Number of previous ART3 0 (0-2) 0 (0-5) 0.378
Number of PCOS3 4 (16.7%) 13 (22.4%) 0.766
Female BMI3 (kg/m2) 27.18 (18.29-35.70) 26.70 (17.80-34.29) 0.654
FSH4 (mIU/ml) 5.35 (1.4-11) 6.8 (2-14) 0.129
LH4 (mIU/ml) 4 (1.5-20) 4.8 (4-21) 0.650
Estradiol4 (pg/ml) 44 (14-300) 45.5 (5-245) 0.956
Antral follicle count (n) 11 (5-20) 11 (5-20) 0.512
Total good sperm (million) 4 (0.1-30) 4 (4-60) 0.473
Stimulation (days) 10 (9-17) 11 (8-14) 0.667
Gonadotropin injections (n) 34 (18-104) 36 (16-80) 0.428
Estradiol on hCG3 day (pg/ml) 2,011 (657-4,300) 1,760 (606-6,100) 0.631
Total oocytes retrieved (n) 11 (5-24) 11 (3-23) 0.396
Mature oocytes (n) 8 (3-20) 7 (1-16) 0.070
Pronuclear number (n) 7 (2-15) 6 (1-13) 0.092
Embryos transferred (n) 4 (2-5) 4 (1-5) 0.182
Having freezed embryos (n, %) 12 (50%) 20 (34.5%) 0.190
Mild-moderate OHSS3 (n, %) 2 (8.3%) 2 (3.4%) 0.289
Calcium (mg/dl)5 9.5 (8.2-10) 9.40 (8.60-10.20) 0.931
Phosphorus (mg/dl)5 3.2 (2.10-4.80) 3.2 (2-4.90) 0.901
Alkaline phosphatase(U/l)5 156 (85-236) 139 (81-350) 0.131
Parathormone (pmol/l)5 2.3(0.7-4.8) 2(0.5-4.30) 0.159
Serum vitamin D5 0.235
(nmol/l) 20.3 (13.4-34) 21.45 (14.80-53)
(ng/ml) 8.13 (5.37-13.62) 8.29 (5.93-21.23)
Follicular vitamin D5 0.433
(nmol/l) 22.95 (13.10-48.70) 25.80 (14.70-74.10)
(ng/ml) 9.19 (5.25-19.51) 10.34 (5.89-29.69)
1 Unless otherwise indicated, values are median (range).

2 Primary infertility describes a couple who have never conceived while secondary infertility follows a previous
pregnancy.
3 ART = assisted reproductive technique; BMI = body mass index; hCG = human chorionic gonadotropin; OHSS
= ovarian hyperstimulation syndrome; PCOS = polycystic ovary syndrome; AFC = antral follicle count.
4 FSH (follicle stimulating hormone), LH (luteinizing hormone) and estradiol were measured on day 3 of the cycle.
5 Calcium, phosphorus, alkaline phosphatase, parathormone, vitamin D in serum and , Vitamin D in follicular fluid
were measured on the day of ovum pick up.
The majority of the women in our investigation were vitamin D deficient, but according to the
ART results, it was considered that vitamin D deficiency does not play a pivotal role in the
outcome of ART. In other words, this study was not able to demonstrate a lack of correlation
between vitamin D and the ART success rate, and did not find that vitamin D deficiency cannot
be a valid predictive value in ART. Prevalent vitamin D deficiency was the main limitation of this
research but the positive point was following the understudy population till 20 weeks of gestation.
In another prospective study by Anifandis et al. (2010) measuring serum and follicular fluid
glucose and 25OH vitamin D levels, and finding their effects on embryo quality in 101 consecutive
women who underwent ART treatment was evaluated. All women were allocated to one of the
three groups according to their follicular fluid vitamin levels. Group A, group B, and group C
with less than 20 ng/ml, 20.1-30 ng/ml and more than 30 ng/ml vitamin levels, respectively. CES,
MSEQ, along with clinical pregnancy were calculated. Serum and follicular fluid vitamin levels
were significantly correlated. The follicular glucose levels were significantly different between
the three groups. Also, a significant negative correlation between follicular vitamin levels and
follicular glucose levels was found (r=-0.25, P<0.05). They proved that follicular vitamin D levels
were negatively correlated to the quality of embryos and a lower possibility of pregnancy was
observed in higher values of this vitamin (Figure 24.3). They proposed that follicular vitamin
levels possibly reflect the quality of embryos and this could help to predict IVF outcome. Also,
they showed that higher serum and follicular fluid vitamin levels along with lower follicular fluid
glucose levels made it less possible to achieve pregnancy in ART. According to their study, an
increase in follicular vitamin levels led to a small and non-significant increase in the number of
collected oocytes. Although the CES was similar between the three groups, the MSEQ of group
Mean score of embryo quality
10
0
0 20 40 60 80
FF vitamin D levels (ng/ml)
Figure 24.3. Correlation between follicular fluid vitamin D and mean score of embryo quality (MSEQ)
(Anifandis et al., 2010, reprinted with permission).
C was significantly lower than that of groups A and B. Clinical pregnancy rates of group C were
significantly lower than groups A and B.
Moreover, in a proteomic analysis of IVF follicular fluid in women 32 years old (Estes et al.,
2009) revealing potential biomarkers of good responders versus poor responders in IVF, it is
observed that in the follicular fluid of the IVF success group expression of DBP is decreased.
The retrospective study on sixty-four egg recipients by Rudick et al. (2011) showed that the effects
of vitamin D may be mediated through the endometrium. They observed that decreased serum
vitamin D levels were associated with lower pregnancy rates in recipients of egg donation. Their
study was conducted proposing that the oocyte donor recipient model is able to separate the
impact of oocyte and endometrium. Their result was in accordance with the findings of Ozkan
et al. (2010), but neither of them investigated the quality of embryos transferred in relation to
the level of vitamin D.
In conclusion, due to limited powerful data about the role of vitamin D in assisted reproduction
and due to small sample sizes in the existing publications it is difficult to draw more solid and
realistic conclusions. Consideration of special problems, like PCOS and endometriosis, deserves
attention in future well-designed studies.
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Aquila, S., Guido, C., Perrotta, I., Tripepi, S., Nastro, A. and Ando, S. 2008. Human sperm anatomy: ultrastructural
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Barrera, D., Avila, E., Hernandez, G., Halhali, A., Biruete, B., Larrea, F. and Diaz, L., 2007. Estradiol and progesterone
synthesis in human placenta is stimulated by calcitriol. Journal of Steroid Biochemistry and Molecular Biology
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Barrera, D., Avila, E., Hernandez, G., Mendez, I., Gonzalez, L., Halhali, A., Larrea, F., Morales, A. and Diaz, L., 2008.
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25. The impact of obesity on ovulation and early
pregnancy: a focus on ovarian function,
fertilization, implantation and early embryo
development
Department of Animal Reproduction, INIA, Avda. Puerta de Hierro s/n, 28040 Madrid, Spain;
bulnes@inia.es
Abstract
Currently, the reproductive function is recognized to be driven by the interaction of endocrine and
metabolic factors that may modulate its genomic clues. The reproductive activity is determined
by endocrine signaling, which is related to metabolic status and energy balance through the
hormones insulin and leptin. Alterations in the pathways regulating the equilibrium between
appetite and satiety give way to obesity, but also to reproductive alterations. Obesity has been
related to decreased fertility, both in males and females. The success of fertility depends mainly
on the success of processes involving ovulation, fertilization, implantation, placentation and
embryo development; processes that seem to be affected in obese females. This chapter reviews
reproductive failures of obese individuals from preovulatory follicle development and ovulation
to completion of placentation and early-embryo development.
Keywords: female reproduction, insulin, leptin, nutrition
401
Summary points
Obesity has been largely related to the pathogenesis of reproductive disorders, although many of the obese
women are multiparous.
Causes of deficiencies in the process of the follicular development and ovulation may be found both at
the systemic level, by hypersecretion of gonadotrophin luteinizing hormone and disequilibrium in the ratio
between follicle stimulating hormone and luteinizing hormone, and at the local level, by hyperleptinaemia/
leptin resistance and hyperinsulinaemia/insulin resistance.
Causes of failures in implantation and early embryo development and placentation are related to
hyperleptinaemia/leptin resistance and hyperinsulinaemia/insulin resistance, as well as to disturbances in
the action of the adipokines.
Future research should be focused on the study of the nutritional components of reproductive failures by the
development of adequate models of female obesity.
Future treatment of obesity-linked reproductive dysfunctions may be based on lifestyle strategies with weight
control, as well as on interventions on diet and exercise habits.
25. The impact of obesity on ovulation and early pregnancy
Abbreviations
GnRH Gonadotrophin releasing hormone
IGFBP Insulin-like growth factor binding protein
IL Interleukins
IUGR Intrauterine growth retardation
NOS Nitric oxide synthase
NPY Neuropeptide Y
PCOS Polycystic ovarian syndromes
SHBG Sex hormone-binding globulin
TNF Tumor necrosis factor
VEGF Vascular endothelial growth factor
25.1 Introduction
Sixty years ago, in 1952, Rogers and Mitchell (1952) reported that obesity was the very common
and most noteworthy feature in around 43% of the women with reproductive disorders. Some
years later, the incidence of menstrual disorders, anovulatory cycles and infertility was confirmed
to be significantly higher in obese than in normal-weight women. If pregnancy is achieved, the
negative influence of overnutrition and obesity may be extended during pre- and post-natal
development of the offspring; a fact firstly highlighted by Cochrane (1965). From these earlier
evidences, there is a huge and increasing amount of both observational and interventional studies
on the links between obesity and reproductive dysfunction.
Obesity has been largely related to the pathogenesis of reproductive disorders, although many
of the obese women are multiparous. Effects vary with age and reproductive status (Metwally et
al., 2007), as depicted in the Figure 25.1. Obese adolescent girls, due to the increased amount
of body fat and the increased secretion of leptin, may display a precocious onset of ovulatory
cyclic activity (menarche) when compared to normal-weight adolescents. Afterwards, women of
childbearing age may be affected by menstrual alterations (from ovulatory irregularities to chronic
anovulation), PCOS and, hence, reduced pregnancy rates and even infertility. Obese pregnant
women may be affected by increased risks of abortion, miscarriage and preterm delivery; finally,
their offspring may be more prone to different diseases.
The influence of obese condition on menarche is afforded in Chapter 10 of the present Handbook.
In agreement with the aim of this Handbook, the objective of the current chapter is to provide
the reader, in a short and focused reviewed manner, with the essential information about the
effects of overnutrition and obese condition on reproductive features in women of childbearing
age; especially during the critical period, including preovulatory follicle development, ovulation,
implantation and placentation, as well as during the early-embryo developmental phase.
Influences of obesity on female reproduction
Juvenile period Childbearing age

Precocious menarche Non-pregnant
Menstrual alterations Polycystic ovarian Reduced pregnancies
ovulatory irregulariities syndromes (PCOS) Infertility
chronic anovulation
Pregnant
Abortion Prenatal programming of adult disease in the offspring
Miscarriage
Preterm delivery
Figure 25.1. Overview of the main reproductive disorders linked to obese condition.
25.2 Influence of obesity on ovarian functionality and ovulation

25.2.1 Influence of the obese condition on reproductive cyclicity and ovulation
Currently, overnutrition and obese condition in adult women are clearly linked to deficiencies
in follicle development and ovulation, even leading to subfertility and anovulatory infertility
(Metwally et al., 2007). Such alterations are increased when obesity is initiated at infantile and
juvenile periods, with obese girls with early menarche showing an increased prevalence of
menstrual disorders (McCartney et al., 2006) and, furthermore, an earlier onset of menopause
(Norman and Clark, 1998).
Causes of deficiencies in the process of the follicular development and ovulation may be found
both at the systemic and local level (Figure 25.2). At the systemic level, obesity has been related
to hypersecretion of gonadotrophin LH and disequilibrium in the ratio between FSH and LH
(Butzow et al., 2000). At the local level, alterations in follicular growth and development have
been hypothesized to be related to hyperleptinaemia/leptin resistance and hyperinsulinaemia/
insulin resistance, both features existing in obese females.
The hormone leptin has a direct effect on the ovary through its receptors within the ovary, the
follicle, and the oocyte. Adequate circulating levels of leptin are essential for ovarian function,
whilst increased concentrations of the hormone have been associated with adverse effects on
granulosa and theca cells affecting follicular development, oocyte maturation and ovulation
(Duggal et al., 2000). Furthermore, leptin has also an indirect effect; high concentrations of the
hormone induce formation of reactive oxygen species, leading to oxidative damage in endothelial
cells (Bouloumie et al., 1999). Such conditions may impair the oxygen supply to the follicle and,
thus, may affect follicular and oocyte maturation (Brannian and Hansen, 2002).
Childbearing age
Non-pregnant
Menstrual alterations Reduced pregnancies
ovulatory irregularities Infertility
chronic anovulation
GnRH E2 Inh P4
GnRH Neurons
Ovary
Anterior LH
pituitary
Systemic level FSH Local level
hypersecretion of LH leptin resistance
disequilibrium ratio FSH/LH insulin resistance
Figure 25.2. Overview of the main causes for alterations in follicular development and ovulation linked to
obese condition.
The excess of insulin affects the ovarian functionality indirectly, mainly by inhibiting
the production of SHBG by the liver; such situation is the first step of a process causing
hyperandrogenism. The decrease in SHBG secretion raises the concentrations of circulating
free steroids (testosterone, dihydrotestosterone and androstenodiol). Immediately, this increase
in circulating free steroids is compensated by a heightened metabolic clearance of these
hormones, which, in turn, causes a compensatory upsurge in androgen synthesis, inducing
hyperandrogenism (Brewer and Balen, 2010).
As also stated in Chapter 19 Lower fertility associated with periconceptional obesity and
underweight of this book, hyperandrogenism is aggravated in obese women (Gambineri et al.,
2002), since adipose tissue has a prominent role in steroid production, storage and metabolism.
Hyperandrogenism has deleterious effects on the reproductive function, both at pituitary
(increased negative feedback diminishing the secretion of gonadotrophins) and ovarian levels
(increased apoptosis in granulosa cells and negative effects on the oocyte), which contribute
to alterations in ovarian cyclicity and ovulation (Brewer and Balen, 2010). Furthermore, the
combination of hyperinsulinaemia and hyperandrogenism in obese women is implicated in the
appearance of a reproductive disease with its own identity, PCOS.
25.2.2 Influence of obesity on the appearance of polycystic ovarian syndrome
PCOS is a reproductive disorder characterized by different neuroendocrine, cellular and

metabolic components causing hyperandrogenism and polycystic ovaries and, therefore, oligo-
or anovulation and infertility.
PCOS has been also associated with obesity, insulin resistance, and dyslipidemia. Prevalence
of PCOS has been found to be 4-fold higher in overweight women (Pasquali, 2006) and, which
is more alarming, in women that had early menarche by obese condition (McCartney et al.,
2006). In fact, high blood concentrations of insulin and leptin, as well as syndromes of resistance
to leptin and insulin, which induce dysfunction of the hypothalamic-pituitary-ovarian axis
have been described as predisposing factors for the development of PCOS (Balen et al., 2009);
mainly hyperinsulinemia. Hyperinsulinemia may cause hyperandrogenemia and, in turns,
hyperandrogenemia causes PCOS (Figure 25.3). Such hypothesis is reinforced by the fact that
weight losses in obese women with PCOS improve insulin resistance condition and allow the
restoration of ovulation and fertility. Moreover, insulin-sensitizing treatments can reduce the
androgens levels in blood and improve both ovulatory function and cyclicity, as well as fertility
rates, independently of weight loss, as revised by Shi et al. (2009).
Childbearing age
Non-pregnant
Polycystic ovarian syndromes (PCOS)
GnRH E2 Inh P4
GnRH Neurons
Ovary
Anterior LH
pituitary
FSH
Systemic level Local level
leptin resistance leptin resistance
insulin resistance insulin resistance
disruption in GnRH secretion hyperandrogenemia
Figure 25.3. Overview of the main causes for appearance of polycystic ovarian syndromes linked to obese
condition.
Appearance of PCOS is also related to concomitant alterations acting at the hypothalamic level
and disruption in GnRH secretion. A primary regulating factor of the GnRH secretion is the
orexigenic NPY, also secreted by hypothalamic neurons. In turns, secretion of NPY is regulated
by leptin and glucose/insulin signaling. In obese women, altered circulating insulin and leptin
levels cause changes in the secretion of NPY and, hence, of GnRH (Baranowska et al., 1999;
Messinis et al., 1999).
25.2.3 Influence of obesity on the response to assisted reproduction techniques
Due to the deficiencies in ovulatory cyclic activity detailed above, most of the affected obese
women are submitted to assisted reproduction treatments. However, if deleterious factors
persist, reproductive malfunction will remain and the success of the induced cycles will be also
compromised (Lintsen et al., 2005).
Assisted reproduction protocols consist of a sequence of actions, with each one of them being a
critical point that compromises the final success of the treatment. In obese women, like in non-
obese women, the first critical point is the ovarian response to the exogenous GnRH treatment.
As we already stated, the follicular growth is altered in obese women, hence, a main cause for
unsuccessful assisted cycles is a low ovarian response to the exogenous hormones (Lashen et al.,
1999). In obese poor-responders, like in non-obese beings, a low follicular growth usually causes
cycle cancellation. If treatment continues, the success of the treatment is compromised by the
collection of fewer oocytes of lower quality (Fedorcsak et al., 2004).
The first option for elapsing low ovarian response would be the administration of higher doses
of exogenous gonadotrophins (Fedorcsak et al., 2004); however, the increased risk of ovarian
hyperstimulation observed in obese women need to be taken into account.
25.3 Influence of obesity on implantation and placentation

Fertility of obese women is also hampered in addition to alterations in follicle/oocyte
development, ovulation and fertilization by failures in implantation and placentation leading
to an increased hazard of miscarriage during the first trimester of pregnancy (Lashen et al., 2004).
Disorders in implantation and placentation may be caused by a hampered embryo developmental
competence and/or by an altered oviduct/uterine environment during first stages of development
(Bellver et al., 2010). Deficiencies in endometrial receptivity and, later, in trophoblast function
have been addressed as main causes.
Primary factors causing failures in implantation and early embryo development and placentation
are relatively unknown, but most of the evidences point out to insulin and leptin resistance
conditions, as well as disturbances in the action of the adipokines (Figure 25.4).
Childbearing age
Reduced pregnancies infertility
Implantation/placentation
Embryo developmental
competence
Oviduct/uterine environment
insulin resistance
leptin resistance
hyperoestrogenaemia
pro-inflammatory cytokines
Figure 25.4. Overview of the main causes for alterations in processes of implantation and placentation linked
to obese condition.
Insulin is actively implicated in endometrial growth, cycle and function; thus, the insulin resistance
may affect implantation and early development. The deleterious effects of hyperinsulinaemia and
insulin resistance during obesity have been related, like in ovulatory alterations, to increased
circulating oestrogens levels. The state of hyperoestrogenaemia can have deleterious effects on
endometrial receptivity, either directly or by reducing IGFBP1 and glycodelin production, which
may alter adhesion at the maternal-fetal interface, which may cause pregnancy losses (Levens
and Skarulis, 2008).
Fertility in obese women with insulin resistance may be even worsened by alterations in the
transport of glucose at the level of the endometrium, due to a diminished expression of the
glucose transporter 4 (Mozzanega et al., 2004).
On the other hand, there is an increasing body of evidences on the main role of leptin and its
receptor LEPR in implantation and uterine receptivity. Leptin and LEPR are expressed in the
oviduct (Duggal et al., 2002) and the endometrium (Duggal et al., 2002). Expression of LEPR
gene is differentially regulated in implantation and inter-implantation endometrial sites. This
fact, suggests a regulatory talk-cross with the embryo. The impact of leptin on implantation has
been related to a direct angiogenic effect, since endothelial cells respond with neovascularization
to stimulation of the leptin receptor (Bouloumi et al., 1998). The hormone leptin has also an
indirect angiogenic activity, since modulates other angiogenic factors, mainly the VEGF and the
endothelial NOS3 (Craig et al., 2005). Possible alterations in the pathway LEP/LEPR-NOS-VEGF
may be related to alterations in implantation and later placentation.
Currently, the role of other adipokines is being highlighted. Pregnancy in obese females is
associated with higher levels of acute phase proteins and pro-inflammatory cytokines like TNF,
IL-1, IL-6 and IL-8 and C-reactive protein (Challier et al., 2008; Founds et al., 2008; Ramsay et
al., 2002; Zhu et al., 2010). The pro-inflammatory stage of obese mothers may negatively affect
pregnancy success (Denison et al., 2010; Zhu et al., 2010).
25.4 I nfluence of obesity on the development of post-implantational embryos

and early fetuses
The objective of the present chapter is focused on the effects of overnutrition and obesity on the
events occurring from preovulatory follicle development and ovulation to placentation and early-
embryo development. However, we cannot leave aside that the effects of obesity persist through
pregnancy and even in postnatal stages by prenatal programming.
Maternal obesity affects metabolism and developmental patterns of the offspring (Figure 25.5).
The fetus from an obese woman is exposed to maternal hyperglicaemia and, as a consequence,
develops hyperinsulinaemia. At birth, offspring have altered body size and weight, depending on
the metabolic status of their mother. Usually, newborns are large for gestational age and obese,
having high amounts of body fat. Moreover, they can manifest visceromegaly, mainly at the heart,
liver and spleen. However, although at a lesser extent, obese mothers may have offspring with
reduced body weight, some of them born at pre-term and some of them born at term but both of
Childbearing age
Maternal obesity
Maternal hyperglicaemia
Foetal hyperinsulinaemia
Alterations in foetal development

Newborns
Large for gestational age
(Some small for gestational age)
Increased depots of body fat
Visceromegaly
Caesarean deliveries
Premature labour
Stillbirth
Neural tube defects
Delayed organ maturation
Figure 25.5. Overview of the main causes and consequences of alterations in processes of embryo/fetus
development and prenatal programming of adult disease.
them, affected by IUGR. Features of the newborns are influenced not only by maternal obesity,
but also by maternal metabolism. Offspring from diabetic obese mothers are frequently obese,
whilst offspring of non-diabetic obese mothers are usually not overweight. In the adulthood,
however, both phenotypes have an increased hazard of becoming overweight.
Other consequences of maternal obesity are a higher percentage of caesarean deliveries because
of overweight and oversized offspring, as well as increases in premature labor and stillbirth rates.
Finally, incidence of neural tube defects and delayed organ maturation are also raised in obese
mothers.
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syndrome. International Journal of Obesity and Related Metabolic Diseases 26, 883-896.
Lashen, H., Fear, K. and Sturdee, D.W., 2004. Obesity is associated with increased risk of first trimester and recurrent
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outcome of superovulation and in vitro fertilization. Human Reproduction 14, 712-715.
Levens, E.D. and Skarulis, M.C., 2008. Assessing the role of endometrial alteration among obese patients undergoing
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2005. Effects of subfertility cause; smoking and body weight on the success rate of IVF. Human Reproduction
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26. Nutraceutical approaches in female infertility:
setting the rationale for treatments tailored to
the patients phenotype and based on selected
molecules
Centro Natalit, Department of Obstetrics & Gynaecology, Vita-Salute University Hospital San
Raffaele, Via Olgettina 60, 20100 Milan, Italy; papaleo.enrico@hsr.it
Abstract
Nutrition affects critical biological pathways in reproduction, and possible roles for nutraceutical
approaches as adjuvants in the treatment of female infertility are being investigated, providing
some preliminary encouraging results. However, well-designed randomized controlled trials
to evaluate their efficacy and safety are still lacking, making it difficult to provide general
recommendations. The aim of this chapter is to present preliminary data that indicate how
specific nutraceutical molecules could influence the ovarian micro-environment in different
pathological conditions, potentially increasing reproductive outcomes. These data emphasize
the importance of tailoring the nutraceutical approach on characterized group of patients and on
single (rather than multiple) micronutrient supplements, so as to increase the rationale for specific
recommendations. We therefore present possible indications for nutraceutical supplementation
in four critical categories of patients that still represent therapeutic challenges in infertility
treatments: polycystic ovary syndrome (i.e. inositol, vitamin B, vitamin C and folic acid), poor
ovarian response (i.e. melatonin, L-arginine and omega 3), ageing (i.e. selenium, aspartic acid and
multiple micronutrients) and endometriosis (i.e. vitamin B, vitamin C, folic acid and elocalcitol).
Keywords: antioxidants, controlled ovarian stimulation, micronutrients, reproductive outcomes,

vitamins
413
Summary points
Therapeutic approaches solely based on hormonal treatments are not optimal for all infertile women. In
particular polycystic ovary syndrome (PCOS), poor ovarian response (POR), ageing and endometriosis still
represent therapeutic challenges in sub/infertility treatments.
Follicular fluid components are crucial for oocyte maturation, and they are related to dietary macro/
micronutrients. Some of them can be implemented by dietary supplementation with nutraceuticals.
Further studies should be conducted on characterized group of patients divided by cause of infertility.
Moreover, therapies should be based on single (rather than multiple) micronutrient supplements.
PCOS is associated (among others) with anovulation, insulin-resistance and luteal phase defects (LPD). In
young PCOS patients with insulin-resistance, inositol could be the first stage of a step-up approach for
improving ovulation. In older patients, more rapidly acting strategies should instead be preferred.
In PCOS patients with LPD, adjuvant vitamin C might be beneficial. In PCOS patients undergoing assisted
reproduction technologies (ART), vitamin B or folic acid should be considered in order to lower oxidative
stress in the follicular fluid.
In the presence of POR, therapeutic approaches should address improved follicular function. In POR women
undergoing ART, melatonin improves fertilization rate. Also L-arginine and omega 3 might positively contribute
to reproductive.
Subfertility starts 15-20 years before menopause, and above 30% of women treated in infertility units are
today aged 38 or more. Ageing women are a group in which multiple micronutrients supplementation is
reasonable. Among others, selenium should be considered, because it is an anti-oxidant and it improves
thyroid function. D-Asp might be useful too.
Inflammation and oxidative stress play crucial roles in the development/progression of endometriosis. Vitamin
C plus vitamin E have synergic effects in lowering peripheral oxidative stress markers. Vitamin B or folate
can lower reactive oxygen species in the follicular fluid. In addition, vitamin C might be beneficial on LPD
associated to endometriosis. Elocalcitol seems a promising molecule that might help control endometriosis
development and reduce peritoneal inflammation.
26. N
utraceutical approaches in female infertility
Abbreviations
ALA Alpha-linoleic acid
ART Assisted reproduction technologies
BMI Body mass index
COS Controlled ovarian stimulation
DCI D-chiro-inositol
FF Follicular fluid
GnRH Gonadotrofine releasing hormoon
Hcy Homocysteine
hGC Human chorionic gonadotropin
IPG Inositolphosphoglycan
LDL Low density lipoproteins
LPD Luteal phase defect
MYO Myo-inositol
POR Poor ovarian response
ROS Reactive oxygen species
VDBP Vitamin D binding protein
26.1 Introduction
Despite the arising interest in nutraceuticals, vitamins, minerals and antioxidants in gynaecological
and obstetrics indications, the major interest in reproductive medicine has been primarily focused
on hormonal treatment for more than three decades. Ovulation induction first, and then the
introduction of COS for multiple follicular development have significantly increased pregnancy
rates, and different stimulation protocols have been developed in the attempt to obtain an optimal
number of oocytes from each treatment cycle. The importance of achieving a good response
to COS is underscored by the fact that the number of oocytes obtained following stimulation
positively correlates with pregnancy rates per cycle (Figure 26.1).
However, hormonal treatments are still one size fits all simplistic approaches, and current
stimulation protocols are not optimal for all patients groups. Indeed, infertility units encounter
a great variety of pathologies and the proportion of patients with a good prognosis is estimated
to be only 30-35%.
45
40
35
Pregnancy rate (%) 30
25
20
15
10
5
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Total number of oocytes retrieved
Figure 26.1. Relationship between number of oocytes retrieved and ongoing pregnancy rate. Controlled
ovarian stimulation for in vitro fertilization should be aimed at obtaining a number of oocytes included in the
optimal range of 8-14, below and above which outcomes are compromised (Fertility Centre, Department of
Obstetrics & Gynaecology, Vita-Salute University Hospital San Raffaele, Milan, Italy).
26.1.1 Therapeutic challenges in PCOS, POR, endometriosis and aged patients
Among other causes of infertility, PCOS, a high BMI, a diminished ovarian reserve, advanced
age and the presence of endometriosis are well-known detrimental factors that hinder ovarian
response and/or treatments outcome. The high prevalence (Figure 26.2) of these poor-prognosis
conditions among couples undergoing ART prompts the search for additional strategies which
may influence fertility treatments and improve their success.
26.2 B eyond the number of oocytes: oocyte quality and the nutraceutical
approach
It is now well established that oocyte quality determines the embryos developmental potential
after fertilization. The assessment of oocyte quality is therefore getting increasing attention, with
the aim to identify the best oocytes, limit embryo overproduction and improve the results of
oocyte cryopreservation programs.
Environmental exposure, oocyte interactions with cumulus cells, and availability of crucial
molecules in the FF, among other factors, play a role in defining oocyte competence. FF, which is
a produced by both by transfer of blood plasma constituents across the blood-follicular barrier
and by secretory activity of granulosa and thecal cells, contains some micronutrients and trace
molecules that are valuable markers of oocyte quality (Revelli et al., 2009). Chemical constituents
26. N
Other
7.4%
Diminished
ovarian reserve
9.2%
Male factor
17.9%
Multiple factors,
female only
9.3%
Multiple factors,
female plus male
14.9%
Tubal factor
9.8%
PCOS
Endometriosis 10.7%
10.2% Unexplained
10.6%
Figure 26.2. Diagnosis among couples undergoing non-donor assisted reproductive technology cycles.
Please note that diminished ovarian reserve is mainly due to advanced age; other include anovulation (other
than polycystic ovarian syndrome (PCOS)), cancer chemotherapies, immunological problems, chromosomal
abnormalities and serious illness (Fertility Centre, Department of Obstetrics & Gynaecology, Vita-Salute University
Hospital San Raffaele, Milan, Italy).
of FF include anti-oxidant factors, vitamins, proteins, peptides, amino acids and carbohydrates
that are related both in quantity and quality to dietary macro and micronutrients.
Therefore, the intake of these substances either as a part of the patients diet or as supplements
can influence their adequate presence and function in the FF. These data suggest that specific,
crucial nutraceutical molecules locally play a role in oocyte survival, growth, maturation and
ovulation. However, the understanding of the functional role of these molecules in physiological
and pathological conditions is still incomplete. Such understanding will be of great importance
for a better characterization of infertile patients phenotype prior to stimulation, in order to
identify groups of women whose treatment could be optimized through supplementation of
specific molecules that are relevant to the pathogenesis of their condition.
In general, there is a deficit of well-designed randomized controlled trials aimed at evaluating

the efficacy and safety of dietary supplements, vitamins and minerals. This makes providing
evidence-based recommendations difficult. Here, on the basis of the available preliminary data,
we discuss the potential roles for specific nutraceuticals in the treatment of critical infertility-
related conditions such as PCOS, POR, aged patients and endometriosis (Table 26.1). Most of
the results we present in this chapter were obtained in non-randomized, uncontrolled studies
Table 26.1. Poor-prognosis causes of female infertility, selected nutraceutical molecules and rationale for
supplementation. The table indicates critical conditions in which the nutraceutical approach should be considered,
which specific molecules could be used and the scientific rationale for their supplementation.
Poor-prognosis female Adjuvant nutraceutical Rationale

factors molecule
Polycystic ovary syndrome inositol insulin-sensitizer

folic acid/vitamin B lowers Hcy levels in follicular fluid
vitamin C improves luteal phase efficiency
Poor ovarian response melatonin anti-oxidant, pro-steroidogenic
L-arginine increases follicular blood supply
omega 3 still unclear
Ageing selenium anti-oxidant, improves thyroid function
aspartic acid elicits hormones synthesis-release
minerals anti-oxidants, others (still unclear)
multiple micronutrients synergic effects, multiple actions
Endometriosis vitamin C plus vitamin E anti-oxidant, improves luteal phase efficiency
folic acid/vitamin B lowers Hcy levels in follicular fluid
elocalcitol reduces development of disease, inhibits inflammation
conducted on small numbers of patients, and further investigations will be needed to analyze
and to confirm these data. We recommend future studies to be conducted on selected groups of
patients and on single (rather than multiple) micronutrient supplements, so as to increase the
rationale for tailored supplementation of selected molecules.
26.3 Polycystic ovary syndrome

With a prevalence of up to 10%, PCOS is the most common endocrinopathy in women of
reproductive age and the first cause of anovulatory infertility. Beside anovulation, PCOS is
also associated with other infertility issues, including impaired embryo-quality, endometrial
hyperplasia, implantation failure and miscarriage. Many features of PCOS, including anovulation
and hyperandrogenism, are driven by compensatory hyperinsulinemia caused by insulin
resistance, which develops in 50-70% of PCOS patients (well beyond what is actually predicted
by BMI).
The molecular mechanisms underlying insulin resistance in PCOS remain elusive; however, a
decrease in the tissue availability of crucial insulin second messengers seems to be involved.
Such post-receptor mediators include the low molecular weight IPGs DCI and MYO (a precursor
that can be converted to DCI intracellularly). Women affected by PCOS present an increased
26. N
urinary clearance of DCI and a decreased release of DCI-IPG mediator, possibly leading to the
impairment of insulin-mediated signaling and to compensatory hyperinsulinemia (Baillargeon
et al., 2006).
26.3.1 Inositol
Inositol is a sugar-like molecule belonging to the vitamin B complex group. Given the function of
IPG in the insulin-mediated signaling and their impaired availability in PCOS patients, possible
roles for exogenous inositol in human reproduction have been investigated. Over the last years,
inositol has therefore become a well-established insulin-sensitizing agent that positively affects
LH secretion (Genazzani et al., 2008), and it has also been found to be positively involved in the
physiological triggering of oocyte maturation (Papaleo et al., 2009a). Consistently, both DCI
(Nestler et al., 2009) and MYO (Gerli et al., 2007; Papaleo et al., 2007) administration to women
with PCOS led to an improvement in ovulation, and to a reduction in serum testosterone levels
and in metabolic parameters such as blood pressure and triglycerides.
Even if an evidence-based scheme of treatment is not available yet, these data suggest that inositol
should be considered as part of a step-up approach in the treatment of women with PCOS. Young
patients whose timeline for achieving pregnancy differs from immediately constitute a well-
defined subset for whom inositol, with its gradual onset of action and decreased potential risk of
multiparity, may be the drug of choice for re-establishing ovulatory menses and fertility. On the
contrary, for infertile patients with PCOS who have an immediate desire for pregnancy, a rapidly
acting agent such as clomiphene would be most appropriate, as recommended by ESHRE/ASRM
Consensus (2007) on infertility treatment related to PCOS.
For those patients with PCOS who are candidate to COS, the nutraceutical approach using
inositol can again be useful. MYO is present in the FF and its concentration correlates with
oocyte quality and embryo developmental potential (Chiu et al., 2002), suggesting that increasing
its availability could positively affect cycle outcomes. Consistently, treatment with MYO and folic
acid (but not folic acid alone) during COS proved to reduce germinal vesicles and degenerated
oocytes at ovum pickup, without compromising total number of retrieved oocytes.
In addition inositol acting as an ovarian insulin-sensitizing agent significantly reduces estradiol

levels at hGC administration, and could therefore decrease the risk of ovarian hyperstimulation
syndrome in PCOS patients (Papaleo et al., 2009b).
26.3.2 Folic acid and vitamin B
The methionine catabolite amino acid Hcy can be oxidized to generate ROS with well-known
detrimental biological effects. Hcy is present in the FF in concentrations that are related
to its plasma levels, and it negatively affects oocyte maturity. As a result, mild to moderate
hyperhomocysteinemia is associated with detrimental effects on reproductive outcomes, and
in patients undergoing IVF-embryo transfer procedure, high levels of Hcy in the FF negatively
correlate with oocyte quality, embryo quality and pregnancy rate (Ocal et al., 2012). In PCOS
patients in particular, the FF concentration of Hcy is a useful marker for fertilization rate, being
inversely related to oocyte and embryo quality after COS (Berker et al., 2009).
Oral administration of either folic acid or vitamin B should therefore be considered in PCOS
patients, in order to lower serum and FF Hcy concentrations and increase the number of mature
oocytes at ovum retrieval (Kilicdag et al., 2005).
26.3.3 Vitamin C
PCOS is a major cause of LPD, a common endocrine disorder associated with infertility and
spontaneous miscarriage that is found in 3-10% of infertile women, and in 35% of women
with repeated abortion. ROS are one of the many causes of LPD: in patients with LPD, levels
of antioxidant substances, such as ascorbic acid, -tocopherol, and erythrocyte glutathione,
were found to be significantly lower than in healthy women, while serum lipoperoxidation was
reported to be elevated (Vural et al., 2000).
The ovary has long been recognized as a site of vitamin C accumulation and turnover, with
the concentration of ascorbic acid reported to be much higher in human FF than in serum.
This suggests that vitamin C may play a role as an antioxidant molecule during folliculogenesis
(Paszkowski et al., 1999). Consistently, ascorbic acid supplementation in patients with LPD
determined an increase in progesterone levels as well as a significant increase in pregnancy rate
(Henmi et al., 2003). This provides rationale for a possible role for vitamin C administration in
patients with PCOS, with the aim to improve their intrinsic defect in luteal phase competence.
26.4 Poor ovarian response

POR to COS indicates a reduction in the follicular response, resulting in a reduced number of
retrieved oocytes. In order to well define the poor response in ART, a Consensus conference was
recently held (Ferraretti et al., 2011). POR is reported in 5-35% of IVF cycles (with a variability
due to differences in definitions used before the Consensus was set) and it represents one of the
major therapeutic challenges in ART: several approaches have been explored, but a single effective
strategy has not yet been established.
The pathogenesis of POR is still uncertain itself. Ovaries responding poorly to COS often show
a reduced number of pre-antral follicles, most frequently linked to the condition of diminished
ovarian reserve. In some cases, however, POR may be associated with suboptimal ovarian
exposure to gonadotrophins, or with the presence of specific FSH-receptor polymorphisms. Thus,
since the underlying and preceding events are still only partially recognized and the number of
FSH-sensitive follicles is likely to be impaired, therapeutic approaches should address improved
function rather than increased numbers of follicles. Moreover, not all POR are similar in terms
of loss of oocyte quality, and patients characteristics other than age and ovarian reserve have not
26. N
yet been properly investigated. Therefore, there is insufficient evidence to support the routine use
of any particular intervention either for pituitary down regulation, COS or adjuvant therapy in
the management of POR patients.
The data we discuss here are indeed preliminary, but if further confirmed, they could represent
interesting strategies for better characterizing and treating POR patients.
26.4.1 Melatonin
Melatonin and its metabolites play essential functions in the human ovary, including that of
receptor-independent free radical scavengers and regulators of gene transcription for antioxidant
enzymes as important as Cu-Zn superoxide dismutase, Mn superoxide dismutase and glutathione
peroxidase (Figure 26.3).
The concentration of melatonin in the FF is higher than in plasma, and it shows a local
dose-dependent blocking effect on the inhibition of oocyte maturation by ROS. As a second
messenger in the LH-receptor mediated signaling pathway, melatonin also has a positive action
on steroidogenesis during the follicular, ovulatory and luteal phase. Among steroid hormones,
androgens in particular have a role in maintaining the survival of follicles before they become
sensitive to gonadotropins, as well as in paracrine signaling within antral follicles (Nardo et al.,
2009). As a result, increased levels of melatonin in FF were shown able to prevent follicular atresia
(Tamura et al., 2009).
Figure 26.3. Schematic illustration of the different functions of melatonin in the human ovary. Melatonin
(M) concentrates in the follicular fluid (FF) and it acts as a scavenger of reactive oxygen species (ROS) in the
liquid phase, a regulator of gene transcription of detoxifying enzymes by granulosa cells, and a regulator of
steroidogenesis by theca cells.
The specific effect of exogenous melatonin on ART outcomes has therefore been investigated,
specifically focusing on poor-prognosis patients who showed a very low fertilization rate
(<50%) in a previous IVF-ET cycle. Daily administration of 3mg melatonin tablets given from
the beginning of the previous menstrual cycle resulted in markedly improved fertilization rate
(Tamura et al., 2008).
In a recent unpublished study from our group, we also observed that the administration of
melatonin and recombinant-LH during COS cycles had a positive effect on ovarian response
and on the number of good quality oocytes retrieved. Therefore melatonin supplementation, for
which no sides effects or infant abnormalities have been observed, could be proposed for both
POR patients and patients with a history of low fertilization rate.
26.4.2 L-arginine
L-arginine is an essential amino acid and a precursor of nitric oxide. Adjuvant L-arginine in the
treatment of POR patients was observed to improve follicular and endometrial blood flow, and
increase number of oocytes collected, number of embryos transferred, endometrial receptivity
and pregnancy rate (Battaglia et al., 1999). These results are however controversial, and the effect
of L-arginine supplementation in non-selected patients was instead proven to be detrimental to
embryo quality and pregnancy rate (Battaglia et al., 2002). Further investigations are thus needed,
and accurate selection and characterization of patients appears fundamental.
26.4.3 Omega 3
Omega 3 are long chain poly-unsaturated fatty acids derived from fish. Dietary intake of omega 3
is associated with its availability in tissues and it has been observed that a higher intake of omega
3 correlates with improved embryo morphology in women undergoing COS (Hammiche et al.,
2011). According to this study, since intake of omega 3 is generally low, either a twice-a-week
consumption of fish or an exogenous supplementation should be prescribed to POR patients, for
whom transferred embryo quality is a crucial prognostic factor.
However, recent data showed that elevated fasting levels of ALA, a member of the omega 3
family, on the morning of ovum pick-up were negatively associated with embryo implantation
and clinical pregnancy rates, with a dose-dependent relationship (Jungheim et al., 2011).
Further investigations are therefore required to determine the potential of ALA as a marker of
decreased omega 3 metabolism, and whether increased circulating ALA could somehow become
detrimental to embryo-implantation.
26. N
26.5 Aged patients

Ageing implies a condition of female subfertility, intended as a state in which the capacity for
fertility is diminished, but not necessarily absent. Beside huge inter-individual variations in all
reproductive ageing events (Te Velde et al., 2002), subfertility usually precedes menopause by
15-20 years (with mean age at menopause in Western Countries of 50-51) (Figure 26.4).
This, together with the social trend of delaying childbearing, explains why in the last decades
there has been an increase in the number of women having fertility treatment in their advanced
reproductive age: above 30% of women treated in infertility units are today aged 38 or more,
and the success rates of ART in this group remains low. Human Fertilization and Embryology
Authority data from 2009 show that the live-birth rate from IVF in the UK was 12.7% in the age
group 40-42, 5.1% in the age group 43-44 and less than 2% in women over 44.
Even if laboratory procedures, such as assisted hatching or blastocyst transfer, may increase the
chance of pregnancy in women with poor prognosis, there is no answer yet to the age-related
decline of female fertility (Figure 26.5).
This explains aged womens growing interest in methods that increase the likelihood of conceiving
a child, and the need for a rational and evidence-based approach to this major cause of infertility.
Infertility in older women is primarily related to:

poor quality of ageing oocytes (chromosomal abnormalities, morphologic abnormalities,
functional abnormalities);
diminished ovarian reserve;
altered hormonal environment resulting in ovulatory dysfunction.
106 Number of follicles Poor quality oocytes
105
Total number of follicles
Sub-fertility
104 Irregular
cycles
Poor quality oocytes (%)
100
103 Menopause
75
102 50
25
101
0 10 20 30 40 50 60
Age (years)
Figure 26.4. Different phases in a womans progressive fertility decline. Subfertility starts around 15-20 years
before menopause; irregular cycles occur around 6-7 years before their definitive cessation.
60
50
40
Rate (%)
30
Pregnancy rate
20 Live birth rate
Singleton live birth rate
10
0
22 26 30 34 38 42 46
Age (years)
Figure 26.5. Reproductive outcomes for women of different ages undergoing non-donor assisted reproductive
technology cycles. Success rates decline steadily from 35 years of age onwards.
26.5.1 Oxidative stress and ovarian ageing
Despite molecular mechanisms responsible for ovarian ageing are still only partially defined, it
seems that they involve the interaction between intrinsically altered follicular cells and follicular
microenvironment (Tatone et al., 2008). In particular, alterations in the number of chromosomes
in the oocyte (aneuploidies) are considered to be the main obstacle to reproductive success.
According to the two-hit hypothesis, oocytes aneuploidy derives from a non-recombination of

homologous chromosomes during fetal life, plus a non-recognition of the failed recombination
during meiosis I in the peri-ovulatory period.
Several age-related, endogenous and exogenous adverse factors are considered to have a role in
the occurrence of the second hit (Eichenlaub-Ritter et al., 2004), by creating a microenvironment
that negatively affects nuclear and cytoplasmic maturation of follicular cells. Among these factors
are impaired follicular oxygenation (Costello et al., 2006) and excessive oxidative stress in the
FF (Carbone et al., 2003). It is therefore likely that an optimal balance between oxygen available
to the oocyte and antioxidants is critical to permit both correct chromosomal alignment and
oocyte maturation. Indeed, severely hypoxic follicles contain oocytes with a high frequency of
aneuploidies (Van Blerkom et al., 1997), and excessive ROS cause atresia of the follicle (Tamura
et al., 2009).
On the other hand, the presence of antioxidant enzyme transcripts at the meiosis II stage in
human oocytes suggests that these defense mechanisms are also important for further oocyte
maturation (El Mouatassim et al., 1999).
26. N
26.5.2 Selenium
Selenium is a mineral that is abundant in meat, sea-food and cereals, but its availability is soil-
dependent and it has been reported that the European intake of selenium is falling. Atoms of
selenium are necessary to form selenocysteine, which is essential for the efficient function of
a variety of enzymes including glutathione peroxidase and deiodinase (or iodide peroxidase).
These enzymes account for two crucial roles of selenium in female reproductive function. On one
hand, selenium and glutathione peroxidase are present in the FF, where their antioxidant activity
seems to be significant for the quality of the follicular milieu.
Interestingly, patients with unexplained infertility had significantly decreased FF Selenium

levels when compared to those with tubal infertility or male factor. Also, the well-established
detrimental effect of tobacco smoking on oxidative stress and infertility seems to be at least partly
mediated by a significant decrease in follicular gluthatione peroxidase activity (Paszkowski et al.,
1995). Selenium supplementation should therefore be considered a crucial molecule in lowering
age-related and possibly environment-driven oxidative stress.
On the other hand, selenium is essential for the conversion of thyroxin in the more active
triiodothyronine (operated by deiodinase). Thus, selenium deficiency might impair thyroid
function. Being both hypothyroidism and subclinical hypothyroidism detrimental on fertility
and obstetric outcomes, the role of Selenium in thyroid hormone conversion is a second crucial
aspect in protecting the reproductive function. The prevalence of hypothyroidism among women
of childbearing age is of 2-4%, while that of subclinical hypothyroidism is even higher, ranging
from 4 to 10% of adults.
Interestingly, thyroid subclinical abnormalities are markedly more common among women
experiencing IVF failures (Bussen et al., 2000). Also, since thyroid diseases often represent
chronic conditions, the prevalence of both hypothyroidism and subclinical thyroid dysfunction
increases with age.
Besides its anti-oxidant properties, selenium supplementation is thus effective in preventing mild
thyroid dysfunctions and in significantly lowering the level of anti-peroxidise autoantibodies
(Toulis et al., 2010), possibly improving implantation and pregnancy rates in aged infertile
women (in whom the underlying thyroid dysfunctions are most frequent).
26.5.3 Aspartic acid
Aspartic acid is an amino acid that holds several crucial functions both in the nervous and in the
endocrine tissues, where it elicits biosynthesis and release of different hormones including GnRH,
LH and progesterone. D-aspartic-acid concentrations in FF is positively related to number of
oocytes retrieved, percentage of mature oocytes and fertilization rate. Interestingly, D-aspartic-
acid concentration is significantly higher in younger women than in older (aged 35 or more)
patients (DAniello et al., 2007). Therefore, supplementation with D-aspartic-acid in order to
restore FF levels similar to those of younger patients might help improving the reproductive
prognosis of aged women undergoing COS.
26.5.4 Minerals and heavy metals
Over the years, long-term nutritional status and environmental exposure to trace elements can
cause relevant metabolic disturbances including deficiencies of micronutrients or heavy metals
toxicities that might both affect reproductive outcomes.
In a study based on the analysis of mercury, selenium and zinc in the hair of women undergoing
COS, it was found that zinc and selenium had a positive correlation with oocyte yield, while
mercury had an opposite, strong detrimental effect on numbers of follicles and oocytes (Dickerson
et al., 2011).
While, on one hand, these data could prompt the opportunity for a more conscious monitoring
of toxic trace-elements intake through contaminated foods, on the other, aged patients might
benefit from long-term supplementation with some beneficial minerals before and during their
infertility treatment (see Section 26.5.5).
26.5.5 Multiple micronutrient supplementation
While further research is still needed to identify specific pathways that may lead to improvements
in treatment of ageing-related sub/infertility, aged patients are perhaps the only group in which a
broad multiple micronutrient supplementation could represent a rational and effective approach.
The reasons for this are several, including the synergic effects of some anti-oxidant molecules
(for example vitamin C plus vitamin E) and the multiple beneficial effects that some other
nutrientshave.
The latter include selenium, which might have multiple beneficial properties on reproduction
(see Section 26.5.4), folate and zinc with both the two having anti-oxidant properties, folate
being also important for oocyte quality and maturation and zinc being implicated in ovulation
(Ebisch et al., 2007).
26.6 Endometriosis
Endometriosis is a gynecological disorder characterized by presence and growth of ectopic
endometrial tissue, with a prevalence that ranges from 2-22% among asymptomatic women and
reaches 35-50% among infertile patients.
A higher concentration of lipid peroxidation markers in the peripheral blood and peritoneal fluid
of women with endometriosis has been identified, suggesting that inflammation and oxidative
stress play crucial roles in the development and progression of endometriosis. In particular, they
26. N
might favor growth of endometrial cells in the peritoneal cavity (Van Landendonckt et al., 2002)
and endometriosis-associated infertility (Jackson et al., 2005). But the role of oxidative stress in
endometriosis seems to be even more crucial: indeed, a low consumption of fruits and vegetables
(important sources of anti-oxidants) apparently places women at a higher risk of endometriosisin
the first place (Parazzini et al., 2004).
Because oxidative stress can have a direct, strong detrimental effect on reproductive cell viability,
oocyte fecundity, implantation, and development of the implanted egg (Agarwal et al., 2005), a
dietary supplementation with some anti-oxidant/anti-inflammatory micronutrients should be
considered part of the treatment of endometriosis.
26.6.1 Vitamin C plus vitamin E
Both vitamin C and its oxidized product dehydroascorbic acid are biologically active, as they
exert anti-oxidant function in the aqueous phase. Vitamin E (a collective denomination for all the
stereoisomers of tocoferols and tocotrienols) is an anti-oxidant molecule that scavenges peroxide
radicals in the hydrophobic phase of LDL and cell membrane polyunsaturated fats, protecting
them from lipid peroxidation. Vitamin C and vitamin E therefore have different phase-specificity,
and a synergic anti-oxidant effect.
A significantly lower basal concentration of serum vitamin E was observed among patients with
endometriosis compared to a control group. Moreover, patients with endometriosis presented
increased lipid peroxidation (and maintained lower vitamin E levels) after COS (Campos Petean et
al., 2008). This could compromise oocyte quality in endometriotic patients, who might therefore
benefit from vitamin E supplementation in terms of improving implantation and pregnancy rates.
In addition, endometriosis is a well-recognized cause of LPD, an infertility-related condition in

which high-dose vitamin C has showed some efficacy (for the role of vitamin C supplementation
in LPD, see Section 26.3.3).
Given these data, a combined intake of vitamin C plus vitamin E might be the best approach for
lowering oxidative stress in women with endometriosis and improve their fertility prognosis.
Indeed, some studies already showed that implementation of vitamin C and vitamin E
consumption in women with endometriosis can improve their oxidative stress markers (Mier-
Cabrera et al., 2008, 2009).
26.6.2 Folic acid and vitamin B
Like PCOS, endometriosis is associated with higher Hcy levels in FF compared with idiopathic
sub-fertile patients, and an inverse association between follicular fluid Hcy levels and embryo
quality has been observed (Ebisch et al., 2006) (see also Section 26.3.2). Hence, patients with
endometriosis represent a population who might benefit from supplementation with folic acid
or vitamin B for the improvement of reproductive outcomes.
26.6.3 Elocalcitol
Several properties of vitamin D in female reproduction are well discussed in other chapters of this
book. In endometriosis, potential roles for vitamin D, VDBP and VDR have all been described,
with possible effects on endometriosis-associated inflammation and infertility.
Some recent promising studies were performed in a validated mouse model of endometriosis
using elocalcitol, a VDR agonist with anti-proliferative and anti-inflammatory properties,
low calcemic liability and a favorable safety profile. Results showed that elocalcitol reduces
endometriosis development in a dose-dependent manner and inhibits peritoneal inflammatory
cell recruitment (Mariani et al., 2012).
The authors suggest that elocalcitol supplementation starting 2-3 weeks before ovulation should
be considered as one possible approach in the prevention of endometriosis recurrences and
endometriosis-related infertility.
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27. Folate and female infertility: folate-
metabolizing pathway in folliculogenesis,
infertility treatment, and implantation
S. Altme1,2, M. Laanpere1, C. Campoy2 and A. Salumets1,3
1Competence Centre on Reproductive Medicine and Biology, University of Tartu, Tiigi 61b, 50410
Tartu, Estonia; 2Department of Paediatrics, School of Medicine, University of Granada, Avda. de

Madrid 11, 18012 Granada, Spain; 3Department of Obstetrics and Gynaecology, University of
Tartu, Puusepa 8, 51014 Tartu, Estonia; signealtmae@ugr.es
Abstract
Folates are important B9 vitamins that are believed to have a crucial role in female reproduction.
Folates are required for one-carbon biosynthetic and epigenetic processes that facilitate the
synthesis and methylation of nucleic acids and proteins. Folates are thus needed during periods
of rapid cell growth and proliferation, which occur in follicular development in the ovaries,
and subsequent pregnancy establishment and embryo development. Insufficient folate intake
has been shown to impair female fertility and early pregnancy in different studies, emphasizing
the necessity of folate during mammalian folliculogenesis and pregnancy establishment. In the
in vitro fertilization procedure, the most successful infertility treatment, folate-deficient women
have shown impaired ovarian function, lower oocyte quality and pregnancy rates. Dietary or
genetically determined folate deficiency may impair the folate-metabolizing pathway, leading
to altered DNA methylation and disrupted DNA integrity, and increased blood homocysteine
levels. Elevated follicular fluid homocysteine levels correlate with poor oocyte maturity, reduced
fertilization rates and poor in vitro embryo quality, while methylenetetrahydrofolate reductase
gene polymorphisms are associated with lower ovarian follicular reserve, diminished response to
ovarian stimulation, and reduced chance of live birth after in vitro fertilization. Previous studies
emphasize that imbalances in folate metabolism and related gene variants may impair female
fertility and affect embryo implantation and successful pregnancy establishment.
Keywords: folate pathway, folliculogenesis, Hcy, implantation, in vitro fertilization
431
Summary points
Folates are required for one-carbon biosynthetic and epigenetic processes that facilitate the synthesis and
methylation of nucleic acids and proteins. Folates are thus needed during periods of rapid cell growth and
proliferation, which occur in follicular development in the ovaries, and subsequent pregnancy establishment
and embryo development.
Insufficient folate intake has been shown to impair female fertility and early pregnancy in different studies,
emphasizing the necessity of folate during mammalian folliculogenesis and pregnancy establishment.
In the in vitro fertilization procedure, folate-deficient women demonstrated impaired ovarian function, lower
oocyte quality and pregnancy rates.
Dietary or genetically determined folate deficiency impairs folate-metabolizing pathway and leads to
homocysteine accumulation, which results in DNA methylation and integrity disruption, slows DNA replication
and causes apoptosis and necrosis of affected cells.
Elevated follicular fluid homocysteine levels correlate with poor oocyte maturity, reduced fertilization rate and
poor in vitro embryo quality, while methylenetetrahydrofolate reductase gene polymorphisms are associated
with lower ovarian follicular reserve, diminished response to ovarian stimulation, and reduced chance of live
birth after in vitro fertilization.
Folate deficiency-induced processes may alter female fertility by interrupting folliculogenesis, fertilization,
implantation and embryo development.
27. Folate and female infertility
Abbreviations
5,10-methyleneTHF 5,10-methylenetetrahydrofolate
5-methylTHF 5-methyltetrahydrofolate
COH Controlled ovarian hyperstimulation
dTMP Thymidine monophosphate
FSH Follicle-stimulating hormone
GCs Granulosa cells
Hcy Homocysteine
MTHFR Methylenetetrahydrofolate reductase
OHSS Ovarian hyperstimulation syndrome
PCFT Proton-coupled folate transporter
RFC Reduced folate carrier
SAM S-adenosylmethionine
27.1 Introduction
More than 10% of couples worldwide are involuntarily childless due to infertility (Boivin et al.,
2007). The prevalence of infertility is increasing, having significant medical, social and financial
implications. Infertility is defined as inability of a couple to become pregnant within a year without
using any contraceptives. Over 72 million women worldwide, aged 20-44, are estimated to be
currently infertile, however, only every second couple seeks medical care for infertility treatment
(Boivin et al., 2007). Infertility treatment is physiologically and psychologically demanding for
the couple, affecting their quality of life and ability to work. Also, the economic demands to both
the couple and society are high. To improve fertility and reduce the treatment costs, it is important
to identify and understand the mechanisms that influence infertility treatment outcome.
There is growing evidence that folates are crucial for human reproduction (Laanpere et al., 2010).
Folates are essential water-soluble B9 vitamins that are required for one-carbon biosynthetic and
epigenetic processes that facilitate the synthesis and methylation of nucleic acids and proteins.
Folate is thus needed during periods of rapid cell growth and proliferation, which occur in
follicular and embryonic development. Indeed, insufficient folate intake has been shown to
impair female fertility and fetal viability in different studies, emphasizing the necessity of folate
during mammalian folliculogenesis and pregnancy establishment (Laanpere et al., 2010).
27.2 Folate-metabolizing pathway

To obtain folates, humans rely solely on dietary sources. The synthetic form of folate, folic acid,
is more stable and can be consumed as nutritional supplements or fortified foods. Intestinal
transport of folates is predominantly mediated by PCFT, but also by other folate transporters
such as RFC (Zhao et al., 2009). During transport through the intestinal mucosa, folates are
converted to 5-methylTHF, which is further mediated by RFC and folate receptors FR, FR,
FR (Laanpere et al., 2010).
Folate functions as an enzyme substrate within the pathway, chemically activating and transferring
one-carbon units. This set of reactions is known as folate-mediated one-carbon metabolism
(illustrated in Figure 27.1). Folate-mediated one-carbon metabolism consists of two intertwined
cycles, one producing dTMP and purine precursors for DNA biosynthesis (called DNA cycle),
and the other producing and utilizing methyl group donor SAM for methylation reactions (called
methylation cycle).
Figure 27.1. Folate-mediated one-carbon metabolism consists of two cycles: DNA biosynthesis and methylation.
Homocysteine is catabolized in the trans-sulfuration pathway. Some genes involved in the pathways are indicated
as ellipses and some of the most studied variations are listed next to the gene. Folate, homocysteine and vitamin
B12 are also indicated. Compound transport into the cell is shown in dotted line (from Laanpere et al., 2011;
published with permission from Elsevier). TCN2 = transcobalamin II, DHF = dihydrofolate, THF = tetrahydrofolate,
SAM = S-adenosylmethionine, MTR = 5-methyltetrahydrofolate-homocysteine methyltransferase, BHMT =
betaine-homocysteine methyltransferase, MTHFR = methylenehydrofolate reductase, Hcy = homocysteine, SAH
= S-adenosylhomocysteine, CTH = cystathionase, FOLR1 = folate receptor 1, SCL19A1 = solute carried family
19, member 1.
5,10-methyleneTHF acts as a critical junction in the folate-metabolizing pathway, as one-carbon

groups can either be used to produce dTMP for nucleic acid synthesis or are directed toward the
methylation cycle by irreversible synthesis of 5-methylTHF mediated by MTHFR. Thus, the two
cycles are competing for folate derived one-carbon groups, especially in a situation of inadequate
folate supply.
27.3 Folate deficiency

Folate deficiency is common in humans and it can occur as a result of poor dietary intake or
malabsorption of folate. Low folate levels could be additionally caused by insufficient levels
of micronutrients necessary for folate metabolism, such as vitamins B2, B6, B12, Fe, and Zn
(Laanpere et al., 2010). One reason for inefficient folate utilization could arise from variations in
folate-metabolizing genes (Laanpere et al., 2010).
Several polymorphisms have been identified in genes involved in folate absorption and folate-
mediated one-carbon metabolism (Table 27.1, Figure 27.1 illustrates some of them). These
variations may modify the beneficial effects of folates and other micronutrients within the folate-
mediated one-carbon metabolism. The most influential polymorphism in folate-metabolizing
pathway in terms of prevalence and impact seems to be 677C/T variation in the MTHFR gene.
It results in an amino acid change at codon Ala222Val, giving rise to an unstable enzyme of
50-60% reduced activity (Frosst et al., 1995). Folate-mediated one-carbon metabolism pathway
with reduced MTHFR activity leads to impaired methylation reactions and accumulation of Hcy
(Harmon et al., 1996).
Hcy is a sulfur-containing amino acid that is synthesized from methionine in the methylation
cycle. High level of Hcy, hyperhomocysteinemia, is a risk factor for several pathologies, including
pathophysiological mechanisms in pregnancy (Laanpere et al., 2010). In normal conditions, Hcy
accumulation is prevented by remethylation of Hcy to methionine and by irreversible conversion
of Hcy to cysteine via transsulfuration (Figure 27.1). In a case of dietary or genetically determined
folate deficiency, both of these cycles are inhibited, which leads to Hcy accumulation (Jacques
et al., 2001).
Folate deficiency increases deoxyuridine monophosphate misincorporation into DNA, disrupts

DNA integrity, slows DNA replication and causes apoptosis and necrosis of the affected cells
(Altme et al., 2011a). Thus, folate deficiency-induced processes may alter female fertility by
interrupting folliculogenesis (oocyte and follicular maturation), subsequent fertilization, and
embryo growth and development (Laanpere et al., 2010). Insufficient folate intake has been
shown to impair fertility in animal models (Mohanty and Das, 1982; Mooij et al., 1992; Willmott
et al., 1968), and cause adverse pregnancy outcomes in humans. Folate deficiency and/or elevated
Hcy levels have been associated with orofacial clefts, Down syndrome, placental abruptions, pre-
eclampsia, spontaneous abortions, intrauterine growth retardation, and pre-term birth (Laanpere
et al., 2010).
Table 27.1. Selection of gene variants affecting folate absorption and metabolism (Modified from Laanpere et
al., 2010. With permission from John Wiley and Sons).
Gene Gene name Polymorphism Phenotypic effect Reference

symbol
CTH cystathionase 1208 G/T (Ser403Ile, higher Hcy levels Altme et al.,
rs1021737) 2010
FOLH1 folate hydrolase 1 1423 C/T (His475Tyr, reduced enzyme activity Devlin et al.,
rs61886492) 2006
FOLR1 folate receptor 1 1816 delC (rs3833748)delC/A haplotype Hcy- Nilsson et al.,
1841 G/A raising effect, lower 2012
(rs1540087) folate
MTHFR methylenetetrahydrofolate 677 C/T (Ala222Val, reduced enzyme Laanpere et al.,
reductase rs1801133) activity, low folate 2010
1298 A/C (Glu429Ala, levels and high Hcy . 4
rs1801131) 677 C/T also DNA

hypomethylation,
reduced uracil
misincorporation
MTR 5-methyltetrahydrofolate- 2756 A/G (Asp919Gly, reduced Hcy Fredriksen et al.,
homocysteine rs1805087) 2007
methyltransferase
MTRR 5-methylenetetrahydrofolate- 66 A/G (Ile22Met, higher Hcy levels Gaughan et al.,
homocysteine rs1801394) 2001
methyltransferase reductase
SLC19A1 Solute carrier family 19, 80 G/A (Arg27His, higher Hcy, impaired Chango et al.,
member 1 rs1051266) folate translocation into 2000; Dufficy
cells et al., 2006
27.4 Folate in folliculogenesis

Folate is required during periods of rapid cell growth and proliferation, which occur during
oocyte and follicular maturation and development.
During maturation, the follicle grows and goes through primordial, primary, secondary, and
preantral stages before it reaches the antral stage that is ready to ovulate (Figure 27.2). In a
resting primordial follicle, non-mature oocyte is surrounded by a single layer of flattened GCs
(Gougeon, 1989). During the early growth phase the proliferating GCs grow larger, and provide
nutrients and different molecular signals to the oocyte (Wandji et al., 1997). Primary GCs start
to secrete mucopolysaccharides forming a layer of glycoproteins and acid proteoglycans around
Figure 27.2. Ovarian follicles at different developmental stages. (a) Primordial follicles (magnification
400), (b) primary follicle (magnification 400), (c) secondary follicle (magnification 400), (d) antral follicle
(magnification 100). Photos with permission by Inger Britt Carlsson.
the oocyte, named zona pellucida (Gougeon, 1989). Further proliferation of GCs and follicular
enlargement results in the formation of a secondary follicle, where two or more layers of GCs
surround the maturing oocyte. Under the influence of gonadotrophins and growth factors the
follicle grows further and surrounding stroma stratifies and differentiates to theca interna and
theca externa with vessels between the two layers (Gougeon, 1989). This enables the follicle to
gain a blood supply, resulting in a direct exposure to factors circulating in the blood, including
folates (Reynolds et al., 1992). In the preantral stage, a fluid-filled antrum starts to develop. From
this point, the GCs proliferate and differentiate to mural GCs in the periphery of the follicle and
to cumulus GCs closest to the oocyte (Gougeon, 1989).
The presence of folate and Hcy in follicular fluid is confirmed, and it has been shown that the
follicular levels correlate with the blood levels of folate and Hcy (Steegers-Theunissen et al., 1993).
It has also been established that folic acid supplementation increases folate levels and decreases
Hcy levels in the microenvironment of maturing oocyte (Boxmeer et al., 2009).
The exact mechanism by which folate metabolism affects ovarian function is not determined. It
is suggested that in folliculogenesis, high levels of Hcy may activate apoptosis and thereby lead
to follicular atresia (Forges et al., 2007). Hcy is a thiol-containing amino acid that induces the
release of reactive oxygen species, which are important for oocyte maturation and fertilization.
However, high levels of reactive oxygen species in the culture medium of embryos can lead
to slow cleavage rates, extensive fragmentation, and low blastocyst formation rate (Laanpere et
al., 2010). In fact, negative correlations between follicular fluid Hcy concentrations and oocyte
maturity and in vitro embryo quality on culture day 3 have been demonstrated (Ebisch et al.,
2006; Szymanski and Kazdepka-Zieminska, 2003). Also, a study on PCOS patients undergoing in
vitro fertilization treatment demonstrated negative correlations between follicular Hcy levels and
fertilization rate as well as oocyte and embryo quality, further indicating that follicular fluid Hcy
may play an important role in the maturation and fertilization of oocytes (Berker et al., 2009). At
the same time, Hcy level in follicular fluid was found in positive correlation with follicle diameter
(Boxmeer et al., 2008). Furthermore, a recent study in IVF-treated women suggests that the effect
of folic acid is crucial during early follicular development, affecting immature follicles (Twigt et
al., 2011), and the regular use of multivitamin supplements including folate decreases the risk of
anovulatory infertility (Chavarro et al., 2008).
In different animal models negative effects of folate deficiency on folliculogenesis and fetal
development have been shown. In super-ovulated rats, deficiency of folates partially inhibited
ovulation (Willmott et al., 1968), while in rhesus monkeys, a folate-restricted diet caused irregular
menstrual cycles with degenerated antral follicles, an increased number of atretic and cystic
follicles, depleted GCs, and reduced or absent corpora lutea (Mohanty and Das, 1982). Further,
golden hamsters fed preconceptionally with a folic acid-free diet were infertile (Mooij et al.,
1992), and murine dams fed preconceptionally and during gestation reduced amount of dietary
folate had profound reductions in pregnancy rates, the number of implanted embryos, and the
number of viable fetuses (Xiao et al., 2005). Also, in bovine, the importance of maternal dietary
folate/B-vitamin status during the periconceptional period has been shown to influence ovarian
follicle, oocyte and preimplantation embryos (Kwong et al., 2010). All these findings in animals
emphasize the importance of folate during mammalian folliculogenesis and fetal development.
27.5 Folate and in vitro fertilization

The IVF procedure is the most successful treatment for various causes of infertility. The procedure
consists of COH with hormones, fertilization of the retrieved oocytes and culturing of embryos,
and the transfer of embryos (Altme et al., 2007). The expected outcome of IVF depends greatly
on the effectiveness of COH, where exogenous gonadotrophins (mostly FSH) are used to induce
the growth and development of multiple follicles. Also, many additional factors may influence
IVF outcomes, such as age, reason of infertility, quality of transferred embryos, etc. Furthermore,
there is growing evidence that infertility treatment outcomes could be modulated by maternal
nutritional status, like B vitamin supplementation (Altme et al., 2011a).
A number of studies have shown that folate-deficient women undergoing COH have lower oocyte
quality, lower pregnancy rates and impaired ovarian function (Berker et al., 2009; Boxmeer et
al., 2009; Ebisch et al., 2006; Forges et al., 2007; Hecht et al., 2009; Rosen et al., 2007). A previous
study of preconception Mediterranean diet in women undergoing IVF treatment demonstrated
positive correlation with red blood cell folate levels and vitamin B6 in blood and follicular fluid,
and ultimately with increased chance of pregnancy (Vujkovic et al., 2010). Interestingly, a recent
study of preconception folic acid use in women undergoing conventional ovarian stimulation
within IVF procedure showed negative effect of low-dose folic acid use in comparison to non-
users (Twigt et al., 2011), as they detected higher estradiol response and higher mean follicle
number in non-users. Their study results are supported by a study on ewes, where higher ovarian
response after gonadotrophin administration was obtained in methyl-donor-deficient ewes
(Kanakkaparambil et al., 2009). They propose that preconception folic acid use attenuates the
ovarian response by affecting only less mature follicles (Twigt et al., 2011). They also suggest that
a possible effect of folates on IVF could be by mediating the ovarian response to gonadotrophins
through interference with FSH receptor, aromatase availability, and an increase in reactive
oxygen species (Twigt et al., 2011). Some results indicate that high folic acid intake may be
harmful for some people (Smith et al., 2008). Increased folic acid intake leads to elevated levels
of naturally occurring folates and unmetabolized folic acid in the blood, which have been related
to decreased natural killer cell cytotoxicity and reduced response to some drugs (Smith et al.,
2008). In pregnant women, a combination of high folate levels and low vitamin B12 status has
been associated with an increased risk of insulin resistance and obesity in their children (Smith
et al., 2008). Further, a very recent study demonstrated that subfertile women taking multiple
micronutrients, not solely folate, during ovulation induction have a higher chance of pregnancy
compared to women on folic acid only (Agrawal et al., 2012).
27.6 Variation in folate-metabolizing pathway genes and COH/IVF

Several polymorphisms have been identified in genes involved in the folate-mediated one-carbon
metabolism, which have been shown to alter the beneficial effect of folates and other B vitamins
that play role in the metabolism of methyl groups and change the flux of folate cofactors between
methyl donor and nucleotide synthesis (Laanpere et al., 2010).
It should be noted that majority of women undergoing infertility treatment take folate supplements,
which could lead to negative/contradicting results in studies analyzing polymorphisms in genes
involved in folate-metabolizing pathway in association with COH/IVF outcomes. Nevertheless,
there are a number of studies demonstrating the influence of gene variation in folate pathway and
IVF treatment outcomes (Table 27.2).
Women with MTHFR 677 CC have been shown to require less gonadotrophin administration
and produce more oocytes in COH than T allele carriers (Thaler et al., 2006), and women with
MTHFR 677 CT demonstrated higher proportion of good quality embryos (Laanpere et al.,
2011), meanwhile 677 TT genotype had a negative effect on the number of oocytes retrieved
following COH (Pavlik et al., 2011). Another study showed that women with MTHFR 1298
minor C allele required more gonadotrophin administration, had higher basal FSH levels and
lower estradiol levels, and produced fewer follicles (Rosen et al., 2007). Unexpectedly, in a study
of women with OHSS (extreme condition of COH) a positive association with the variant 677 T
Table 27.2. Polymorphisms and controlled ovarian hyperstimulation and in vitro fertilization outcomes in infertile
women (Modified from Altme et al., 2011a. With permission from Oxford University Press).
Gene Variation Phenotypic difference of variation

symbol
MTHFR 677C/T (Ala222Val, rs1801133) women with 677 CT have higher proportion of good
1298A/C (Glu429Ala, rs1801131) quality embryos1, 677 CC women require less
1793G/A (Arg594Gln, rs2274976) gonadotrophin and produce more oocytes2,3, 677 T
associated with ovarian hyperstimulation syndrome4
women with 1298 C allele require more gonadotropin
administration, have higher basal follicle-stimulating
hormone levels and lower estradiol levels, and produce
fewer follicles5
FOLR1 1314G/A (rs2071010) no association with COH/IVF1
1816C/delC (rs3833748)
1841G/A (rs1540087)
1928C/T (rs9282688)
TCN2 776C/G (Arg259Pro, rs1801198) no association with COH/IVF1
CTH 1208G/T (Ser403Ile, rs1021737) 1208 GT less frequent among IVF patients1
SLC19A1 80G/A (His27Arg, rs1051266) 80 GA less frequent among IVF patients1
1 Laanpere et al., 2011.

2 Pavlik et al., 2011.
3 Thaler et al., 2006.
4 Dulitzky et al., 2002.
5 Rosen et al., 2007.
allele was found, though the sample size was very small (Dulitzky et al., 2002). While this finding
was not replicated in another study of bigger sample size (Machac et al., 2006).
27.7 Folate in implantation and early pregnancy

The importance of folate, especially adverse effects of elevated Hcy levels in embryo implantation
and pregnancy has also been suggested.
A recent study on human material demonstrated that folic acid is important in a number of
crucial early stages of pregnancy, including extravillous trophoblast invasion, angiogenesis, and
secretion of matrix metalloproteins (important players in implantation process) (Williams et al.,
2011). High folate intake and plasma levels have also been associated with an increased likelihood
of having twins in IVF patients (Haggarty et al., 2006). Further, preconception supplementation
with folate and vitamin B12 has been associated with lower incidence of miscarriages in women
planning pregnancy (Zetterberg, 2004). Meanwhile, high Hcy concentrations associated with
low pregnancy and implantation rates and high abortion rates in IVF patients (Haggarty et al.,
2006; Pacchiarotti et al., 2007), and with defective chorionic villous vascularization in women
with recurrent pregnancy loss (Nelen et al., 2000). An embryotoxic effect of Hcy has been
demonstrated on post-implantation rat embryos in culture (Vanaerts et al., 1994). Further, Hcy
induces trophoblast apoptosis, reduces human chorionic gonadotrophin secretion in vitro (Di
Simone et al., 2003), and increases contractions in isolated myometrium derived from pregnant
women (Ayar et al., 2003), which all may cause spontaneous abortion.
Hcy pathophysiology has also been associated with enhanced blood clotting (Colucci et al.,
2008). Thus Hcy role in pregnancy establishment may be in disturbing the required balance
between coagulation, anticoagulation, and fibrinolysis during the process of implantation when
blastocyst-derived syncytiotrophoblasts penetrate the spiral arteries to breach endometrial blood
vessels, establishing thereby the uteroplacental circulation (Altme et al., 2011b).
27.8 V ariation in folate-metabolizing pathway genes and pregnancy

establishment
Several studies have analyzed the impact of maternal polymorphisms in folate-metabolizing
pathway genes on folate and Hcy status in pregnancy establishment either spontaneously or with
IVF treatment (see Table 27.3 for more data). MTHFR 677 TT homozygotes were found more
frequently among female patients that had experienced several consecutive IVF implantation
failures (Azem et al., 2004; Qublan et al., 2006) and in women with recurrent pregnancy loss
(Goodman et al., 2006; Nair et al., 2011). Meanwhile, a number of studies have found no such
associations (Coulam et al., 2006; Dobson et al., 2007; Ivanov et al., 2007; Martinelli et al., 2003;
Settin et al., 2011; Simur et al., 2009).
In reality, many countries recommend folic acid supplementation during pregnancy, in addition
to the existence of folate fortified foods, which can mask the negative effects of polymorphisms
in folate-metabolizing pathway, and thereby lead to negative/contradicting results. For instance,
it is known that individuals carrying the MTHFR 677 T allele have increased Hcy levels, and with
additional folate administration normal Hcy levels are achieved (Fohr et al., 2002).
A previous study of 10 different polymorphisms in folate pathway genes in women with

unexplained infertility found that MTHFR 677 CT genotype was more prevalent among
control women than infertile women (Altme et al., 2010). Also a recent study on IVF patients
demonstrated that MTHFR 677 CT heterozygous individuals had increased chance of pregnancy
(Laanpere et al., 2011). Additionally, 677 CT genotype, rather than CC, has been associated with
an increased chance of having had a previous IVF pregnancy, and a live birth in the current
treatment cycle (Haggarty et al., 2006). Further, heterozygous polymorphisms MTHFR 1793
GA and SLC19A1 80 GA have been associated with decreased number of previously failed IVF
Table 27.3. Polymorphisms studied in relation to implantation and pregnancy establishment either spontaneously
or with in vitro fertilization treatment.
Gene Variation Phenotypic difference of variation

symbol
MTHFR 677C/T (Ala222Val, rs1801133) 677 TT more frequent in women with consecutive
1298A/C (Glu429Ala, rs1801131) IVF implantation failure1,2 and recurrent pregnancy
1793G/A (Arg594Gln, rs2274976) loss3,4, IVF patients with 677 CT have higher chance
of pregnancy and live birth5,6 and is less frequent in
IVF patients with unexplained infertility7, 1793 GA
associated with a lower percentage of previously failed
IVF treatments6
FOLR1 1314G/A (rs2071010) 1816 CdelC and 1841 GA associated with raised risk of
1816C/delC (rs3833748) pregnancy loss6
1841G/A (rs1540087)
1928C/T (rs9282688)
TCN2 776C/G (Arg259Pro, rs1801198) no association with pregnancy in IVF patients6
CTH 1208G/T (Ser403Ile, rs1021737) 1208 GT associated with increased chance of pregnancy
and a smaller number of previously failed IVF cycles6
SLC19A1 80G/A (His27Arg, rs1051266) 80 GA associated with decreased number of previously
failed IVF treatments6
1 Azem et al., 2004.

2 Qublan et al., 2006.
3 Goodman et al., 2006.
4 Nair et al., 2011.
5 Haggarty et al., 2006.
6 Laanpere et al., 2011.
7 Altme et al., 2010.
treatments, meanwhile CTH 1208 GT with increased chance of pregnancy (Laanpere et al., 2011).
These findings are in line with the theory of heterozygous advantage (overdominance), where
genetic variation is maintained in a population due to stronger viability and reproductive fitness
among heterozygotes (Hansson and Westerberg, 2002). The concept of heterozygous advantage
is not new in reproduction, as has been seen in domestic sheep, where heterozygous advantage
in genes that affect fecundity is clear (Gemmell and Slate, 2006).
27.9 Conclusions
Published data suggest a significant role of folates in the pre- and periconceptional period and
the following pregnancy establishment. Adequate folate-mediated one-carbon metabolism
supports DNA synthesis, repair and integrity, and provides methyl groups for DNA methylation,
thereby establishing chromosome stability and required gene expression. These processes
are essential for female reproductive functions and thus sufficient folate intake and adequate
folate-mediated one-carbon metabolism seems to be crucial for female fertility (Figure
27.3). Indeed, numerous studies conducted in animal models and observations in humans
confirm that folate deficiency, elevated Hcy concentrations, and related gene variants impair
ovarian reserve and folliculogenesis, inhibit ovulation, worsen oocyte and embryo quality and
compromise implantation process and pregnancy establishment. However, the studies regarding
polymorphisms in folate-metabolizing pathway could have been undervalued, as women are
advised to take folate supplements during pre- and periconceptional period and pregnancy.
Even though, the influence of a single polymorphism on a phenotype may be too subtle, but it
may become evident when coexisting with other polymorphic variants and/or in case of folate
deficiency. There is need for further experimental and interventional human studies in order to
clarify folate-gene interactions, and to optimize dietary recommendations for maternal pre- and
periconceptional periods. It also remains to be studied further whether folate supplementation,
proven to prevent pathophysiological mechanisms in pregnancy, can improve female fertility and
increase infertility treatment success rates.
Dietary or genetically determined folate deficiency

impairs folate-mediated one-carbon metabolism
Folate& Hcyaffect:
DNA synthesis (replication, DNA and protien methylation DNA damage protection
mitosis, meiosis, cell (chromosome stability and (antioxydant protection,
proliferation) gene expression) oxidative stress, apoptosis)
Impaired folliculogenesis (follicular atresia,

oocyte maturation)
Fertilisation
Embryo quality
Compromised implantation and pregnancy
establishment
Pregnancy-related complications
Figure 27.3. Schematic overview of factors affecting folate metabolism, the genomic and cellular responses
caused by altered folate status, and subsequent outcomes that influence female fertility. Hcy = homocysteine.
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28. Dietary intolerance and endometriosis: an
immunological link in the pathogenesis of an
enigmatic disease?
Y. Muscat Baron
Consultant Obstetrician and Gynaecologist, Mater Dei University Hospital, Msida, Malta;
yambaron@go.net.mt
Abstract
The pathogenesis of endometriosis remains enigmatic. Not one theory explains all cases
of endometriosis, however, there is a growing consensus that the initiation of endometriosis
is retrograde menstruation. The consolidation of endometriotic colonization appears to be
mediated through altered immunological processes in the peritoneal milieu. The diverse
findings in the immunological reaction to endometriosis suggest several etiological agents. Some
workers have looked into dietary intolerance focusing on the various components in the diet
that may act as etiological agents which alter the bodys immunological response. This altered
immunological response may contribute to the establishment and progression of endometriosis.
Studies in their varied forms, as in the animal model, in vitro and observational studies have been
carried out, so as to elucidate a connection between dietary components and the pathogenesis
of endometriosis. Also analyzed in significant detail are the common immunological mediators
between nutrition and endometriosis. These studies indicate that the linkage of dietary intolerance
and endometriosis is complex and is further compounded with the multifaceted mediation of
the immunological process. There is variance in the connection between dietary intolerance
and endometriosis, possibly due to multifarious variables including differences in population
nutritional components, genetic and immunological characteristics. Endometriosis may indeed
be the final common pathway of various inflammatory processes reacting differentially to a
diverse number of elements, some of which may include dietary intolerance. Dietary modification
to alleviate symptoms and attenuate the progression of endometriosis may have to be tailored to
the pattern of the patients dietary intolerance.
Keywords: dietary intolerance, endometriosis, immunity
449
Y. Muscat Baron
Summary points
Components in the diet may act as etiological agents which alter the bodys immunological response to
retrograde menstruation initiating endometriosis.
There may be a diverse number of dietary elements which may be implicated in the genesis of endometriosis.
The diverse dietary components may affect individuals in a differential manner.
The link between dietary intolerance and endometriosis may be mediated through the intercession of
multifaceted immunological processes.
Epigenetics and environmental factors may act as common denominators for hormonal and immunological
aberrations in endometriosis
Endometriosis may be the final common pathway of various inflammatory processes affecting peritoneal
immunity.
Nutritional modification to alleviate symptoms of endometriosis may have to be tailored to the pattern of the
patients dietary intolerance.
28. D
ietary intolerance and endometriosis
Abbreviations
HR Hazard ratio
IF- Interferon-gamma
IL Interleukin
MCP-1 Monocyte chemokine protein-1
NF Nuclear factor-kappa beta
OR Odds ratio
TNF Tumor necrosis factor-alpha
28.1 Introduction
Endometriosis is the presence of endometrial implants outside the uterine cavity. The pathogenesis
of endometriosis remains elusive to the extent that some workers have looked at dietary etiologies
possibly mediated through altered immunological processes (Muscat Baron et al., 2011, 2012;
Parazzini et al., 2004; Trabert et al., 2011).
Although no single theory can explain all cases of endometriosis, the most commonly accepted
initiation of the disease is Sampsons theory of retrograde menstruation expounded in 1927 (Figure
28.1). Retrograde menstruation has been noted in 76 to 90% of women (Seli and Arici, 2003).
Retrograde Fallopian Retrograde

menstrual flow tube menstrual flow
through Fallopian through Fallopian
tubes tubes
Endometrial Uterus
cavity
Normal
menstruation
Figure 28.1. Coronal section through uterus demonstrating retrograde menstrual flow the initiation of
endometriosis.
Y. Muscat Baron
Despite such a high occurrence of retrograde menstrual flow, a much lower prevalence of
endometriosis of 7-10% suggests that additional factors such as altered immunological peritoneal
surveillance may determine susceptibility to the disease (Seli and Arici, 2003). Once colonies of
endometriosis gain a foothold onto the peritoneum the disease may spread across the peritoneal
cavity (Figure 28.2).
Endometriosis is associated with altered cell-mediated and humoral immunity. Natural killer cell
activity appears deficient in cases with endometriosis allowing inadequate removal of refluxed
menstrual debris may play a role in the genesis of endometriotic peritoneal implants. Paradoxically,
the immune cells found in peritoneal fluid appear to encourage endometriotic implantation
rather than inhibit its development. Although it is unclear whether these immunologic alterations
are a cause or a consequence of endometriosis, they appear to play an important role in allowing
endometriosis implants to persist and progress (Oosterlynck et al., 1991). Endometriotic implants
under the right conditions will spread across the pelvis peritoneum to eventually involve the
pelvic viscera and abdominal contents (Figures 28.3-28.6).
It is well known that appropriate dietary intake is responsible for the maintenance of the
immunological equilibrium, in humans and animals. An adequate balance of vitamins, elements,
lipids, proteins and nucleic acids play an important role in the regulation of cellular and humoral
immune responses to the development of endometriosis.
This review will deal with the influence the various dietary components may have on the diverse
elements of bodys immunological response which may catalyze the progression of endometriosis.
Retrograde
flow
Anteverted Rectosigmoid
uterus colon
Cervix
Endometriotic spots
and implants
posterior to uterus
Figure 28.2. Sagittal section of uterus showing retrograde flow through Fallopian tubes with deposition of
endometriotic implants posterior to the uterus.
28. D
Ovarian
endometriosis
Endometriotic
spots posterior to
uterus and pouch
Anteverted of douglas
uterus
Bladder
Endometriosis on
rectosigmoid colon
Cervix
Figure 28.3. Sagittal section of uterus with pelvic regions affected by peritoneal endometriosis. Retrograde flow
through the Fallopian tubes with deposition of endometriotic implants posterior to the uterus.
Figure 28.4. Endometriosis on external surface of left ovary containing a chocolate cyst.
Y. Muscat Baron
Figure 28.5. Ovarian cyst ruptured. Chocolate fluid exuding out of left ovarian endometrioma.
Figure 28.6. Laparoscopic excision of ovarian endometrioma. After evacuation of ovarian endometrioma, the
cyst wall is peeled off to reduce risk of recurrence of endometriosis.
28.2 Evidence linking diet and endometriosis

There is a paucity of evidence associating diet with the occurrence of endometriosis. In a study
by Muscat Baron et al. (2011), dietary intolerance to various foodstuffs was assessed by asking
a comprehensive questionnaire on gastro-intestinal symptoms and dietary intolerance. In this
28. D
study 57 women who complained of gynecological symptoms and/or complained of infertility

underwent laparoscopy in effort to reach a diagnosis. These women were recruited sequentially
into the study so as to avoid bias (age range 20-50 years) (Tables 28.1 and 28.2).
Prior to the laparoscopy the questionnaire was filled in, enquiring on occurrence of gynecological
symptoms, dietary intolerance, gastrointestinal and general complaints in these women. The
frequency of symptoms was defined as occurring on a regular basis at least once a week. The
questionnaire was done pre-operatively to avoid bias where the observers knowledge of the
diagnosis could influence the results of questionnaire. In 15 of the women the whole questionnaire
including all the parameters, was asked again by a different observer to validate the methodology.
The correlation of the results obtained in the latter group when comparing the results of the same
questionnaire asked twice was r=0.94.
Differences in dietary intolerance were noted between women who were diagnosed as having
pelvic endometriosis and women who did not have endometriosis (Table 28.3). These differences
did not reach statistical significance possibly because the study was not powered sufficiently
to show this. However across practically all foodstuffs the trend was for dietary intolerance as
evidenced by the increased occurrence of gastro-intestinal symptoms to occur more commonly
in the group of women with endometriosis (Figure 28.7).
These differences in dietary intolerance were most marked in four groups of foodstuffs.
Foodstuffs containing gluten registered a dietary intolerance of 43% in the group of women
with endometriosis as compared to 26% in the control group (Figure 28.8; Table 28.3). A similar
Table 28.1. Demographic characteristics of patients recruited for this study (Muscat Baron et al., 2011).
Number of patients Age Laparoscopy Laparotomy
No endometriosis 34 44.69.2 32 2
Endometriosis 23 3910.2 21 2
Table 28.2. Main reason for laparoscopy (Muscat Baron et al., 2011).
No endometriosis group Endometriosis group
Dysmenorrhoea/dysparuenia 12 10
Ovarian cyst 4 1
Infertility 10 8
Abdominal pain 8 4
Total 34 23
Y. Muscat Baron
Table 28.3. Dietary intolerance to various types of food (Muscat Baron et al., 2011).
No endometriosis Endometriosis
Number of patients 34 23
Bread 9 (26.5%) 10 (43.5%)
Pizza 9 (26.5%) 10 (43.5%)
Pasta 5 (14.7%) 5 (21.7%)
Red meat 0 3 (13.0%)
White meat 2 (5.8%) 0
Chicken 1 (2.9%) 0
Fish 1 (2.9%) 0
Dairy products 5 (14.7%) 9 (43.8%)
Milk 4 (11.8%) 7 (30.4%)
Yoghurt 1 (2.9%) 3 (13.0%)
Vegetables 8 (23.5%) 11 (48.8%)
30
Percentage of patients
20
10
0
Endometriosis Control group
Figure 28.7. Dietary intolerance to all foodstuffs in endometriosis group versus control group (Muscat Baron
et al., 2011).
40
35
30
25
20
15
10
5
0
Figure 28.8. Intolerance to wheat products in endometriosis group versus control group (Muscat Baron et al.,
2011).
28. D
pattern was noted for dairy products with 43.8% in the women diagnosed with endometriosis
versus 14.7% in the women without endometriosis (Table 28.3). For vegetables the percentage
of dietary intolerance was 48.8% in the women with endometriosis against 23.5% in the control
group. Intolerance to red meat was found in three women endometriosis, however none were
noted in the women without endometriosis (Table 28.3).
Below is a letter from one woman attending the Malta Endometriosis Support Group. This
woman suffered from severe symptoms attributed to endometriosis and following a meeting
at the support group avoided red meat, starch and dairy products in an effort to alleviate her
symptoms.
Since our last meeting my life changed. The following day I decided to completely
change my diet, stopped all red meat, dairy products and wheat (which used to be
a big part of my diet). After a week I had my usual, ugly and painful period of just
one and a half day. I simply thought that all was as always, but after the usual period
I continued to have black spots every day. I could only describe it like old cobwebs
coming out and cleaning me from the inside!! In fact after all the spring clean ;o) I
had a normal, healthy, red period that lasted for 4 days!! I havent had such a good
period for a decade!! I went to my gynea and explained my new diet and all that
had happened and asked if I should be worried but she confirmed my positive
thoughts and in fact I should be happy because most probably I found the solution
for my pain. This month I had no sign of endo and I am sooooooo HAPPY!!!!!
This letter was sent more than a year ago and since then this member of the Malta Endometriosis
Support Group has narrowed her dietary restriction to red meat and wheat products. The
remarkable improvement in her symptoms attributed to endometriosis persists.
The findings from our study (Muscat Baron et al., 2012) suggest that dietary intolerance has a
tendency to occur in women with endometriosis to a varying range of foodstuffs. This pattern of
dietary intolerance in these women with endometriosis may suggest variance in the immunological
reaction to the various antigens and molecular characteristics in foodstuffs. This variance in
immunological reaction of women with endometriosis may be reviewed by analyzing the medical
literature for the varied cell mediated and humoral reactivity to the different constituents of
foodstuffs.
28.3 Protein ingestion, and endometriosis

The connection between protein ingestion and the development of endometriosis through altered
cell-mediated immunity has been assessed in the animal model. L-carnitine administration
to female mice has been shown to alter the cellular and growth factor profile in the uterus
and peritoneum towards a pattern similar to that of clinical endometriosis (Vassiliadis and
Athanassakis, 2011). L-carnitine is an amino acid manufactured in the body from the essential
Y. Muscat Baron
amino acids lysine and methionine. L-carnitine helps carry fatty acids into the mitochondria in
cells so as to catalyze the conversion of these acids to energy.
L-carnitine treatment resulted in a significant increase of macrophages and to a lesser degree

an increase of T-cells, while elevated levels of IF- and TNF were detected in both serum and
peritoneal fluid compared to controls. Although levels of L-carnitine measured in mouse serum
samples showed an increase as compared to controls, levels of acyl-L-carnitine measured in
the murine peritoneal fluid samples showed a decrease similar to that measured in peritoneal
fluid samples from patients with stage IV endometriosis. These results indicate that L-carnitine
administration to female mice alters the cellular and growth factor profile in the uterus and
peritoneum towards a pattern similar to that of clinical endometriosis (Vassiliadis and
Athanassakis, 2011) (Figure 28.9).
In our study three women described intolerance to red meat leading to a variety of gastro-
intestinal symptoms.
28.4 Endometriosis, gluten and Celiac disease

Another protein that may be associated with the pathogenesis of endometriosis is gluten. In
our study, foodstuffs containing gluten registered a dietary intolerance of 43% in women with
endometriosis as compared to 26% in the control group. Gluten is a protein composite of the
gliadin and glutelin found in foods processed from wheat and related grain species, including
barley and rye. Gliadin is the etiological agent leading to gluten enteropathy, also known as
Celiacs Disease.
Ingested gliadin, the triggering agent of the disease on crossing the epithelial barrier may
elicit a harmful T cell-mediated immune response. This seems to be mediated through an
increased secretion of chemokines and cytokines, mainly of IL-6, IL-8, IL-10, TNF, growth-
related oncogene, macrophage cytokine-1 and macrophage-derived chemokine. Maturation is
L-carnitine
Cell mediated Growth factor

immunity profile
Increase in Tumour necrosis

Interferon-gamma
T-cells factor-alpha
Figure 28.9. L-carnitine in the mouse model influencing cell mediated immunity and growth factor profile
(Vassiliadis and Athanassakis, 2011).
28. D
accompanied by a greater capacity to stimulate proliferation of allogenic T cells and significantly

reduced endocytic activity (Palova-Jeninkova et al., 2005) (Figure 28.10).
In gluten enteropathy, immunoregulatory invariant natural killer cells rapidly produce IL-4 and
other cytokines that suppress a Th1 response and are also deficient in some autoimmune diseases.
Natural killer T-cells in Celiacs subjects were shown to be reduced to 30% of those in normal
subjects (Grose et al., 2008) (Figure 28.10). Similar deficiencies in natural killer cell activity has
been noted in women with endometriosis.
In vitro treatment of peripheral blood mononuclear cells with gliadin inhibited natural killer cell
activity in patients with Celiacs Disease. On the other hand, incubation with gliadin-induced
cytotoxic cell activity of peripheral blood monocytes against the natural killer-resistant target
cells (Castany et al., 1995).
A linkage between endometriosis and Celiacs Disease has been demonstrated. Serological testing
of 120 women diagnosed with endometriosis was compared to 1,500 healthy volunteers (Aguiar
et al., 2009). Nine of the 120 women in the study group were tissue transglutaminase positive and
four women had anti-endomysium antibodies. Four of these patients were submitted to intestinal
biopsy, which revealed Celiacs Disease in three cases. The overall Celiac Disease prevalence
among the population control group was 1:136 women (0.66%), whereas in the endometriosis
group it was 2.5%. This was the first study reporting the increased prevalence of Celiacs Disease
among women with endometriosis.
Another large study by Stephannson et al. (2011), identified 11,097 women with Celiac Disease.
Subsequent follow-up of these Celiac subjects compared 118 individuals diagnosed with
endometriosis to 399 matched controls. Patients with Celiac Disease were at increased risk of
subsequent endometriosis (HR=1.39; 95% CI=1.14-1.70). The absolute risk of endometriosis
Gluten
Cell mediated Pro-inflammatory

immunity cytokines
Growth-related
Interleuken-6,
Reduction in natural oncogene,
interleuken-8,
killer cells T-cells macrophage
interleuken-10,
Reduced endocytic cytokine-1
tumour necrosis
T-cell activity macrophage-derived
factor-alpha chemokine
Figure 28.10. Gluten enteropathy influencing cell mediated immunity and growth factor profile activity (Grose
et al., 2007; Palova-Jeninkova et al., 2005).
Y. Muscat Baron
in patients with Celiac Disease was 112/100,000 person-years with an excess risk of 31/100,000
(Stephannson et al., 2011).
28.5 Dairy products and endometriosis

In our study dairy products intolerance was commoner in the women with endometriosis with
43.8% versus 14.7% in the women without endometriosis. As regards milk intolerance, lactose
has been shown to block galectin-3 which is a beta-galactoside-binding protein implicated in
diverse biological processes.
Galectin-3 is also relevant in the inflammatory response which may be relevant in the peritoneal
reaction to endometriotic implantation. Galectin-3 is expressed by virtually all immune and
inflammatory cell types. A large body of work has demonstrated the role of galectin-3 in
regulation of the functions of these cells. In animal studies the use of galectin-3-deficient mice
has provided additional evidence for this proteins contribution to the inflammatory response. It
has been postulated that galectin-3 may be a therapeutic target for various inflammatory diseases
(Hsu et al., 2009).
T-cell functions appear to be intimately regulated by galectin-3. Galectin-3 exerts extracellular

functions including cell activation, adhesion, induction of apoptosis, and formation of lattices
with cell surface glycoprotein receptors. These factors may be relevant to the initial implantation
of endometriosis.
Consistent with the presence of galectin-3 in intracellular locations, several functions have been
described for this protein inside T cells. These include inhibition of apoptosis, promotion of cell
growth, and regulation of T-cell receptor signal transduction (Hsu et al., 2009). The impact on T-cell
function may be related to the establishment and progression of endometriosis (Figure28.11).
A lactose-binding protein, MCF-pl5-L exhibited an evident dose-dependent monocyte

chemotactic activity for monocytes and macrophages. The biological functions of MCF-pl5-L
include prolonging the life span of macrophages, probably by inhibiting apoptosis of macrophages,
and stimulate the production of TNF from macrophages (Yamanaka et al., 2000). Galectin-3
induces a human monocyte migration in vitro in a dose-dependent manner, and it was found to
be chemotactic at high concentrations, but chemokinetic at low concentrations (Sano et al., 2000).
As referred to in Section 28.6, non-digestible oligosaccharides may have a protective effect in

cows milk allergy to casein as evidenced from animal studies (Shouten et al., 2012).
28. D
Lactose blocks galectin-3
Galectin-3 responsible for
Cell mediated immunity
T-cell function
T-cell activation Human monocyte
promotion of migration
T-cell growth
Figure 28.11. Lactose blocks galectin-3 which is implicated in diverse biological processes including the
inflammatory response to endometriosis (Hsu et al., 2009).
28.6 Carbohydrates and endometriosis

Non-digestible carbohydrates found in plants may influence bowel immunity, especially in the
area of the gut-associated lymphoid tissue. Recent data now provide evidence that prebiotics
such as inulin/oligofructose modulate functions of the immune system. In animal studies besides
the essential nutrients, non-essential food constituents such as inulin/oligofructose primarily
activated immune cells in Peyers patches including interleukin-10 production and natural killer
cell cytotoxicity (Watzl et al., 2005) (Figure 28.12).
Savaris and Do Amaral (2011) in a study of 25 women with endometriosis compared to 20

controls showed that the mean total daily calorie intake in the women in the endometriosis group
was significantly higher than that of the women in the control group (P=0.005). With respect to
Non-digestible
carbohydrates
Cell mediated Growth factor

immunity profile
Increased Increased secretion

natural killer of interleukin-10
cell cytotoxicity
Figure 28.12. Non-digestible carbohydrates such as inulin/oligofructose in animal studies influenced cell
mediated immunity and growth factor profile (Watzl et al., 2005).
Y. Muscat Baron
nutrient intake, the only statistically significant difference found referred to a higher intake of
fiber in the endometriosis group.
Non-digestible carbohydrates have been shown to have a protective effect against casein allergy
in cows milk allergic mice. The oligosaccharide diet to cows milk allergic mice strongly reduced
the development of the acute allergic skin response (Shouten et al., 2012),
28.7 Fats and endometriosis

Fish oil consumption has been associated with symptom improvement in studies of women with
primary dysmenorrhea and with decreased endometriosis risk in autotransplantation animals.
The relation between dietary fat intake and the risk of endometriosis, was analyzed from data of
the Nurses Health Study II. Dietary fat was assessed via food frequency questionnaire in 1991,
1995 and 1999. During the 586 153 person-years of follow-up, 1,199 cases of laparoscopically
confirmed endometriosis were reported. Although total fat consumption was not associated
with endometriosis risk, those women in the highest 20% of long-chain omega-3 fatty acid
consumption were 22% less likely to be diagnosed with endometriosis compared with those with
the lowest fifth of intake (95% CI=0.62-0.99; P=0.03). In addition, those in the highest quintile
of trans-unsaturated fat intake were 48% more likely to be diagnosed with endometriosis (95%
CI=1.17-1.88; P=0.001). These data suggest that specific types of dietary fat are associated with
the incidence of laparoscopically confirmed endometriosis (Missmer et al., 2010). Savaris and
Do Amaral (2011) showed that women diagnosed with endometriosis had a significantly higher
intake of polyunsaturated fatty acids than in the control group (P<0.05).
Another study by Gazvani et al. (2001) demonstrated in vitro survival of endometrial cells from
women with and without endometriosis was significantly reduced in the presence of high omega
3:omega 6 polyunsaturated fatty acids ratios compared with cells incubated in the absence of
fatty acids, in balanced omega 3:omega 6 polyunsaturated fatty acids ratios, and in high omega
3:omega 6 polyunsaturated fatty acids ratios. Endometrial cells from women with endometriosis
secreted higher concentrations of IL-8, especially in the presence of high omega-3:omega-6
polyunsaturated fatty acids ratios. Omega 3 polyunsaturated fatty acids may have a suppressive
effect on the in vitro survival of endometrial cells and omega 3 polyunsaturated fatty acids may
be useful in the management of endometriosis by reducing the inflammatory response and
modulating cytokine function (Gazvani et al., 2001).
Animal studies have shown that dietary fish oil results in altered lymphocyte function and
in suppressed production of proinflammatory cytokines by macrophages. Consumption of
fish oils diminishes lymphocyte proliferation, T-cell mediated immunity, natural killer cell
activity macrophage-mediated cytotoxicity, monocyte and neutrophil chemotaxis, major
histocompatibility class II expression and antigen presentation, production of pro-inflammatory
cytokines (IL-1 and -6, TNF) and adhesion molecule expression (Calder, 1998). This has also
been shown in humans, whereby supplementation of the diet of healthy human volunteers with
28. D
fish oil-derived n-3 polyunsaturated fatty acids results in decreased monocyte and neutrophil
chemotaxis and decreased production of proinflammatory cytokines. Fish oil feeding has also
been shown to ameliorate the symptoms of some animal models of autoimmune disease (Calder,
1998) (Figure 28.13).
Eicosapentaenoic acid inhibits production of cyclooxygenase metabolites of arachidonic acid.

The effect of oral administration of pure eicosapentaenoic acid on endometriosis in the animal
model, was studied by supplementing the diet of Japanese white rabbits with eicosapentaenoic
acid (100 mg/kg/day, experimental group) or with a 5% gum arabic solution (control group). The
experimental group had endometriosis surgically induced. Peritoneal fluid prostaglandin E2,
prostaglandin F2-alpha and IL-1- concentrations in the experimental rabbits and controls were
measured before and after induction of endometriosis by laparotomy. Following the treatment,
eicosapentaenoic acid levels in plasma and peritoneal fluid increased significantly. However, no
differences were seen in arachidonic acid levels. Peritoneal fluid prostaglandin E2 and IL-1-
concentrations increased significantly after induction of endometriosis in the control group. In
contrast, they were not significantly changed after endometrial implantation in the experimental
group. Peritoneal fluid prostaglandin F2-alpha concentrations were lower in the eicosapentaenoic
acid group than in the controls, but there were no significant differences. This study suggests
that eicosapentaenoic acid may modulate the prostaglandin release in peritoneal endometriosis
(Yano, 1992).
It is not only the absolute increase in fish oils ingestion that may act as a deterrent against
endometriosis. The relationship between lipid metabolism and inflammatory reactions in
endometriosis may be due to imbalance in fat intake leading to the recent increase of endometriosis.
Another animal study investigated the anti-inflammatory effect of n-3 eicosapentaenoic acid
compared with n-6 linoleic acid in an endometriosis rat model. Rats were fed a diet with
Fish oil ingestion
Cell mediated Proinflammatory

immunity cytokines
Decreased
lymphocyte proliferation, Decreased
T-cell mediated immunity interleukin-1
macrophage-mediated interleukin-6
cytotoxicity, tumour necrosis
monocyte chemotaxis factor
neutrophil chemotaxis,
Figure 28.13. Human consumption of fish oil-derived n-3 polyunsaturated fatty acids results in decreased cell-
mediated immunity activity and decreased production of proinflammatory cytokines (Calder, 1998).
Y. Muscat Baron
eicosapentaenoic acid (n=9) or with n-6 linoleic (n=9) for 2 weeks. Two weeks after feeding, the
uterus was autotransplanted to the peritoneum to construct an endometriosis model. Feeding was
continued for a total of six weeks. Two and four weeks after autotransplantation, three rats of each
group were killed and evaluated. Endometriotic lesions were morphologically evaluated and their
fatty acid composition was examined. Gene expression in these tissues was evaluated by cDNA
microarray analysis and quantitative real-time reverse transcriptase-polymerase chain reaction.
In the eicosapentaenoic acid group, the n-3:n-6 ratio in each tissue significantly increased and the
thickening of the interstitium, an active site for inflammation in endometriosis, was significantly
suppressed (0.300.09 mm [eicosapentaenoic acid group] vs. 0.770.23 mm (n-6 linoleic acid
group). The mRNA of metalloproteinases, IL-1, IL-1r, prostaglandin E synthase, and NF were
reduced in the eicosapentaenoic acid group (Netsu et al., 2008).
28.8 Environmental contaminants and epigenetics

The features of estrogen dependence and immune modulation of endometriosis have also
implicated environmental contaminants in the pathogenesis of the disease. Previous work in
nonhuman primates has shown that exposure to the dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin
is associated with an increased prevalence and severity of endometriosis (Rier and Foster, 2003).
Further experiments in the animal model have implicated dioxin and dioxin-like compounds
in this disease. Moreover combined with the endocrine/immunological-toxicant hypothesis,
evidence has accumulated that endometriosis may be an epigenetic disease. This evidence
ties epigenetics and environmental factors which may include dietary intolerance as common
denominators for hormonal and immunological aberrations in endometriosis (Guo, 2009).
28.9 Immunity and endometriosis

Alterations in cellular immunity may result in inadequate removal of ectopic endometrial
cells from the peritoneal cavity. This may be deduced by the increased number and activation
of peritoneal macrophages and the paradoxically decreased T-cell and natural killer cell
cytotoxicities in the presence of endometriosis natural killer activity and the cytotoxicity against
autologous endometrial cells have been shown to decrease in women with endometriosis and
correlate with the severity of the disease. Decreased cytotoxicity to endometrial cells in women
with endometriosis is therefore thought to be due to a defect in natural killer activity, but is also
partially because of a resistance of the endometrium to natural killer cytotoxicity (Oostelynck et
al., 1991). Variation of the innate immunity mediated by natural killer cells may actually promote
impairments or disrupt functions of adaptive immunity, which can contribute to development
and progression of endometriosis (Sikora et al., 2011) (Figure 28.14).
Increased levels of several cytokines and growth factors which are secreted by either immune and
endometrial cells seem to promote implantation and growth of ectopic endometrium by inducing
proliferation and angiogenesis. Immunoreactivities of both chemokines IL-8 and MCP-1 have
28. D
Cell mediated immunity
Immunity and endometriosis
Growth factor Antibodies

profile
Figure 28.14. The immunological response to endometriosis encompasses alterations in the cell mediated
immunity, antibody formation and growth factor profile.
been shown to be significantly increased in the epithelial cells of ectopic endometrial tissues
compared with those of normal endometrium. These findings suggest that IL-8 and MCP-1 may
be involved in the pathogenesis of endometriosis (Ulukus et al., 2008). Serum levels of monocyte
chemokine protein-1 were found to be significantly higher in patients with endometriosis. These
results imply the potential of MCP-1 measurements for the diagnosis of endometriosis (Agic et al.,
2008). TNF of peritoneal fluid is believed to have important pro-inflammatory and angiogenic
activities in the complex mechanisms of development of peritoneal endometriotic lesions.
Impairment of macrophage function supports the theory that an inappropriate immunological
reaction of the peritoneal milieu to retrograde ejected endometrium may play a part in the initial
phases of endometriotic implants (Calhaz-Jorge et al., 2000) (Figure 28.14).
In addition to the impaired capacity of the immune cells to mediate endometrial cell removal,
inherent resistance of the ectopic endometrial cells against immune cells is another interesting
concept in the pathogenesis of endometriosis. In the initial stages of endometriosis the activated
peritoneal fluid natural killer cells can be intensively eliminated, thus providing conditions for
the survival of ectopic endometrial cells and the development of the disease (Eidukaite et al.,
2006). Sera from patients with endometriosis suppressed natural killer cell zeta expression, which
resulted in suppression of these cell IF- induction (Bohler et al., 2007). Increased expression
of CD94/NKG2A in peritoneal natural killer cells may mediate the resistance of endometriotic
tissue to natural killer cell-mediated lysis, thus contributing to the progression of endometriosis
(Galandrini et al., 2008).
Endometriosis has also been considered to be an autoimmune disease, since it is often associated
with the presence of autoantibodies, other autoimmune diseases, and possibly with recurrent
immune-mediated miscarriage. Patients with endometriosis also exhibit autoantibodies reactive
with cellular proteins; endometrial membrane proteins exhibited the greatest reactivity, followed
by nuclear antigens. A spectrum of auto-immunity has been noted when in subcellular fractions,
patients with stage III endometriosis exhibited significantly more immunoreactivity than did
stage II patients, which in turn was greater than that observed in stage I patients (Bohler et al.,
2007) (Figure 28.15).
Y. Muscat Baron
Immunity and
endometriosis
Cell mediated Growth factor profile Antibodies

immunity
Decreased Interleukin-8
natural killer monocyte
cell cytotoxicity chemotactic protein-1 Autoantibodies in
T-cell cytotoxicity interleukin-10 endometriosis
endometrium resistance tumour necrosis
to cytotoxicity factor alpha
Figure 28.15. Alterations in the cell mediated immunity, antibodies and growth factor profile due to
endometriosis.
Undoubtedly a wide variety of immune reactions are associated with endometriosis. This suggests
that possibly endometriosis is the final common pathway of a diverse number of inflammatory
instigators, including dietary intolerance.
28.10 Comprehensive dietary studies and endometriosis

There are few studies that assess the whole gamut of dietary components and the occurrence of
endometriosis. This may be relevant as the immunological reactivity may vary across populations
and according to the staple diet of the populations. Parazzini et al. (2004) collected data from two
case-control studies with a total of 504 women laparoscopically diagnosed with endometriosis
conducted in Northern Italy between 1984 and 1999. These were compared with a control
group of 504 women (median age 34 years, range 20-61) admitted for acute non-gynecological,
non-hormonal, non-neoplastic conditions. Compared to women in the lowest tertile of intake,
a significant reduction in risk emerged for higher intake of green vegetables (OR=0.3 for the
highest tertile of intake) and fresh fruit (OR=0.6), whereas an increase in risk was associated
with high intake of beef and other red meat (OR=2.0) and ham (OR=1.8). Consumption of milk,
liver, carrots, cheese, fish and whole-grain foods, as well as coffee and alcohol consumption, were
not significantly related to endometriosis. These findings do not concur with those shown in our
study (Muscat Baron et al., 2011). This may be due to differences in dietary composition, additives
to foodstuffs and epigenetics of the populations concerned.
Trabert et al. (2011), from Seattle, USA, found a different pattern of dietary intakes associated
with endometriosis. They evaluated dietary risk factors for endometriosis in a population-
based case-control study. Cases involved 284 subjects aged 18-49 years with newly diagnosed,
surgically confirmed endometriosis between 1996 and 2001. The controls recruited were 660
randomly selected age-matched female subjects without a history of endometriosis. Nutrients
and selected food groups were assessed using the Womens Health Initiative Foodstuff Frequency
28. D
Questionnaire. Increased total fat consumption was associated with decreased endometriosis risk
(fourth quartile vs. lowest: OR=0.5, 95% CI=0.2-1.0, P=0.12). Increased -carotene consumption
of fruit was associated with increased risk (-carotene third quartile vs. lowest: OR=1.7, 95%
CI=1.1-2.6; fourth quartile vs. lowest: OR=1.6, 95% CI=1.0-2.5, P=0.16; fruit OR=1.5, 95%
CI=1.0-2.3, P=0.04). In our study an increased proportion of women with endometriosis did
complain of gastro-intestinal symptoms on ingesting vegetables (Muscat Baron et al., 2012).
There was also a trend of increased endometriosis risk associated with the consumption of dairy
products, but this association was not statistically significant for the highest tertile. A similar
pattern of dietary intolerance to dairy products was demonstrated in the women diagnosed with
endometriosis in our study (Muscat Baron et al., 2012).
28.11 Conclusion
The interface of dietary intolerance and endometriosis is indeed complicated and is further
compounded with the mediation of the immunological process. Endometriosis may indeed be
the final common pathway of various inflammatory processes reacting to a diverse number of
elements some of which in the particular subject may manifest themselves as dietary intolerance.
In this vein, dietary modification to alleviate the symptoms of a woman suffering from
endometriosis may have to be tailored to the pattern of the patients gastro-intestinal symptoms
as experienced when ingesting certain food products.
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29. Gastro-intestinal symptoms in women with
pelvic endometriosis
Y. Muscat Baron
Consultant Obstetrician and Gynaecologist, Mater Dei University Hospital, Msida Malta;
yambaron@go.net.mt
Abstract
Gastro-intestinal symptoms frequently overlap with the clinical presentation of endometriosis.
The psychological profile of patients with endometriosis may promote upper gastro-intestinal
symptoms such as gastro-esophageal reflux and dyspepsia. As a consequence, neuroendocrine-
immune imbalance in response to high levels of perceived stress in women diagnosed with
endometriosis has been demonstrated. The most common site of non-genital endometriosis is
the lower intestinal tract. Lower intestinal symptoms, such as loose stools, tenesmus, constipation
and abdominal pain may mimic or co-exist with those of endometriosis. The overlap of lower
gastro-intestinal symptoms with those of endometriosis frequently lead to misdiagnosis or
delayed diagnosis. Moreover, there appears to be an increased incidence of inflammatory bowel
disease and irritable bowel syndrome in women diagnosed with endometriosis. A major portion
of the intestinal tract is in close anatomical proximity to the female genital tract. Through
neuroendocrine and immunological intermediaries, the upper gastro-intestinal system may
also interact with the physiology of the female genital system. These variables have directed
some workers to suggest an interrelationship between both systems including the occurrence
of pathology. Gastro-intestinal symptoms may act as a guide to dietary modification which may
result in improvement in the symptoms of endometriosis and an attenuation of its progression.
Keywords: upper gastro-intestinal symptoms, lower gastro-intestinal symptoms endometriosis
471
Y. Muscat Baron
Summary points
Gastro-intestinal symptoms frequently overlap with those of endometriosis.
The psychological profile of patients with endometriosis may promote upper gastro-intestinal symptoms.
Lower intestinal symptoms may mimic or co-exist with those of endometriosis.
The overlap of lower gastro-intestinal symptoms with those of endometriosis frequently lead to misdiagnosis
or delayed diagnosis.
There appears to be an increased incidence of inflammatory bowel disease and irritable bowel syndrome in
women diagnosed with endometriosis.
Dietary modification tailored to the patients gastro-intestinal symptoms may result in improvement in the
symptoms of endometriosis and an attenuation of its progression.
29. Gastro-intestinal symptoms in women with pelvic endometriosis
Abbreviations
BMI Body mass index
HAMA Hamilton rating scale for anxiety
OR Odds ratio
SSRI Selective serotonin re-uptake inhibitor
STAI State-trait anxiety inventory
29.1 Introduction
Endometriosis is the presence of endometrial tissue outside the uterus, mainly colonizing
the ovaries, utero-sacral ligaments and pelvic peritoneum. This condition is assuming greater
importance with an incidence of 7% to 10% of women and endometriosis affects one fourth
of young women under the age of 30 years. More than 80% of women complaining of chronic
pelvic pain have been diagnosed as having endometriosis and subfertility in 20-50% of women
has been associated with endometriosis. Moreover endometriosis has been diagnosed in 20-
50% of women who were completely asymptomatic, unaware that they had this pelvic condition
(Mounsey, 2006).
The enigmatic pathogenesis of endometriosis has led to the development of several theories, but
none have been proven conclusively. The elusiveness of its pathology has directed some workers
to search beyond the female genital tract and focus their efforts at the gastro-intestinal system, the
small and large intestine being in close anatomical proximity to the female genital tract (Figure
29.1) (Muscat Baron et al., 2011, 2012; Parazzini et al., 2004). The overlap of symptoms between
both the gastro-intestinal and endometriosis does in fact impact clinical practice and in several
cases leads to delayed or misdiagnosis.
Both upper and lower gastro-intestinal symptoms appear more prevalent in women diagnosed
with endometriosis (Figure 29.2). Specific symptoms and frequent medical consultation are
associated with endometriosis and appear useful in the diagnosis. Endometriosis may coexist
with or be misdiagnosed as pelvic inflammatory disease, inflammatory bowel disease or irritable
bowel syndrome (Ballard et al., 2008). From both the anatomical and symtomatological point
of view, it may be relevant to address upper and lower gastro-intestinal symptoms as separate
entities as their pathological linkage with endometriosis may be different.
29.2 Upper gastro-intestinal symptoms and endometriosis

Gastric emptying does not appear to be affected by the menstrual cycle, however abdominal
symptoms appear more commonly during the follicular phase. The latter finding may be due to
Y. Muscat Baron
Fallopian tube
Retrograde menstrual
flow through
Uterus fallopian tubes
Adhesions between
uterus and
rectosigmoid colon
Cervix due to endometriosis
Rectosigmoid
colon
Lumen of rectosigmoid Adhesions
colon
Endometriotic
implant
Figure 29.1. Sagittal section: retroposed uterus due to endometriosis-induced adhesions between rectosigmoid
colon and posterior aspect of uterus.
60
50
40
30
20
10
0
Figure 29.2. Upper and lower gastrointestinal symptoms in endometriosis group versus control (Muscat Baron
et al., 2011).
the longer transit time in the small bowel during the follicular phase. Gastric emptying did not
change significantly between the follicular and luteal phases indicating that the normal menstrual
cycle has no effect on gastric motility (Bjrnsson et al., 2006).
The anatomical distance between the upper gastro-intestinal tract and the female genital tract
may prima facie, appear unrelated without any anatomical or physiological connection. In a
study by Muscat Baron et al. (2011), however, symptoms such as heartburn and dyspepsia were
more commonly found in women with endometriosis as compared to a group of women without
endometriosis. This was a prospective trial involving 57 menstrual women who had undergone
laparoscopic examination of the pelvis for a diverse number of abdominal and gynecological
symptoms and complaints such as infertility. These women were recruited sequentially, so as to
avoid bias and were asked a comprehensive questionnaire with included information on gastro-
intestinal symptoms, gynecological symptoms, dietary intolerance and general symptoms.
23 women were diagnosed as having endometriosis, while in the other 34 this diagnosis was
excluded during laparoscopy.
As shown by Table 29.1, the upper gastro-intestinal symptoms of heartburn and dyspepsia were
significantly more common in the endometriosis group (P<0.001). The question arose as to why
two apparently anatomically disparate systems, that is the upper gastro-intestinal tract and the
female genital system, should influence each other. The explanation to the above findings may
possibly be sought out through a combination of psychological and dietary reasons as evidenced
from the other findings in this study.
While addressing the presence of psychological disorders, a significant number of women

(11/23; P<0.03) in the endometriosis group stated that they required anti-anxiolytic and/or anti-
depressant therapy for symptoms related to significant anxiety or depression (Table 29.2).
Psychological disorders in women have been significantly detected in women suffering from
endometriosis. In a prospective study by De P. Sepulcri and Do Amaral (2009), out of 104 women
diagnosed with pelvic endometriosis 87.5% of women complained of mild anxiety in 24% and
severe in 63.5% of the subjects recruited. Positive correlations between pain intensity and anxiety
symptoms, were obtained using STAI (state, P=0.009; trait, P=0.048) and HAMA (P=0.0001).
Anxiolytic treatment with benzodiazepines such as clonazepam has been used in women with
Table 29.1. Upper gastro-intestinal symptoms in women with and without endometriosis (Muscat Baron et al.,
2011).
Number of Heartburn Dyspepsia Belching Bloating Total of

patients occurrences
No endometriosis 34 9 5 6 13 33
Endometriosis 23 19* 14* 4 9 46
* P<0.001.
Table 29.2. Anxiety and depression in women with and without endometriosis (Muscat Baron et al., 2011).
Number of patients Anxiety/depression
No endometriosis 34 7
Endometriosis 23 11*
* P<0.03.
Y. Muscat Baron
endometriosis. A number of these women required prolonged treatment with serotonin SSRIs
(De P. Sepulcri and Do Amaral, 2009).
Depression has also been noted to be prevalent in women with endometriosis, a high proportion
of which require anti-depressants. De P. Sepulcri and Do Amaral (2009) showed that 86.5%
of patients with pelvic endometriosis complained of depressive symptoms (mild in 22.1%,
moderate in 31.7%, and severe in 32.7%) Depression was been detected in 86% of the women
with endometriosis in the presence of chronic pelvic pain (Lorenatto et al., 2006). Psychosomatic
symptoms, such as work inhibition, dissatisfaction, and sadness, were observed at a significantly
higher rate in the group with abdominal pain (Lorenatto 2006) (Figure 29.3).
The psychological profile of these women may have been moulded from a very young age. The
cyclical experience of the symptoms of dysmenorrhea and menorrhagia from adolescence, may
have conditioned these women to acquire certain personality traits as a reaction to the cyclical
physical and consequent psychological suffering they sustained (Sepulchri and Do Amaral,
2009). High pain scores are correlated to lower quality of life. Patients with higher pain scores
demonstrate lower quality of life status in psychological and environmental perspectives resulting
in an inverse relationship was observed between pain scores and psychological dimension of
quality of life (r=-0.310, P=0.02) (Souza et al., 2011).
Once the diagnosis of endometriosis is established, the fear of infertility may set in, further
compounding the psychological profile. If infertility does occur, then depressive symptoms are
more likely to appear. In a large study comparing fertile women (n=4,905) to infertile women
(n=1,031), self-reported depression was more common in women with endometriosis (OR=5.43,
CI=4.01-7.36) (Herbert et al., 2010). Many of the women suffering from endometriosis are well
versed in their condition and with easy access to medical literature, the risk of inflammatory
bowel disease and ovarian cancer has now become universally known to most women suffering
Psychological profile of
women with endometriosis
Anxiety Depression Psychosomatic

symptoms
Fear of
infertility/
Anticipation ovarian cancer/ Work inhibition Dissatisfaction Sadness
of symptoms inflammatory
bowel disease
Figure 29.3. Psychological profile of women with endometriosis.
from endometriosis (Jess et al., 2012). All these factors aggravate the tenuous psychological status
of these women.
Neuroendocrine-immune imbalance in response to high levels of perceived stress has been

alluded to as a reaction to principal clinical symptoms of endometriosis. Higher levels of
cortisol and prolactin are often associated with stress. Lima et al. (2006) have shown that serum
prolactin levels were significantly higher in infertile women with stage III-IV endometriosis
(28.92.1 ng/ml) than in healthy controls (13.22.1 ng/ml). Similarly, serum cortisol levels were
significantly elevated in infertile women with stage III-IV endometriosis (20.11.3 ng/ml) than
in controls (10.51.4 ng/ml) (Lima et al., 2006). Elevated perception of stress has been noted
to trigger or aggravate the incidence or exacerbation of diseases, such as inflammatory bowel
disease, immunological cutaneous conditions, or pregnancy complications such as spontaneous
miscarriage and pre-eclampsia (Figure 29.4).
The connection between psychological status and the upper gastrointestinal tract are well
established. The impact of psychological stress and gastric mucosa changes were well expounded
by William Beaumont in 1833. Beaumont, hailed as the Father of Gastric Physiology, carried out
observations and experiments on an individual in his employ, Alexis St. Martin, who had sustained
a gastric fistula followed gunshot wound to the stomach. Beaumont noted that the gastric mucosa
exposed in the stomach fistula instantly reddened when Alexis St. Martin was angered, connecting
the neuroendocrine-emotional status with gastric physiology (Beaumont, 1833).
Heartburn and dyspepsia are well known symptoms related with psychological disorders. Gastro-
esophageal reflux disease can be anatomically traced back to disorders of the gastro-esophageal
junction, however psychological factors can play an important role in the process of heartburn.
Secretion of central
neurotransmitters
Increased prolactin and
cortisol secretion
Dietary indiscretion
Hypersecretion
of gastric acid
Stomach
Nonsteroidal
anti-inflammatory
drugs irritating
gastric mucosa
Figure 29.4. Factors influencing gastric acid secretion.
Y. Muscat Baron
It is conceivable that well defined personality factors modulate the effect of stress on the gastro-
esophageal junction, just as they can influence the perception and assessment of symptoms.
Gastric acid hypersecretion, gastric and small intestinal motor disorders interacting with
psychological and neurohormonal factors all may play a role in the pathogenesis of dyspepsia.
Patients with proven gastro-esophageal reflux disease demonstrate greater proximal extension
of acid during reflux episodes. These patients describe a shorter history of symptom onset, worse
anxiety scores, and more endoscopic findings compatible with gastritis (Shapiro et al., 2012).
A vast array of modulators support the endocrinological connection linking the hormone
secretion with altered secretion of gastric acid in the stomach. These central neurotransmitters
and/or neuromodulators may excite or inhibit gastric acid secretion. Excitatory neuromodulators,
such as acetylcholine, thyrotropin releasing hormone, GABA and oxytocin, have been cited.
Conversely, noradrenaline, adenosine, bombesin, calcitonin-gene related peptide, corticotropin
releasing factor, beta-endorphin, neurotensin, neuropeptide Y, insulin-like growth factor II and
prostaglandins have been shown to be inhibitory. Several of these mediators have been noted
in endometriosis. In deep infiltrating endometriosis, where severe and frequent chronic pelvic
pain is commonly encountered, significantly more nerve fibers are detected histologically, than
in superficial peritoneal endometriotic lesions. The deep infiltrating endometriotic lesions
were shown to be innervated abundantly by sensory nerve fibers utilizing acetylcholine and
norepinephrine as neurotransmitters (Wang et al., 2009).
Psychological stress is also related to injudicious consumption of several dietary components that
may irritate the upper gastro-intestinal tract. Psychological variables (somatization, neuroticism,
state and trait anxiety) and binge eating are significant predictors of coexistent gastro-intestinal
disorders. Diet and nutrition research suggests that vitamins, minerals, and other nutrients are
important underpinnings of general physical and mental health. Moreover, these nutrients may
even be useful in treating mood dysfunction by providing a more favorable risk-benefit ratio
than contemporary psychotropic agents (Frazier et al., 2009). Anxiety has been shown to lead
to excessive ingestion of benzodiazepines facilitating gastro-esophageal reflux by reducing lower
esophageal sphincter pressure. Depressive symptoms treated with clomipramine was associated
with an increased risk of esophageal reflux (OR=4.6, 95% CI=2.0-10.6) in a duration- and dose-
dependent manner (Van Soest et al., 2007) (Figure 29.5).
The BMI of women who experience depression is significantly higher than non-depressed women.
Meta-analyses confirm a reciprocal link between depression and obesity. Obesity increases the
risk of self-confirmed depression, and for clinically diagnosed depression. In addition, depression
and its treatment were found to be predictive of developing obesity. Weight gain commences
early during the first 6 weeks of nortriptyline treatment, reached on average 1.2 kg at 12 weeks
leading to 0.44% BMI increase (Uher et al., 2011). Moreover the chronic consumption of
nonsteroidal anti-inflammatory agents to counter endometriosis-induced dysmenorrhea and
menorrhagia may lead to irritation of the gastric mucosa to the extent of developing gastric and
duodenal ulceration. There does seem to be enough evidence to indicate that administration of
Secretion of central
neurotransmitters
Oesophagus
Gastro-oesophageal
sphincter relaxes with
benzodiazepines
stomach
Gastro-oesophageal
sphincter relaxes with
tricyclic antidepressants
Figure 29.5. Factors influencing gastro-esophageal reflux disease.
nonsteroidal anti-inflammatory drugs could be considerably reduced and adverse effects avoided
if practitioners were persuaded to change their prescribing practices (Bloor and Maynard, 1996).
It is relevant to mention that upper gastro-intestinal symptoms are common occurrences in the
general population. In Western countries, 10-40% of the adult population experience heartburn
and dyspepsia, although estimates vary according to the diagnostic criteria used (Kang, 2004).
The risk of gastro-esophageal reflux disease increases with age, rising sharply above the age of 40.
More than 50% of those afflicted are between the ages of 45 and 64 and these symptoms impact
quality of life (Kang, 2004).
Upper gastro-intestinal symptoms have been related to both dietary indiscretion and psychological
stress both of which may, for a variety of reasons, be commonly encountered in women with
endometriosis.
29.3 Lower gastro-intestinal symptoms and endometriosis

The most common site of non-genital endometriosis is the lower intestinal tract, which accounts
for approximately 80% of all endometriosis cases not affecting the female genital tract (Lewis
and Nezhat, 2007). The inflammatory and fibrotic reaction to endometriosis condition the
symptomatology of lower intestinal tract affected by endometriosis (Figure 29.6). Intestinal
endometriosis presents as crampy pain, flatulence, painful tenesmus, hyper-peristalsis, progressive
constipation, diarrhea, diarrhea alternating with constipation, and occasionally rectal bleeding
(Figure 29.6).
Y. Muscat Baron
Dense adhesions
Retroverted uterus between uterus and
rectosigmoid colon
due to endometriosis
Rectosigmoid colon
Cervix
Adhesions
Lumen of rectosigmoid Endometriotic
colon implant
Figure 29.6. Retroverted uterus with obliteration of Pouch of Douglas due to endometriosis-induced adhesions
between rectosigmoid colon and posterior aspect of uterus.
Endometriotic implants to the bowel may occur in 3-37% of women affected by endometriosis,
the sigmoid colon being the most common site of localization. Due to the fibrotic changes
intestinal endometriosis may become refractory to hormonal therapy, which makes surgery the
only option for many women (Figure 29.7). Following surgery, both gynecological symptoms,
such as dysmenorrhea, dyspareunia and intestinal symptoms, such as constipation, diarrhea and
rectal bleeding improve in affected women throughout the post-operative follow-up (Lewis and
Nezhat, 2007) (Figures 29.7 and 29.8).
In the study by Muscat Baron et al. (2012), lower gastro-intestinal symptoms in the form of
diarrhea and loose stools were more commonly found in women who were diagnosed with
endometriosis (Table 29.3). As opposed to the upper gastro-intestinal tract, both the small and
to a greater extent the large bowel is in close proximity with the female genital tract. Both systems
throughout their physiological functioning are likely to influence each other. Constipation also
occurred more commonly in the group diagnosed with endometriosis however this did reach
statistical significance. Diarrhea did occur in a significant manner in the endometriosis group
compared to the control group (Table 29.3).
During the menstrual cycle the hormonal fluctuations and the actual physiological and anatomical
changes experienced by the female genital tract are likely to influence adjacent intestinal tract.
The slowly elevating levels of estrogen in the follicular phase have been noted to affect intestinal
motility. Small intestinal motility appears to decrease during the follicular phase of the menstrual
cycle. Small bowel transit was faster in the luteal phase (75.7 min) compared with the follicular
phase (99.3 min). However there is no difference in large bowel transit time (Bjornsson, 2006).
Severe constipation is a disorder largely confined to young women. The possibility that menstrual
related factors contribute to disturbed gastrointestinal motor function has been raised. Turnbull et
Figure 29.7. Ovarian endometriosis. (a) Right ovary. 7 cm ovarian endometrioma in Pouch of Douglas. (b, c)
Brown chocolate-like fluid aspirated from ovarian endometrioma.
Round
Round Uterus ligament
ligament
Left Right
ovary ovary
Endometriotic
Recto-sigmoid implant
lumen
Adhesions
Figure 29.8. Posterior view: ovarian endometriomas (chocolate cysts) with adhesions to rectosigmoid colon
and uterus. Risk for bowel perforation during surgical dissection.
Y. Muscat Baron
Table 29.3. Lower gastro-intestinal symptoms in women with and without endometriosis (Muscat Baron et al.,
2011).
Number of Constipation Diarrhea Flatulence Mucus in

patients stools
No endometriosis 34 14 5 14** 0
Endometriosis 23 18 10* 9 2
* P<0.05; ** P<0.02.
al. (1989) have assessed relationships between symptom severity and orocaecal transit during the
menstrual cycle in a group of 14 constipated women and a series of control groups, to determine
whether phases of the menstrual cycle were associated with alteration in symptoms or transit.
Repeated orocaecal transit measurements in the 4 study groups showed no consistent differences
between groups or during the menstrual cycle (mean change weeks 1-4, -1020 min). These
findings did not support the hypothesis of a progesterone related effect upon orocaecal transit in
either normal or constipated women (Turnbull et al., 1989). This, however, does not exclude the
possibility that the caecal-anal transit time is not affected by progesterone.
Endometriosis may influence lower gastro-intestinal function and pathology. Abdominal

distension and worsening constipation due chronic large bowel obstruction has been associated
with endometriosis. Through the mediation of prostaglandins, intestinal function may be
associated with dysmenorrhea. In this light, dietary supplementation with polyunsaturated fatty
acids employed and has been shown to alleviate the menstrual pain. This may possibly be through
the conversion of linoleic acid, via gamma-linolenic acid, to dihomo-gamma-linolenic acid (a
precursor of anti-inflammatory prostaglandin E1) in dysmenorrheic subjects (Wu et al., 2008).
In a study by Luscombe et al. (2009), a significantly larger proportion of women with

endometriosis than control subjects complained of abdominal bloating (96% vs. 64%). Women
with endometriosis noted to have abdominal bloating, complained of severe discomfort (30%
vs. 0%), and the necessity to wear loose clothing during bloating (87% vs. 38%). Similar to our
study, the experiences of cyclically related diarrhea and constipation were more frequent with
endometriosis. Lower abdominal girth measurements changed significantly across menstrual
cycle phases. Women with endometriosis complained of greater variation of abdominal girth
measurements throughout the menstrual cycle (Luscombe et al., 2009).
Rectovaginal endometriosis with obliteration of the Pouch of Douglas is a severe variant of

endometriosis presenting symptoms with dysmenorrhea, pelvic pain and dyspareunia. In a study
by Griffiths et al. (2007), the presence of dyschesia gave a likelihood ratio of 1.27 (95% CI=0.56-
2.89) with a predictive prevalence of rectovaginal endometriosis of 37%. Apareunia and nausea
or abdominal bloating were particularly strong markers for rectovaginal disease with a predictive
prevalence of 87% and 89%, respectively (Griffiths et al., 2007).
Rectovaginal endometriosis is typically associated with severe fibrosis and obliteration of the
pouch of Douglas, frequently involving the ovaries in the mesh of tough adhesions (Figure
29.8). Key molecules associated with the transforming growth factor family and its regulatory
mechanisms are recognized to regulate peritoneal wound repair and adhesion formation.
Transforming growth factor 1 induced increase in proteinase-activated receptor 2 expression may
be a complex mechanism that augments the progression of the disease. It appears that proteinase-
activated receptor 2 plays a significant roles in pathogenesis of endometriosis (Chegini, 2008).
A number of mediators have been linked to both gastrointestinal symptoms, such as constipation
and endometriosis. Large intestinal contractility is influenced by acetylcholine release through
activation of 5-hydroxytrytophan receptors on cholinergic nerves and is effective in patients with
constipation. Patients with intestinal endometriosis can present with constipation and this was
successfully treated with the 5-hydroxytrytophan receptor agonist prucalopride enhances large
intestinal contractility (Lefebvre et al., 2010).
In the study by Muscat Baron et al. (2012), flatulence was more common in the women who did
not have endometriosis (Table 29.3). These women had gynecological complaints not related
to endometriosis, but this may still have impacted on intestinal function leading to flatulence.
Flatulence has been noted to occur in inflammatory bowel disease. Moreover this symptom may
be a reflection of the dietary pattern of the individual. It may well be that the ingestion of green
vegetables was commoner in this cohort of women as opposed to the group of women who had
endometriosis. This finding may shed light on the pathogenesis in the relation of dietary intake
and endometriosis.
29.4 Gastro-intestinal symptoms, menstrual cycle and endometriosis

As referred to earlier, there lies a great overlap between the symptoms of endometriosis and
gastro-intestinal symptoms related to conditions pertaining to the gastro-intestinal tract. In
fact, endometriosis has been referred to as the great masquerader. The menstrual cycle may also
impact on gastro-intestinal function. As confirmed in the general literature, the greater frequency
of menstruation in our study in patients with endometriosis increased the likelihood of increased
frequency of related gastro-intestinal symptoms (Table 29.4).
Abdominal symptoms are significantly more pronounced at the beginning of the follicular phase
(Bjornsson, 2006). One-third of otherwise asymptomatic women may experience gastrointestinal
symptoms at the time of menstruation, and almost 50% of women with irritable bowel syndrome
report a perimenstrual increase in symptoms. Epigastric pain, nausea, and diarrhea are more
prevalent at the time of menses in women complaining of bowel dysfunction. Patients complaining
of bowel dysfunction indicate that stomach pain was higher throughout the menstrual cycle
Y. Muscat Baron
Table 29.4. Duration with standard deviation of menstrual cycle in women with and without endometriosis
(Muscat Baron et al., 2012).
Number of patients Menstrual cycle (days standard deviation)
No endometriosis 34 30.47.4
Endometriosis 23 26.02.6*
* P<0.008.
however cramping pain was commoner in the perimenstrual phase (Heitkemper et al., 2003)
(Figure 29.9).
Intestinal motility may be associated with the genesis of endometriosis and conversely
endometriosis may influence intestinal motility. Preclinical studies have shown experimental
animals had significantly more colonic damage, myeloperoxidase activity, and white cell count
numbers than controls did. Experimental animal studies demonstrated an increased tension in
the longitudinal muscle, which correlated with white blood cell numbers and colonic damage.
Mabrouk et al. (2002) have shown that in deep infiltrating endometriosis, hypertonicity of
the internal anal sphincter was found in 20 of 25 patients. Almost half of the patients had an
increase of the threshold of desire to defecate, and seven patients had a reduction of the anal
sphincter squeeze pressure. According to the responses to the defecatory function questionnaire,
incomplete evacuation was the most common symptom (Mabrouk et al., 2012).
Dietary intake may impact on premenstrual symptoms. Soy products and isoflavone intake have
been shown not to alter Moos Menstrual Distress scores significantly during premenstrual phase
(Nagata et al., 2004). Conversely ingestion of total, saturated and monounsaturated fats were
significantly correlated with change in Moos Menstrual Distress scores and subscale pain in the
premenstrual phase after controlling for the covariates. Intake of cereals/potatoes/starches was
significantly inversely correlated with a change in total Moos Menstrual Distress scores in the
premenstrual phase (Nagata et al., 2004).
Gastro-intestinal symptoms
during menstrual cycle
Epigastric pain,
Abdominal symptoms nausea, and
increased diarrhoea increased
during follicular phase during menses
Figure 29.9. Gastro-intestinal symptoms during menstrual cycle.
During menstruation, women with irritable bowel syndrome using oral contraceptives display
less cognitive, anxiety, and depression symptoms (P<0.05), but no differences were seen for most
symptoms of irritable bowel syndrome (Heitkemper et al., 2003). The symptoms pertaining to
irritable bowel syndrome correlate with whether women complained of alternating constipation
and diarrhea. In this study, all irritable bowel syndrome symptoms except diarrhea were highest
in the alternating bowel motion group and lowest in the diarrhea group, with the constipation
group either intermediate or close to the alternating group (Heitkemper et al., 2003).
Endometriosis may mimic inflammatory bowel disease. Prostaglandins, released by the

endometrium at menstruation, cause contraction of uterine smooth muscle, resulting in
the cramping pain of dysmenorrhea. Prostaglandins are also an important component of the
inflammatory process in active inflammatory bowel disease, increasing contractility of intestinal
smooth muscle resulting in diarrhea and abdominal pain.
Crohns disease complicated by multiple stenoses and internal fistulas has been clinically
misdiagnosed as small bowel endometriosis. Patients perimenstrual symptoms may demonstrate
mechanical subacute ileus which later on becomes continuous, and gradually increased in severity.
Intestinal endometriosis and Crohns disease can also occur concurrently, whereby endometriosis
of the terminal ileum seems to be the most common site affected. The diagnosis is often only
made after surgical resection of the diseased segment (Figure 29.8).
Ulcerative colitis may also co-exist with endometriosis. Women with endometriosis have been
shown to have an increased risk of inflammatory bowel disease overall. Restricting analyses
to women with surgically verified endometriosis suggested strong associations with ulcerative
colitis (standardized incidence ratios=1.8; 95% CI=1.4-2.3) and Crohns disease (standardized
incidence ratios=1.7; 95% CI=1.2-2.5). The risk of inflammatory bowel disease in women with
endometriosis was increased even in the long term, hence suggesting a genuine association
between the diseases, which may either reflect common immunological features (Jess et al., 2012)
(Figure 29.10).
Endometriosis
and bowel disorders
Increased incidence Increased incidence

of inflammatory of irritable
bowel disease bowel syndrome
Increased incidence Increased incidence

of ulcerative colitis of Crohns disease
Figure 29.10. Endometriosis and bowel disorders.
Y. Muscat Baron
The distinction between the diagnosis of endometriosis and inflammatory bowel disease
is clinically relevant. Non-steroidal anti-inflammatory drugs may relieve symptoms of
dysmenorrhea, however, they often are contraindicated in inflammatory bowel disease due to the
risk of bowel perforation. Both gynecologists and gastro-intestinal specialists need to be aware of
the interphase between endometriosis and gastro-intestinal conditions so as to reach the correct
diagnosis and institute the appropriate treatment.
29.5 Conclusion
There appears to be considerable overlap between the symptoms of endometriosis and gastro-
intestinal disease. Similar to the menstrual cycle, endometriosis may influence gastro-intestinal
symptoms related to bowel disease. Meticulous investigation of womens symptoms is required
to avoid delay or possibly misdiagnosis. A delay or misdiagnosis may further exacerbate the
psychological background of anxiety and depression both of which are prevalent in women with
endometriosis. Moreover, gastro-intestinal conditions and endometriosis may co-exist requiring
a multi-disciplinary approach to treatment. This approach may also include dietary modulation
as guided by the womens gastro-intestinal symptoms which in turn may attenuate or possibly
prevent the progression of endometriosis.
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30. The role of vitamin D for conception, polycystic
ovary syndrome, endometriosis and the
menstrual cycle
Department of Obstetrics, Gynecology and Reproductive Medicine, Hannover Medical School,
Carl-Neuberg-Strasse 1, 30625 Hannover, Germany; vonversen-hoeynck.frauke@mh-hannover.de
Abstract
The past decade has seen a renewed interest in the sunshine vitamin vitamin D, because new
evidence demonstrates benefits beyond bone health and an increase in the prevalence of vitamin
D deficiency in the general population and in populations at risk, e.g. infants, adolescents,
women of childbearing age and elderly. The well-established function of this steroid hormone is
to maintain calcium and phosphorus homeostasis, and to promote bone mineralization. It has
become the focus of many studies in recent years since vitamin D can have a significant influence
on the growth and differentiation of a variety of tissues, and decreases the risk of many chronic
illnesses including common cancers, autoimmune, infectious and cardiovascular diseases. The
discovery that many tissues and cells in the body express a vitamin D receptor has provided new
insights into the function of the vitamin. Reduced fertility rates and fertility capacity, diminished
mating success, gonadal insufficiency, reduced aromatase gene expression, low aromatase activity,
hypogonadism, uterine hypoplasia, impaired folliculogenesis and infertility have been described
in vitamin-D-deficient animal models. Also, it has been reported that vitamin D affects placental
steroidogenesis, expression of placental lactogen and decidualization of the endometrium.
Confirmation of experimental human and animal observations establishing an association of
vitamin D deficiency with adverse reproductive outcomes by high quality randomized clinical
trials is still lacking. Therefore the objective of this chapter is to summarize the metabolism,
epidemiology, and treatment of vitamin D deficiency but especially to focus on its role in
reproductive health.
Keywords: vitamin D, reproduction, fertility, menstruation
489
Summary points
Vitamin D receptor and 1-hydroxylase are present on cells of the ovary, uterus, placenta, testis and pituitary
gland, suggesting paracrine/autocrine functions of 1,25-dihydroxyvitamin D3 in reproductive tissues.
Vitamin D deficiency is a worldwide epidemic and often clinically unrecognized.
Vitamin D deficiency in women and men might be associated with lower fertility and an increased risk for
polycystic ovary syndrome.
The best method for determining a persons vitamin D status is to measure a 25-hydroxyvitamin D3
concentration.
Good evidence shows that fortified foods and supplemental vitamin D increase 25-hydroxyvitamin D3
concentrations.
Current data on reproductive health outcomes in humans rely on animal or laboratory studies and can
therefore not determine causality.
Contradictory results of observational studies can be explained by methodological, genetic, ethnic and
racial differences. Latitude of residence and season may account to the observed discrepancies in several
reproductive health outcomes.
Experimental and observational data have to be confirmed in large-scale randomized clinical trials to help
clarify the benefits and risks of vitamin D in reproductive outcomes as well as the optimal amount of vitamin
D supplementation.
30. Vitamin D for conception, PCOS, endometriosis and the menstrual cycle
Abbreviations
1,25(OH)2D 1,25-dihydroxyvitamin D
25(OH)D 25-hydroxyvitamin D
AMH Anti-Mllerian hormone
BMI Body mass index
DBP Vitamin D-binding protein
DHEAS Dehydroepiandrosteronsulfat
IL-8 Interleukin-8
IOM Institute of Medicine
30.1 Introduction
The vitamin D endocrine system plays an essential role in calcium homeostasis and bone
metabolism, but research during the past two decades has revealed a diverse range of biological
actions that include induction of cell differentiation, inhibition of cell growth, immunomodulation,
and control of other hormonal systems. Vitamin D itself is a prohormone that is metabolically
converted to the active metabolite, 1,25(OH)2D3, that activates its cellular receptor (VDR) and
alters the transcription rates of target genes responsible for the biological responses (Figures
30.1 and 30.2). The presence of 1-hydroxylase and the VDR indicates autocrine/paracrine
functions of 1,25(OH)2D3 in target cells of the reproductive system. Vitamin D deficiency is
now considered an epidemic and women in their reproductive period are a special population
at risk. This chapter focuses on several recent developments that extend our understanding of
the complexities of vitamin D metabolism and actions with respect to reproductive health and
fertility (Figure 30.3).
30.2 Classification of vitamin D status

Low vitamin D status is now a health concern in many countries that raises awareness and calls
for actions regarding the revision of dietary reference values, the advice regarding the risk of
sun exposure and the implementation of fortification and supplementation programs. Recent
evidence demonstrates that the prevalence of vitamin D deficiency in women of childbearing
age is surprisingly high (Datta et al., 2002; Holmes et al., 2009). The vitamin D status is mainly
assessed by the measurement of serum 25(OH)D3 concentration (Holick, 2009). Historically,
vitamin D sufficiency was defined as the serum concentration of 25(OH)D3 sufficient to prevent
rickets in children and osteomalacia in adults. Levels of 25(OH)D3 >25 nmol/l (to convert to
UVB (95%) Skin Liver Kidneys:

endocrine pathway
Pro-vitamin D
(7-dehydrocholesterol)
Vitamin D3 25(OH)D3 1,25(OH)2D3,

(cholecalciferol) (calcidiol) (calcitriol)
Diet (5%) Extrarenal synthesis: Target cells &

paracrine & organs
autocrine pathways
Figure 30.1. Schematic representation of the synthesis and metabolism of vitamin D. Cholecalciferol as
the main source (about 95%) of vitamin D3 is photochemically synthesized in the skin. There, pro-vitamin
D3 (7-dehydrocholesterol) conversion to pre-vitamin D3, is followed by isomerization into cholecalciferol.
Cholecalciferol is subsequently hydroxylated in the liver to form 25-hydroxycholecalciferol (calcidiol). In the kidney
and extrarenal tissues, further hydroxylation turns 25-hydroxycholecalciferol (25(OH)D3) into the biologically
active secosteroid hormone calcitriol (1,25(OH)2D3).
H2C OH
CH2 CH2
VDR
CH2
H
CH2
Ligand
HO OH VDR X = Coregulators
VDRE VDR = Vitamin D receptor
VDRE = Vitamin D response elements
RXR VDR VDR RXR RXR = Retinoid-X receptor
VDRE RNA Pol II X X
DNA RNA Pol II
Nucleus
mRNA
Cytoplasm
Figure 30.2. The vitamin D receptor (VDR) is a member of the nuclear steroid receptor superfamily and an
intracellular transcription factor, located on chromosome 12q. Linked to 1,25(OH)2D3, binding to the hormone
ligand-binding domain and associated with the retinoic acid receptor via dimerization domains, the VDR-complex
binds to vitamin D response elements (in the promoter of 1,25(OH)2D3-responsive genes (Deeb et al., 2007).
Immune system Pancreas

Fertility Chronic Pituitary gland Insulin secretion
inflammation Insulin resistance
Ovary
Steroidogenesis
of sex hormones
PCOS Vitamin D Placenta
Anti-mllerian
hormone
Folliculogenesis
Male fertility
Endometrium Bone Androgen synthesis
Endometriosis Implantation Calcium balance Spermatogenesis
Menstruation mineralisation Sperm maturation
Figure 30.3. Overview of vitamin D and its suggested classical and non-classical effects.
ng/ml divide by 2.5) can do so and most experts use this level as a conventional cut-off for
defining the lower limit of adequacy of vitamin D status. Investigators have argued that a plasma
25(OH)D3 concentration of greater than 75 nmol/l is appropriate to define vitamin D sufficiency
based on associations between plasma 25(OH)D3 and plasma PTH concentration, calcium
absorption, bone turnover markers, and bone mineral density (Bischoff-Ferrari et al., 2006;
Dawson-Hughes et al., 2005; Holick, 2007; Hollis, 2005). Figure 30.4 shows a simplistic scheme
for the classification of vitamin D status. However, these levels are probably not sufficient to
provide the more recently discovered clinical benefits of vitamin D and precisely defining vitamin
D deficiency or insufficiency remains still a matter of great debate. To date most of the data
supporting higher target concentrations are based on cross-sectional, or observational cohort
data with need for further evidence from randomized clinical trials. The upper limit of adequacy
has also been questioned. However, supraphysiologic, potentially toxic levels are defined as a
25(OH)D3 concentration above 150 nmol/l (Holick, 2007; Mulligan et al., 2010).
30.3 Vitamin D, female fertility and conception

Infertility has been recognized as a public health issue by the World Health Organization with
an estimated median prevalence of 9% worldwide and a high demand for infertility services
(Boivin et al., 2007). As 30-40% of the cases is either caused by male or female factors for
the remaining 20-40% both partners or unknown factors contribute to the childlessness. An
association between vitamin D and fertility has been suggested after a seasonal distribution in
human natural conception with peak conception rates in summer in northern countries with
strong seasonal contrast in luminosity has been demonstrated (Rojansky et al., 1992). Season may
have an impact, not only on sperm quality, but on ovulation rate as well as endometrial receptivity
(Halloran, 1989; Rojansky et al., 1992). Because of the expression of VDR in germinal epithelium,
it has been proposed that the ovary is a target organ for 1,25(OH)2D3 raising the possibility
200 500
Intoxication
150 375
60 150
50 125
Sufficiency
(optimal range)
40 100
30 75
20 50
Deficiency
10 25
0 0
ng/ml nmol/l
Figure 30.4. Vitamin D status is mainly assessed by measurement of serum 25(OH)D3 concentration. The figure
shows a simplistic scheme for its classification.
that this active metabolite of vitamin D3 plays a role in modulating ovarian activity (Dokoh et
al., 1983). It has been shown that vitamin D cooperates with estradiol in labilizing lysosomes,
which weakens the tunica albuginea and enhances ovum release during ovulation. It might also
facilitate egg transport and fertilization in the oviduct (Rojansky et al., 1992). In fact, it stimulates
steroid hormone production (progesterone, estradiol and estrone) in human ovarian tissue and
placenta (Barrera et al., 2007; Parikh et al., 2010) and regulates human chorionic gonadotropin
and placental lactogen expression in human syncytiotrophoblasts (Barrera et al., 2008). The
expression and activity of the estrogen biosynthesis catalyzing enzyme P450 are stimulated by
1,25(OH)2D3 (Sun et al., 1998). AMH, a marker of gonadal status, is produced in the granulosa
cells of the ovaries. The AMH gene promoter contains a vitamin D response element. Serum
AMH positively correlates with 25(OH)D3 in females and change in AMH level positively
correlates with the magnitude of change in vitamin D levels after vitamin D supplementation
(Dennis et al., 2012; Merhi et al., 2012). Vitamin D deficiency might therefore be associated
with lower ovarian reserve in late-reproductive-aged women and supplementation might help to
improve fertility outcome.
The hormone has also been suggested to be involved in uterine physiology. Molecular analyses of
the vitamin D system in normal endometrium and its regulation have received limited attention.
Stumpf and colleagues hypothesized that the presence of VDR in the uterus and corpus luteum
may point to a possible role in endometrial receptivity (Stumpf, 1988; Stumpf and Denny, 1989).
Human cycling, early pregnant and ectopic endometrium is now recognized as one of the
extrarenal sites of vitamin D synthesis and action since 1-hydroxylase is expressed in human
endometrial stromal cells (Evans et al., 2006; Vigano et al., 2006). HOXA 10 expression, an
important modulator for the development of the uterus and the endometrium, is also regulated
by calcitriol in human endometrial stroma cells (Du et al., 2005).
Data from animal studies investigating the significance for fertility and reproductive capacity,
demonstrate that 25(OH)D3-deficient female rats had reduced fertility rates, decreased litter
sizes and compromised mating behavior (Halloran and DeLuca, 1980). Supplementation with
1,25(OH)2D3 lead to successful mating and gave rise to normal, healthy litters. These results
indicate vitamin D and not calcium is responsible for reduced reproductive capacity (Kwiecinksi
et al., 1989a). VDR-null model mice showed hypergonadotropic hypogonadism with decreased
aromatase activities in the ovary, uterine hypoplasia and impaired folliculogenesis (Kinuta et al.,
2000). Similar to VDR knockout mice ablation of 1-hydroxylase is accompanied by abnormal
ovarian follicle development, uterine hypoplasia and infertility (Panda et al., 2001; Yoshizawa
et al., 1997). However, when serum calcium and phosphorus were normalized by a rescue diet
the defective phenotype including dysfunction in the hypothalamic-pituitary-ovarian axis and
ovarian angiogenesis were reversed suggesting not a direct effect of vitamin D deficiency but
rather an indirect effect mediated by extracellular calcium and phosphorus (Johnson and DeLuca,
2001; Sun et al., 2010).
For information on vitamin D and assisted reproductive technology we would like to refer the
reader to Chapter 24 Vitamin D and assisted reproductive technology in this book.
30.4 Vitamin D and male fertility

In 30-40% of infertile couples the underlying cause is the male factor (Forti and Krausz, 1998).
Importance of vitamin D for male reproduction has been demonstrated in several animal studies
(Bouillon et al., 2008; Inpanbutr et al., 1996; Kinuta et al., 2000). The expression of the VDR was
shown in the smooth muscles of the epididymis, spermatogonia and Sertoli cells indicating a role
of vitamin D in spermatogenesis and sperm maturation in rats (Johnson et al., 1996; Merke et al.,
1985). The genomic and non-genomic vitamin D effects on rat testis have been reviewed in great
detail elsewhere (Zanatta et al., 2011).
One of the VDR null mutant mice models is characterized by an infertile phenotype with decreased
sperm count, decreased motility, histological abnormalities of the testis and hypergonadotropic
hypogonadism showing elevated serum levels of LH and FSH (Kinuta et al., 2000). Vitamin
D deficiency in rats leads to reduced sperm counts, impaired motility and a 45% reduction in
successful matings, as well as a decreased fertility rate that is reduced by 73% (Kwiecinski et al.,
1989b).
In humans, VDR is expressed in the ejaculatory duct, germ cells and spermatozoa (Blomberg
Jensen et al., 2010), as well as in human testicular tissue homogenates (Habib et al., 1990). An
ultrastructural localization of VDR showed that VDR is located in the sperm nucleus and mid-
piece of the sperm (Aquila et al., 2008; Corbett et al., 2006).
Vitamin D exerts its mechanisms and functions on the male reproductive processes by affecting
cholesterol efflux, protein phosphorylation (Aquila et al., 2008) and by increasing intracellular
Ca2+ levels, motility and acrosin activity, suggesting an effect of vitamin D in capacitation and
sperm survival (Aquila et al., 2009). In a study including 300 healthy men, Blomberg-Jensen et
al. (2011) showed that men with vitamin D deficiency had lower numbers of motile, progressive
motile and morphologically normal spermatozoa compared to vitamin D-replete men. This effect
was independent of calcium levels. In contrast, a study in 307 healthy men addressing the same
question found opposite results (Ramlau-Hansen et al., 2011). However, in a phase II, randomized,
double-blind, placebo-controlled trial, treatment of 121 male patients with chronic prostatitis or
pelvic pain syndrome with a synthetic derivate of vitamin D (elocalcitol) showed reduced levels
of IL-8 in semen. This finding suggests an improvement of semen quality and sperm motility by
treatment with elocalcitol (Tiwari, 2009). Optimal sperm function may thus depend on a direct
effect of vitamin D. However, if this effect is indeed independent of calcium homeostasis has to
be addressed in further studies. A decrease in testosterone levels is presumably responsible for
many aspects of male infertility. Vitamin D levels have been associated with androgen levels in
several different studies (Pilz et al., 2011; Wehr et al., 2010), so that vitamin D substitution by
increasing testosterone levels could possibly positively influence male fertility (Pilz et al., 2011).
30.5 Vitamin D and menstrual cycle

During the human menstrual cycle a mid-cycle rise in the serum level of 1,25(OH)2D3 with
a near doubling of its concentration compared to early follicular levels has been described by
several investigators (Buchanan et al., 1986; Gray et al., 1982; Pitkin et al., 1978), whereas other
studies could not reproduce this effect (Baran et al., 1980; Muse et al., 1986).
The described peak was observed without a significant change in serum calcium levels (Gray et
al., 1982), as well as unchanged biochemical indices of bone turnover (Tjellesen et al., 1983). This
mid-cycle elevation of 1,25(OH)2D3 was not observed in women taking oral contraceptives (Gray
et al., 1982) suggesting that mid-cycle endogenous estrogen elevation promotes the formation of
1,25(OH)2D3 from 25(OH)D3 (Buchanan et al., 1986). It was shown that vitamin D intake was
significantly higher during mid-luteal phase suggesting a regulation of food intake by menstrual
cycle hormones (Martini et al., 1994). In conclusion of these findings the serum concentrations of
1,25(OH)2D3 in women must be interpreted in the context of the stage of menstrual cycle. There
is scientific evidence indicating that women with premenstrual syndrome have an underlying
calcium dysregulation and vitamin D deficiency (Thys-Jacobs, 2000). Concerning these aspects
we would like to refer the reader to Chapter 8 Vitamin D3 and other nutrients in the premenstrual
syndrome in this book. In a randomized treatment study comparing calcium plus vitamin D with
dydrogesterone and placebo both dydrogesterone and calcium plus vitamin D treatments were
more effective than placebo in promoting womens well-being and were able to improve the mean
scores of general health questionnaires (Khajehei et al., 2010).
30.6 Vitamin D and polycystic ovary syndrome

PCOS is one of the most common endocrine disorders affecting women of reproductive age
and is characterized by hyperandrogenism, menstrual disturbances, and polycystic ovaries on
ultrasound. Besides ovarian dysfunction contributing to oligo-/anovulation and infertility, its
clinical manifestations may include obesity, increased insulin resistance and compensatory
hyperinsulinemia (Anonymous, 2004). An inverse correlation between vitamin D levels and
metabolic risk factors in patients with PCOS, e.g. insulin resistance, BMI, waist-to-hip-ratio,
triglycerides, total testosterone and DHEAS and a positive correlation with insulin sensitivity
has been demonstrated in several studies (Li et al., 2011; Wehr et al., 2009; Yildizhan et al.,
2009). Associations between VDR polymorphisms and the development of PCOS as well as
insulin resistance have been suggested in studies with only modest sample sizes (Chiu et al.,
2001; Mahmoudi, 2009; Ranjzad et al., 2011, 2012; Wehr et al., 2011b). Explanations on possible
mechanism of VDR variants in the pathogenesis of PCOS include effects on luteinizing hormone,
sex hormone binding globulin levels and testosterone (Ranjzad et al., 2011; Wehr et al., 2011b).
Research in this area is growing and studies on further genes involved in vitamin D synthesis,
hydroxylation and transport and their role in PCOS are currently under investigation (Wehr
et al., 2011b). Positive effects on insulin secretion, lipid profile, menstrual cycle and follicular
development and a decrease of fasting and stimulated glucose and C-peptide levels were
shown in clinical trials with either vitamin D supplementation or administration of vitamin
D3 analogues (Kotsa et al., 2009; Rashidi et al., 2009; Selimoglu et al., 2010; Thys-Jacobs et
al., 1999; Wehr et al., 2011a). Limitations of most studies include a rather small sample size, a
quite heterogeneous experimental set-up and as great confounder the presence of obesity. The
association of hypovitaminosis D with features of PCOS may be associated with obesity but not
with the presence of PCOS since lower serum levels of 25(OH)D3 were shown in obese PCOS
patients than in non-obese (Mahmoudi et al., 2010; Yildizhan et al., 2009). Vitamin D deficiency
has recently been considered as a potential risk factor for obesity (Foss, 2009). Treatment with
vitamin D could recompense vitamin D deficiency, increase vitamin D level and decrease BMI.
Vitamin D supplementation could play an important role in the treatment of PCOS patients,
not only to improve insulin sensitivity and fertility. More randomized intervention trials seem
necessary to determine the potential usefulness of vitamin D supplementation on different
features of PCOS.
30.7 Vitamin D and endometriosis

Endometriosis is characterized by ectopic endometrium and an impairment of immunologic
mechanism and inflammatory responses has been suggested to be involved in the pathogenesis.
The genes encoding for 1-hydroxylase and the VDR show an enhanced expression in ectopic
endometrium of women suffering from endometriosis (Agic et al., 2007; Vigano et al., 2006).
Women with endometriosis have higher serum levels of 25(OH)D3 than healthy ones (Hartwell
et al., 1990; Somigliana et al., 2007). An analysis of VDR gene polymorphisms (ApaI, TaqI, FokI,
BmsI) including 132 women with endometriosis and 133 controls found relatively similar VDR
polymorphism genotype frequencies in cases and controls suggesting no role of gene variants in
the pathophysiology of the disease (Vilarino et al., 2011). Insignificantly higher serum and lower
peritoneal DBP concentrations were detected in an observational study of 26 women with and
17 women without endometriosis (Borkowski et al., 2008). However, about 3 times higher DBP
levels associated with the GC*2 allele product that was in greater concentration in serum pools, as
well as in single validation samples in 56 women with endometriosis compared to healthy controls
were detected in another study. Explanations of this finding include an influence of vitamin D on
local activity of immune cells and cytokines maintaining endometriosis and an insufficiency to
activate macrophages phagocytotic function in those carrying the GC*2 polymorphism (Faserl
et al., 2011; Sibai et al., 2005).
30.8 Prevention and supplementation of vitamin D deficiency

Our skin is part of the vitamin D endocrine system. A number of other natural factors can
limit or regulate the cutaneous production of vitamin D, including aging, increased melanin
pigmentation, season and latitude (MacLaughlin and Holick, 1985; Matsuoka et al., 1991).
Clothing and sunscreen reduce the cutaneous generation of vitamin D3 (Matsuoka et al.,
1992). Adequate sunlight exposure is the most cost-effective means of obtaining vitamin D.
Unfortunately, vitamin D does not naturally occur in adequate amounts in unfortified foods with
some exceptions, e.g. oily fish (e.g. salmon) and fish liver oil. The treatment of choice for vitamin
D deficiency is cholecalciferol (vitamin D3). For every 100 IU vitamin D3 supplementation
administered on a daily basis for 4+ months, 25(OH)D3 levels rise by 1.25-2.5 nmol/l (Heaney et
al., 2003; Vieth, 1999). Dietary recommendations are available in many European countries, the
US and also globally (Lanham-New et al., 2011; Ross et al., 2011). However, most countries have
no recommendations for ethnic minority groups, where low vitamin D status and deficiency are
more common (Ross et al., 2011). Thus, the optimal intake for woman in their reproductive years
is not known (Specker, 2004). Therefore, current recommendations for daily vitamin D intake
(e.g. IOM 400 IU for 0-12 month, 600 IU for 1 to 70 years old, pregnant/lactating women, and
800 IU for adults older than 70 years of age) might be too low to ensure non-skeletal benefits and
might not maintain 25(OH)D3 at the desired level for many individuals as is in discussion by
several scientists (Heaney and Holick, 2011; Ross et al., 2011). More information on consequences
of both low and high intakes of vitamin D will be provided only by large scale clinical trials that
are still missing.
30.9 Summary and conclusions

Traditionally, vitamin D has been associated primarily with bone health. It is now known
that adequate vitamin D status is important for optimal function of many organs and tissues
throughout the body, including the reproductive system. VDRs are present on a large variety of
cell types, including cells of the ovary, uterus, placenta, testis and pituitary gland. The presence
of the VDR and 1-hydroxylase suggests paracrine/autocrine functions of 1,25(OH)2D3 in
reproductive tissues. Vitamin D deficiency or insufficiency is prevalent in practically every

segment of population, including women in their reproductive years. This worldwide pandemic
remains generally unrecognized and untreated. Vitamin D deficiency in women and men might
be associated with lower fertility and an increased risk for polycystic ovary syndrome. Critically
evaluating the evidence regarding the benefit of vitamin D on reproductive functions and outcomes
in humans is difficult. The bulk of current data is based on observational, epidemiological studies,
which are useful for generating hypotheses but not for proving causality. It is particularly difficult
to tease out the effects of confounding variables that relate both to reproductive health and to
vitamin D status, such as physical activity, milk or calcium intake, and adiposity. Few of the
observational associations have been confirmed by randomized controlled trials, and many of the
interventional studies of vitamin D also included calcium supplementation. Future clinical trials
will help clarify the benefits and risks of vitamin D supplementation in conditions, e.g. PCOS and
endometriosis that were discussed in this chapter.
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31. Polycystic ovary syndrome and the metabolic
syndrome
School of Womens and Infants Health, The University of Western Australia, King Edward
Memorial Hospital, 374 Bagot Road, Subiaco, 6008 Perth Western Australia, Australia;
tamara.hunter@health.wa.gov.au
Abstract
Polycystic ovary syndrome (PCOS) affects a large number of women and manifests as
reproductive, psychological and metabolic health problems. It has been associated with a higher
prevalence of the metabolic syndrome, which is a major risk factor for cardiovascular disease.
This review examines the different components of the metabolic syndrome in relation to PCOS
and the management strategies that optimise the health of a woman with PCOS.
Keywords: polycystic ovary syndrome, metabolic syndrome, insulin resistance, dyslipidemia,

obesity, hypertension, cardiovascular disease
505
Summary points
Prevalence of the metabolic syndrome is increased in the presence of polycystic ovary syndrome (PCOS).
There is a greater prevalence of insulin resistance in women with PCOS compared to women without PCOS.
The mechanism has yet to be elucidated.
Women with PCOS are more frequently reported to have dyslipidemia than non-PCOS women. The typical
profile is one of elevated triglyceride levels, elevated low density lipoprotein cholesterol (LDL-C) and reduced
high density lipoprotein (HDL-C) levels.
There is an increased prevalence of hypertension in women with PCOS, however this is controversial as many
of these cases are confounded by obesity.
Clinical evaluation of the metabolic syndrome amongst PCOS patients should include assessment of blood
pressure, waist circumference and/or body mass index, complete fasting lipid profile (total cholesterol, LDL-C,
HDL-C and triglycerides) and oral glucose tolerance test.
Management of the metabolic syndrome in women with PCOS should include lifestyle intervention programs
as first line, followed by targeted medical therapies should these fail.
31. Polycystic ovary syndrome and the metabolic syndrome
Abbreviations
AMPK Adenosine monophosphate activated-protein kinase
BMI Body mass index
HDL-C High density lipoprotein cholesterol
LDL-C Low density lipoprotein cholesterol
TZD Thiazolidinediones
31.1 Introduction
PCOS affects a large number of women of reproductive age and is a major health concern. Although
the reproductive effects are highly emphasized, PCOS has major metabolic consequences across
the lifespan of a woman.
31.1.1 Definition of polycystic ovary syndrome
PCOS is the most common endocrine disorder in women. It encompasses a broad range of
signs and symptoms that indicate ovarian dysfunction and has been most recently defined by
the Rotterdam criteria (Anonymous, 2004a,b) as two out of three of the features demonstrated
in Table 31.1.
31.1.2 Definition of the metabolic syndrome
The metabolic syndrome is a constellation of factors that increase the risk of adverse cardiovascular
health and include both lipid and non-lipid abnormalities that are of metabolic origin (Reaven,
2002). Numerous diagnostic criteria for the metabolic syndrome have been suggested (Alberti
et al., 2006; Balkau and Charles, 1999; Expert Panel, 2001), but no single recognized guideline
exists (Table 31.2).
Table 31.1. Diagnostic criteria for polycystic ovary syndrome.
Diagnostic criteria (2 out of 3 of the following)1
1. Oligo- or anovulation
2. Clinical and/or biochemical signs of hyperandrogenism
3. Polycystic ovaries
Plus exclusion of other etiologies (congenital adrenal hyperplasia, androgen-secreting tumors, Cushings syndrome
1 The Rotterdam ESHRE/ASRM-sponsored PCOS consensus workshop group, 2004.
Table 31.2. Definitions for the metabolic syndrome.
IDF1 WHO2 EGIR3 NCEP4
Weight Central obesity WHR>0.9 male; Waist circumference Waist circumference

BMI>30 kg/m2 WHR>0.85 female >94 cm male; >102 cm male;
or raised waist or BMI >30 kg/m2 >80 cm female >88 cm female
circumference (using
ethnic values)
Dyslipidemia TG>1.7 mmol/l; TG>1.69 mmol/l; TG>2.0 mmol/l and/ TG>1.7 mmol/l;
HDL-C<1.03 mmol/l HDL-C<0.9 mmol/l or HDL-C<1.0 mmol/l HDL-C<1.03 mmol/l
male, male, male,
HDL-C <1.26 mmol/l HDL-C<1.0 mmol/l HDL-C<1.26 mmol/l
female female female
Blood SBP>130 mmHg; BP>140/90 mmHg BP 140/90 mmHg BP>130/85 mmHg
pressure DBP>85 mmHg
Insulin Fasting Diabetes or, impaired Fasting Fasting
resistance BSL>5.6 mmol/l glucose tolerance or, BSL>6.1 mmol/l BSL>6.1 mmol/l
insulin resistance
BMI = body mass index; BP = blood pressure; BSL = blood sugar level; DBP = diastolic blood pressure; HDL-C =
high density lipoprotein cholesterol; SBP = systolic blood pressure; TG = triglycerides; WHR = waist:hip ratio. 1
International Diabetes Federation (Alberti et al., 2006).
2 WHO (1999).
3 European Group for the study of Insulin Resistance (Balkau and Charles, 1999).
4 US National Cholesterol Education Program (Expert Panel, 2001).
Regardless of the diagnostic criteria used, the fundamental components include insulin resistance,
dyslipidemia (abnormal triglycerides, LDL-C and HDL-C), central obesity (as determined by
waist-hip ratio or in some cases BMI (weight kg/height m2) and hypertension (Alberti et al., 2006;
Balkau and Charles, 1999; Expert Panel, 2001; Reaven, 2002).
31.2 The metabolic syndrome in polycystic ovary syndrome

The metabolic syndrome has been reported to occur at an increased prevalence (43-47%)
(Anonymous, 2012; Apridonidze et al., 2005) in patients diagnosed with PCOS when compared
to the background rate of 24% in women in general (Moran et al., 2010; Palomba et al., 2009)
(Figure 31.1a). This increased prevalence in PCOS women is still present when comparing BMI-
matched populations (Figure 31.1b).
a PCOS Control Odds ratio Odds ratio

Study or subgroup Events Total Events Total Weight M-H, fixed, 95% CI Year M-H, fixed, 95% CI
Faloia, 2004 10 50 3 20 2.4% 1.42 [0.35, 5.80] 2004
Dokras, 2005 45 129 442 1887 26.0% 1.75 [1.20, 2.55] 2005
Vrbikova, 2005 1 69 0 73 0.3% 3.22 [0.13, 80.36] 2005
Alvarez-Blasco, 2006 8 32 19 72 6.2% 0.93 [0.36, 2.42] 2006
Leibel, 2006 7 36 0 21 0.4% 10.93 [0.59, 201.92] 2006
Welt, 2006 74 418 7 64 7.1% 1.75 [0.77, 3.99] 2006
Coviello, 2006 18 49 8 165 1.6% 11.40 [4.55, 28.53] 2006
Carmina, 2006 23 282 2 85 2.0% 3.69 [0.85, 15.96] 2006
Shroff, 2007a 92 258 13 110 8.3% 4.14 [2.20, 7.78] 2007
Caliskan, 2007 15 182 5 182 3.2% 3.18 [1.13, 8.94] 2007
Shroff, 2007b 6 24 4 24 2.1% 1.67 [0.40, 6.87] 2007
Cussons, 2008 62 168 91 883 13.0% 5.09 [3.48, 7.45] 2008
Shaw, 2008 58 104 125 286 20.8% 1.62 [1.03, 2.55] 2008
Attuoua, 2008 17 107 4 100 2.5% 4.53 [1.47, 13.98] 2008
Gulcelik, 2008 20 60 7 60 3.3% 3.79 [1.46, 9.82] 2008
Cheung, 2008 52 288 1 98 0.9% 21.37 [2.91, 156.79] 2008
Total (95% CI) 2256 4130 100.0% 2.88 [2.40, 3.45]
Total events 508 731
Heterogeneity: Chi2=45.40, df=15 (P<0.0001); I2=67%
Test for overall effect: Z=11.51 (P<0.00001) 0.001 0.1 1 10 1000
Lower risk for PCOS Higher risk for PCOS
b PCOS Control Odds ratio Odds ratio
Study or subgroup Events Total Events Total Weight M-H, fixed, 95% CI Year M-H, fixed, 95% CI
Faloia, 2004 10 50 3 20 14.7% 1.42 [0.3.5, 5.80] 2004
Alvarez-Blasco, 2006 8 32 19 72 37.6% 0.93 [0.36, 2.42] 2006
Shroff, 2007b 6 24 4 24 12.9% 1.67 [0.40, 6.87] 2007
Attuoua, 2008 17 107 4 100 14.9% 4.53 [1.47, 13.98] 2008
Gulcelik, 2008 20 60 7 60 20.0% 3.79 [1.46, 9.82] 2008
Total (95% CI) 273 276 100.0% 2.20 [1.36, 3.56]

Total events 61 37
Heterogeneity: Chi2=6.47, df=4 (P=0.17); I2=38%
Test for overall effect: Z=3.23 (P=0.001) 0.001 0.1 1 10 1000
Lower risk for PCOS Higher risk for PCOS
Figure 31.1. (a) Metabolic syndrome prevalence in women with and without polycystic ovary syndrome
(PCOS). (b) Subgroup analysis of metabolic syndrome prevalence in women with and without PCOS with body
mass index-matched populations (Adapted from Moran et al., 2010).
Apridonidze et al. (2005) demonstrated that the majority of women with PCOS present clinically
with at least one component of the metabolic syndrome. In their cohort, low HDL-C occurred
most commonly (68%), followed by elevated BMI and waist-hip ratio (67%), hypertension (45%),
elevated triglycerides (35%) and elevated fasting blood glucose levels (4%). Some of the possible
theories for this close association between PCOS and the metabolic syndrome include insulin
resistance underlying both syndromes, obesity and central adiposity being major contributors
to these conditions and vascular and coagulation abnormalities being primary pathogenic
causes of both (Essah et al., 2007). Insulin resistance is the most well supported theory linking
the two conditions. Certainly cardiovascular disease risk, one of the major comorbidities of
insulin resistance, is higher in both PCOS and metabolic syndrome (Anonymous, 2012). This
is thought to be an inflammatory atherothrombotic state, either associated with or initiated
by insulin resistance, that leads to an elevation of several serum proinflammatory substances

including C-reactive protein, fibrinogen, white blood cells, plasminogen activator inhibitor-1
and endothelin-1, which impairs endothelial function, reduces vasoreactivity and promotes
subclinical atherosclerosis (Essah et al., 2007; Jean Hailes Foundation, 2011; Wild et al., 2010).
Due to the overlap in features and comorbidities of PCOS and the metabolic syndrome, it is
difficult to determine any causal pathway in the pathogenesis of these syndromes. Bearing this
in mind, each of the criteria for metabolic syndrome can still be examined individually in the
context of PCOS.
31.2.1 Insulin resistance and polycystic ovary syndrome
There is a greater prevalence of insulin resistance in women with PCOS compared to women
without PCOS (Ehrmann et al., 1999; Moran et al., 2010). Insulin resistance is believed to occur
in approximately 60-80% of women with PCOS and in 95% of obese PCOS women (DeUgarte
et al., 2005; Jean Hailes Foundation, 2011; Wild et al., 2010). Alarmingly, insulin resistance and
type 2 diabetes mellitus are increasingly prevalent amongst adolescents with PCOS (Hart et
al., 2011; Jean Hailes Foundation, 2011; Wild et al., 2010). Insulin resistance in PCOS patients
is independent of, and also additive with obesity they act synergistically to impair insulin
sensitivity (Norman et al., 2001; Wild et al., 2010). Although obese PCOS women are more likely
to display insulin resistance it is important to note that lean women with PCOS also have increased
risk of insulin resistance (Moran et al., 2010). Early onset hyperglycemia and rapid progression to
type 2 diabetes mellitus is also documented in women with PCOS (Jean Hailes Foundation, 2011)
and it is also a recognized risk factor for the development of gestational diabetes.
The precise mechanism of insulin resistance in this population has yet to be elucidated. It may be
due to increased peripheral insulin resistance, insulin hypersecretion and elevated B-cell function
(Dunaif, 1997; Holte et al., 1994; Moran et al., 2010). Hyperandrogenism and genetic propensity
have also been proposed as causative factors (Dunaif, 1997).
31.1.2 Dyslipidemia and polycystic ovary syndrome
Women with PCOS are more frequently reported to have dyslipidemia than non-PCOS women
(Anonymous, 2012; Essah et al., 2007; Legro et al., 2001). The typical profile is one of elevated
triglyceride levels, elevated low density lipoprotein cholesterol (LDL-C) and reduced high density
lipoprotein (HDL-C) levels, leading to a higher LDL-C to HDL-C ratio(Legro et al., 1999). This
profile is typical of what would be expected in those individuals with metabolic syndrome and
confers a higher cardiovascular disease risk.
It is hard to separate out the confounding effects of obesity on dyslipidemia, however it is reported
that lean, as well as obese PCOS patients display dyslipidemia (Essah et al., 2007). This is most
commonly in the form of elevated triglycerides and a reduced HDL-C, which is an especially
strong predictor of cardiovascular disease in women. Hyperinsulinemia has also been associated
with lipid abnormalities in women with PCOS. This is typically elevated triglycerides and low
HDL-C levels
One study, however (Legro et al., 2001), has reported a significant elevation of LDL-C as the
predominant lipid abnormality in PCOS women, independent of obesity. This same study also
reported an elevation in the HDL-C in obese PCOS women that was also independent of other
factors, such as alcohol, smoking and level of exercise.
Some investigators have suggested that hyperandrogenism affects lipid and lipoprotein levels
in PCOS women (Anonymous, 2012; Legro et al., 2001). Altered levels of triglycerides, HDL-C
and LDL-C found in PCOS women are more severe in those who display hyperandrogenism
(Anonymous, 2012). In the study by Legro et al. (2001) circulating androgen levels were associated
with elevated triglyceride levels in PCOS women.
31.1.3 Central adiposity and polycystic ovary syndrome
The typical phenotypic appearance of a woman with PCOS is one of obesity. There is, however,
widespread variability in the prevalence of overweight (i.e. BMI 25-30 kg/m2) and obese (BMI
>30 kg/m2) PCOS women, as well as women of normal weight. It is also reported that there is
significant geographical differences with the incidence of overweight PCOS women being 10%
in Italy to 37% in Kuwait (Anonymous, 2012). The highest prevalence of obesity is reported in
Australia and USA, where 61-76% of women with PCOS are obese (Anonymous, 2012).
The distribution of body fat in PCOS is typically central and upper body fat, when compared
with weight-matched controls. This is the same whether the woman is overweight or normal
weight. One study demonstrated that PCOS women with a BMI <25 kg/m2 (i.e. normal weight)
had significantly greater intra-abdominal pre-peritoneal and visceral fat accumulation when
compared to lean non-PCOS women (Table 31.3). The subcutaneous fat mass remained similar
between the two groups (Yildirim et al., 2003).
Table 31.3. Ultrasound measurements of subcutaneous, pre-peritoneal and visceral fat thickness for polycystic
ovary syndrome (PCOS) patients versus control groups (Adapted from Yildrim et al., 2003).
Fat tissue Fat thickness (mm, mean standard deviation) P-value
PCOS (n=30) Control (n=30)
Subcutaneous 10.39.9 8.95.1 NS1

Pre-peritoneal 8.35.8 4.92.4 0.0001
Visceral 16.713.9 9.44.3 0.0001
1 NS = non-significant.
It is known that obesity, particularly central adiposity, is associated with PCOS, but its causal
association is yet to be determined, due to the intimate relationship between obesity, insulin
resistance and dyslipidemia. Certainly in the study by Yildirim et al. (2003), multiple regression
analysis determined that visceral and intra-abdominal fat mass contributed to high serum
triglycerides and fasting insulin levels. Mean HDL-C levels also correlated negatively with visceral
fat and intra-abdominal fat mass in this non-obese PCOS population. This same relationship was
not found with subcutaneous fat mass (Yildirim et al., 2003). Other studies have also determined
that greater abdominal or visceral adiposity is associated with greater insulin resistance, which
could exacerbate the reproductive and metabolic abnormalities in PCOS (Lord et al., 2006).
Abdominal adiposity in PCOS worsens with weight gain to increase the risk of cardiovascular
death, even after adjusting for BMI (Wild et al., 2010).
31.1.4 Hypertension and polycystic ovary syndrome
It is suggested that there is an increased prevalence of hypertension in women with PCOS when
compared with the general population (Bentley-Lewis et al., 2011), however this is controversial.
A factor complicating the interpretation of these studies is that obesity, which is common in
PCOS and is an independent risk factor for hypertension, is not consistently considered as a
variable (Bentley-Lewis et al., 2011). In those studies that have controlled for BMI or weight, there
are as many supporting PCOS as an independent risk for hypertension as those who suggest that
there is no significant difference when looking at BMI-matched controls.
Most premenopausal women with PCOS are normotensive, however it is reported that when
compared with controls, PCOS women do have higher ambulatory daytime systolic and mean
arterial pressures and a higher prevalence of labile blood pressure, independent of the degree of
insulin resistance (Essah et al., 2007). This may represent a prehypertensive state. Overweight
adolescent women with PCOS demonstrate, from an early age, abnormalities in nocturnal
blood pressure regulation (Essah et al., 2007). As women with PCOS age, there is a recognized
increased incidence of hypertension. Postmenopausal women who were diagnosed with PCOS
have a 2-fold increased incidence of hypertension when compared to age-match controls
(Bentley-Lewis et al., 2011).
PCOS is also correlated with an increased risk of pregnancy-induced hypertension and pre-
eclampsia in women when compared to non-PCOS matched pregnancies (Bentley-Lewis et al.,
2011). Once again, some studies have matched for BMI, whereas some have not.
31.3 Assessing metabolic syndrome in the polycystic ovary syndrome patient

Given the association of PCOS with metabolic syndrome, all PCOS women should be screened
for all of the components of it. Clinical evaluation should include assessment of blood pressure,
waist circumference and/or BMI, complete fasting lipid profile (total cholesterol, LDL-C, HDL-C
and triglycerides) and oral glucose tolerance test. Some guidelines recommend assessing other
cardiovascular risk factors including c-reactive protein and homocysteine levels (Jean Hailes
Foundation, 2011; Wild et al., 2010), due to the increased risk for cardiovascular disease morbidity
and mortality amongst women with PCOS (Wild et al., 2010).
31.4 M
anagement of the metabolic syndrome in polycystic ovary syndrome
patients
The treatment of PCOS is multifactorial and aims to improve all facets of the syndrome including
the biochemical and clinical hyperandrogenism, the reproductive outcomes as well as the
metabolic features of the disease process.
A combination of lifestyle modification together with medical management will optimize the
metabolic syndrome that goes hand in hand with PCOS.
31.4.1 Lifestyle modifications
Obesity and central fat deposition is intimately involved in the pathogenesis of insulin resistance,
dyslipidemia and hypertension as well as other pathological processes of PCOS (such as ovulatory
dysfunction and subfertility), therefore weight loss is a key component of the management of
PCOS (Dunaif, 1997; Jean Hailes Foundation, 2011; Wild et al., 2010).
Weight loss, achieved through lifestyle interventions (diet, exercise and behavior modification),
is recommended to decrease central adiposity, improve lipid profiles, reduce insulin resistance
and reduce risk factors for cardiovascular disease and type 2 diabetes mellitus in overweight
and obese PCOS women (Jean Hailes Foundation, 2011). It is suggested that a reduction in the
circulating insulin levels is the mechanism for the weight-loss associated reproductive benefits
(Dunaif, 1997).
Ongoing lifestyle modification will also reduce the likelihood of weight gain in a population of
women who have a greater propensity for an increased rate of weight gain as well as a higher
overall weight when compared to non-PCOS women.
Lifestyle interventions, independent of weight loss, are also recognized as improving hypertension,
insulin resistance and dyslipidemia (Jean Hailes Foundation, 2011). It is therefore recommended
as first line therapy for all women with PCOS and particularly for those women who display
features of the metabolic syndrome (Table 31.4).
Many of the studies that have been done assessing the effectiveness of lifestyle interventions
on metabolic risk factors are small and quite heterogeneous, so it is difficult to be certain
about the effects. One recent systematic review compared intervention programs (single and
combined interventions) with minimal treatment (Moran et al., 2011). There was a suggested
improvement in testosterone, hirsuitism, waist circumference, waist-to-hip ratio, fasting insulin,
Table 31.4. Lifestyle strategies for managing dyslipidemia in polycystic ovary syndrome (adapted from Wild
et al., 2010).
Dietary feature Modification % estimated low density

lipoprotein reduction
Reduced saturated fat reduce to 7% of total energy intake 8-10

Reduced trans fat reduce to 1% of total energy intake 2
Reduced dietary cholesterol reduce to <200 mg/d 3-5
Added plant stanols/sterols add plant stanols (2 g/d) 6-10
Added dietary fiber add viscous fiber (5-10 g/d) 3-5
Reduced weight reduce by 7-10% 5-8
Total improvement 25-35%
oral glucose tolerance test results and percentage weight change, but no significant difference in
BMI, free androgen index, serum hormone binding globulin, fasting glucose or lipid profile. A
recommendation has therefore been made to include lifestyle interventions (single or combined)
in management of women with PCOS (Jean Hailes Foundation, 2011). It is also suggested that
these interventions should precede or at the very least accompany pharmacological treatment
(Jean Hailes Foundation, 2011).
The dietary interventions should be aimed at weight loss in overweight or obese PCOS women.
There should also be attention to diet for weight maintenance in lean PCOS women. Suggested
dietary changes include a hypocaloric, low saturated fat, increased mono and polyunsaturated
fat diet. The macronutrient composition has not been shown to make any appreciable difference
(Jean Hailes Foundation, 2011; Wild et al., 2010).
The optimal exercise interventions should include a minimum 30 min of moderate-intensity

exercise daily (Wild et al., 2010). Examples include walking or swimming. Moderate aerobic
exercise is known to enhance glucose utilization within the body, improve insulin sensitivity,
and improve cardiovascular risk factors such as weight, dyslipidemia and blood pressure (Jean
Hailes Foundation, 2011). Although the evidence in PCOS women is less robust, exercise is
recommended in the management of PCOS, especially in obese women.
31.4.2 Medical management
The main limitations to lifestyle interventions as the mainstay of treatment of the metabolic
complications of PCOS include difficulty in maintaining changes over a long period of time
especially in the absence of organized programs. Medical therapies therefore have a substantial
role in managing the metabolic syndrome in PCOS.
Insulin sensitizers
Metformin and TZDs have been suggested as options for treating insulin resistance/
hyperinsulinemia (Li et al., 2011; Marshall and Dunaif, 2012), and metformin administration,
theoretically, has been suggested to collectively improve the individual components of the
metabolic syndrome (Palomba et al., 2009).
The mechanism of action of metformin is incompletely understood, but through the AMPK
activation, hepatic glucose production is reduced and thereby increasing insulin sensitivity
in peripheral tissues, as well as lowering plasma free fatty acids (Marshall and Dunaif, 2012;
Palomba et al., 2009). TZDs work by decreasing glucose production and increasing peripheral
tissue sensitization to insulin. It also helps to preserve beta cell function in the pancreas.
Although minimal, a recent meta-analysis found that metformin reduced BMI to a significantly
greater extent than TZDs (Li et al., 2011) and researchers agree that body weight gain is indeed
an adverse effect of TZDs (Li et al., 2011; Marshall and Dunaif, 2012).
Dyslipidemia can be controlled using metformin and TZDs (Li et al., 2011; Marshall and Dunaif,
2012; Palomba et al., 2009; Wild et al., 2010). Metformin was shown to be superior to TZDs in
reducing triglyceride concentrations (Li et al., 2011), but there are no significant differences
when considering LDL-C or HDL-C. Some studies suggest that metformin does not result in
any appreciable difference in LDL-C or HDL-C and should not be used to treat these in isolation
(Wild et al., 2010).
It is important to note that these medications are not without adverse effects. Metformin is well
known to cause gastric upset and certainly significantly more than TZDs (Li et al., 2011). Slow
titration of metformin to the desired dose does lessen this and the side effect is known to abate
with time. Despite improving insulin resistance, TZDs have been associated with increased bone
fracture risk and exacerbation of pre-existing congestive heart failure, as well as recent concerns
regarding bladder cancer (Marshall and Dunaif, 2012) so is not recommended for first line
cardiovascular disease prevention (Wild et al., 2010).
Although insulin sensitizing drugs do appear to have widespread actions in managing many
of the metabolic and reproductive features of PCOS, not all researchers support its blanket use
(Marshall and Dunaif, 2012) and suggest vigilance in detecting the conditions for which this
treatment is warranted: insulin resistance, metabolic syndrome and dyslipidemia.
Cholesterol lowering drugs
Cholesterol lowering therapy should be reserved for those PCOS women who have increased
LDL-C and/or non-HDL-C (Wild et al., 2010). Again, this should be used in conjunction with
lifestyle intervention programs encouraging weight loss. Only statins have been adequately
studied in women with PCOS. It is suggested (Wild et al., 2010) that they will also diminish
insulin resistance and may well assist in controlling the chronic inflammation and improve
endothelial dysfunction that is associated with increased cardiovascular disease risk. Severe
dyslipidemia may warrant polypharmacotherapy such as the addition of fibrates, nicotinic acid
and omega-3 fatty acids (Wild et al., 2010).
Anti-hypertensives
It is recommended that anti-hypertensives are introduced for a blood pressure of at least 140
mmHg systolic or 90 mmHg diastolic. Once again this should be in combination with lifestyle
modifications (Wild et al., 2010).
Obesity management
Several studies have considered the use of weight loss medications in women with PCOS.
Sibutramine and Orlistat have shown variable weight loss and sibutramine, combined with a low
calorie diet, has resulted in improved insulin resistance and triglycerides (Wild et al., 2010). It is
recommended, however, that due to the limited clinical usage in PCOS, these drugs are not used
for weight loss in this population (Moran et al., 2009; Wild et al., 2010).
Bariatric surgery has been shown to be effective for weight loss and improve insulin resistance,
hypertension and dyslipidemia in PCOS women (Wild et al., 2010). It is a suggested option for
morbidly obese PCOS women (Moran et al., 2009), especially where lifestyle interventions have
failed. The benefits should however be balanced against the risks of the surgery including bowel
obstruction, infection, nutritional deficiencies and a 0.1-1% mortality risk.
31.5 Conclusion
The clinical implications of the disease processes associated with PCOS are broad and significant.
Type 2 diabetes, cardiovascular disease and obesity are all prominent global health issues and are
intimately linked with the metabolic syndrome in PCOS. With increasing obesity exacerbating
the incidence and severity of PCOS and the knowledge that weight loss will improve all aspects
of PCOS, lifestyle intervention programs will remain first line management, with focused medical
therapies to be instituted with resistant or severe disease or when compliance to intervention
programs is not sustained. The algorithm in Figure 31.2 demonstrates a suggested plan for
management of the metabolic syndrome in PCOS.
Clinical evaluation includes

blood pressure
body mass index/waist to hip ratio
Identication of the lipid prole
metabolic syndrome oral glucose tolerance test/fasting insulin
in PCOS
Dietary
hypocaloric
low saturated fat
increased mono/polyunsaturated fat
Lifestyle modication Exercise
program 30 minutes
(single or combined moderate intensity
intervention) every day
Psychological
support to optimise engagement/adherence to interventions
Insulin sensitizers
metformin
thiazolidinodiones
Cholesterol lowering drugs
statins
Medical management brates, nicotinic acid, omega 3 fatty acids
Anti-hypertensives
Obesity management
medical therapy
surgery
Figure 31.2. Flow chart for management of the metabolic syndrome in polycystic ovary syndrome (PCOS).
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32. Diabetes mellitus and polycystic ovary
syndrome: implications for diet and nutrition
N. Mendoza1 and M.J. Cancelo2
Department of Obstetrics and Gynecology, University of Granada, Maestro Montero 21, 18004
Granada, Spain; University Hospital, Alcal de Henares, C/Donante de Sangre, Guadalajara
19002, Spain; nicomendoza@telefonica.net
Abstract
Diabetes mellitus is a common and heterogeneous disease that involves metabolic problems and
predispose to the cardiovascular events. It is a complex disease that is subject to environmental
influences, mainly dietary, which makes it similar to other gynecological and obstetrics processes,
such as polycystic ovary syndrome and gestational diabetes. So, the interest of the diagnosis of
them is, not only improve fertility but to prevent possible future medical complications. For this
purpose, simple methods to quantify insulin sensitivity are available, and we propose reasonable
treatment with dietary measures to improve not only the metabolic changes, but also clinical
hyperandrogenism and fertility.
Keywords: diabetes mellitus, insulin resistance, lifestyle, polycystic ovary syndrome
521
Summary points
There is an increased prevalence of insulin resistance (IR) in anovulatory women.
Women with polycystic ovary syndrome and IR have an increased risk of diabetes mellitus.
Clinical evaluation of the IR should be done with a simple technique such as homeostatic model assessment.
Weight loss represents one of the main preventative strategies for patients with chronic anovulation and IR.
The diet should be low calorie, low saturated fat, rich fiber and needs of psychological reinforcement and
physical exercise to improve compliance.
With inositol supplementation an improvement in insulin sensibility, ovulation and spontaneous pregnancies
have been observed.
32. D
iabetes mellitus and polycystic ovary syndrome
Abbreviations
DM Diabetes mellitus
HOMA Homeostatic model assessment
IR Insulin resistance
32.1 Introduction
Both DM and PCOS are a complex and heterogeneous disease that involves reproductive
difficulty as well as metabolic problems. More evidence is being found that suggests that PCOS
predisposes individuals to metabolic disorders, such as gestational or adult onset diabetes. The
fact that these diseases share predisposing genes speaks of a common origin from a genetic point
of view (Mendoza, 2011; Figure 32.1). Therefore, it is recognized that changes in lifestyle should
be the first steps in a patient with PCOS or gestational diabetes to prevent DM (Thessaloniki
PCOS Consensus, 2008).
31.1.1 Definitions
According to a panel of experts from the American Diabetes Association (Gabir et al, 2000),
metabolic glucose changes are classified in impaired fasting glucose, impaired glucose tolerance
or DM (Table 32.1).
The expert conference by the European Society for Human Reproduction and Embryology and
the American Society for Reproduction Medicine recommended that PCOS be defined when at
least two of the following three features were present (Rotterdam PCOS Consensus, 2004):
oligo- and/or anovulation;
hyperandrogenism;
polycystic ovaries.
These criteria also recognize that other causes of androgen excess and related disorders should
be excluded before confirming the diagnosis of PCOS. This definition is more generic and
will probably increase its already high prevalence: it is the most common endocrinopathy in
women and affects 7-14% of women of childbearing age worldwide. (De Groot et al, 2011). It
was proposed that insulin resistance (IR) is the pathophysiological basis for this syndrome. The
primary interest in diagnosing these patients is not to assist in conception, but to prevent possible
future medical complications, including DM and cardiovascular disease (Pasquali et al, 2011).
Central nervous system

Dopamine
Hypothalamus Immunitary system
GnRH
Hypofisis AMH
TNGa
LH FSH IL-1
Granulose cell
Teca cell LHR
FSHR
AMPc AMPc Oocite
P450c17
Cholestrol P450sc P450aromatase
Estrogens
3HSD Androgens Inositol Ca2+
Ca2+ IGF-1, IGF-2 Androgens
ESR Meiosis
Inositol NRIP1
IR
VEGF
IGF-2R Testosterone Nucleus
PG2F
5 - reductase endotelin 1 Endotelial cells
Insuline
SHBG dihyrotestosterone
Figure 32.1. Endocrine, paracrine and autocrine pathways involved in the etiopathogenesis of polycystic ovary
syndrome.
AMH = anti-Mllerian hormone; 3HSD = 3 hydroxysteroid dehydrogenase; ESR = estrogen receptor; FSH
= follicle-stimulating hormone; IGF1 = insulin-like growth factor-1; IGF2 = insulin-like growth factor-2; IGF2R =
insulin-like growth factor-2 receptor; IL1 = interleukin 1; IR = insulin receptor; LH = luteinizing hormone; LHR =
luteinizing hormone receptor; NRIP1 = nuclear receptor-interacting protein-1; PGF2 = prostaglandin F2; SHBG
= sex hormone-binding globulin; TNF = tumor necrosis factor alpha; VEGF = endotelial growth factor.
Table 32.1. Metabolic glucose changes definitions suggested by the American Diabetes Association (Gabir
et al, 2000).
Impaired fasting glucose Fasting glucose between 100 and 126 mg/dl (5.6-7 mmol/l).
Impaired glucose tolerance Glycaemia between 140 and 200 mg/dl (7.8-11 mmol/l) 2 h after the
administration of 75 g of glucose.
Diabetes mellitus Clinical diabetic symptomatology and blood glucose above 200 mg/dl
(11.1mmol/l)
Fasting glucose above 126 mg/dl (7 mmol/l). This value must be confirmed on
a second occasion and on a different day
Glycaemia above 200 mg/dl (11.1 mmol/l) 2 h after the administration of
75g of glucose. This value must be confirmed on a second occasion and on
a different day
32. D
32.2 R elationship between anovulation, insulin resistance and diabetes

mellitus
It has been found that certain influences during fetal life, such as PCOS, can result in metabolic
diseases in the adult via epigenetic mechanisms caused by the exposure of the fetus to an excess
of androgens (Diaz-Garcia et al, 2011; Hickey et al, 2006).
Despite the common genetic origin of PCOS and DM and the fact that numerous studies
have linked PCOS with the highest risk of gestational diabetes, a 2009 meta-analysis did not
find sufficient evidence to confirm this hypothesis (Toulis et al, 2009), likely because the new
definition of PCOS includes other phenotypes that are exempt from this risk. In this sense, the
main link between PCOS and cardiovascular disease is IR. However, in women with PCOS,
other risk markers, such as homocysteine and the C-reactive protein, also increase; additionally,
the associated hyperandrogenism is already an independent risk factor of cardiovascular disease
(Dokras, 2008).
IR refers to a decrease in the capacity of insulin to exert its biological action in its primary target
tissues. This concept is so important that it is currently considered to be a common characteristic
of multiple diseases, such as DM, dyslipidemia, and hypertension, which together form the basis
of metabolic syndrome (Barth, 2011).
A considerable proportion of women with PCOS display IR. The mechanisms responsible for this
insensitivity are currently unknown, although it has been postulated that IR results from altered
adipose and muscular cell glucose uptake, which is caused by a genetic defect that is independent
of obesity. Although IR does not occur exclusively in obese women with PCOS, the incidence is
more frequent among these patients because PCOS may be an additional cause of IR (Baranova
et al, 2011). In addition, the potential for genetic predisposition indicates that the patients direct
family members may also be affected by this condition, and they must be warned of this risk
(Mukherjee et al, 2010).
Similarly, a frequent association between IR and hyperandrogenism has been observed, although
the cause and effect relationship is not well understood. A chronic androgen effect on the
physiology of the adipocyte and the homeostasis of glucose has not been detected, although tests
have indicated that hyperinsulinism tends to be a cause rather than a result of excess androgens
(Baptiste et al, 2010).
In practice, it is important to recognize the occurrence of IR among women with PCOS (Table
32.2). Furthermore, various studies have attempted to demonstrate that anovulatory patients
with some extent of IR display greater difficulty in follicle development with exogenous FSH
than women with normal ovulation or who do not have IR. The short-term benefit of an
insulin decrease during anovulation has been repeatedly demonstrated, both in women with an
appropriate diet and in women who were treated with pharmaceutical agents. Thus, some clinics
Table 32.2. Insulin resistance suspected signs.
Anovulation and hyperandrogenism

Prior gestational diabetes
Relatives with diabetes mellitus
Obesity
Upper waist more than 88 cm
Hyperglycemia
Hyperlipidemia
Hypertension
Adolescent with persistent anovulation
prefer to delay fertility treatment until the patients have reached an appropriate weight or have
leveled out their insulin numbers (Moran et al, 2011).
A number of publications have expressed the possibility that a woman with PCOS may display
a greater risk of suffering cardiovascular disease or DM at later age. In this context, it would be
helpful to investigate these effects in future studies. However, even with this knowledge gap, there
is existing evidence of the relationship between these effects: type 2 evidence for adult DM and
early arterial disease, and type 3 evidence for hypertension or dyslipidemia (Pasquali et al, 2011).
Clearly, IR and hyperinsulinism are important factors related to PCOS due to the etiological
implications and because different therapeutic strategies should be used with these markers. We
should keep in mind that for certain patients who seek an anovulation consult, their families
should be warned, and kept under long-term monitoring due to the special risk of suffering from
diabetes or arteriosclerosis.
32.3 Diagnosis of insulin resistance in anovulatory women

The hyperinsulinemic euglycemic clamp technique is considered to be the gold standard for
quantifying sensitivity to insulin. However, the difficulty of performing this technique in clinical
practice has led to the development of other, simpler techniques (Table 32.3). For example,
HOMA is a method based on a mathematical model that provides a semi-quantitative measure
of the sensitivity to insulin. The normal range of HOMA values is between 2 and 3, although there
is no consensus to establish a limit to begin treatment (Matthews et al, 1985).
32. D
Table 32.3. Methods to diagnose insulin resistance.
Basal glucose/insulin <4.5

Basal insulin >16 mUI/l
Abnormal oral glucose test
HOMA = basal insulin (mcU/ml) basal glucose (mg) / 405 (normal<3.2)1
Insulin sensibility = basal insulin / 91 0.05551 (normal 1)
HOMA cell = 360 basal insulin / basal glucose (mg) 63 (normal=100%)
1 HOMA = homeostatic model assessment.
32.4 Insulin resistance and pregnancy

IR, DM and obesity may lead to infertility and to a higher risk of miscarriage. Management
of anovulation in these patients involves diet and exercise as well as standard approaches to
ovulation induction.
On the other hand, pregnancy is characterized as a temporary condition of IR that occasionally

leads to gestational diabetes or complicates pre-existing diabetes. Some authors have found an
association between PCOS and glucose intolerance during pregnancy. In the absence of insulin
measurements, a risk marker for diabetes mellitus among patients with PCOS may be the
appearance of certain disorders related to carbohydrate metabolism during pregnancy. In fact,
it is known that the appearance of glucose intolerance during pregnancy increases the risk of
suffering from adult diabetes by 50%. As a result, a positive result in routine examinations in
clinical obstetrics to identify patients with glucose intolerance not only achieves an improvement
of the perinatal outcome, but can also be considered to be a cardiovascular and metabolic
risk marker. A history of PCOS can be considered to be a risk marker of gestational diabetes
based on the available evidence without implying other pregnancy complications, such as fetal
macrosomia, when good metabolic control is performed by the pregnant woman and she does
not excessively increase her weight (Kjerulff et al., 2011). In addition, pregnancies in IR, DM and
obese women have increased rates of preeclampsia, gestational diabetes, cesarean section and
perinatal mortality and morbidity (Diaz-Garcia et al., 2011; Kjerulff et al., 2011; Thangaratinam
et al., 2012).
32.5 T he importance of diet in women with diabetes mellitus or insulin

resistance and anovulation
It is estimated that half of the women with PCOS are overweight or obese, with the majority of
them having a fat distribution in the mid-section or abdomen, which is an independent risk
factor of DM. However, IR is also present in thin women, who are also at a higher metabolic
risk that should be corrected through caloric restriction. In fact, weight loss represents one of
the main preventative strategies for patients with PCOS, regardless of whether they are obese
(Garruti et al, 2009; Wu et al, 2012).
We have strong evidence indicating that lifestyle changes (diet and exercise, Table 32.4) can
obtain better PCOS clinical outlook and consequences, mainly by stabilizing the IR but also by
stabilizing the P-450 and 17-hydroxylase activity, which would improve the hyperandrogenism
symptoms. In fact, lifestyle changes would be the first recommendation we make to a patient who
is overweight and has PCOS, who wants to re-establish ovulation and attempt pregnancy, or who
wishes to prevent complications in the long run (Moran et al., 2011).
The weight loss that is produced under these measures not only increases the sensitivity to
insulin but also decreases the level of androgens and increases that of SHBG. As a result of these
changes, ovulation is re-established in women with irregular menstrual cycles, and spontaneous
pregnancies occur. Similarly, it has been recorded that a weight loss of 5-10% can be enough to
reach those goals. However, a hypocaloric diet may not be sufficient if it is not accompanied by
support for dietary compliance or through other hygienic or pharmacological means. This issue
arises because the excess of androgens and the hyperinsulinism prevailing in patients with PCOS
make it difficult to lose weight and greatly compromise patients success during the medium- or
long-term course of a diet (Moran et al, 2011).
Undoubtedly, lack of compliance is the most important obstacle preventing the hypocaloric diet
from being effective. Reinforcement can sometimes be obtained by helping patients understand
that caloric restriction is a slow means of obtaining their goals and by helping build their patience
with information on the risks that can occur in the short, medium, and long term: in other words,
advising them that their ovulation, pregnancy, and cardiovascular risk depend on their diet.
For that reason, it is also important that the diet not be overly aggressive, given that compliance
is less likely when great effort is required to continue it. Similarly, the distribution of essential
nutrients should be balanced, without altering or omitting the well-proportioned distribution of
each of them; that is, the diet should contain 55% carbohydrates, 15% protein, 25% fat (preferably
unsaturated), and a minimum of 5% fiber (Panidis et al, 2008).
Table 32.4. Characteristics of the diet for patients with diabetes mellitus or polycystic ovary syndrome.
Hypocaloric
Low in saturated fat
Cessation of alcohol and tobacco consumption
Well-proportioned distribution of essential nutrients: 55% carbohydrates, 15% protein, 25% unsaturated fat,
at least 5% fiber
Accompanied by psychological reinforcement or aerobic regular exercise to improve compliance
32. D
It appears that diets that reduce the ingestion of carbohydrates improve the metabolic results
in patients with IR, and we believe that these diets would also perform well for women with
PCOS (Reaven et al, 2005). However, we have conducted several randomized controlled trials
that compare the effects of different types of diets in these patients, and none of them appear to
improve either weight loss or reproductive results. (Moran et al, 2011).
The main reinforcement for maintaining compliance with a diet is accompanying it with physical
exercise, preferably aerobic and regular. On its own, physical exercise increases the glucose
capacity and sensitivity to insulin and decreases body weight and abdominal fat, among other
benefits. Thus, this activity reduces the risk of metabolic syndrome and DM (Crosignani et al,
2003; Knowler et al, 2002; Moran et al, 2011).
From a strictly reproductive perspective, excess weight limits the probability of becoming
pregnant and increases that of suffering a miscarriage. The exact mechanism that produces
this effect is unknown, but various hypotheses have been proposed, such as the diminution of
SHBG or the increase in the amount of FSH required to obtain adequate follicular recruitment.
Accordingly, in a patient who is obese, has PCOS, and is planning to undergo some type of
fertility treatment, the first recommendation is that of emphasizing lifestyle, especially weight
loss. This factor should be emphasized, ideally until the patients body mass index falls below 30-
35 kg/m2 (Balen et al., 2007). In addition to exercising, ceasing alcohol and tobacco consumption
should be emphasized. In those persons who are extremely obese, treatment accompanied by
pharmacological or surgical remedies can be considered in addition to diet and exercise (Nybacka
et al., 2011; Palomba et al., 2010; Thessaloniki Consensus, 2008).
In addition, diet significantly reduced the risk of gestational diabetes (and hypertension) without
adverse fetal outcomes. In a recent systematic review, the diet recommended for reducing weight
gain in pregnancy was a balanced, hypocaloric and low glycemic diet with unprocessed whole
grains, fruits, beans and vegetables. It is also important to include high intake of dietary fiber,
which decreases the concentrations of triglycerides, and this, in turn, is known as a factor to
reduce the incidence of pre-eclampsia by up to 70% (Thangaratinam et al., 2012).
32.6 Importance of the consumption of inositol

It has been suggested that an inositol deficit could be behind PCOS. The myo-inositol
hexaphosphate, commonly known as phytic acid, is found in nature in wheat seeds, citrus,
nuts, and legumes. Phytic acid is part of the vitamin B group, and it works intimately with
choline, participating at the post-receptor level as a second messenger. Monogastric animals,
such as humans, have little phytase in their digestive apparatus, which is why inositol is mostly
metabolized by bacteria in the intestinal flora. However, despite not being an essential nutrient
in our diet, the consumption of this compound reaps certain benefits (Table 32.5). For example,
in plasma, phytic acid acts as a hypolipidemic agent, minimizing the risk of MS, and it has
antioxidant properties that conveniently prevent the development of cancers and cellular damage.
Table 32.5. Summary of D-chiro-inositol benefits in patients with polycystic ovary symdrome undergoing fertility
treatments.
Improvement of the insulin sensibility

Increase of spontaneous ovulation
Increase of spontaneous pregnancies
Regularity of menstrual cycles
Decrease of the dose of follicle stimulating hormone required for fertility treatments
Increase of mature oocytes
Improvement of the quality of the embryos
Improvement in lipid profile
Weight reduction
Additionally, this molecule is hypoglycemic because it delays the digestion and absorption of
starch, which decreases the need for insulin and reduces the risk of DM. The daily ingestion is set
at approximately 1000 mg, but for the prevention or treatment of MS, the consumption of doses
of up to 2,000 mg is recommended (Ciotta et al., 2011; Galazis et al., 2011; Le Donne et al., 2012).
At a reproductive level, inositol has been detected in the ovarian follicle, and it appears to
intervene in the meiosis of the oocytes. We have little data about the effects of a deficit in this
molecule, but it has been observed that supplementing the diet with D-chiro-inositol, its galenic
form, has been associated with ovulation and spontaneous pregnancies. In fact, in several clinical
assays, reproductive variables have improved, such as the amount of FSH required, the number
of mature oocytes, and the quality of the embryos in patients with PCOS undergoing fertility
treatments. Improvements with respect to lipid profile and excess weight were also obtained
(Galazis et al., 2011; Papaleo et al., 2009).
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33. Hypocaloric diets in overweight and obese
patients with polycystic ovary syndrome
University of Modena and Reggio Emilia, IRCSS, Azienda Ospedaliera S. Maria Nuova of
Reggio Emilia, Unit of Obstetrics & Gynecology, Viale Risorgimento, 42123 Reggio Emilia, Italy;
stefano.palomba@asmn.re.it
Abstract
Polycystic ovary syndrome (PCOS) is a heterogeneous disorder. The majority of women with
PCOS, regardless of weight, have a form of insulin resistance that is intrinsic to the syndrome.
Obese women with PCOS have an added burden of insulin resistance related to their adiposity.
Thus, adequate nutritional status is a critical determinant of the onset and maintenance of normal
reproductive function. Weight loss should always be recommended as first-line approach in the
treatment of obese and overweight PCOS women, since it significantly improves hormonal and
metabolic abnormalities, and may favor spontaneous ovulation improving fertility in the majority
of patients. In addition, weight loss associated with a moderate physical activity is desirable
because physical exercise improves the reduction of the insulin resistance. Unfortunately, it is
very difficult to achieve these goals in the clinical practice, especially in PCOS patients.
Keywords: diet, lifestyle programs, obesity, PCOS, polycystic ovary syndrome, weight loss
533
Summary points
Weight loss should always represent the first-line approach in the treatment of obese and overweight polycystic
ovary syndrome (PCOS) women, since it significantly improves hormonal and metabolic abnormalities, and
may favor spontaneous ovulation improving fertility in the majority of patients.
The composition of the optimal diet for PCOS patients is not yet known.
Normal-weight or lean PCOS patients, probably, could benefit of an increased physical activity that may
improve insulin sensitivity.
The real efficacy of lifestyle changes in normal-weight or lean patients with PCOS is unknown.
Weight loss associated with a moderate physical activity is desirable because physical exercise improves the
reduction of the insulin resistance.
It is very difficult to achieve these goals in the clinical practice, especially in PCOS patients.
33. Hypocaloric diets in overweight and obese patients with PCOS
Abbreviations
AEPS Androgen Excess and PCOS Society
ASRM American Society for Reproductive Medicine
BMI Body mass index
CC Clomifene citrate
DM Diabetes mellitus
ESHRE European Society or Human Reproduction and Embryology
GL Glycemic load
HOMA-IR Homeostasis model of assessment-insulin resistance
NICHD National Institute of Child Health and Human Development
NIH National Institute of Health
PCO Polycystic ovaries
RCT Randomized controlled trial
SET Structured exercise training
33.1 Introduction
PCOS is a heterogeneous disorder characterized by hyperandrogenism, ovarian dysfunction, and
polycystic ovarian morphology. Its definition remains somewhat controversial (Table 33.1). Three
main criteria have been proposed: the NIH/NICHD criteria proposed in April 1990 (Zawadzki
and Dunaif, 1992), the ESHRE and the ASRM criteria proposed in May 2003 (Rotterdam ESHRE/
ASRM-Sponsored PCOS Consensus Workshop Group, 2004), and AEPS criteria published in
2009 (Azziz et al., 2009).
The 1990 NIH criteria (Zawadzki and Dunaif, 1992) indicated the presence of hyperandrogenism
and/or hyperandrogenemia and chronic anovulation with exclusion of related ovulatory or other
androgen excess disorders (e.g. thyroid dysfunction, hyperprolactinemia, androgen-secreting
neoplasms, or non-classic adrenal hyperplasia) as PCOS features. The presence of PCO, with
morphology assessed by ultrasound, was not included as a diagnostic criterion, because
considered a finding with a low specificity (Polson et al., 1988).
In 2003, an international conference in Rotterdam, the Netherlands (Rotterdam ESHRE/ASRM-

Sponsored PCOS Consensus Workshop Group, 2004), proposed to revise the PCOS criteria.
In particular, the ESHRE/ASRM conference proceedings expanded the definition of PCOS,
establishing that it could be diagnosed, even after the exclusion of related disorders, by at least
two of the following features: (1) oligo- or anovulation; (2) clinical and/or biochemical signs of
hyperandrogenism; and (3) PCO. All women considered to be affected by PCOS, according to
Table 33.1. Diagnostic criteria proposed for polycystic ovary syndrome.
Criteria NIH1 Rotterdam2 AE-PCOS3
Oligo-anovulation + +/ +/
Clinical and/or biochemical signs of hyperandrogenism + +/ +
Polycystic ovaries +/ +/ +/
1 Zawadzki & Dunaif, 1992.

2 Rotterdam ESHRE/ASRM-Sponsored PCOS consensus workshop group, 2004.
3 Task Force on the Phenotype of the Polycystic Ovary Syndrome of The Androgen Excess and PCOS Society,
2009.
the NIH criteria, would also be identified through the Rotterdam criteria, which included women
with PCOS excluded by the NIH criteria: those with PCO affected by hyperandrogenism and
ovulatory cycles or chronic anovulation and normal androgen levels.
In 2009, an expert panel of the AEPS, after an extensive literature review, recommended that
PCOS should be considered an androgen excess disorder characterized by alteration in androgen
biosynthesis, utilization, and/or metabolism in women. Thus, the AEPS criteria for PCOS are
fulfilled by the combination of hyperandrogenism with ovarian dysfunction or PCO or both
(Azziz et al., 2009).
As a consequence, the phenotypes considered to represent the disorder may vary according to the
definition used, and several authors (Azziz, 2006) questioned the points of agreement between
the new phenotypes according to the ESHRE/ASRM criteria and the features classically related
to the syndrome. Ovulatory women with PCO seem to be less insulin-resistant than anovulatory
women with PCO (Adams et al., 2004). Patients with PCO, chronic anovulation, and normal
androgen levels are not insulin-resistant (Barber et al., 2007) and only women who fulfill the
NIH criteria have been demonstrated to be at high risk for glucose intolerance (Sam and Dunaif,
2003) and metabolic syndrome (Apridonidze et al., 2005).
Notwithstanding the extreme variability of the diagnostic criteria adopted, the main clinical
features related to the syndrome, albeit not pathognomonic, are menstrual disorders, ano
vulatory infertility, and signs of hyperandrogenism (Rotterdam ESHRE/ASRM-Sponsored PCOS
Consensus Workshop Group, 2004). Moreover, PCOS is characterized by a number of other
clinical traits and long-term sequelae.
The reduced fertility observed in PCOS patients cannot be attributed to anovulation alone.
Other factors seem to reduce the fertility in these women, including lower oocyte and/or embryo
quality, defects in endometrial development, and implantation abnormalities (Palomba et al.,
2008a). In addition, pregnant women with PCOS seem to have a significantly higher risk for
miscarriage, gestational DM, pregnancy-induced hypertension, pre-eclampsia and poor infant

outcome (Palomba et al., 2008a).
The pathogenesis of PCOS is still unclear, and a complex of genetic and environmental factors
may be involved. In particular, family studies demonstrated that PCOS is significantly more
prevalent among family members than in the general population (Legro and Strauss, 2002).
Among first-degree female relatives of patients with PCOS, 35% of premenopausal mothers
and 40% of sisters were affected by the disorder (Kahsar-Miller et al., 2001). These rates are
significantly higher than the 6-7% observed in the general population (Azziz et al., 2004). Other
than PCOS itself, increased androgens, insulin secretion, and insulin resistance also appear to
be under significant genetic control, as established by family studies (Goodarzi 2008; Legro et
al., 2002a,b). Several genes have been logically chosen as candidates, but most of these studies
were performed in small cohorts and confounded by several biases. Thus, no particular gene is
universally recognized as importantly contributing to PCOS risk, and several environmental
factors probably modulate the clinical expression of genetic predisposition. In particular, obesity,
insulin resistance, prenatal androgen excess during early gestation, birth weight, precocious
pubarche, dietary intake, lifestyle, and stress may act on the genetic substrate.
The majority of women with PCOS, regardless of weight, have a form of insulin resistance that
is intrinsic to the syndrome (Dunaif, 1997). Obese women with PCOS have an added burden
of insulin resistance related to their adiposity. Thus, adequate nutritional status is a critical
determinant of the onset and maintenance of normal reproductive function (The ESHRE Capri
Workshop Group, 2006).
Low birth weight modifies the relationship between insulin resistance and PCOS and seems to
increase the risk of developing PCOS. Diet composition, eating disorders, and psychological
stress seem to be related to the syndrome, even if no causality link has been clearly demonstrated
(Altieri et al., 2013; Barr et al., 2011).
The molecular mechanisms of insulin resistance in PCOS differ from those in other common
insulin-resistant states, such as obesity and type 2 DM (Dunaif, 1997). In fact, insulin resistance
occurring in PCOS patients is not evident in all tissues, but it seems to have a tissue peculiarity
(Legro et al., 2004). Specifically, in skeletal and adipose tissue, insulin resistance led to a decreased
glucose uptake and increased lipolysis, respectively, whereas, the ovary, adrenal, liver, and skin
seem to remain insulin sensitive (Legro et al., 2004). Furthermore, recently a peripheral insulin
resistance, which is related to ovarian (Palomba et al., 2006a) and uterine (Palomba et al., 2006b;
2006c) abnormalities detected in PCOS patients, has been demonstrated.
Because insulin resistance with compensatory hyperinsulinemia is widely considered the pivotal
feature of PCOS, and obesity and overweight worsen that feature significantly, the weight loss,
obtained with lifestyle programs, is universally considered the gold standard treatment for obese/
overweight patients with PCOS (Figure 33.1). To this regard, almost all available guidelines
(Thessaloniki ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group, 2008) consider the
Lifestyle modifications
Peripheral tissues
Skeletal muscle
Insulin sensitivity
CNS Liver improvement
Intestine Glucose uptake
Regulation of Gluconeogenesis FFA oxidation
LH secretion (?) SHBG Glucose use
Insulin sensitivy Adipocytes

Glucose uptake
Free androgens
Hyperinsulinemia Long-term sequelae
Adrenal tissue
Adrenal androgen production
Hyperandrogenemia Endometrial abnormalities
Theca cells Endometrium

Hirsutism Glucose uptake
Androgen production
Follicular development
Ovarian disfunction Granulosa cells
Estrogen production Ovary
Menstrual disorders Infertility
Figure 33.1. Potential mechanisms of action for lifestyle modifications.
weight loss the first therapeutic step for treating the overweight/obese patients with PCOS before
to start drug intervention. This non-pharmacologic approach has been widely accepted also in
the clinical practice by worldwide gynecologists and endocrinologists (Cussons et al., 2005).
In the current chapter the importance of the alterations in body weight and composition in PCOS
will be treated. The pivotal role of the hypocaloric diet in overweight/obese patients with PCOS
will be analyzed with particular regard for its clinical efficacy. The mechanism of action will be
also elucidated. Finally, the future researches on this crucial issue will be discussed.
33.2 Alterations of the body weight in polycystic ovary syndrome

Obesity is encountered in 30-70% of women affected by PCOS, and its presence significantly
modifies both clinical and laboratory expression of the syndrome (Cupisti et al., 2008). Moreover,
women with PCOS have been shown to have higher amounts of visceral fat compared to healthy
controls even when they are normal weight (Kirchengast and Huber, 2004). Visceral adiposity
further worsens the underlying insulin resistance and insulin resistance-associated reproductive
(Balen et al., 1995) and metabolic features (Ehrmann et al., 2006).
Several evidences demonstrated a relationship between obesity and infertility (Norman et al.,
2004). In fact, obesity is often related to insulin resistance and the associated hyperinsulinemia
plays a pivotal role in ovarian steroidogenesis and androgen blood transport and/or activity in
the target tissues.
Obesity worsens the presentation of PCOS. Obesity and, in particular, abdominal obesity in
adolescence and adulthood, and weight gain after adolescence is a predictor of the development
of hirsutism and menstrual disturbances in PCOS (Laitinen et al., 2003). In addition, obese PCOS
women are more likely to suffer from hirsutism and infertility than normal-weight PCOS subjects
(Balen et al., 1995).
A wide study on PCOS women has shown that the sterility rate is about 40% higher in women
with a BMI higher than 30 compared to those with a BMI lower than 30 (Balen et al., 1995).
Only 12-22% of these obese women have regular menstrual cycles vs. 28-32% of normal-weight
women (Balen et al., 1995).
The importance of obesity in the pathophysiology of PCOS is definitely confirmed by the fact
that even modest to moderate weight loss may improve all features of PCOS, including insulin
resistance, lipid abnormalities, androgen excess, menses and ovulatory rates and favor the
complete recovery of the PCOS phenotype in some of them (Wright et al., 2004) (see below).
Based on these considerations, the initial counseling of obese/overweight PCOS women is crucial
(Figure 33.2).
33.3 Clinical effects of the hypocaloric diet in polycystic ovary syndrome

Treatment of PCOS includes addressing reproductive, metabolic, and psychological features.
From a reproductive perspective, reducing biochemical and clinical hyperandrogenism, regulating
menstrual cycles, restoring ovulation and reproductive function, and improving reproductive
outcomes are important. On the other hand, from a metabolic perspective, addressing insulin
resistance and the metabolic syndrome are important in reducing long-term metabolic morbidity.
Finally, from a psychological perspective, addressing factors including self-esteem and dysthymia
is critical to improving motivation for effective lifestyle change.
Preconceptional counseling in women with PCOS should identify risk factors for reproductive
failure and correct them prior to treatment initiation. In this respect, it is imperative to recognize
the presence of obesity and its centripetal distribution.
Primary care services should ensure that all women of childbearing age have the
opportunity to optimize their weight before pregnancy. Advice on weight and
lifestyle should be given during family planning consultations, and weight, body
mass index and waist circumference should be regularly monitored.
Women of childbearing age with a BMI 30 should receive information and

advice about the risks of obesity during pregnancy and childbirth, and be
supported to lose weight before conception.
Women with a BMI 30 wishing to become pregnant should be advised to take

5 mg folic acid supplementation daily, starting at least one month before
conception and continuing during the first trimester of pregnancy.
Health professionals should take particular care to check that women with a
booking BMI 30 are following advice to take 10 micrograms Vitamin D
supplementation daily during pregnancy and while breastfeeding.
CMACE/RCOG Joint Guideline, 2010
Figure 33.2. Counseling for obese/overweight polycystic ovary syndrome patients (CMAC/RCOG joint
guideline, 2010).
The most recent guidelines recommended weight loss as first-line therapy in obese women with
PCOS seeking pregnancy (Thessaloniki ESHRE/ASRM-Sponsored PCOS Consensus Workshop
Group, 2008). This recommendation is based on the extrapolation from the benefits of weight
loss seen in multiple other conditions, such as DM and CVD, as well as recognition of obesitys
association with poor reproductive outcome and with a high incidence of complicated pregnancy.
The specific effects of hypocaloric diet in overweight and obese women with PCOS is difficult to
understand, since diet an physical activity are often combined in lifestyle modifications programs
(see Section 33.4). However, it has been proven that diet, exercise and appropriate lifestyle
programs, either alone or combined with anti-obesity pharmacologic options, are critical for the
management of overweight and obese PCOS patients.
In the short-term, the therapeutic targets of dietary management of PCOS are weight loss, the
amelioration of symptoms such as acne and hirsutism, and improvement of fertility in those who
wish to fall pregnant. In the long-term, dietary management needs to focus on the increased risk
of type 2 DM, CVD and certain cancers, associated with this condition. Here we will focus on
the short-term effects of the hypocaloric diet in PCOS patients.
An important point is that a minimal amount of weight loss of about 5-10% over as little as 4
weeks is sufficient for improving the presentation of PCOS despite subjects remaining clinically
overweight or obese (Norman et al., 2004) and for restoring a normal menstrual cycle in PCOS
obese women with amenorrhea (Huber-Buchholz et al., 1999).
Weight loss improves ovary function in obese PCOS women, probably by affecting obesity related
hyperinsulinemia (Huber-Buchholz et al., 1999). Weight loss also improves the pregnancy rate in
untreated obese PCOS patients (Clark et al., 1995) and in PCOS women who underwent fertility
treatment (Clark et al., 1995, 1998).
In overweight/obese women with hyperandrogenism and PCOS, weight loss decreases abdominal
fat, hyperandrogenism and insulin resistance and improves lipid profiles, menstrual cyclicity and
fertility, and risk factors for DM and CVD (Clark et al., 1995; Moran et al., 2003); moreover,
lifestyle modification, through diet and exercise programs in obese subjects with PCOS, improves
not only reproductive (Norman et al., 2004; Clark et al., 1998) but also psychological outcomes
(Legro et al., 2004).
With respect to androgens, most studies demonstrated reduction in either total or free testosterone,
and some demonstrated reduction in adrenal androgens (Moran et al., 2009). Levels of SHBG
were improved in all of the longer term studies, with only one short-term intervention failing to
show improvement (Moran et al., 2009). Clinically, improvement in hirsutism was documented
in a number of studies. Menstrual function and ovulation improved in all the studies reporting
this end point. Pregnancy or conception was measured in six studies, but all without controls
(Moran et al., 2009).
Metabolic improvements in fasting insulin, glucose, and glucose tolerance were seen in all
studies in which they were measured (Moran et al., 2009). Total cholesterol and triglycerides
improved with weight reduction, such as plasminogen activator inhibitor-1 and free fatty acids,
while C-reactive protein did not change (Moran et al., 2009). Furthermore, more recently it
was demonstrated that overweight and obese women with PCOS may respond disparately to
weight loss, ranging from the persistence of the PCOS phenotype to partial or even complete
recovery from it, and these effects seemed to be totally independent of the degree of weight loss,
treatment duration, and improvement in insulin resistance (Pasquali et al., 2011). Pretreatment
abdominal (visceral) adiposity and, particularly, androstenedione plasma levels appear to be the
most important predictive factors of outcomes (Pasquali et al., 2011).
A recent systematic review of RCTs aimed to compare lifestyle treatment to minimal or no

treatment in women with PCOS demonstrated, after data synthesis of six RCTs for an overall
population of 164 subjects, that lifestyle intervention improves body composition, clinical
and biochemical hyperandrogenism, and insulin resistance. No beneficial effect of lifestyle
intervention was detected on glucose tolerance or lipid profiles (Moran et al., 2011).
Furthermore, the studies evaluated were very heterogeneous since the lifestyle treatment
consisted in diet, exercise, behavioral or combined treatments. In particular, three studies
compared physical activity to minimal dietary and behavioral advice or no advice, whereas other
three studies compared combined dietary, exercise and behavioral interventions to minimal
intervention (Moran et al., 2011).
There were no studies assessing the fertility primary outcomes of pregnancy, live birth and
miscarriage and no data for meta-analysis on ovulation or menstrual regularity. However, lifestyle
intervention provided benefits when compared to minimal treatment in terms of secondary
reproductive, anthropometric and reproductive outcomes. In particular, a significant benefit on
total testosterone levels, hirsutism, body weight, waist circumference and fasting insulin was
detected. No effect of lifestyle on BMI, free androgen index, SHBG, glucose or cholesterol levels
was observed (Moran et al., 2011).
Even if a significant effect of lifestyle intervention was observed on clinical features of

hyperandrogenism, no data for acne is actually available.
33.3.1 Comparison among different hypocaloric diets
An optimal diet is one that not only prevents nutrient deficiencies by providing sufficient nutrients
and energy for human growth and reproduction, but that also promotes health and longevity and
reduces the risk of diet-related chronic diseases, such as risks of type 2 DM, CVD and certain
cancers (Marsh et al., 2005).
Weight loss improves insulin sensitivity and reduces the risk for conversion from impaired
glucose tolerance to DM. Furthermore, the composition of the diet, independent of weight loss,
may also influence insulin sensitivity.
Several hypocaloric diets have been proposed (Table 33.2). Unfortunately, the composition of
the optimal diet for PCOS patients is not yet known, and research into the dietary management
of PCOS is lacking and most studies have focused on energy restriction rather than dietary
composition per se.
High-fat diet
Animal studies demonstrated that a diet high in fat, particularly saturated fat, may lead to insulin
resistance, whereas in humans, data regarding changes in dietary fat intake were inconclusive
(Riccardi and Rivellese, 2000). In RCTs, no significant changes in insulin sensitivity were observed
in patients with type 2 DM, such as in non-diabetic subjects, after high fat diet (Vessby, 2000). On
the contrary, epidemiological studies suggested an association between high fat intake and reduced
insulin sensitivity (Mayer-Davis et al., 1997), and increased risk of developing type 2 DM.
Table 33.2. Different hypocaloric diets.
High protein diet (>25% proteins)

High carbohydrate diet (55% carbohydrates)
High fat diet (>30% fat)
Low glycemic index diet
Data on high fat diet in PCOS women are few. One study showed an increased intake of
polyunsaturated fatty acid resulted in a significant increase in fasting glucose levels, but no effect
in insulin levels, blood lipids, testosterone or sex hormone-binding globulin levels (Katcher et al.,
2009). More recently, it was demonstrated that altering meal composition improves testosterone
levels in women with PCOS, but a high-fat Western meal was related to lower insulin and glucose
levels in comparison with a low-fat high-fiber meal (Katcher et al., 2009). Notwithstanding the
hypothetical benefits of that dietary regimen, a major concern is the risk of weight gain due to
high energy density. Thus, this is relevant in overweight and obese PCOS women, which need
weight loss; whereas it could be applicable for other indications in non-obese PCOS subject.
High-protein diet
In the last years, a new interest has been addressed in high-protein diets for weight loss, DM
management and for women with PCOS.
A number of studies in women with PCOS, overweight and obese subjects, as well as those with
hyperinsulinaemia and type 2 DM have failed to show significant long-term benefits of a high-
protein diet on weight loss or insulin sensitivity (Moran et al., 2003; Stamets et al., 2004). In
particular, in women with PCOS, a hypocaloric diet resulted in a significant weight loss and an
improvement in metabolic and reproductive abnormalities, even if a high-protein diet was no
more effective than a high-carbohydrate diet (Moran et al., 2003; Stamets et al., 2004).
On the other hand, in patients with PCOS, a hypocaloric diet supplemented with protein reduced
body weight, fat mass, serum cholesterol, and apoprotein B more than the diet supplemented with
simple sugars (Kasim-Karakas et al., 2009).
Moreover, in a successive 8-week study (Toscani et al., 2011), comparing the effect of a high
protein diet with normal protein diet in PCOS patients, it was found that calorie reduction,
rather than protein content, affects body composition and hormonal profile, ameliorating the
anthropometric and clinical phenotype.
More recently, in a prospective 6-month controlled study, Srensen et al. (2012) demonstrated
that the replacement of carbohydrates with proteins in ad libitum diets improves weight loss and
glucose metabolism by an effect independent of the weight loss.
Low-glycemic index diet
In overweight subjects without PCOS but with high postprandial insulinemia, diets with a low
GL have been shown to facilitate weight reduction and weight maintenance (Marsh et al., 2010).
Reducing postprandial insulin concentrations has been postulated to increase fat oxidation
(and spare carbohydrate stores) for several hours after a meal and to reduce hunger, overeating,
and weight gain. Because the majority of women with PCOS show a marked compensatory
hyperinsulinema after carbohydrate ingestion, there may be specific advantages of diets with a
low-glycemic index for these patients.
In a recent prospective study (Marsh et al., 2010), an ad libitum, low-glycemic index diet with
or without metformin therapy may provide an additional advantage over and above that of a
conventional healthy diet in managing the underlying insulin resistance, cardiovascular risk, and
irregular menstrual patterns in women with PCOS who are overweight but not morbidly obese.
33.3.2 Hypocaloric diet versus physical activity
Hypocaloric diet is frequently combined to physical activity and only very few studies have
compared the efficacy of both interventions.
Recently, we published a prospective study (Palomba et al., 2008b) aimed to compare in a clinical
setting the effects on reproductive functions of two different lifestyle interventions in obese
infertile PCOS patients who chose to undergo SET or a hypocaloric hyperproteic diet program.
The efficacy of both interventions was demonstrated in terms of improvements in menstrual
cyclicity and fertility, whereas a trend towards higher pregnancy and cumulative pregnancy rates
was observed after SET (Palomba et al., 2008b). Moreover, both SET and hypocaloric hyperproteic
diet, even if with peculiar mechanisms, induced hormonal and metabolic improvements within
12 weeks, without further changes at 24 weeks (Palomba et al., 2008b). In particular, significant
improvements in androgens level (only after diet) and insulin resistance indexes (for both
interventions, but to a great extent for SET) were observed (Palomba et al., 2008b). Thus, in both
cases, it was hypothesized that insulin sensitivity improvement itself is the pivotal factor involved
in the restoration of ovarian function, even if for the SET program an additional action on visceral
fat and skeletal muscle was demonstrated (Palomba et al., 2008b).
33.3.3 Hypocaloric diet versus other drugs
Few data are available regarding the comparison between hypocaloric diet and pharmacologic
therapy in patients with PCOS.
A recent prospective parallel RCT (Palomba et al., 2008a) was published with the aim to compare
the lifestyle modification, consisting in diet and exercise program, with medical treatment of
PCOS patients CC, metformin, and CC plus metformin, in obese patients with PCOS. After an
8-month period, metformin or metformin plus CC did not cause a significant weight loss or an
improvement in the endocrine status of PCOS women. On the other hand, lifestyle modification
improved menstrual cycles and hormonal pattern, avoiding the side effects of medical treatment
for PCOS. Of note, the pregnancy rate after diet and exercise program was of 20%, whereas that
obtained after medical treatment ranging from 12.2 to 14.4% (Karimzadeh and Javedani, 2010).
33.3.4 Hypocaloric diet as co-treatment
Several studies investigated the effects of hypocaloric diet, alone or integrated into a more
complex strategy for lifestyle modification, in combination with other therapeutic options on
the PCOS population.
Some evidences suggested integrated programs including the combination of diet and anti-obesity
drugs (Florakis et al., 2008; Panidis et al., 2008). In particular, an hypocaloric diet plus sibutramine
resulted in significant weight loss in overweight and obese women with PCOS (Florakis et al.,
2008). Sibutramine showed a greater weight loss and improvement in hyperandrogenemia and
insulin sensitivity after 6 months of treatment. In addition, it increased compliance to diet and it
was well tolerated. Similarly, orlistat administration, combined with diet for 24 weeks, resulted
in an acute significant weight loss and improvement of insulin resistance and hyperandrogenism
in obese women with PCOS (Panidis et al., 2008).
The effects of diet combined with metformin were also studied. Early studies (Glueck et al., 2008;
Goldenberg et al., 2005) were specifically addressed to evaluate the metabolic and hormonal
effects of the integrated programs, whereas most recent studies (Ladson et al., 2011; Otta et al.,
2010) investigated also reproductive aspects.
In an early study (Glueck et al., 2008; Goldenberg et al., 2005), it was demonstrated that
metformin plus diet had beneficial effects in women with PCOS. However, these metabolic
effects were much more marked in women in the top vs. the bottom quintile for insulin
resistance. Women with PCOS in the bottom insulin-resistant quintile, conventionally thought
not to respond optimally to metformin-diet, nevertheless experienced significant metabolic
and menstrual benefits. Successively, Glueck et al. (2008) in an observational study suggested
that PCOS women who developed type 2 DM vs. those who remained free of disease had
higher pretreatment glucose and insulin resistance, and less reduction of HOMA-IR after the
combination metformin-diet.
The most recent study by Otta et al. (2010) demonstrated that metformin has an additive effect to
diet and exercise to improve parameters of hyperandrogenism and insulin resistance. Additionally,
a small decrease in body weight through lifestyle changes could be enough to improve menstrual
cycles in insulin-resistant women with PCOS (Otta et al., 2010). Unfortunately, the superiority
of the combination of lifestyle and metformin in improving the PCOS phenotype was not
successively confirmed (Ladson et al., 2011). In fact, the addition of metformin to lifestyle therapy
produced little reproductive or glycemic benefit in women with PCOS.
Finally, preliminary experiences suggested that lifestyle interventions could induce an ovarian
sensitization to fertility drugs (Palomba et al., 2010) (Figure 33.3).
In a RCT, Palomba et al. (2010) demonstrated that a short-term lifestyle modification program
of 6 weeks had reproductive benefits in overweight and obese patients with PCOS who were
Study start Follow-up Study end
1st week 2nd week 3rd week 4th week 5th week 6th week
Group A
SET plus hypocaloric diet
Group B Observation
CC administration (150 mg daily)
Group C
Baseline clinical, Two-week clinical, Reproductive

biochemical and biochemical and outcomes
ultrasound ultrasound assessment
assessments assessments
40 *
*P=0.012 vs. groups A and B
Menstrual bleeding (%)
30
Group A
20 11/32 Group B
Group C
10
4/32 3/32
0
Figure 33.3. Effects of hypocaloric diet as co-treatment.
resistant to CC. In particular, two weeks of SET and a hypocaloric diet improved ovarian response
to CC, with an ovulation rate of 37.5% and a restoration of menstrual cycle in 34.4% of patients,
who were previously anovulatory after administration of a maximal dose of CC, and with relative
risks of about 4.0 and 3.4 for ovulation and menstrual cycle restoration, respectively.
Unpublished data by a 10-year retrospective analysis of overweight/obese PCOS women

undergoing weight loss before gonadotrophin controlled ovarian stimulation for non-IVF and
IVF cycles reported a better response to the stimulation in women who lost weight. Prospective
data on the same issue are still in progress, but preliminary results seem to confirm the
observational findings.
33.4 Main concerns to hypocaloric diet in polycystic ovary syndrome

Drop-out is one of the main concerns in studies on lifestyle modifications in PCOS patients
(Table 33.3). In fact, programs that involved lifestyle modifications are related to a very low
compliance, even if they were followed for a short-term period (Moran et al., 2003). In addition,
weight loss studies indicate that PCOS women have reduced retention rates (Clark et al., 1998;
Stamets et al., 2004) with drop-outs significantly higher than non-PCOS patients (Bowen et al.,
Table 33.3. Drop-out rates in lifestyle modification programs.
Study Follow-up (months) Drop-out rate (%)
Palomba et al., 2008b 6 35

Stamets et al., 2004 1 26
Clark et al., 1998 6 23
Clark et al., 1995 6 27
2004; Farnsworth et al., 2003) over a four-month period (30% versus less than 10% in PCOS and
non-PCOS subjects, respectively). This point is particularly important since many patients who
have to lose weight from dietary weight loss programs will eventually regain the weight outside of
a clinical research setting. In fact, the real goal is the long-term weight loss maintenance, i.e. the
reduction >10% of body weight and its maintaining for at least 1 year. To this regards, considering
that fertility depends on the number of ovulatory cycles, it is clear that lifestyle modifications
cannot be proposed in a poorly compliant infertile PCOS patient who desires to conceive as
soon as possible.
The low compliance to lifestyle modification programs is probably due to the poor adherence
to the hypocaloric diet. In a clinical trial (Palomba et al., 2008b), we detected a compliance
significantly higher in PCOS patients who underwent SET in comparison with those treated with
hypocaloric diet, with a drop-out rate of 15 and 35%, respectively.
Thus, several tips and tricks should be adopted to increase the compliance to diet program. First,
the patients management should be tailored on the basis of their personal choice. Secondly,
a well monitored and low aggressive hypocaloric hyperproteic diet should be offered. Thirdly,
an integrated lifestyle program can be useful. In particular, the use of multiple-short bouts of
exercise may facilitate greater participation in exercise programs in obese populations. Regular
aerobic exercise can, independently of weight loss, improve insulin sensitivity and thus the clinical
features in PCOS (Andersen et al., 1999). Higher intensity exercise may be more efficacious in
altering body composition and improving insulin sensitivity than aerobic endurance exercise
alone. Fourthly, periodic interactive group education meetings should be offered.
Finally, it is clear that it is possible to realize the long-term weight loss maintenance only with a
multidisciplinary approach. In fact, women with PCOS recognize the importance of diet, but few
received dietary advice from a registered dietitian. The dietary information women with PCOS
received was often from an unregulated source (Jeanes et al., 2009).
Beyond the low retention of dietary programs, women with PCOS often report difficulties losing
weight, although some studies have not shown this to be the case (Pasquali et al., 2000). Moreover,
some authors showed a heterogeneity in the responsiveness to long-term lifestyle intervention

(Pasquali et al., 2011).
33.5 Future researches

In literature, data assessing the efficacy of lifestyle modification programs on several clinical
and/or biochemical endpoint in PCOS patients are widely available. Moreover, the term lifestyle
modification programs include diet, exercise, behavioral or combined treatments. Only few
studies have assessed the impact of each component on PCOS features, because the different
arms were not controlled for each specific intervention. Thus, at the moment, we know the
importance of hypocaloric diet in PCOS, but further studies needed to evaluate its effects are
single intervention and the role of combined interventions.
The composition of the optimal diet for PCOS patients is not yet known. This point is particularly
important to hypothesize a role in the treatment of normal-weight or lean PCOS patients that are
also, even if less, insulin resistant. These patients, probably, could benefit of an increased physical
activity that may improve insulin sensitivity. Moreover, the real efficacy of these lifestyle changes
in this subgroup of PCOS patients is unknown.
33.6 Conclusions
Weight loss should always represent the first-line approach in the treatment of obese and
overweight PCOS women, since it significantly improves hormonal and metabolic abnormalities,
and may favor spontaneous ovulation improving fertility in the majority of patients. In addition,
weight loss associated with a moderate physical activity is desirable because physical exercise
improves the reduction of the insulin resistance. Unfortunately, it is very difficult to achieve these
goals in the clinical practice, especially in PCOS patients.
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Index
Index
A ART 322, 354, 389, 416

AAM 69, 72, 81, 157, 159 and obesity 407
abdominal fat 89 and vitamin D deficiency 396
abdominal symptoms 483 at advanced age 423
adipokines 409 arteriosclerosis 526
adipose tissue 85 aspartic acid 425
and age 27 assisted reproduction technologies
as endodrine organ 317 See:ART
adiposity 512 atherosclerosis 242
adrenarche 82 atresia 36, 437
age at menarche See:AAM autonomic nervous system (ANS) 52
alcohol 116, 131, 184, 305 awareness
alkylphenols 364 of folic acid benefits 298, 307
alpha-linoleic acid (ALA) 422
amenorrhea 37, 125, 210, 262, 540 B
definition 259 bariatric surgery 335, 375, 516
AMH 42, 494 behavioral change 338
AN 61, 89, 184, 215, 257 bile acid metabolism 248
mortality rate 259 biliopancreatic limb 375
anandamide 140, 144 binge eating 140, 185, 197, 478
androgens 228, 525 biped locomotion 88
anemia 282, 375 bisphenol A (BPA) 364
postpartum 380 blood
anhedonia 184 clotting 441
anorexia nervosa See:AN glucose 54
anovulation 37, 248 oxygen level dependent (BOLD) 178
anti-epileptic medication 40 BMI 158, 248
anti-hypertensives 516 during adulthood 90
anti-inflammatory drugs 479 healthy range for pregnancy 316
anti-mullerian hormone See:AMH male 26
anti-obesity drugs 545 BN 215, 257
antioxidant 23, 277, 424 definition 260
antiphospholipid antibodies 44 body fat 24, 264, 511
antral follicle count (AFC) 42 content 158
antropometry 161 body mass index See:BMI
anxiolytic treatment 475 body weight 86, 158
apoptosis 283 low ~ 213
appetite 115, 116, 172, 192 bone mineralization 260
control 112 bowel dysfunction 483
stimuli 173 brain response 173
suppression by exercise 218 gender differences 175
arsenic 369 to reward anticipation 182
Index
breast development 159 cholecystokinin See:CCK

bromelain 353 cholesterol 241, 515
bulimia nervosa See:BN testing 245
CL 37, 50
C climate 72
cacao clomiphene 419
active ingredients 141 citrate (CC) 544
beans 140 coagulation factors 231
pharmacology 143 cocaine 184, 185
cadmium 368 co-enzyme Q10 (CoQ10) 29
caffeine 131, 144 constipation 480
calcaemia 125 controlled ovarian hyperstimulation
calcitriol 125 See:COH
calcium 98, 279 controlled ovarian stimulation See:COS
absorption 102, 127 copper 278
deficiency 113 Corpus Luteum See:CL
metabolism 127 corticolimbic
regulating hormones 99 network 185
supplementation 97, 101 reward regions 182
calciuria 125, 127 cortisol 53, 477
caloric cows milk allergy 462
depletion 143 craving 61, 199
intake 171, 197, 215, 265 chocolate 139
carbohydrate cue-elicited 147
consumption 116 cultural factors 149
rich diet 117 food 139
cardiovascular disease See:CVD gender differences 148
cardiovascular reactivity 49 hormonal fluctuations 145
l-carnitine 29 post-menopause 145
-carotene 467 prevalence 141
catch up growth 162 critical fat mass 87
CCK 195, 217 Crohns disease 485
Celiacs Disease 459 CVD 52, 241, 509
childlessness 356 risk factors 513
Chlamydia trachomatis 42 cytotoxicity 464
chocolate
ambivalent views 148 D
and guilt 146 dairy 457
orosensory properties 145 D-chiro-inositol (DCI) 419
perimenstrual craving 139 decidua 391
psychoactive effects 142 dehydroepiandrosterone (DHEA) 29
cholecalciferol 390 deoxyhemoglobin 173
Index
depression 60 shedding 37
de-satiating effect 195 endometriosis 39, 350, 426, 451, 473, 497
diabetes mellitus See:DM associated infertility 427
diet 215 non-genital 479
dietary behavior 130 rectovaginal 482
dietary intolerance 454 endometrium 191
dietary restraint 147 energetic costs
guidelines prior to conception 334 of gestation and lactation 87
hypocaloric 545 energy
hypocaloric hyperproteic 544 availability 212
dioxin 464 deficiency 209
DM 310, 523 deficit 210, 216
type 2 510 intake 114, 196
DNA 435 enterocyte senescence 376
synthesis 283 environmental contaminants 464
dopamine 184, 185, 193 epigenetic modifications 331
Down syndrome 19 epoxy resins 364
drive for thinness (DT) 218 ergocalciferol 390
drugs 306 estradiol 191, 244
dyslipidemia 510 implants 194
dysmenorrhea 123, 128, 476 estrogen 52, 242, 318
dyspepsia 474 affecting intestinal motility 480
dysphoria 97, 144 anorexigenic effect 179
dysthymia 539 cardioprotective effects 57
colon reabsorption 246
E receptors 193
early pregnancy loss (EPL) 234 ethnicity 301
eating disorder 257 excessive
inventory 217 dieting 318
not otherwise specified See:ED-NOS nutrition 19
ectopic endometrial cells 465 exercise 24, 71, 212, 218, 514, 529
ED-NOS 215, 261 acute 219
educational level 303 structured training (SET) 544
eicosapentaenoic acid 463 exogenous gonadotrophins 41
elocalcitol 428, 496 extravillous trophoblast (EVT) 285
embryo implantation 191
encephalization 88 F
endocrine disruption 84 FA 39, 278, 295, 420
compounds (EDCs) 361 high intake 439
effects 364 periconceptional supplement 296
endometrial preconceptional supplement 297
cells 50 supplementation and maternal age 300
Index
fat food
free mass (FFM) 213 appeal 178
intake 114, 462, 542 contamination 366
fatness 161 cravings 112
fear of ~ 199 cues 174
underestimation 337 intake 193
fatty acids 28 intolerance 455
metabolism 20 packaging 362
omega-3 132 FSH 36, 83, 404
fear of fatness 199 fuel receptors 319
fecundability 35 fusiform 179
ratio (FR) 321
female athlete triad 211, 266 G
ferritin 280, 376 GABA 59, 113
fertility galattorrea 229
decision to reduce ~ 25 gamma-aminobutyric acid See:GABA
seaonal variation 493 gastric acid 379
treatment 307, 315 gastric emptying 474
fetal macrosomia 527 gastro-esophageal reflux 478
FF 393, 416, 437 gastro-intestinal
fiber intake 128, 242, 246 hormones 219
fight-or-flight response 52 symptoms 473
fish oil 462 genetic influence 84
flatulence 483 geographical location 302
folate gestational diabetes 330, 338, 527
deficiency 379, 433 ghrelin 92, 174, 195, 210, 218
effects of deficiency 435 gliadin 458
maternal ~ status 287 glucose
metabolism 435 intolerance 536
proton-coupled transporter (PCFT) 434 tolerance 331
reduced carrier (RFC) 434 gluten 458
folate-mediated one-carbon metabolism 434 glycemic
folic acid See:FA index 131
follicle 19 load (GL) 543
development with exogenous FSH 525 GnRH 36, 51, 82, 210, 248, 319
folliculogenesis 438 goitre 232
stimulating hormone See:FSH gonadotrophins 50, 439
follicular gonadotropin releasing hormone
atresia 36 See:GnRH
fluid See:FF granulosa cell 228
phase 37, 55, 175 green vegetables 466
recruitment 529 guilt 140
Index
H hypoleptinemia 264
Hcy 288, 419, 435, 438 hypothalamic ovarian feedback 158
embryotoxic effect 441 hypothalamic-pituitary-adrenal axis
HDL-C 243, 244, 510 See:HPA axis
heartburn 474 hypothalamic-pituitary-gonadal axis
heavy metals 366 See:HPG axis
hedonia 184 hypothalamic-pituitary-ovarian axis
hemoglobin 375, 377 See:HPO axis
hemosiderin 377 hypothalamus 172, 219
hepcidin 377 hypothyroidism 425
herbal and ovulatory dysfunction 233
placebo effects 355 fetal 226
practitioners 355 prepubertal 226
product registration 355 hypothyroid women 231
products 346, 348 hypothyroxinemia 225
products for female fertility 352 hysterosalpingography (HSG) 43
heroin 184 hysteroscopy 43
HHCY 279, 284, 287
high-density lipoprotein cholesterol I
See:HDL-C ID 225, 232
hirsutism 539, 541 deficient areas 231
homeostatic model assessment (HOMA) immune cells 452
526 immunological response 452
homocysteine See:Hcy implantation 283, 407, 440
hormonal disturbances 113 impotence 354
hormone therapy (HT) 241 incentive sensitization 147
HOXA10 392, 494 industrialization 24
HPA axis 53 infant mortality 23
HPG axis 50, 317 infertility 35, 348, 433
immaturity 262 inflammatory
HPO axis 209 bowel disease 486
hyperandrogenism 405, 511, 525, 536, 541 instigators 466
hyperglicaemia 409 ingestive behavior 175
hypergonadotropic hypogonadism 495 inositol 419, 529
hyperhomocysteinemia See:HHCY insemination 40
hyperinsulinemia 162, 409, 510 insula 200, 219
hyperoestrogenaemia 408 insulin 406
hypertension 58, 338, 512 growth factor 1 (IGF-I) 266
pregnancy induced 512 insulinemia 543
hypocalcemia 97 peripheral resistance 537
hypocaloric diet 528, 542 resistance (IR) 408, 418, 510, 525, 537
hypogonadotropic hypogonadism 163, 263 sensitivity 497
Index
sensitizing drugs 515 lipoprotein cholesterol 242

intestinal motility 484 cyclic changes 245
intrauterine growth retardation See:IUGR in postmenopausal women 249
in vitro fertilization See:IVF lipoprotein metabolism 242
iodine longevity 25
deficiency See:ID low birth weight (LBW) 23, 282
repletion 229, 232 low-density lipoprotein cholesterol
supplementation 227 See:LDL-C
iron 280, 376 lower intestinal tract 479
requirement during pregnancy 381 LPD 212, 420, 427
irritable bowel syndrome 485 luteal phase 37, 55, 175
IUGR 162, 280 defect See:LPD
IVF 389, 438, 546 hyper-reactivity 60
and vitamin D deficiency 396 luteinizing hormone See:LH
cycle 393
M
K macrophages 460
Kallmanns syndrome 263 magnesium 130, 143
kisspeptin 211 male fertility 283
manganese 103, 130
L maternal malnutrition 379
lactose intolerance 460 meal
laparoscopic adjustable gastric band (LAGB) size 193
330 timing 219
laparoscopy 43, 455 melatonin 421
L-arginine 422 menarche 69
LDL-C 243, 244, 510 and body weight 90
lead 368 early ~ and risk of diseases 90
learned associations 199 health effects 70
LEPR 408 precociuos 403
leptin 91, 163, 196, 210, 248, 264, 404 menopause
receptor See:LEPR earlier onset 404
levo-thyroxine (LT4) 233 menorrhagia 230, 476
LH 37, 83, 210, 264, 404, 422, 497 menses 69
pulsatility 218 mercury 369, 426
lifestyle changes 336, 337, 513, 528 metabolic
compliance 547 clearance rate (MCR) 228
effectiveness 513 fuel hypothesis 318
lipiodol 227 syndrome 507, 525
lipolysis 537 metformin 515, 544, 545
lipophilicity 361 methionine cycles 285
Index
methylenetetrahydrofolate reductase O
See:MTHFR obesity 39, 84, 161, 283, 315, 403
methylxanthines 144 and PCOS 511, 539
metrorrhagia 230 childhood 89
micronutrients 275 drug treatment 334
milk 105 prior to pregnancy 329
mitochondrial function 29 oligomenorrhea 37, 212, 261
molimina 59 omega 3 fatty acids 422
monetary reward 181 oocyte 436
mood age 40
enhancing neurotransmitters 144 aneuploidy 424
state 144 atresia 30
swings 115 quality 317, 416
symptoms 98 orbitofrontal cortex (OFC) 174
Moos Menstrual Distress scores 484 orexigenic effects 195
morning sickness 299 organochlorine substances 361
MTHFR 435, 441 orocaecal transit 482
multiparous women 308 osteoporosis 128, 209, 260
multiple micronutrient supplementation 426 ovarian
multivitamin supplements 378 controlled hyperstimulation (COH)
muscle metabolism 216 438, 439
myo-inositol (MYO) 419 controlled stimulation (COS) 415
cycle in in rats 191
N dysfunction 233, 536
neural tube defects See:NTD infertility 329
neuroimaging 173, 200 ovariectomy 124
neuropeptide Y See:NPY poor response (POR) 420
neurotransmitters 478 reserve 40
nicotine 184 steroidogenesis 539
nocturnal blood pressure regulation 512 steroids 171, 185
nomadic populations 21 overweight 86
non-milk extrinsic sugar (NMES) 115 ovulatory phases 50
nonylphenol 365
NPY 194, 211, 407 P
NTD 278, 287, 295, 308 pain relief 353
nurturing 25 parasympathetic nervous system (PNS) 52
nutraceuticals 415, 417 parathyroid hormone See:PTH
nutrient intake 112, 114 parity 307
nutritional requirements 21 PCOS 24, 86, 262, 329, 352, 361, 406, 418,
nutrition in utero 26 497
definition 507, 523, 535
in families 537
Index
peak bone mass attainment 266 care 332, 336

peak height velocity 161 diet 280
pelvic preeclampsia 275, 286, 527
infection 43 pregnancy 275
minimum ~ breadth for menarche 88 premenstrual
peptide YY See:PYY dysphoric disorder See:PMDD
periconceptional period 280 syndrome See:PMS
peritoneum 452 prepubertal fat mass 162
personality disorders 259 primordial follicles 36
pesticides 365 progesterone 42, 52, 58, 196
phthalates 365 challenge test 261
physical activity 265, 320 withdrawal effect 59
phytic acid 529 progestin 171
phytoestrogen 129 prolactin 477
phytomedical research 350 prolactinoma 264
phytotherapy 346 propylthiouracile (PTU) 226
placenta 285 protein 162, 165, 457
placental deficiency 23
endovasculature 287 high-protein diet 543
ischemia 286 intake 127
placentation 407 psychological disorders 475
plasma lipid profile 245 psychophysiological stress 52
PMDD 59, 97, 111, 112, 124 PTH 98, 126
PMS 60, 111, 496 pubertal hormonal transition 89
and diet 116 public health interventions 339
symptoms 97, 123 PUFA 28, 462, 482
treatment 124 PYY 211, 218, 219
polycarbonate plastics 364
polycystic ovarian syndrome See:PCOS R
polymenorrhoea 37 radiation 40
polymorphisms 439 RDA 215
polyploidy 19 reactive oxygen species See:ROS
polyunsaturated fatty acids See:PUFA recommended dietary allowance See:RDA
postcoital test (PCT) 43 red meat 457, 466
postpartum depression 129 intolerance 378
Pouch of Douglas 482 resting metabolic rate (RMR) 218
Prader-Willi Syndrome 174 retinoic acid (RA) 288
prebiotics 461 retrograde menstruation 451
precocious puberty 229, 537 reward satiation 185
precocity 69 ROS 277, 283
preconception Roux-en-Y gastric bypass See:RYGB
behaviours 309 rubella 41
Index
RYGB 375 sweet food 197

and anemia 377
dietary guidelines 380 T
TCM 345, 350
S teenage pregnancies 90
satiety telarche 161
biomarker 217 testosterone 497
hormones 218 thelarche 160
seafood 369 theobromine 144
selenium 277, 425 thiazolidinediones (TZDs) 515
selenocysteine 425 thyroid hormone (TH) 225
self-esteem 539 thyroid stimulating hormone (TSH) 229
semiallogenic conceptus 276 and poor IVF 234
serotonin 113, 116, 130, 197 thyrotropin releasing hormone See:TRH
selective reuptake inhibitor (SSRI) 113 thyroxine (T4) 230
SES 72, 164 time-to-pregnancy (TTP) 320
sex hormone binding globulin See:SHBG negative lifestyle variables 322
sexual maturation 81 tonic inhibitory effect 192
SHBG 38, 405, 528 traditional Chinese medicine See:TCM
sibutramine 545 transferrin 377
smoking 305, 425 transitional society 164
social stratification 165 TRH 229
socioeconomic status 302, 336 See:SES tests 232
soy 129 tryptophan 114, 116, 143
sperm 496 tubal infertility 38, 353
chromatin integrity 26 tunica albuginea 494
count and birth weight 26
count decrease 364 U
motility 354 undernourishment 266
spermatogenesis 392, 495 underweight 316, 322
spontaneous abortion 24 uterine pathologies 39
starch 457
statins 515 V
stress 354 VDR 390, 491
stressor vegetarian diet 128, 216
cognitive 55 ventromedial hypothalamus (VMH) 193
laboratory 55 visceral fat 538
physical 55 visceromegaly 409
subcutaneous fat mass 511 visual food cues 173, 200
subfertility 35 vitamin A 279, 288
causes 351 vitamin B 427, 529
subsistence diet 21 vitamin B6 130, 284, 287, 438
Index
vitamin B12 279

deficiency 379
vitamin C 277, 420, 427
vitamin D 98, 126, 389
25-hydroxyvitamin-D serum levels 126
and sperm quality 392
binding protein (DBP) 397
deficiency 100, 491
effects of deficiency 391
metabolites in follicular liquid 392
receptor See:VDR
sources 132
supplementation 127, 498
vitamin E 277, 427
vitamins 216
Von Willebrands disease (aVWD) 231
W
water retention 104
weaning size 25
weight gain in post-menopause women 171
weight loss 330, 537
and conception 333
and PCOS 541
cultural barriers 338
maintenance 335
medications 516
pre-pregnancy 332
X
xanthine alkaloids 131
Z
zinc 278, 288
About the editors
Caroline J. Hollins Martin PhD MPhil BSc ADM PGCE RMT RM RGN MBPsS, is a Professor
in Midwifery in the College of Health and Social Care at the University of Salford, United
Kingdom. Her background has encompassed a career in womens reproductive health that
spans 26 years; the first 11 of these were spent as a clinical midwife in Ayrshire (Scotland) and
15 teaching and researching womens reproductive health within universities. She is a NMC
Registered Midwife and Lecturer/Practice Educator. She is also a graduate and postgraduate in
psychology and a Member of the British Psychological Society (MBPsS). Her research interests
lie in social psychology that relates to womens reproductive health, with much of her work
relating to obstructing autonomy, evidenced-based practice and providing choice and control
to childbearing women. More recently focus has shifted to developing useful tools for maternal
health practitioners to use in clinical practice. For example, the Birth Participation Scale (BPS)
to assess fathers fears and needs in relation to childbirth and the Birth Satisfaction Scale (BSS)
to assess mothers perceptions of their birth experience. Current research interests lie in shaping
perinatal bereavement care, outcomes of maternal activity during labour and the effects of music
upon womens stress levels. She has published numerous peer-reviewed original research papers
and presented her work extensively at leading conferences. She is an associate editor for womens
reproductive health papers submitted to the Journal of Nurse Education in Practice.
Colin R. Martin RN BSc PhD YCAP CPsychol CSci AFBPsS is Chair in Mental Health at the
University of the West of Scotland, Paisley, United Kingdom. Professor Martin is a Chartered
Health Psychologist and a Chartered Scientist. Having originally trained in mental health
nursing, he specialised in the addictions and following further training, worked as a community
psychiatric nurse and then as an addictions counsellor in the NHS. On completion of BSc and
PhD degrees in psychology, Professor Martin worked in senior management posts in the NHS
followed by academic posts in the UK and the Far East, during which he conducted original
research on the relationship between mental health and many aspects of chronic disease. He has
published over 100 scientific papers in psychology, biology, medical and nursing journals and has
a long-standing research interest in reproductive psychology and perinatal mental health. He is
editor of the influential Journal of Reproductive and Infant Psychology.
Olga B.A. van den Akker BSc PhD AFBPsS, C.Psychol is Professor of Health Psychology and
Head of Department of Psychology at Middlesex University in London, United Kingdom. Since
completing her BSc in Psychology at the University of East London, she was awarded an MRC
studentship at St Georges Hospital Medical School, University of London to carry out a PhD
investigating the Psychophysiology of the menstrual cycle. This was followed by post doctoral
research at the Institute of Psychiatry and St. Marys hospitals London, and senior academic posts
565
at the University of East London, University of Birmingham and Aston University. She was an
HFEA Advisor, Editor in Chief of the Journal of Reproductive & Infant Psychology and has been a
member of the executive committees of the British Infertility Counseling Association, the British
Fertility Society and the Society for Reproductive & Infant Psychology. She is currently on the
editorial board of the Journal of Reproductive Psychology, Biomed Central and Expert Reviews
of Obstetrics and Gynecology. Her research interests cover a variety of Health Psychology areas
with a focus on reproductive health psychology, using qualitative, quantitative and experimental
research techniques. She has secured funding on a number of research grants and was awarded
a prestigious 5 year Senior Research Fellowship by the NHS R&D. Olga has authored two books
and has written numerous book chapters, two Government commissioned reports and well over
a 100 publications in scientific journals, presented over 70 conference presentations and appears
regularly in the media.
Victor R. Preedy BSc, PhD, DSc, FSB, FRCPath, FRSPH FRSC is Professor at Kings College
London and also at Kings College Hospital. He is attached to both the Diabetes and Nutritional
Sciences Division and the Department of Nutrition and Dietetics. He is also Director of the
Genomics Centre and a member of the School of Medicine. Professor Preedy graduated in 1974
with an Honours Degree in Biology and Physiology with Pharmacology. He gained his University
of London PhD in 1981. In 1992, he received his Membership of the Royal College of Pathologists
and in 1993 he gained his second doctoral degree, for his outstanding contribution to protein
metabolism in health and disease. Professor Preedy was elected as a Fellow to the Institute of
Biology in 1995 and to the Royal College of Pathologists in 2000. Since then he has been elected
as a Fellow to the Royal Society for the Promotion of Health (2004) and The Royal Institute of
Public Health (2004). In 2009, Professor Preedy became a Fellow of the Royal Society for Public
Health and in 2012 a Fellow of the Royal Society of Chemistry. In his career Professor Preedy has
carried out research at the National Heart Hospital (part of Imperial College London) and the
MRC Centre at Northwick Park Hospital. He has collaborated with research groups in Finland,
Japan, Australia, USA and Germany. He is a leading expert on the science of health. He has
lectured nationally and internationally. To his credit, Professor Preedy has published over 570
articles, which includes 165 peer-reviewed manuscripts based on original research, 90 reviews
and over 50 books and volumes, of which several Human Health Handbooks.

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cycle, periconception and fertility

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Setting the scene

3. P sychophysiological changes in the menstrual cycle phases 47

Puberty, menarche and the menstrual cycle

5. Menarcheal onset and body composition 79

6. Calcium intake and premenstrual syndrome 95

11. Neuroimaging menstrual cycle associated changes in appetite 169

14. Iodine deficiency: female menstrual cycle to conception 223

15. The menstrual cycle and lipid levels 239

16. Menstrual patterns in adolescents with eating disorders 255

18. Prevalence and predictors of periconceptional folic acid use 293

21. Herbal medicine and fertilisation 343

22. Diet containing endocrine-disruptors and reproductive health 359

23. Anemia, Roux-en-Y gastric bypass and conception 373

27. Folate and female infertility: folate-metabolizing pathway in folliculogenesis, infertility

28. Dietary intolerance and endometriosis: an immunological link in the pathogenesis of an

29. Gastro-intestinal symptoms in women with pelvic endometriosis 471

31. Polycystic ovary syndrome and the metabolic syndrome 505

About the editors 565

1.1 Reproduction and natural selection in humans

1.2 Reproduction in women of different ages

Country Life expectancy female Births per female

Chad 46.7 4.93

Because of these critical age-related changes, it is necessary to consider the nutritional

1.3 What is the minimum diet for fertility in young women?

Nutrient Okra, 100 g Millet, 1 cup cooked Milk, 1 cup

Protein 4% 12% 16%

1.3.2 Energy expenditure versus calorie intake and fertility

1.3.4 Does poor nutrition cause spontaneous abortion?

1.3.5 Infertility in malnourished women

1.3.6 Excess calories as a cause of infertility in developed countries?

So if severe under-nutrition is a cause of infertility in the poorest of nations, is over-nutrition a

1.4 Parental investment in size of offspring and decreasing fertility

Table 1.3. Male body mass index and infertility.1

Body mass index Pregnancy Nine months infertility Total

Less than 18.5 9 0 (0%) 9

Birth weight (g)

1.6.1 Fatty acid supplementation

1.6.2 Supplementation with mitochondrial antioxidants: l-carnitine, coQ10

1.6.3 DHEA-S supplementation

1.6.4 Endurance and aerobic interval training

1.6.5 Suggestions for future work in counteracting ovarian ageing

LE1 5WW, United Kingdom; tarek.gelbaya@uhl-tr.nhs.uk

Keywords: fertility, fecundity, reproduction, subfertility, ovulation, endometriosis

2.2 Female reproductive physiology

An intact hypothalamic-pituitary-ovarian-uterine axis is the cornerstone of a healthy female

Table 2.1. Relative prevalence of the aetiologies of infertility (%).

Male factor 25-40

2.3 Factors affecting female fertility

Ovulation dysfunction is usually indicated by absence of menstruation (amenorrhoea),

Table 2.2. WHO classification of ovulatory disorders.

Incidence Biochemistry Causes

Class I: Hypothalamic pituitary 10% Low FSH/LH/oestrogen Kallmanns syndrome

Hyperprolactinemia impairs ovulation and requires cranial MRI to exclude pituitary

2.3.2 Tubal factor

2.3.4 Uterine factors

2.3.5 Life style factors

2.4 Prognostic factors of female fertility

3. P sychophysiological changes in the menstrual cycle phases 47

11. Neuroimaging menstrual cycle associated changes in appetite 169

14. Iodine deficiency: female menstrual cycle to conception 223

16. Menstrual patterns in adolescents with eating disorders 255

18. Prevalence and predictors of periconceptional folic acid use 293

22. Diet containing endocrine-disruptors and reproductive health 359

23. Anemia, Roux-en-Y gastric bypass and conception 373

27. Folate and female infertility: folate-metabolizing pathway in folliculogenesis, infertility

28. Dietary intolerance and endometriosis: an immunological link in the pathogenesis of an

29. Gastro-intestinal symptoms in women with pelvic endometriosis 471

31. Polycystic ovary syndrome and the metabolic syndrome 505