Best Practice & Research Clinical Obstetrics & Gynaecology

Vol. 17, No. 2, pp. 211 –229, 2003

doi:10.1053/ybeog.2003.359, www.elsevier.com/locate/jnlabr/ybeog

Male infertility

E. Iammarrone MD
Medical Doctor

R. Balet MD

Medical Doctor
The Bridge Centre, 1 St Thomas Street, London Bridge, London SE1 9RY, UK

A. M. Lower FRCOG
Medical Director
Isis Fertility Centre, Colchester CO4 9YA, UK

C. Gillott BS c (Hons)
Embryologist
Centre for Reproductive Medicine, St Bartholemew’s Hospital, London EC1A 7BE, UK

J. G. Grudzinskas* FRCOG , MD

Professor
Department of Obstetrics and Gynaecology, St Bartholomew’s Hospital, London EC1A 7BE, UK

Infertility affects 13 – 18% of couples and growing evidence from clinical and epidemiological
studies suggests an increasing incidence of male reproductive problems. The pathogenesis of male
infertility can be reflected by defective spermatogenesis due to pituitary disorders, testicular
cancer, germ cell aplasia, varicocele and environmental factors or to defective sperm transport
due to congenital abnormalities or immunological and neurogenic factors. Recent studies suggest
an increased incidence of genetic disorders related to male infertility which may affect different
levels, interfering with germ cell generation and maturation or leading to the production of non-
functional spermatozoa. The identification of genetic causes of male infertility raises the issue of
the transmission of defects to the offspring, a situation that is becoming more important given the
increasing use of intracytoplasmic sperm injection (ICSI), a procedure in which the natural
selection of the spermatozoa is by-passed. Fertilization can occur in vitro using ejaculated,
epididymal or testicular spermatozoa, either fresh or frozen– thawed, providing opportunities
hitherto not possible for men to be genetic fathers.

Key words: male infertility; unexplained male infertility; assisted reproductive technologies;
intracytoplasmic sperm injection; oligo-azoospermia; male genetic infertility.

* Corresponding author. Tel.: þ44-207-7403-3363; Fax: þ44-207-7403-8552.

E-mail address: jggrudzinskas@thebridgecentre.co.uk (J. G. Grudzinskas).

1521-6934/03/$ - see front matter Q 2003 Elsevier Science Ltd. All rights reserved.
212 E. Iammarrone et al

CLINICAL INVESTIGATION

The following three aspects of male fertility should be considered in management

strategies (Table 1).

1. The history may identify the causes of the infertility and detect the presence of
reversible factors (drugs, smoking, endocrine diseases).
2. Clinical examination may detect the presence of abnormal genitalia (hypospadias,
atrophic testes and varicoceles). Varicoceles have been controversially associated
with male infertility and despite numerous studies of the pathophysiology, the
benefits of surgical treatment are still unclear.1,2
3. Semen analysis, endocrine and sperm antibody tests are useful in the identification of the
aetiology of male infertility. The follicle stimulating hormone (FSH) level is an
indicator of sperm production, while testosterone and luteinizing hormone (LH)
levels are indicative of Leydig cell dysfunction.3

Antisperm antibodies (ASAs) can impair spermatozoal function at different

stages: sperm motility, cervical mucus penetration and oocyte penetration. In the
most severe cases, intracytoplasmic sperm injection (ICSI) is the only treatment.4
The screening tests for ASAs are the immunobead test (IBT) and the mixed anti-
globulin reaction (MAR) test5,6, both being performed on a fresh semen sample. To
be valid at least 200 motile spermatozoa have to be available for counting. The IBT
is able to detect the presence of antibodies on the sperm surface. At least 50% of
the motile spermatozoa need to be coated with immunobeads (polyacrylamide
spheres with covalently bound rabbit anti-human immunoglobulins) before the test
can be considered to be clinically significant.7 The MAR test is diagnostic of
immunological infertility when 50% or more of the motile spermatozoa have
adherent particles.8 Corticosteroid therapy has been used in these men, but ICSI is
now the primary treatment of choice.9

Table 1. Clinical evaluation for male infertility.

History Duration of infertility

Previous conceptions
Intercourse
General illness
Alcohol, smoking
Examination Testicular size (4.0–5.5 cm long)
Identification of vas deferens at the neck of the scrotum
Identification of the epididymis behind the testis
Identification of varicocele
Investigation Semen analysis
Endocrine tests: FSH, LH, testosterone
Sperm antibody tests: MAR, IBT

FSH, follicle-stimulating hormone; LH, luteinizing hormone; MAR, mixed anti-globulin reaction, IBT,
immunobead test.
 Male infertility 213

Table 2. Nomenclature for semen variables.

Normospermia Normal ejaculate (as defined by reference values)

Oligozoospermia Sperm concentration ,20 million/ml
Asthenozoospermia ,50% spermatozoa with forward progression or
,25% spermatozoa with rapid progression
Oligoasthenoteratozoospermia Disturbance of all three variables
Teratozoospermia ,30% spermatozoa with normal morphology
Azoospermia No spermatozoa in the ejaculate
Aspermia No ejaculate

Source: WHO (1999).12

SEMEN ANALYSIS

Semen analysis represents the initial test for evaluating male-factor infertility. Semen
analysis includes examination of the spermatozoa and the seminal fluid, semen being a
mixture of spermatozoa suspended in a secretion from the testis and epididymis that is
combined with secretions from the prostate, seminal vesicles and bulbourethral glands.
The nomenclature for semen constituents is described in Table 2. During the initial
macroscopic examination the following characteristics are considered:

1. Liquefaction. A normal semen sample liquefies within 60 minutes at room

temperature, but usually this occurs within 15 minutes.
2. Appearance. A normal sample has a homogenous grey-opalescent appearance. It can
be less opaque if the sperm concentration is very low and red-brown when red
blood cells are present.
3. Volume. In normospermia the volume is 1.5 – 5 ml of semen/ejaculation.
4. Viscosity. The viscosity can be evaluated by gentle aspiration into a wide-bore 5 ml
pipette. The semen is then allowed to drop by gravity. A normal sample leaves the
pipette as small drops.
5. pH. Semen pH is normally slightly alkaline (range 7.2 –8.0).
6. Sperm concentration. Normozoospermic values are derived from large population
studies that established a statistically significant lower conception rate when the
sperm concentration was less than 20 million/ ml.10,11,12
7. Sperm motility. The motility of spermatozoa can be classified as:
* Class A: rapid progressive motility.
* Class B: slow or sluggish progressive motility.
* Class C: non-progressive motility.
* Class D: immotility.

Normal semen samples have 50% or more motile sperm, most of these exhibiting
good to excellent forward progression up to 3 hours after ejaculation.
8. Sperm morphology. Normal sperm have an oval head with a well defined acrosomal
region that comprises 40 –70% of the head area. The midpiece must be axially
attached. The tail should be uniform, slightly thinner than the midpiece, uncoiled and
free from bends or twists. The proportion of normal forms should be $ 30%.
9. Cellular elements other than spermatozoa. A normal ejaculate should not contain more
than 5 million round cells/ml and the number of leukocytes should not exceed
214 E. Iammarrone et al

1 million/ml. The round cell population includes round spermatids, spermatocytes,

spermatogonia and exfoliated epithelial cells.

Although semen analysis is routinely used to evaluate the male partner, the
numerical aspects of sperm parameters to discriminate between fertile and infertile
men are yet to be agreed universally. A recent study13 has shown that although the
sperm assessment (concentration, motility, morphology) can help to distinguish
between fertile and infertile men, none of the parameters is diagnostic of infertility. The
authors classified the sperm analysis into three ranges: subfertile, fertile and
indeterminate fertility. The subfertile ranges were defined as a sperm concentration of
, 13.5 million/ml, motility , 32%, normal morphology , 9%; the fertile ranges were
associated with a sperm concentration of . 48 million/ml, motility . 63%, normal
forms . 12%. The values between these ranges indicate an indeterminate fertility.

PATHOGENESIS OF MALE INFERTILITY

Male infertility can be considered as a syndrome that results from many congenital or
acquired illnesses but most infertile men are healthy and have no symptoms (Table 3).
Ejaculated sperm can be abnormal because of testicular dysfunction or impaired
stimulation of normal testis or because the sperm produced do not develop normally
or are damaged in the extratesticular genital tract. Moreover, there can be azoospermia
as the result of a defect of sperm migration. Inflammation and infections of the
reproductive tract, such as prostatitis, can be a cause of male infertility due to
detrimental effects on the quality of the sperm.14 In particular, mediators of the
inflammatory reaction can be responsible for oxidative stress of the seminal plasma,
which is known to impair the sperm parameters.15
Mechanical infertility is classified on the basis of ejaculatory dysfunction such as
retrograde ejaculation and failure of ejaculation and it is always related to a problem
with coitus (erections, vaginal penetration and ejaculation). Erectile dysfunction can be
corrected with Viagrae in the majority of men.16
It has also been postulated that the increased incidence of male reproductive
problems can be related to genetic disorders17,18, such as the testicular dysgenesia
syndrome (TDS) as a result of disruption of embryonal programming and gonadal
development during life. The syndrome is characterized by genital tract abnormalities,
testicular cancer, reduced semen quality and subfertility. The recent rise in the
prevalence of TDS may be linked to endocrine disrupters affecting genetically
susceptible individuals.19

UNEXPLAINED MALE INFERTILITY

Unfortunately, in nearly 50% of infertile men it is not possible to find a cause of

infertility and this situation has been defined as unexplained or idiopathic. However, in
this group of men, an alteration of the sperm parameters is frequently present, such as
decreased sperm concentration, diminished motility and morphological abnormalities.
Sperm maturation is a complex process involving events that occur in the
seminiferous epithelium and during the sperm migration through the seminiferous
tubules and epididymis. The membrane and nuclear remodelling that occurs during
 Male infertility 215

Table 3. Causes of men infertility.

A. Primary gonadal disorders

Congenital: Y chromosome deletions
XYY male
Klinefelter’s syndrome
Cryptorchidism
Congenital anorchia
Noonan’s syndrome
Myotonic muscular dystrophy
Sickle cell disease
Varicocele
Androgen insensitivity syndrome
5a reductase deficiency
Acquired: Orchitis: mumps pyogenic, traumatic
Malignant neoplasma: germ cell, leukaemia, lymphoma
Trauma
Torsion
Castration
Systemic illness (e.g. renal failure, hepatic cirrhosis,
cancer, liver disease)
Retroperitoneal fibrosis
Drugs (e.g. cytotoxic drugs, alkylating agents, alcohol,
marijuana, anti-androgens, ketoconazole,
spironolactone, histamine receptor antagonist)
Environmental toxins (e.g. dibromochloropropane,
carbon disulphide, cadmium, lead, mercury,
environmental oestrogens, phyto-oestrogens)
Irradiation
Hyperthermia
B. Hypothalamic-pituitary disorders
Congenital: Congenital GnRH deficiency (Kallmann’s syndrome)
Haemochromatosis
Multiorganic genetic disorders (Prader-Willi syndrome,
Laurence-Moon-Beidl syndrome, familiar cerebral ataxia)
Acquired: Pituitary and hypothalamic tumours and cysts
Infiltrative disorders (sarcoidosis, histiocytosis,
tuberculosis)
Trauma, post-surgical, post-irradiation
Vascular (infarction, aneurysm)
Hormonal (hyperprolactinaemia, androgen, oestrogen
and cortisol excess)
Drugs (opioids and psychotropic drugs, GnRH agonists
or antagonists)
Systemic disorders: Chronic illnesses
Obesity
Nutritional deficiencies
Disorders of sperm transport (post-testicular)
Epididymal dysfunction (drugs, infection)
Abnormalities of the vas deferens (congenital absence,
Young’s syndrome, infection, vasectomy)
Ejaculatory dysfunction (spinal cord disease,
autonomic dysfunction, premature ejaculation)

GnRH, gonadotrophin releasing hormone.

216 E. Iammarrone et al

LH FSH
Negative feedback Negative feedback

Leydig cells Testosterone Sertoli cells Inhibin

Spermatogenesis

Spermatogonia (diploid) Spermatogonia are situated close to the basement

membrane between the Sertoli cells. Multiple mitosis
produces other spermatocytes or primary
spermatocytes

Primary spermatocyte
Secondary spermatocyte
Spermatocytes divide by meiosis to form four
spermatids. During this event there is: a random
separation of homologous chromosomes and the
crossing over of genetic material. Each primary
spermatocyte gives rise to two secondary
spermatocytes

Spermatids (haploid) The transit from spermatid to spermatozoa is the

process during which the cell changes to a highly
differentiated spermatozoon

Spermatozoa The cytoplasm extensions with the Sertoli

cells are broken and the spermatozoa are released.
The spermatozoa maturation occurs during the transit
through the epididymis, where the secreted proteins
confer their potential for mobility and fertilization

Figure 1. Spermatogenesis cycle. The spermatogenesis cycle takes 70 days. Spermatogenesis is under the
control of two somatic cells: the Leydig cells which have LH receptors and Sertoli cells which have FSH
receptors. LH stimulates Leydig cell synthesis of testosterone, which acts on Sertoli cells together with the
FSH to support all of the phases of spermatogenesis. Testosterone exerts a negative feedback control over LH
secretion, while inhibin, secreted by Sertoli cells, exerts a negative feedback control over FSH secretion. All
aspects of Sertoli cell function seem to depend on adequate stimulation by FSH, which plays a central role on
the control of testicular function.3

these events leads to sperm differentiation, the acquisition of motility and the ability of
spermatozoa to undergo the zona-induced acrosome reaction, this latter requiring an
increase in membrane fluidity.20 The cycle of spermatogenesis is shown in Figure 1.
Many of the sperm alterations present in idiopathic infertility have recently been
related to an alteration of the process of sperm maturation and, in particular, to
the presence of high levels of reactive oxygen species (ROS). ROS are metabolites of
oxygen and include the superoxide anion, hydrogen peroxide, the hydroxyl radical, the
hydroperoxyl radical and nitric oxide. ROS are important mediators of normal sperm
 Male infertility 217

function and are involved in the induction and development of sperm hyperactivation,
capacitation and acrosome reaction.21,22 However, an increased production of ROS
results in oxidative damage to cellular lipids, proteins and DNA.23,24 This leads to
a decreased sperm motility, damage to the acrosome membranes and inability of the
sperm to fertilize the oocyte or produce a viable pregnancy. Normally the seminal plasma
possesses antioxidant scavengers and enzymes that may be deficient in some patients.25
In a recent study Gil-Guzman et al26 found that there was a variation in ROS
production in different stages of spermatozoa maturation. In particular, ROS
production was significantly higher in immature spermatozoa with abnormal head
morphology and cytoplasmic retention and lowest in immature germ cells and mature
spermatozoa. The cytoplasmic retention is the result of defective spermatozoa
maturation. During the last stage of spermatogenesis, there is a membrane remodelling
and dehydration of the cytoplasm of the spermatids that release the residual body into
the Sertoli cell. If this process does not occur, there will be the production of testicular
spermatozoa with cytoplasmic retention that can be responsible for the production of
increased levels of toxic oxygen radicals.27 This situation may result in damage to
mature spermatozoa by immature spermatozoa during the sperm migration from the
seminiferous tubules to the epididymis. Research tests, such as chemiluminescent
procedures using probes such as luminol or lucigenin, are currently under investigation
for measuring ROS production.28
Mutations in mitochondrial DNA (mtDNA) have also been associated with poor
sperm quality and in particular with decreased motility.29 Because there is an exclusive
maternal inheritance of mtDNA, there is the interesting possibility that some types of
male infertility can be inherited only through the female line. However, in ICSI
treatment the use of mature spermatozoa does not alter the uniparental pattern of
inheritance of mtDNA.30,31

OLIGO-AZOOSPERMIA AND GENETIC ABNORMALITIES

Severe oligozoospermia and azoospermia can be associated with genetic abnormalities

such as constitutive chromosome abnormalities, microdeletions of the Y chromosome
(AZF: azoospermia factor region) and mutation in the cystic fibrosis transmembrane
conductance regulator (CFTR) gene. The chromosomal and genetic disorders
responsible for male infertility are summarized in Table 4.
The prevalence of families with more than one infertile member suggests that
genetic causes for infertility are common and that mutations of genes different from
those present in the AZF region can be involved. These genes may control the synthesis
of the androgen receptor, FSH and LH, and may be responsible for the regulation of
spermatogenesis or sperm motility.32

Chromosome abnormalities

Dohle et al33 analysed the genetic abnormalities of 150 men having a sperm analysis that
showed , 1 million motile sperm/ml. An abnormal karyotype was found in 10.6% of
men with severe oligozoospermia or azoospermia. Numerical sex chromosome
abnormalities were present in 63% of all cytogenetic abnormalities and were associated
with azoospermia. Autosomal anomalies, such as Robertsonian and reciprocal
translocations and inversions, were present in men with oligospermia.
218 E. Iammarrone et al

Table 4. Chromosomal and genetic disorders responsible for male infertility.

A. Chromosome abnormalities: Found in 10.6% of men with oligo-azoospermia

Klinefelter’s syndrome 47, XXY
B. Gene defects
Microdeletion of the Y chromosome Area of azoospermia factor closest to
the centromere on long arm of Y chromosome
Regions:
AZFa (5%), AZFb (16%), AZFc (60%)
Genes:
DBY, USP9Y for AZFa
RBMY1 for AZFb
DAZ for AZFc
Azoospermia only occurs when Yq is
entirely deleted
Cystic fibrosis Autosomal recessive
Defective gene on chromosome 7
66% of men with congenital bilateral absence
of the vas deferens are CF carriers

Klinefelter’s syndrome (47, XXY), one of the most common sex chromosome
abnormalities, has a prevalence of 0.1% in the general population. It has been found in
11% of azoospermic and 0.7% of oligospermic men.34 These men have small testes,
gynaecomastia, incomplete androgenization and infertility. Seminiferous tubule
hyalinization is present and an increase in the Leydig cell density is seen in the testis.

Microdeletions of the Y chromosome

Microdeletions of the Y chromosome long arm (Yq) represent the most frequent
genetic abnormality seen in severe testicular disorders, different clinical conditions
depending upon the localization and extent of the deletion. A higher prevalence of
microdeletion is found in patients affected by idiopathic severe oligozoospermia (14%)
or in idiopathic non-obstructive azoospermia (16%). Microdeletions can involve
different regions of the AZF region on the Y chromosome, namely: region (5%), AZFb
region (16%) and AZFc region (60%). Larger microdeletions involving two or three AZF
regions are seen in 14% of cases. In the remaining 5% of cases, the microdeletions are
located in regions not overlapping AZFa, b or c (Figure 2).35
Interesting data have been derived from the analysis of the so-called genotype –
phenotype relationship. The phenotype of patients with Y microdeletions is defined on
the basis of seminal analysis and diagnostic testis histology. Only when the entire Yq

Figure 2. Diagrammatic representation of the areas responsible for male fertility on the long arm of the Y
chromosome.
 Male infertility 219

region is deleted, is the seminal phenotype always azoospermic. Deletions in AZFa and b
are more frequently associated with azoospermia than severe oligozoospermia, but
AZFb deletion can be, rarely, associated with moderate oligozoospermia.36,37 At
present there is no evidence that a relationship exists between the localization of the
microdeletion and the testicular phenotype. It is only possible to distinguish between
the different forms of azoospermia, Sertoli cell-only syndrome (SCOS), hyposperma-
togenesis, spermatogenic arrest and obstructive forms by a diagnostic biopsy or fine
needle aspiration cytology.38
All infertile patients with a sperm count of , 5 million/ml should be screened for
microdeletions, despite the presence of other causes of testicular damage, since
patients affected by cryptorchidism or varicocele are known to have a high prevalence
of microdeletions. These findings suggest that the testicular damage observed in such
patients can be related to the deletion of the Y chromosome and not to the abnormal
position of the testis or to the varicocele itself.39,40
FSH concentrations in patients with microdeletions, although higher than in
control groups, are lower than in patients with similar tubular alterations but
without microdeletions.38
Thus, it is not possible to make a prognosis about the presence of spermatozoa in
the testis of a man with a Y microdeletion (except for those with deletions of the entire
AZFa – c region) either from the clinical presentation (idiopathic versus non-idiopathic),
or from clinical parameters (testicular volume, hormonal concentrations).

Cystic fibrosis

Congenital bilateral absence of the vas deferens (CBAVD) occurs in 0.1% of men and in
30 –50% of azoospermic patients. It is caused by failure of the Wolffian (mesonephric)
duct development and it is characterized by varying degrees of absence of the vas
deferens, seminal vesicle and the ejaculatory duct. Mutations of the CFTR gene are
associated with obstruction of the male genital tract (CBAVD) but not with
spermatogenic failure.
It has been suggested that CBAVD patients are heterozygotes for a severe mutation
in one allele in combination with a mild CFTR gene mutation in the other, thus causing a
reduced concentration of CFTR protein. Impaired CFTR protein function may be
responsible for a defective but not absent chloride excretion, resulting in the absence of
the vas deferens, but not in pulmonary or pancreatic insufficiency.41 Approximately 66%
of men with CBAVD have at least one CFTR gene mutation, while only 7% of men
without CBAVD have a mutation.
Before undertaking assisted conception for CBAVD, it is important to screen both
partners for the cystic fibrosis (CF) mutation. If both partners are positive, there is a
25% risk of a child affected by CF, a condition characterized by chronic lung disease,
pancreatic exocrine insufficiency, an increase in the concentration of sweat electrolytes
and obstructive azoospermia. Because of the possible presence of a genetic cause of
male infertility, ICSI treatment raises the dilemma of the transmission of the disorder to
the next generation and genetic counselling is recommended.

AZOOSPERMIA

Azoospermia affects 2% of infertile men or can be classified as obstructive and non-

obstructive.
220 E. Iammarrone et al

In obstructive azoospermia, the obstruction can be present at different points: the

vas deferens, the epididymis and the rete testis. If the obstruction is present in the vas
deferens, spermatozoa can be retrieved from the vas deferens, the epididymis and the
testis. If the obstruction is present in the epididymis, spermatozoa can be retrieved
from the epididymis. In the absence of spermatozoa from the epididymis, it will be
necessary to retrieve the spermatozoa from the testicle.
In non-obstructive azoospermia or secretory azoospermia, there is defective
spermatogenesis that can be due to pituitary insufficiency or primary testicular failure.
Azoospermia can also be classified as pre-testicular (LH-FSH deficiency), testicular
(seminiferous failure) or post-testicular (obstructive azoospermia) according to the
pathophysiological mechanisms involved.
Richard et al42 analysed the medical records of 153 men with azoospermia in order
to define the most important parameters for detecting and differentiating obstructive
from non-obstructive azoospermia. They found that men with obstructive
azoospermia had significantly lower serum FSH, LH and prolactin levels and a larger
testicular long axis, compared with non-obstructive azoospermia cases. In particular,
91% of men with obstructive azoospermia had FSH levels of 7.6 mIU/l or less, while
76% of men with non-obstructive azoospermia had FSH levels greater than 7.6 mIU/l.
Regarding testicular measurements, in 78% of men with obstructive azoospermia the
long axis was 4.6 cm or greater while it was less than 4.6 cm in the non-obstructive
form. The analysis of LH and prolactin failed to reveal a significant difference between
the two groups.
Thus, men with diagnostic parameters indicative of non-obstructive azoospermia
should not consider undergoing therapeutic sperm retrieval without a prior diagnostic
testicular biopsy unless it is agreed that the procedure will serve a diagnostic as well as a
therapeutic purpose. Men with diagnostic parameters indicative of obstructive
azoospermia can confidently undergo sperm retrieval or reconstruction procedures
without a prior biopsy. Unfortunately, although the FSH levels and testicular long axis
measurements are predictive of obstructive versus non-obstructive azoospermia, they
are not indicative of a successful sperm retrieval.43
Men with azoospermia, a normal ejaculate volume and FSH levels may have
obstructive or non-obstructive azoospermia44 and in these patients a diagnostic
testicular biopsy is recommended.
Recent studies have indicated that serum inhibin B levels may have a predictive value for
the presence of testicular spermatozoa in azoospermic men. Inhibin is a polypeptide
secreted by Sertoli cells and is responsible for feedback regulation of spermatogenesis to
the pituitary gland, inhibin having a negative feedback on the anterior pituitary gland to
maintain functional levels of FSH secretion.45 The regulation of inhibin B production
changes during life. After birth, there is an inhibin B peak, probably related to the
proliferating activity of the Sertoli cells. Afterwards, inhibin B levels decrease and remain
low until puberty, when they rise again. In adults the inhibin B production is sufficient to
maintain detectable serum concentrations depending on spermatogenic activity.46 In
infertile men inhibin B decreases and FSH increases and this can reflect spermatogenic
damage, with spermatogenic arrest at the earlier stage having the lowest inhibin B levels.47
Brugo-Olmedo et al48 analysed serum inhibin B and FSH levels prospectively in non-
obstructive azoospermic men in relation to the presence of spermatozoa on testicular
sperm extraction (TESE), percutaneous epididymal sperm aspiration (PESA) or semen
analysis. They found that patients with non-obstructive azoospermia had significantly
higher levels of serum FSH and significantly lower levels of inhibin B. Only inhibin B
 Male infertility 221

levels had a predictive value in relation to the presence of spermatozoa on TESE, being
significantly higher in patients who had spermatozoa.

REPRODUCTIVE TECHNOLOGIES FOR MALE INFERTILITY

A small proportion of infertile men have causes that are treatable, only 1 –2% of
infertile men being candidates for hormonal gonadotrophin therapy because of
hypothalamic-pituitary insufficiency.
No treatment to date has been proven to increase semen quality and in the majority
of cases assisted fertilization techniques are the only effective treatment to overcome
multiple sperm deficiencies. These techniques include ICSI, (developed most recently),
partial zona dissection (PZD) and subzonal sperm injection (SUZI) (Table 5). ICSI is the
most efficient treatment for male factor infertility (Table 6).49,50 The ICSI procedure
involves the injection of a single sperm into a mature oocyte using a micropipette. The
spermatozoa can be obtained either after ejaculation or aspiration from the testis or
epididymis. When ejaculated spermatozoa are used the semen characteristics (sperm
count, motility, morphology) do not seem to affect fertilization, embryo quality and
pregnancy rate in ICSI treatment.51 – 53 The fertilization rate using ejaculated
spermatozoa ranges between 60.7 and 76%.53,54 A review of the world literature
reported an average fertilization rate of 64% with ejaculated sperm, 62% with
epididymal sperm and 52% with testicular sperm.55 The clinical pregnancy rate from a
1998 database showed a live birth rate of 32.2% compared with 33.2% for IVF.56
The ICSI procedure has been associated with a reduced blastocyst development and a
higher miscarriage rate.57,58 These finding have been related to the poor quality of the
injected spermatozoa or the physical damage that may occur with the injection.59,60

Table 5. Micromanipulation technologies.

1 ICSI: a single spermatozoon is placed in the cytoplasm of the mature oocyte

2 PZD: the zona pellucida is pierced in two places using a sharpened glass pipette. The spermatozoa
can traverse the two holes to fertilize the oocyte
3 SUZI: the spermatozoa are selected on the basis of the morphology, aspirated into a glass injection
pipette and then placed in the perivitelline space

Table 6. Indications for ICSI.

Ejaculated sperm
Repeated fertilization failure after conventional IVF-ET
Ejaculatory disorders (electroejaculation, retrograde ejaculation)
Oligozoospermia (,20 million/ml)
Asthenozoospermia (,40% with progressive motility, of which at least 10% are of type A motility)
Teratozoospermia (,14% normal forms)
Antisperm antibody
Globozoospermia
Surgically retrieved sperm

IVF, in vitro fertilization; ET, embryo transfer.

222 E. Iammarrone et al

However, a recent randomized controlled study showed that pregnancy rates are not
different when compared with IVF treatment (32.8 versus 31%).61 The incidence of
ectopic pregnancy after ICSI is statistically significantly lower compared with IVF
treatment (1.2% versus 3.6%).62 The increasing use of ICSI has raised some concerns
regarding the association with an increased risk of birth defects and other
complications. Since this technique is an invasive procedure, many potential risks
have to be taken into consideration:

† Natural spermatozoa selection is bypassed and there is the possibility of injecting

spermatozoa carrying a genetic defect.63
† There is the possibility of introducing unknown material into the cytoplasm, e.g.
debris, oil, traces of polyvinyl pyrimidine (PVP).
† The oocyte may be damaged during the injection.

Follow-up studies on the fetal karyotype of babies born after ICSI have revealed that
in comparison with the general neonatal population there is:

† An increase in de novo sex chromosome aneuploidy (0.6% instead of 0.2%) and in

structural autosomal abnormalities (0.4% instead of 0.07%).
† An increased number of inherited structural aberrations (mostly from the
infertile father).

The overall data (8319 liveborn ICSI children) do not indicate a higher rate of
malformation in ICSI children than in naturally conceived children.64 Most of the
malformations found in ICSI offspring have been attributed to prematurity secondary to
multiple births, one of the more common defects being hypospadias.65 Recent studies
addressing the developmental outcome of ICSI-born children at 1 and 2 years did not
reveal any excess incidence of abnormalities, thus providing reassurance that there is no
evidence of any procedure-induced abnormality.64,66
In about 30% of men with non-obstructive azoospermia, no spermatozoa can be
found in the testicular tissue and spermatid injection is currently being assessed as a
technique for achieving pregnancy.67,68 The fertilization rate varies in relation to the stage
of maturation, being 54% with elongating and elongated mature spermatids and 17%
with round spermatids.69 The use of sperm precursors has raised some concerns such as:

† Difficulties in identifying, classifying and isolating the precursor cells.

† The fertilization ability depends on the phase of development of the germ cells.
† The issue of genomic normality of these sperm precursors.

Furthermore, in four pregnancies resulting from the use of spermatids for ICSI,
there were two cases of major malformations with one fetus being affected by trisomy
9.70 The injection of oocytes with spermatids has been banned in the UK by the Human
Fertilization and Embrology Authority (HFEA).

SURGICAL SPERM RECOVERY

Since the introduction of ICSI, the surgical retrieval of epididymal and testicular
spermatozoa has become a routine technique in azoospermic men. Surgical sperm
recovery is a necessary procedure in cases of irreversible obstructive azoospermia,
 Male infertility 223

Table 7. Indications for MESA and PESA.

Congenital bilateral absence of the vas deferens

Vasectomy or failed vasectomy reversal
Post-inflammatory obstruction
Inoperable ejaculatory ducts or distal vasal obstruction
Radical cysto-prostatectomy

MESA, microsurgical epididymal sperm aspiration;

PESA, percutaneous epididymal sperm aspiration.

primary testicular failure (non-obstructive azoospermia) and in some cases of

ejaculatory disorder. Men with obstructive azoospermia usually have normal
spermatogenesis and sperm recovery is indicated if reconstructive surgery is not
feasible or if the surgery was unsuccessful. The techniques are: microsurgical epididymal
sperm aspiration (MESA), Percutaneous epididymal sperm aspiration (PESA) and
testicular sperm aspiration or extraction (TESA and TESE). The indications for surgical
sperm recovery are listed in Tables 7 and 8.
PESA is the method of choice in irreparable obstructive azoospermia, MESA being
indicated when it is necessary to perform a vasoepididymostomy. TESE is the only
treatment available for non-obstructive azoospermia.71
The recovered spermatozoa can be used fresh or can be cryopreserved for a future
ICSI cycle. An analysis of the outcome of ICSI treatment in men affected by
azoospermia showed that in those with obstructive azoospermia the fertilization,
implantation and pregnancy rates were similar to those obtained in men who had
undergone ICSI with ejaculated sperm. In men with non-obstructive azoospermia there
were lower fertilization, embryo quality and pregnancy rates compared with those for
obstructive azoospermia (64 versus 73%; 35 versus 56%, 13 versus 47%, respectively).
These findings can be explained by the defective spermatogenesis present in men with
non-obstructive spermatogenesis.72
Pasqualotto et al73 analysed the outcome of 198 ICSI cycles with epididymal and
testicular sperm obtained from patients with obstructive and non-obstructive
azoospermia. They did not observe any difference in the fertilization and embryo
transfer rates with respect to obstructive versus non-obstructive causes of
azoospermia. However, there were lower fertilization and pregnancy rates and higher
abortion rates in testicular sperm compared with the epididymal sperm retrieval cycles.
Epididymal or testicular motile sperm resulted in a lower abortion rate than epididymal
or testicular immotile sperm.

Table 8. Indications for TESA and TESE.

Non-obstructive azoospermia (incomplete Sertoli cell only, maturation arrest,

severe hypospermatogenesis)
Obstructive azoospermia (no sperm in the epididymis, rete testis blockage)
Complete sperm immobility
Anejaculation
Complete terato/necrozoospermia

TESA, testicular sperm aspiration; TESE, testicular sperm extraction.

224 E. Iammarrone et al

For the last 2 years, we have performed surgical extraction of sperm using PESA or
TESE followed by elective cryopreservation. This increases flexibility both for the
couple and the medical team and avoids unnecessary treatment for the female partner if
sperm is not recovered. All procedures are performed in the Day Case Surgery Unit
under local anaesthesia. A spermatic chord block is achieved with the injection of 10 ml
of 0.5% xylocaine followed by a local infiltration of the scrotal skin with 1 ml of 2%
lignocaine. This will provide reasonable analgesia for most patients. In some cases, it is
necessary to add a light intravenous sedation. PESA is performed with a 23 G needle
connected to a 20 ml syringe where suction is applied.74 For TESE we use a Cook Trucut
and, from a single entry, the whole testis is sampled with an average of 20 samples. PESA
is the initial procedure in all cases of obstructive azoospermia. If no sperm is found
following 5 – 6 attempts on one side (generally selected as being the side where a
distended epididymis is palpable), we proceed to TESE on the same side. The procedure
continues until motile sperm have been identified microscopically. The second testis is
sampled in the same way if no sperm are identified microscopically in the theatre.
Patients are discharged 1– 3 hours later depending on whether sedation was necessary,
painkillers and antibiotics being given for 5 days. Patients inform us of their follow-up by
sending back a questionnaire. To date no complications that did not permit all patients to
resume normal activity within 5 days have resulted from these procedures. Our results
are summarized in Table 9 and show an overall sperm recovery rate of 80%.
In cases of non-obstructive azoospermia we were able to find sperm in 11 out of 18
cases. Preliminary results for ICSI performed with epididymal or testicular sperm from
January 2000 to August 2001 are reported in Table 10.
The use of non-ejaculated spermatozoa raises the question of the possibly unknown
effects in offspring. A possible risk of using epididymal or testicular spermatozoa in ICSI
is incomplete DNA condensation of the chromatin, implying that the spermatozoa are
more sensitive to damage by toxic agents.75 Ramos et al76 found that the integrity of
DNA in the motile fraction of epididymal or testicular semen of patients with
obstructive azoospermia was similar to that in fertile donors. They concluded that
motility is a reliable marker for intact spermatozoa when selecting cells for use in ICSI.
Our recent experience at St Bartholomew’s Hospital using immotile sperm for
injection has shown that it is only the fertilization rate, and not the cleavage and
clinical pregnancy rates, which are significantly affected (Table 11). Whilst there was

Table 9. Results of PESA and TESE at the Bridge Centre, London UK (2000–2001).

Aetiology of azoospermia Number of cases Successful sperm retrieval n (%)

Previous vasectomy 17 17 (100)

CBAVD 4 4 (100)
Sertoli cell only syndrome 1 0
Maturation arrest 0
Other obstructive 19 17 (89)
Other non-obstructive 18 11 (61)
Anejaculation 1 1(100)
Unknown 11 7 (63)
Total 71 57 (80)

PESA, percutaneous epididymal sperm aspiration; TESA, testicular sperm extraction; CBAVD,
congenital bilateral absence of the vas deferens.
 Male infertility 225

Table 10. Results for ICSI performed with epididymal or testicular sperm at the Bridge Centre, London
UK (January 2000–August 2001).

Fresh Frozen

Epididymal Testicular Epididymal Testicular

Number of cases 2 2 23 19
Positive b hCG 1 – 9 3
Clinical pregnancy (%) 50 – 34.5 15.7

ICSI, intracytoplasmic sperm injection; hCG, human chorionic gonadotrophin.

a negative trend when using testicular rather than epididymal sperm, significant
differences were not seen (Table 12).
It has been established that frozen – thawed spermatozoa can be used to fertilize
oocytes by ICSI, although this process may influence chromatin structure which may
affect the decondensation of the sperm nucleus after injection into the oocyte.77
It has been shown that the fertilization rate is the same using fresh or cryopreserved
and thawed epididymal spermatozoa (58% for fresh and 57% for cryopreserved
spermatozoa). Testicular spermatozoa have a decreased fertilization capacity after
cryopreservation when compared with freshly retrieved spermatozoa (52 versus 71%).
The pregnancy rate is unaffected by cryopreservation in both epididymal and testicular
spermatozoa.78

SUMMARY

Male infertility has become one of the most common conditions treated at tertiary level
fertility centres. Although semen analysis is the first test in clinical evaluation of male

Table 11. Embryonic development related to motility parameters of surgically retrieved sperm at St
Bartholomew’s Hospital, London.

Motile Non-motile

Number of egg collections (EC) 72 14

Mean number of eggs injected (^SD) 8.9 (^3.9) 9.9 (^4.8)
Mean number of eggs fertilized (^SD) 4.5 (^2.6) 2.9 (^1.5)
Fertilization rate (%(^SD)) 54.0 ^ 23.2 29.8 ^ 15.6a
Mean number of embryos cleaved (^SD) 4.2 (^2.6) 2.2 (^1.4)
Cleavage rate (%(^SD)) 94.1 (^15.8) 77.0 (^29.7)
Number of embryo transfer events 69 13
Mean number of embryos transferred (^SD) 1.9 (^0.7) 1.7 (^0.6)
Number of clinical pregnanciesb 19 3
Clinical pregnancy rate/EC (%) 26.4 21.4

SD, standard deviation.

a
P ¼ 0.007.
b
Presence of fetal heart action on ultrasound.
226 E. Iammarrone et al

Table 12. Embryonic development related to origin of surgically retrieved sperm: epididymal or testicular.

Epididymal Testicular

Number of egg collections (EC) 60 26

Mean number of eggs injected (^SD) 8.7 (^3.7) 9.6 (^4.5)
Mean number of eggs fertilized (^SD) 4.4 (^2.5) 4.0 (^2.9)
Fertilization rate (%(^SD)) 52.3 (^25.7) 41.7 (^24.6)
Mean number of embryos cleaved (^SD) 4.3 (^2.5) 3.3 (^2.5)
Cleavage rate (%(^SD)) 85.2 (^31.7) 84.5 (^27.3)
Number of embryo transfer events 58 24
Mean number of embryos transferred (^SD) 1.8 (^0.5) 1.8 (^0.7)
Number of clinical pregnanciesa 16 6
Clinical pregnancy rate/EC (%) 26.7 23.1
a
Presence of fetal heart action on ultrasound.

infertility, no single parameter analysed is predictive of fertility. Recent studies have

raised the importance of ROS in the pathogenesis of idiopathic male infertility and the
possible inheritance of infertility through the maternal mutation of the mtDNA.
Genetic disorders, such as constitutive chromosome abnormalities, microdeletions of
the Y chromosome and mutations in the CFTR gene, have been related to severe
oligospermia and azoospermia. Further studies are needed to define which genes
control male fertility and this information may lead to the development of gene therapy.
To date, assisted reproductive technology, in particular intracytoplasmic sperm
injection (ICSI), is the only effective treatment in most cases of male infertility.
Currently, it is possible to obtain sperm even from azoospermic men, with good
fertilization and pregnancy rates. The use of immature spermatozoa needs more
investigation. Long term follow-up studies on ICSI-born children evaluating the
potential risk resulting from this procedure are ongoing, although the present data do
not indicate a higher rate of malformations with respect to naturally conceived children.

Practice points

† clinical investigation for male infertility should include

(i) history
(ii) clinical examination
(iii) semen analysis, endocrine and sperm tests
† all infertile men with a sperm count , 5 million/ml should be screened
for karyotype þ microdeletion in the Y chromosome
† mutations of the CFTR gene are associated with obstruction of the male
genital tract but not with spermatogenic failure
† FSH level is predictive of obstructive versus non-obstructive azoospermia
† ICSI is the most efficient treatment for male factor infertility
† the overall data do not indicate a higher rate of malformation in ICSI children
compared with naturally conceived children
 Male infertility 227

Research agenda
† sperm parameters and tests to discriminate between fertile and infertile men
are needed
† the genes involved in the control of male fertility need to be identified
† the use of spermatids in azoospermic men should be investigated
† long term follow-up of children born after ICSI is required

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