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El Samra et al.

Alexandria Bulletin 165


Bull. Alex. Fac. Med. 42 No.1, 2006.
SMOKING INCREASES DNA DAMAGE IN MALE INFERTILE
PATIENTS IN EGYPT
Mohamed El Samra
1
, Abdel Rahman Zahran
2
and Mohamed Elsawy
3,**

Department of Obstetrics and Gynecology, Alexandria University
1
.Department of Urology, Alexandria
University
2
Department of Clinical Pathology, Alexandria University
3
.
ABSTRACT
Background: Environmental factors, have been shown to have a deleterious effect on spermatogenesis. Cigarette
smoking is a major source of environmental pollution. Many studies have suggested that cigarette smoking is
associated with altered semen quality, but conclusions about the extent of the deleterious effects vary widely.
Sperm nuclear chromatin abnormalities /DNA damage could occur at the time of, or be the result of, DNA packing
at spermatogenesis. Environmental stress, gene mutations, and chromosomal abnormalities can disturb the highly
refined biochemical events that occur during spermatogenesis. This can ultimately lead to an abnormal chromatin
structure that is incompatible with fertility. However, the exact mechanism by which chromatin abnormalities/DNA
damage arise in human spermatozoa are not precisely understood.
Methods: We have conducted a study on 50 infertile males, of whom 25 are smokers, to assess the degree of sperm
DNA damage using the COMET assay and correlate our findings with other semen parameters.
Conclusion: In the present study, the percentage of severe DNA fragmentation was significantly higher in smoker
infertile group compared to no smokers and control groups.
INTRODUCTION
Many environmental factors, in the broadest sense
of the term, physical, chemical and psychological
factors have been identified as having a strongly
deleterious effect on spermatogenesis. Some of these
factors have increased during the course of the last
fifty years, due to the agricultural and industrial
development of mankind. The postulated secular,
geographical and inter-individual variation in semen
quality has been partly attributed to environmental
factors, including estrogens, pesticides, phthalates,
polychlorinated biphenyls (PCBs), air pollution and
tobacco smoke acting in pre- and perinatal life or
directly on sperm after puberty.
(1-3)

According to the World Health Organization,
approximately one third of the worlds population
older than 15 years of age smokes. Tobacco
smoking is recognized as a general health hazard,
and evidence indicates that in both men and
women, cigarette smoking affects reproductive
health more than does consumption of caffeine or
alcohol.
(4)
The combustion of tobacco yields about
4,000 chemical compounds. In the gaseous fraction,
carbon monoxide, nitrogen oxide, ammonia, and
hydrocarbons are found, whereas the main
component of the particulate phase is composed of
aggregates of nicotine. Polycyclic aromatic
hydrocarbons activate a proapoptotic pathway,
(5)

which leads to damage of oocytes and reduced
fertility. Given that cigarette smoke contains more
than 30 chemical agents known to be mutagens,
aneugens, or carcinogens in model systems, direct
deleterious effects on human embryos and female
and male germ cells are plausible.
(6)

Many studies have suggested that cigarette
smoking is associated with altered semen quality,
but conclusions about the extent of the deleterious
effects vary widely. Affected variables include
motility, sperm concentration, total sperm count,
semen volume, and morphology.
(7-10)
Cigarette
smoking was associated with reduced sperm quality
including lower total sperm count combined with
increased abnormal morphology and decreased
citrate concentration (increased pH).
(11)
A negative
association was found between decreased sperm
quality and smoking.
(12)
Cigarette smoke contains
several chemical agents, many of which are
carcinogenic or mutagenic. These agents affect the
production and function of healthy normal sperm via
different mechanisms. Smoking has also been found
to affect accessory glands (prostate, epididymis, and
seminal vesicles).
(13)
Significantly reduced citrate
concentrations were observed in smokers, indicating
impaired prostate function.
(11)

Cigarette smoke may decrease male fertility
through a direct effect on the testis and its ability to
produce progressively motile, vital sperm.
The nuclear status of sperm cells is determined by
two major events that occur during spermatogenesis:
acquisition of the final nuclear shape and the
replacement of somatic-type histones by protamines
(sperm-specific basic nuclear proteins) leading to
highly packaged chromatin. Sperm DNA is
organized in a specific manner to keep the chromatin
in the nucleus compact and stable. It is packed into a
tight, almost crystalline status that is at least six
times more condensed than mitotic chromosomes. It
occupies nearly the entire nucleus volume, whereas
somatic cell DNA only partly fills the nucleus.
(14)

This DNA organization not only permits the very
tightly packaged genetic information to be
transferred to the egg, but also ensures that the DNA
is delivered in a physical and chemical form that
allows the developing embryo to access the genetic
information.
(15)

Sperm nuclear chromatin abnormalities / DNA
damage could occur at the time of, or be the
166 Smoking Inc DNA Damage in Male Infertile Pat. El Samra et al.
Bull. Alex. Fac. Med. 42 No.1, 2006.
result of, DNA packing at spermatogenesis.
(16)

Environmental stress, gene mutations, and
chromosomal abnormalities can disturb the highly
refined biochemical events that occur during
spermatogenesis. This can ultimately lead to an
abnormal chromatin structure that is incompatible
with fertility. However, the exact mechanism(s) by
which chromatin abnormalities/DNA damage arise
in human spermatozoa are not precisely understood.
A causal relationship between cigarette smoking
and impaired reproductive function is highly
suspected because smokers inhale a host of toxins
such as nicotine, carbon monoxide, cadmium, and
other mutagenic compounds. Studies results indicate
that cigarette smoking is significantly correlated
with increased levels of seminal OS, as evidenced
by a significant reduction in ROS-TAC scores.
It has been recently reported that ROS-TAC scores
decrease as a result of an imbalance between levels
of ROS and antioxidants in semen.
(17)
This can be
attributed to the significant increase in seminal ROS
levels. Studies indicate that a variety of mechanisms,
particularly oxidative stress, conspires to induce
DNA strand breaks in the male germ line,
particularly in cases of male sub fertility associated
with poor semen quality. Such samples frequently
show depressed fertilization rates in vitro in
association with the DNA damage,
(18)
presumably as
a consequence of collateral peroxidative damage to
the sperm plasma membrane. Such membrane
damage is physiologically important since it
constitutes a protective mechanism designed to
ensure that spermatozoa with severely damaged
genomes cannot participate in the normal process of
fertilization.
The aim of this work was directed to study sperm
morphology and sperm nuclear DNA damage in a
group of infertile males who smoke cigarettes on
regular bases. The results were compared with
infertile non smokers and fertile non- smokers
individuals.
METHODS
Semen samples: Approval for the study was
obtained from our institutional review board. Human
semen samples from 50 males presenting to the
andrology clinic at Alexandria University Hospital,
were grouped as follows: Group A: 25 smoker
infertile males.
Group B: 25 non smoker infertile males. As a
control group (group C), 10 normal fertile males
were chosen for the study. All specimens were
collected by masturbation at the clinical andrology
laboratory after an abstinence period of 72 hours.
After liquefaction, semen analysis was performed
using a computer-assisted semen analyzer to
measure sperm concentration, percent motility, and
motion characteristics. Smears were prepared for the
assessment of sperm morphology. Myeloperoxidase
staining was performed to evaluate leukocyte
concentration in the specimen.
Sperm morphology: Air-dried seminal smears
stained with Diff-Quik (Baxter Scientific, Miami,
Fla) were scored for sperm morphology according to
WHO guidelines and Krugers strict criteria. A total
of 200 cells were scored for normal and abnormal
forms at 1000X magnification.
Comet assay: The modified alkaline Comet assay
for sperm
(16)
was carried out on the prepared
samples. A slide was prepared for each of the
patients and controls. Fully frosted slides (Trevigen
inc., Gaithersburg, MD, USA) were covered with
100 ml 0.5% normal melting point agarose. a
coverslip added and the agarose allowed to solidify.
The coverslips were removed and 1X10
3
cells in 10
ml BWW were mixed with 90 ml 0.5% low melting
point agarose (Sigma) and used to form the second
layer. The slides with coverslips removed were then
placed in lysis buffer for 1 h [2.5 M NaCl, 100 mM
NaEDTA, 10 mM Tris, 1% Triton X-100 and 10%
dimethyl sulphoxide (Sigma), pH 10]. The slides
were then incubated overnight at 37C in 100 ml/ml
proteinase K (Sigma) added to the lysis buffer. After
draining the proteinase K solution from the slides,
they were placed in a horizontal electrophoresis unit
filled with fresh alkaline electrophoresis solution
containing 300 mM NaOH and 1 mM EDTA
(Sigma), pH 12.5, for 20 min to allow the DNA from
the cells to unwind. Electrophoresis, for 10 min was
performed at room temperature, at 2 V (0.714 V/cm)
and 300 mA, obtained by adjusting the buffer level.
The slides were then washed with a neutralizing
solution of 0.4 M Tris (Sigma), pH 7, to remove
alkali and detergents. After neutralization, the slides
were stained with 50 ml 20 mg/ml ethidium bromide
(Sigma) and covered with a coverslip. All steps were
carried out under yellow light to prevent further
DNA damage. Two hundred cells from each slide
were selected randomly and analysed by image
analysis software from Scion imaging corp.
Observations were made at magnification 3400
using an epifluorescent microscope (Olympus BH2).
Statistical analysis was carried out on the values
obtained for the percentage head DNA of each cell.
Percentage head DNA is the percentage of DNA
which remains in the head of the Comet after
specified electrophoresis times and so indicates the
amount of undamaged DNA. DNA containing strand
breaks is drawn away from the Comet head into the
tail.
Statistical analysis: The Student t-test was used to
compare groups and fractions. Coefficients of
correlation were calculated using Spearmans rank
correlation analysis. These correlations were
considered clinically meaningful at the r >0.1. The
sample size in this study is sufficient to determine
whether the correlation is significantly greater than
the null hypothesis of r= 0.1 (alternative hypothesis,
r>0.05) with 90% power. All hypothesis tests were
El Samra et al. Alexandria Bulletin 165
Bull. Alex. Fac. Med. 42 No.1, 2006.
two-tailed with statistical significance assessed at
the P <.05 level. Statistical computations were
calculated using Microsoft excel.
RESULTS
Seminal fluid parameters: The range of sperm
count was between 0-191 x10
6
/ml with a mean of
23.7 40.8 x10
6
/ml in group A, the range was
between 5.6-85 x10
6
/ml with a mean value of 48.8
22.5 x10
6
/ml in group B and the range was 43.6 -
98.7 x10
6
/ml with a mean value of 79.3 17.6
x10
6
/ml in group C. The sperm count was
significantly lower in group A compared to the other
two groups, and in group B compared to group C (P
= 0.000). (table I, figure 1).
The Range of grade A motility was between 0-
45% with a mean percentage 6.9 12.0% in group
A, the range was between 0- 80% with a mean
percentage 19.5 21.1% in group B and the range
was between 40- 80% with a mean percentage 55.9
14.4% in group C. The percentage of grade A
motility was significantly lower in group A
compared to the other two groups, and in group B
compared to group C (P = 0.000) (table II, figure 2).
The Range of grade B motility was between
0 - 50% with a mean percentage 13.7 14.2% in
group A, the range was between 7- 48% with a mean
percentage 23.3 11.3% in group B and the range
was between 10- 36% with a mean percentage 23.5
8.6% in group C. The percentage of grade B
motility was significantly lower in group A
compared to the other two groups. (P = 0.015) (table
II, figure 2).
The Range of grade C motility was between 2-
46% with a mean percentage 21.1 11.2% in group
A, the range was between 5- 45% with a mean
percentage 23.1 10.3% in group B and the range
was between 5- 19% with a mean percentage 11.6
4.2% in group C. The percentage of grade C motility
was significantly higher in group A and group B
compared to group C (P = 0.012) (table II, figure 2).
The Range of grade D motility was between 8-
98% with a mean percentage 59.6 25.7% in group
A, the range was between 5- 75% with a mean
percentage 33.7 20.4% in group B and the range
was between 2- 21% with a mean percentage 9.0
6.6% in group C. The percentage of grade D motility
was significantly higher in group A compared to the
other two groups, and in group B compared to group
C (P = 0.000) (table II, figure 2).
The range of abnormal forms of sperm was
between 28- 100% with a mean percentage 68.8
19.8% in group A, the range of abnormal forms of
sperm was between 15- 61% with a mean percentage
41.5 13.1% in group B and the range of abnormal
forms of sperms was between 12- 36% with a mean
percentage 24.1 7.9% in group C. The percentage
of abnormal forms of sperms was significantly
higher in group A compared to the other two groups,
and in group B compared to group C (P = 0.000)
(table III, figure 3).
Quantitation of DNA damage in sperms
The range of mild DNA fragmentation was
between 0- 63% in group A with a mean 18.8
16.2%; the range was between 15- 72% with a mean
51.6 14.9% in group B and the range was
between 60-88% with a mean 76.0 9.5% in group
C. The percentage of mild fragmentation was
significantly lower in group A compared to the other
two groups, and in group B compared to group C (P
= 0.000) (table IV, figure 4).
The range of moderate fragmentation was between
0- 65% with a mean percentage 33.8 15.7% in
group A, the range was between 12- 57% with a
mean 38.5 11.5% in group B and the range was
between 12- 38% with a mean 21.8 9.1% in group
C. The percentage of moderate fragmentation was
significantly higher in groups A and B compared to
group C (P = 0.005) (table IV, figure 4).
The range of severe fragmentation was between 5-
100% with a mean 46.9 26.9% in group A; the
range was between 1- 35% with a mean 9.0 7.2%
in group B and the range was between 0- 6% with a
mean 2.2 1.6% in group C. The percentage of
severe fragmentation was significantly higher in
group A compared to the other two groups (P =
0.000) (table IV, figure 4).
Correlation between grades of DNA
fragmentation and semen parameters (sperm
count, sperm morphology and motility) in
different groups
In group A, there were no significant differences
between grades of DNA fragmentation and sperm
count, percentage of abnormal forms and motility
except that the mean percentage of grade D motility
was significantly higher in severe DNA
fragmentation (65.44 23.63%) compared to
moderate grade (41.5 27.32%) (P = 0.049) (table
V and figures 5-7).
In group B, sperm count was significantly lower
in moderate grade (39.75 23.76 x10
6
/ml)
compared to mild grade (60.24 14.95 x 10
6
/ml)
(P = 0.020). There was no significant difference
between mild and moderate DNA fragmentation as
regards percentage of abnormal sperms. Grade A
motility was significantly lower in moderate grade
(8.43 5.79 x10
6
/ml) compared to mild grade (33.64
25.2 x 10
6
/ml) (P = 0.001). Grade C motility was
significantly higher in moderate grade (27.5 10.2
x10
6
/ml) compared to mild grade (17.55 7.67 x
10
6
/ml) (P = 0.013). Grade D motility was
significantly higher in moderate grade (44.64 18.1
x10
6
/ml) compared to mild grade (19.73 13.76 x
10
6
/ml) (P = 0.001). There was no significant
difference between DNA fragmentations and grade
B motility (P = 0.053) (table VI and figures 8-10).
166 Smoking Inc DNA Damage in Male Infertile Pat. El Samra et al.
Bull. Alex. Fac. Med. 42 No.1, 2006.
Table I. Comparison between the three groups
as regards sperm count
Sperm count
(x10
6
/ml)
Group A
(Smoker, infertile)
(n = 25)
Group B
(Non-smoker, infertile)
(n = 25)
Group C
(Control group)
(n = 10)
Range 0-191 5.6-85 43.6-98.7
Mean SD 23.7 40.8 48.8 22.5 79.3 17.6
F-test P = 0.000*
LSD
Significance between:
A & B A & C B & C
P* is significant if < 0.05

Table II. Comparison between the three groups
as regards grades of motility
Grades of
motility (%)
Group A
(Smoker, infertile)
(n = 25)
Group B
(Non-smoker, infertile)
(n = 25)
Group C
(Control group)
(n = 10)
F-test
Grade A (%)
Range 0-45 0-80 40-80 P = 0.000*
Mean SD 6.9 12.0 19.5 21.1 55.9 14.4
(A & B), (A &C)
(B & C)
Grade B (%)
Range 0-50 7-48 10-36 P = 0.015*
Mean SD 13.7 14.2 23.3 11.3 23.5 8.6 (A & B), (A &C)
Grade C (%)
Range 2-46 5-45 5-19 P = 0.012*
Mean SD 21.1 11.2 23.1 10.3 11.6 4.2 (A &C)(B & C)
Grade D (%)
Range 8-98 5-75 2-21 P = 0.000*
Mean SD 59.6 25.7 33.7 20.4 9.0 6.6
(A & B), (A &C)
(B & C)


Table III. Comparison between the three groups as regards
percentage of sperm morphology
Abnormal forms of
sperms (%)
Group A
(Smoker, infertile)
(n = 25)
Group B
(Non-smoker, infertile)
(n = 25)
Group C
(Control group)
(n = 10)
Range 28-100 15-61 12-36
Mean SD 68.8 19.8 41.5 13.1 24.1 7.9
F-test P = 0.000*
LSD
Significance between:
A & B A & C B & C
P* is significant if < 0.05


Table IV. Comparison between the three groups
as regards DNA fragmentations
Grades of DNA
fragmentation (%)
Group A
(Smoker, infertile)
(n = 25)
Group B
(Non-smoker, infertile)
(n = 25)
Group C
(Control group)
(n = 10)
F-test
Mild (%)
Range 0-63 15-72 60-88 P = 0.000*
Mean SD 18.8 16.2 51.6 14.9 76.0 9.5 (A&B),(A&C)(B&C)
Moderate(%)
Range 0-65 12-57 12-38 P = 0.005*
Mean SD 33.8 15.7 38.5 11.5 21.8 9.1 (A & C), (B &C)
Severe (%)
Range 5-100 1-35 0-6 P = 0.000*
Mean SD 46.9 26.9 9.0 7.2 2.2 1.6 (A &B)(A & C)

El Samra et al. Alexandria Bulletin 165
Bull. Alex. Fac. Med. 42 No.1, 2006.
Table V. Comparison between grades of DNA fragmentations
and sperm analysis in group A
DNA fragmentation N Mean SD t-test
Sperm count Moderate 6 28.55 28.87 0.777
(x10
6
/ml) Severe 18 22.81 45.70
Percentage of abnormality
Moderate 6 58.33 17.47 0.107
Severe 18 73.38 19.40
Motility
Grade A Moderate 6 14.33 14.60 0.100
Severe 18 4.89 10.67
Grade B Moderate 6 23.83 11.84 0.054
Severe 18 11.00 13.77
Grade C Moderate 6 25.33 12.39 0.229
Severe 18 18.94 10.50
Grade D Moderate 6 41.50 27.32 0.049*
Severe 18 65.44 23.63

Table VI. Comparison between moderate grades of DNA fragmentations
and sperm analysis in group B
DNA fragmentation N Mean SD t-test
Sperm count Mild 11 60.24 14.95 0.020*
(x10
6
/ml) Moderate 14 39.75 23.76
Percentage of abnormality
Mild 11 37.36 12.02 0.167
Moderate 14 44.71 13.34
Motility
Grade A Mild 11 33.64 25.20 0.001*
Moderate 14 8.43 5.79
Grade B Mild 11 28.18 11.30 0.053
Moderate 14 19.43 10.10
Grade C Mild 11 17.55 7.67 0.013*
Moderate 14 27.50 10.20
Grade D Mild 11 19.73 13.76 0.001*
Moderate 14 44.64 18.10

23.7
48.8
79.3
0
20
40
60
80
100
x 10
6
/ml
GroupA (Smoker,
infertile)
GroupB (Non-smoker,
infertile)
GroupC (Control group)

Fig 1. Comparison between the three groups as regards sperm count
166 Smoking Inc DNA Damage in Male Infertile Pat. El Samra et al.
Bull. Alex. Fac. Med. 42 No.1, 2006.
6.9
19.5
55.9
13.7
23.3
23.5
21.1
23.1
11.6
59.6
33.7
9
0
20
40
60
80
%
Grade A Grade B Grade C Grade D

Fig 2. Comparison between the three groups as regards grades of motility

68.8
41.5
24.1
0
20
40
60
80
100
%
GroupA (Smoker,
infertile)
GroupB (Non-smoker,
infertile)
GroupC (Control group)

Fig 3. Comparison between the three groups as regards percentage of sperm morphology


18.8
51.6
76
33.8
38.5
21.8
46.9
9
2.2
0
20
40
60
80
%
Mild Moderate Severe

Fig 4. Comparison between the three groups as regards DNA fragmentations

















Fig 5. Comparison between grades of DNA fragmentations and sperm counts in group A
28.55
22.81
0
10
20
30
40
50
10
6
/ml
Moderate Severe
DNA fragmentation
El Samra et al. Alexandria Bulletin 165
Bull. Alex. Fac. Med. 42 No.1, 2006.
58.33
73.38
0
20
40
60
80
100
%
Moderate Severe
DNA fragmentation

Fig 6. Comparison between grades of DNA fragmentations and percentage of abnormality in group A

14.33
4.89
23.83
11
25.33
18.94
41.5
65.44
0
20
40
60
80
%
Grade A Grade B Grade C Grade D
Moderate Severe

Fig 7. Comparison between grades of DNA fragmentations and sperm motility in group A

60.24
39.75
0
10
20
30
40
50
10
6
/ml
Mild Moderate
DNA fragmentation

Fig 8. Comparison between grades of DNA fragmentations and sperm count in group B

37.36
44.71
0
20
40
60
80
100
%
Mild Moderate
DNA fragmentation

Fig 9. Comparison between grades of DNA fragmentations and percentage
of abnormal form of sperms in group B
166 Smoking Inc DNA Damage in Male Infertile Pat. El Samra et al.
Bull. Alex. Fac. Med. 42 No.1, 2006.
33.64
8.43
28.18
19.43
17.55
27.5
19.73
44.64
0
20
40
60
80
%
Grade A Grade B Grade C Grade D
Mild Moderate

Fig 10. Comparison between grades of DNA fragmentations and sperm motility in group B

DISCUSSION
Smoking has a detrimental effect on sperm qual-
ity, most significantly sperm concentration, motility,
and morphology.
(7,19-22)
In addition, cigarette
smoking has been correlated with poor sperm
function in sperm penetration assays.
(20,23)

Furthermore, paternal smoking has been associated
with a significant increase in the percentage of
spermatozoa with DNA damage
(20, 24-26)
and a higher
risk of birth defects and childhood cancers in the
offspring.
(27-29)
On the other hand, a handful of
studies have found no association between smoking
and sperm quality,
(30,31)
sperm function,
(32)
or sperm
nuclear DNA damage.
(33)
Such contradictory data
could be due in part to the fact that the studies were
conducted on two different populations: healthy
fertile men and infertile men. Another important
source of conflict among studies may be the
difficulty in adjusting for confounding factors such
as exposure to other toxins, socioeconomic status,
and abnormalities of genital examination.
The aim of this work was directed to study sperm
morphology, hormonal profile and sperm nuclear
DNA damage in a group of infertile males who
smoke cigarettes on regular bases. The results were
compared with infertile non-smokers and non-
smoker fertile individuals.
Many studies have suggested that cigarette
smoking is associated with altered semen quality,
but conclusions about the extent of the deleterious
effects vary widely. Affected variables include
motility, sperm concentration, total sperm count,
semen volume and morphology.
(7-10, 2, 34-36)
In the current study, the sperm count was
significantly lower in smoker infertile group
compared to the other two groups, and in non-
smoker infertile group compared to control group.
Grade A sperm motility was significantly lower in
smoker infertile group compared to the other two
groups, and in non-smoker infertile group compared
to control group. Grade B motility was significantly
lower in smoker infertile group compared to the
other two groups. Grade C motility was significantly
higher in smoker infertile group and non-smoker
infertile group compared to control group. Grade D
motility was significantly higher in smoker infertile
group compared to the other two groups, and in non-
smoker infertile group compared to control group.
The percentage of abnormal forms of sperms was
significantly higher in smoker infertile group
compared to the other two groups, and in non-
smoker infertile group compared to control group.
There was no significant difference between the
three studied groups as regards age.
Similarly, Lewin et al, 1991; Sofikkitis et al 1995
and Zinaman et al 2000 reported that cigarette
smoking may result in decreased sperm count, lower
sperm motility and a reduced percentage of
morphologically normal sperm.
(32,20,37)

Vine et al
(7)
and Kunzle et al
(11)
found a negative
association between decreased sperm quality and
smoking only among men 22 years of age or older.
In younger men, sperm and sperm quality were not
reduced.
On the other hand Saleh et al
(38)
found no
significant difference in standard sperm parameters
(sperm count ,motility and abnormal form) between
infertile smokers and infertile non-smokers, and the
overall correlation of these parameters with smoking
was not statistically significant. This observation is
consistent with the conclusion of Vine et al
(39)
that
the association between cigarette smoking and
standard sperm parameters is stronger among studies
of healthy men for the general population than
among men from infertility clinics. These mean that
the detrimental effect of smoking, on standard sperm
parameters and levels of sperm DNA damage may
be masked because of the infertility status.
Also, Trummer et al 2002
(40)
in their study
including around 500 smokers and 500 non-smokers
found no effect of smoking on standard semen
parameters.
The mechanisms by which cigarette smoking
affect semen quality are not fully understood. The
fact that nicotine and its water-soluble metabolite
cotinine are detectable in the seminal plasma of
smokers suggests that other harmful components of
tobacco smoke would pass through the blood-testis
barrier.
(41)
Using the immunoperoxidase method,
El Samra et al. Alexandria Bulletin 165
Bull. Alex. Fac. Med. 42 No.1, 2006.
Zenzes et al
(42)
demonstrated the presence of adducts
formed between benzopyrene and sperm DNA in
smoking men. Lower motility has also been
associated with abnormalities in the ultra structure of
the flagellum and the axonemal structures of the
sperm tail.
(43)

Pacifici et al.
(44)
found that total motility of
spermatozoa was significantly and negatively
correlated with concentrations of cotinine and
hydroxycotinine in the seminal plasma. The
mechanisms responsible for the correlation between
the plasma concentration of cotinine and reduced
sperm function parameters remain unexplained.
Zavos et al
(45)
investigated the effect of smoking
on the ability of seminal plasma to maintain sperm
viability and found that seminal plasma from
smokers had a strong detrimental effect on motility
of spermatozoa from nonsmokers. Washing of sperm
from smokers and exposure to seminal plasma from
nonsmokers restored motility.
(45)

In the present study, the percentage of severe
DNA fragmentation was significantly higher in
smoker infertile group compared to the other two
groups.
This observation is supported by Sun et al.
(46)
His
study was conducted on a group of male partners in
an IVF program, which indicated that the smokers
had a significant higher percentage of spermatozoa
with DNA damage than the non-smokers.
Although smoking may not be a primary cause for
sperm DNA damage in normal subjects, it may be an
important factor mediating the DNA damage in
spermatozoa with altered or abnormal chromatin
structure, which are commonly found in infertile
men. In this context, the additional increase of sperm
DNA damage in infertile smokers may be caused, at
least in part, by the increased levels of seminal
oxidative stress.
(33)

It is known that cigarette smoke contains a large
amount of oxidants that could lead to oxidative
DNA damage in sperm.
(47)
This is supported by
recent reports showing that oxidative stress not only
results in damage to the sperm plasma membrane
but also to the damage of sperm nuclear DNA by
causing high frequencies of single and double-strand
DNA breaks.
(48,49,50)
The most direct evidence
suggesting the involvement of oxidative sperm DNA
damage in male infertility is the finding that infertile
men contained higher level of 8-OHdG in sperm
than control subjects.
(49)

Kodama et al
(51)
found that the levels of 8-OHdG
in sperm DNA in 19 infertile patients were
significantly higher than in the control. Shen et al
(52)

found that the 8-OHdG levels in sperm DNA was
positively correlated to the number of sperm cells
with head defects, and inversely correlated to sperm
density, sperm number, sperm motility and the
number of sperm cells with normal morphology.
(53)
Therefore, these previous data supported strongly
the involvement of oxidative DNA damage caused
by smoking in the pathological process of male
infertility.
Using cotinine concentration as the biomarker of
smoking exposure, a significant correlation was
found by Shen et al
(52)
between seminal cotinine
concentrations and sperm 8-OHdG levels in 28
healthy smokers.
Cigarette smoke contains several chemical agents
many of which are carcinogenic or mutagenic. These
agents affect the production and function of healthy
normal sperm via different mechanisms. Smoking
has also been found to affect accessory glands
(prostate, epididymis, and seminal vesicles).
(13)

Reduced citrate concentration was observed in
smokers, indicating impaired prostate function.
Cigarette smoke may decrease male fertility through
a direct effect on the testis and its ability to produce
progressively motile, vital sperm.
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