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
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
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
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)
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