Effect of cigarette smoking on morphological characteristics of sperm

Original article: Effect of cigarette smoking on morphological characteristics of sperm Dr. Mahadeo Maroti Zalke 1, Dr. Noorin Bhimani 2, Dr. Pushpa Pazare 3 1, 2 Assistant Professor, Dept. of Physiology, Lokmanya Tilak Muncipal Medical College, Mumbai, India 3 Professor, Dept. of Physiology, GS Medical College, Mumbai, India Corresponding author: Dr Noorin Bhimani Date of submission: 14 Aug 2014 ; Date of publication : 22 September 2014 Abstract: Background: Our aim was to study the cigarette smoking and its correlation with human semen quality Material and methods: 300 male volunteers of age group 21 to 40 years, including 150 smokers and 150 nonsmokers were recruited for the study, following stringent exclusion criteria. Smokers further divided into subgroups according to number of cigarettes smoked per day as mild ( 10), moderate (11-20) and heavy smokers ( 21). Sperm motility and morphology were studied according to WHO manual. Results: Statistical analysis using ANOVA test showed that smokers had significantly lower percentage of spermatozoa with motility (grade a and b') and morphologically normal form. The percentage of sperms with head defects, midpiece defects and tail defects were also significantly higher in smokers (P<0.0001). These parameters were worse in the heavy smoking groups. Conclusion: There is inverse dose response relationship between cigarette smoking and sperm quality. Key words: cigarette smoking, sperm motility and morphology Introduction: Cigarette smoking is very common in spite of worldwide anti-smoking campaigns. According to World Health Organization, approximately one third of world s population older than 15 years of age smokes. The highest prevalence of smoking is observed in young males during their reproductive period (46% smokers between 20 and 39 years). 24 prevalence of smoking is 45% in men and 7% in women in urban India. 11, 25 Material and methods: The present study of semen analysis was conducted in a tertiary care hospital with the prior permission of the Ethics Committee. The study involved 300 healthy male volunteers within the age group of 21-40 years, out of which 150 were nonsmokers and 150 were smokers. Smokers further categorized as mild, moderate and heavy smokers depending upon the number of cigarettes smoked per day. The participants were selected based on the selection criteria. A detailed explanation regarding the procedure was given to the subjects and voluntary informed written consent was taken to participate in the study. Selection Criteria: 27, 17 The subjects who had never smoked throughout their life were labeled as nonsmoker. The subjects were classified as smokers if they claim to have smoked regularly (at least one cigarette per day) for at least one year before their clinical examination. Smokers were further categorized into three groups as:- Mild smokers - who smoked < 10 cigarettes per day. Moderate smokers - who smoked 11-20 cigarettes per day. 169

Heavy smokers - who smoked > 21cigarettes per day. Exclusion criteria: 19, 13, 20 The subjects having history of systemic diseases (e.g. TB, DM, Leprosy, Mumps), surgery on genital organ, sexually transmitted diseases (e.g. Gonorrhea, syphilis), exposure to radiation, alcohol consumption, inflammatory diseases of genital organs (e.g. Epididymitis, orchitis), developmental anomalies of genito-urinary system. (e.g. Undescended testis ), any medication, varicocele, genital tract infection, hormonal treatment, drug abuse or addiction, working near hot furnaces or in petrochemical industries were excluded from the study. A detailed history was taken and complete physical examination was carried out. Detailed examination of genitals was done to rule out any abnormalities. An appointment for semen analysis was given to the subjects. Instructions regarding abstinence from sexual intercourse as well as masturbation for a period of 3-5 days was given to subjects. 28 Subjects were called in the morning at 9.00 am after 3-5 days of sexual abstinence at the Semen Analysis Laboratory. Sample collection: Clean, sterile, warm and dry 50 ml wide mouth glass container was given to the subject for collection of the semen. The semen samples were obtained by masturbation in the proximity of laboratory. Adequate privacy was given Observations and results: to subjects for masturbation. The patients were instructed to wash their hands and genitalia and allow it to dry before sample collection. The first part of the ejaculate contains higher sperm concentration hence subjects were advised to report if sample collection was improper. The samples were allowed to liquefy for 20-30 minutes protecting it from extremes of temperature i.e below 20 0 C and above 40 0 C. The semen analysis was carried out after the liquefaction as per the guidelines laid by World Health 6, 28 Organization. Sperm motility: At least 5 microscopic fields were assessed in a systemic way to classify 200 spermatozoa. The motility of each spermatozoon is graded as grade a, b, c, or d according to WHO criteria. Normal value is 50% or more motile (grades a + b ) or 25% or more with grade a. Percentage in each motility grade was calculated and noted down. Sperm morphology: Seminal smear fixation and staining (Papanicolaou stain) and examination done according to WHO guidelines. Statistical Analysis: All the values were presented as mean + SD. The data were statistically analyzed by using one way analysis of variance (ANOVA) and Tukey Kramer (post test) wherever necessary. A p value of less than 0.05 is considered to be significant. 7 Table I : Comparison of percentage of motile sperms in different groups. Group Percentage of motile sperm ANOVA Nonsmoker 73.90 14.73 Mild smoker 67.62 11.48 Moderate smoker 57.22 11.86 Heavy smoker 42.56 6.91 P < 0.0001 significant 170

Nonsmoker Vs Mild smoker P < 0.05 Significant Nonsmoker Vs Moderate smoker P < 0.001 Significant Nonsmoker Vs Heavy smoker P < 0.001 Significant Mild smoker Vs Moderate smoker P < 0.01 Significant Mild smoker Vs Heavy smoker P < 0.001 Significant Moderate smoker Vs Heavy smoker P < 0.001 Significant Fig I: Mean percentage of motile sperms in different groups. 80 70 60 50 % 40 30 20 10 0 73.9 67.62 57.22 NON MILD MODERATE 42.56 HEAVY Table II : Comparison of percentage of grade a motile sperms in different groups. Groups Percentage of grade a motile sperms ANOVA Nonsmoker 60.92 14.78 Mild smoker 54.88 11.03 Moderate smoker 44.34 9.50 P < 0.0001 Significant Heavy smoker 32.84 6.35 Nonsmoker Vs Mild smoker P < 0.05 Significant Nonsmoker Vs Moderate smoker P < 0.001 Significant Nonsmoker Vs Heavy smoker P < 0.001 Significant Mild smoker Vs Moderate smoker P < 0.001 Significant Mild smoker Vs Heavy smoker P < 0.001 Significant Moderate smoker Vs Heavy smoker P < 0.001 Significant 170 171

Fig II: Mean percentage of grade a motile sperms in different groups. 70.00 60.00 50.00 40.00 % 30.00 20.00 60.92 54.88 44.34 32.84 10.00 0.00 NON MILD MODERATE HEAVY Table III: Comparison of percentage of grade b motile sperms in different groups. Groups percentage of grade b motile sperms ANOVA Nonsmoker 7.4 6.17 Mild smoker 5.5 5.08 Moderate smoker 5.44 4.44 Heavy smoker 3.24 3.15 P < 0.0001 Significant Nonsmoker Vs Mild smoker P > 0.05 Not significant Nonsmoker Vs Moderate smoker P > 0.05 Not significant Nonsmoker Vs Heavy smoker P < 0.001 Significant Mild smoker Vs Moderate smoker P > 0.05 Not significant Mild smoker Vs Heavy smoker P > 0.05 Not significant Moderate smoker Vs Heavy smoker P > 0.05 Not significant Fig III: Mean percentage of grade b motile sperms in different groups. 8 7 6 5 % 4 3 2 1 0 7.4 5.5 5.44 NON MILD MODERATE 3.24 HEAVY 171 172

Table IV: Comparison of percentage of morphologically normal sperms in different groups. ANOVA Percentage of morphologically normal Sperms Group Nonsmoker 66.98 13.56 Mild smoker 54.9 7.80 P < 0.0001 significant Moderate smoker 51.62 7.60 Heavy smoker 39.42 6.99 Nonsmoker Vs Mild smoker P < 0.001 Significant Nonsmoker Vs Moderate smoker P < 0.001 Significant Nonsmoker Vs Heavy smoker P < 0.001 significant Mild smoker Vs Moderate smoker P > 0.05 Not Significant Mild smoker Vs Heavy smoker P < 0.001 Significant Moderate smoker Vs Heavy smoker P < 0.001 Significant Fig IV : Mean percentage of morphologically normal sperms in different groups. 70 60 50 40 % 30 20 66.98 54.9 51.62 39.42 10 0 NON MILD MODERATE HEAVY Table V: Comparison of percentage of sperms having head defects in different groups. Percentage of sperms having head defects ANOVA Group Nonsmoker 16.79 5.38 Mild smoker 17.74 3.78 P < 0.0001 significant Moderate smoker 18.6 4.11 Heavy smoker 22.54 3.99 172 173

Nonsmoker Vs Mild smoker P > 0.05 Not Significant Nonsmoker Vs Moderate smoker P > 0.05 Not Significant Nonsmoker Vs Heavy smoker P < 0.001 significant Mild smoker Vs Moderate smoker P > 0.05 Not Significant Mild smoker Vs Heavy smoker P < 0.001 Significant Moderate smoker Vs Heavy smoker P < 0.001 Significant Fig V: Mean percentage of sperms having head defects in different groups. 25 20 % 15 10 16.79 17.74 18.6 22.54 5 0 NON MILD MODERATE HEAVY TABLE VI: Comparison of percentage of sperms having midpiece defects in different groups. Group Percentage of sperms having midpiece defects ANOVA Nonsmoker 14.37 9.27 Mild smoker 22.34 5.57 Moderate smoker 23.96 6.71 Heavy smoker 31.76 5.16 P < 0.0001 significant Nonsmoker Vs Mild smoker P < 0.001 Significant Nonsmoker Vs Moderate smoker P < 0.001 Significant Nonsmoker Vs Heavy smoker P < 0.001 Significant Mild smoker Vs Moderate smoker P > 0.05 Not Significant Mild smoker Vs Heavy smoker P < 0.001 Significant Moderate smoker Vs Heavy smoker P < 0.001 Significant 173 174

Fig: VI Midpiece defects in different groups. 35 30 25 20 % 15 10 14.37 22.34 23.96 31.76 5 0 NON MILD MODERATE HEAVY TABLE VII: Comparison of percentage of sperms having tail defects in different groups. Group Percentage of sperms having tail defects ANOVA Nonsmoker 1.86 3.22 Mild smoker 5.06 3.29 Moderate smoker 5.8 3.82 Heavy smoker 6.36 3.46 P < 0.0001 significant Nonsmoker Vs Mild smoker P < 0.001 Significant Nonsmoker Vs Moderate smoker P < 0.001 Significant Nonsmoker Vs Heavy smoker P < 0.001 significant Mild smoker Vs Moderate smoker P > 0.05 Not Significant Mild smoker Vs Heavy smoker P > 0.05 Not Significant Moderate smoker Vs Heavy smoker P > 0.05 Not Significant Fig VII: Mean percentage of sperms having tail defects in different groups. 7 6 5 4 % 3 5.06 5.8 6.36 2 1 1.86 0 NON MILD MODERATE HEAVY Discussion: compared to nonsmokers. Difference was statistically Sperm motility: The percentage of motile sperms was significant. In addition, when the percentage of 73.90 + 14.73 in nonsmokers, 67.62 + 11.48 in mild motile sperms of different groups was compared by smokers, 57.22 + 11.86 in moderate smokers and using Tukey-Kramer test, the difference between 42.56 + 6.91 in heavy smokers. The percentage of nonsmoker and mild smoker, nonsmoker and motile sperms was decreased in smokers as 174 175

moderate smoker, nonsmoker and heavy smoker was statistically significant. The percentage of grade a motile sperms was 60.92 + 14.78 in nonsmokers, 54.88 + 11.03 in mild smokers, 44.34 + 9.50 in moderate smokers and 32.84 + 6.35 in heavy smokers. There was decrease in the percentage of grade a motile sperms in smokers as compared to nonsmokers. The difference was significant. The difference between nonsmoker and mild smoker, nonsmoker and moderate smoker, nonsmoker and heavy smoker was significant. Also it was significant between mild and moderate, mild and heavy, moderate and heavy smokers. The percentage of grade b motile sperms was 7.4 + 6.17 in nonsmokers, 5.5 + 5.08 in mild smokers, 5.44 + 4.44 in moderate smokers and 3.24 + 3.15 in heavy smokers. There was decrease in the percentage of grade b motile sperms in smokers as compared to nonsmokers. The difference was statistically significant. The difference between nonsmoker and heavy smoker was significant. Gaur DS et al. found decrease in sperm motility in smokers as compared to nonsmokers. They also found decrease in progressive sperm motility in mild, moderate and heavy smokers as compared to nonsmoker. They concluded that decrease in progressive sperm motility can be early indicator of reduction in semen quality in smokers. 4 Results obtained by Ramlau-Hansen CH et al. 18, Rantala ML and Koskimies AI 19, and Vine et al. 26 are conclusive with the present study. Pasqualotto FF. et al. 17, Trummer et al. 24, Vogt HJ et al. 27, Godfrey B. 5 and Dikshit RK et al. 2 found no statistically significant difference in the sperm motility in smokers as compared to nonsmokers. Mechanism by which cigarette smoke affects sperm motility is not fully understood. Results of the study by Zavos PM et al. showed that high intensity of cigarette smoking severely affected the ultrastructure of the flagellum and more specifically the axoneme of human spermatozoa. In general, the cross-sections of spermatozoa from nonsmokers had normal axonemal arrangements, which consist of two central fibers surrounded by the nine fibers doublets and nine course outer fibers. The most severe abnormality noted in the axoneme of spermatozoa from smokers was complete disappearance of one or more of the nine fiber doublets and one or more of central fibers. Abnormalities or reduction in the number of axonemal fibers could have significant effects on progressive motility of spermatozoon. This study shows that smoking clearly has a degenerative effect on spermatozoa. Most important, one of the direct effects of smoking that can be qualitatively measured is the production of abnormalities induced on the flagellar structure, which tend to bring about deficiencies in the motility of sperms. 29 Lower values for the sperm motility in smokers may be caused by disturbances in epididymal sperm maturation process secondary to secretory dysfunction of Leydig and Sertoli cells. Testosterone and androgen-binding protein have important roles in the epididymal sperm maturation process. It is appreciated that epididymal sperm passage involves a cascade of events that lead to development of sperm capacity for forward progression and fertilization. Dysfunction of epididymis in smokers will likely have detrimental effects on various cyto-structural modifications and biochemical changes that spermatozoa normally undergo during epididymal maturation, and may result in a decrease in sperm motility and fertilizing capacity. 23 170 176

Cigarette smoking in man may affect his reproductive capacity by disturbing normal testicular steroidogenesis, spermatogenesis and epididymal function through adrenomedullary and adrenal cortical hyperactivity as a result of induced stress. This may explain the disorder in both the sperm motility and morphologic characteristics. 22 Kulikauskas V. et al. opined that reduction of sperm motility in smokers may be due to the presence of inhibitors of choline acetyltransferase (which facilitates the sperm motility) in smoke condensates. 13 Radioactive particles present in the cigarette smoke are known to move with tobacco smoke to testicular tissue and spermatogonia. As the testicular tissue and spermatogonia are sensitive to radiation it may lead to decrease in the sperm motility. 21 Sperm Morphology: The percentage of morphologically normal sperms was 66.98 + 13.56 in nonsmokers, 54.9 + 7.80 in mild smokers, 51.62 + 7.60 in moderate smokers and 39.42 + 6.99 in heavy smokers. There was decrease in the percentage of morphologically normal sperms in smokers as compared to nonsmokers. The difference was statistically significant. In addition, when the percentage of morphologically normal sperms of different groups was compared by using Tukey-Kramer test, difference between nonsmoker and mild smoker, nonsmoker and moderate smoker, nonsmoker and heavy smoker was significant. The difference was also significant in mild and heavy, moderate and heavy smokers. In the present study, percentage of sperms with head defects was 16.79 + 5.38 in nonsmokers, 17.74 + 3.78 in mild smokers, 18.6 + 4.11 in moderate smokers and 22.54 + 3.99 in heavy smokers. There was increase in the percentage sperms with head defects in smokers as compared to nonsmokers. The difference was significant. In addition, when the percentage of sperms with head defects of different groups was compared by using Tukey-Kramer test difference between nonsmoker and heavy smoker, mild smoker and heavy smoker, moderate smoker and heavy smoker groups was significant. The percentage of sperms with midpiece defects was 14.37 + 9.27 in nonsmokers, 22.34 + 5.57 in mild smokers, 23.96 + 6.71 in moderate smokers and 31.76 + 5.16 in heavy smokers. There was increase in the percentage of sperms with midpiece defects in smokers as compared to nonsmokers. The difference was significant. In addition, when the percentage of sperms with midpiece defects of different groups was compared by using Tukey-Kramer test difference between nonsmoker and mild smoker, nonsmoker and moderate smoker, mild smoker and heavy smoker groups was significant. The difference was also significant in mild and heavy, moderate and heavy smokers The percentage of sperms with tail defects was 1.86 + 3.22 in nonsmokers, 5.06 + 3.29 in mild smokers, 5.8 + 3.82 in moderate smokers and 6.36 + 3.46 in heavy smokers. There was increase in the percentage sperms with tail defects in smokers as compared to nonsmokers. The difference was significant. In addition, when the percentage of sperms with tail defects of different groups was compared by using Tukey-Kramer test difference between nonsmoker and mild smoker, nonsmoker and moderate smoker, nonsmoker and heavy smoker was significant. Zavos PM et al. studied 15 nonsmokers and 29 smokers for effect of cigarette smoking on semen. 171 177

They found percentage of morphologically normal sperms was decreased in smokers as compared to nonsmokers. They found significant increase in specific abnormalities of sperms such as head, midpiece, and tail among the smokers as compared to nonsmokers. 29 Chia SE et al. 1, Evans HJ et al. 3, Kunzle R. et al. 14, Shaarawy and Mahmoud 22, Sofikitis N. et al. 23, and Vine et al. 26 found similar results. Gaur DS et al. studied 200 men for effect of smoking on semen. Study population was divided into nonsmoker and smokers. Smokers further divided into mild smokers (1-20 cigarettes per day), moderate smokers (21-40 cigarettes per day) and heavy smokers (> 41 cigarettes per day) depending on the number of cigarettes smoked per day. They found significant increase in the abnormal forms of sperms in smokers compared to nonsmokers. 4 Ozgur K. et al. found significantly increased tail abnormalities in heavy smoker as compared to nonsmokers. 16 Jedrzejczak P. et al. studied 41 nonsmokers and 17 smokers for the effect of cigarette smoking on semen. They found percentage of sperm with head defects and cytoplasmic droplets were significantly higher in smokers as compared to nonsmokers. 10 Few researchers obtained results which differ from the present study. Pasqualotto FF. et al. 17, Dikshit RK et al. 2, Trummer et al. 24, Vogt HJ et al. 27, Godfrey B. 5, Hassa H. et al. 8, Kulikauskas V. et al. 13, Osser S. et al. 15, Rodriguez- Rigau LJ et al. 20, and Saaranen M. et al. 21 Although an association between smoking and measures of semen quality has been noted, the mechanism by which cigarette smoking might affect sperm morphology is not known. Evidence suggests that nicotine and/or other compounds in cigarette smoke may be involved. Nicotine exposure in rats leads to atrophy of testes and impaired spermatogenesis. The fact that nicotine and cotinine, a metabolite of nicotine, are detectable in semen suggests that perhaps other components of tobacco smoke are also able to enter the semen by crossing the blood-testis barrier and affects sperm development. For example, polonium-210 and α- emitting radioelement, capable of damaging DNA, are detected at higher concentration in the semen of smokers as compared with nonsmokers. 26 There is small but significant correlation between cotinine concentration in seminal plasma and abnormal sperm morphology. Damage to the germ cells induced by testicular toxicants might affect other cells in the testis e.g. Sertoli, peritubular, or Leydig cells. 17 Direct or indirect effect of cigarette smoke on Leydig and\or Sertoli cell function, testicular microcirculation and oxidative balance alters the sperms morphology. 23 Cigarette smoking in man may affect his reproductive capacity by disturbing normal testicular steroidogenesis, spermatogenesis and epididymal function through adrenomedullary and adrenal cortical hyperactivity as a result of induced stress. This may explain the disorder in both the sperm morphologic characteristics and motility. 22 The sperm morphological abnormalities and the lower values for the sperm motility in smokers may be caused by disturbances in spermatogenesis or epididymal sperm maturation process secondary to secretory dysfunction of Leydig and Sertoli cells. Testosterone and androgen-binding proteins have important role in the epididymal sperm maturation process. It is appreciated that epididymal sperm passage involves a cascade of events that lead to development of sperm capacity for forward progression and fertilization. 172 178

Dysfunction of epididymis in smokers will likely have detrimental effects on various cyto-structural modifications and biochemical changes that spermatozoa normally undergo during epididymal maturation, and may result in abnormal sperm morphology. 23 Animal studies have shown that rats exposed to cigarette smoke for 7-10 weeks found a secretary deficiency of Leydig cells and Sertoli cells, leading to impaired epididymal sperm maturation, and leads to formation of abnormal sperms. 17 Cigarette Smoking causes increased serum level of noradrenaline, which can cause increase aromatization of testosterone to estradiol in Sertoli cells. Estradiol might impair spermatogenesis by several mechanisms. It inhibits the biosynthesis of testosterone. Estradiol has been reported to increase the synthesis, inhibit the degradation and potentiate the synaptic activity of catecholamines. The high concentration of catecholamines might cause ischemia in seminiferous tubules causing impaired spermatogenesis, ultimately affecting sperm morphology. 12 Radioactive particles are known to move with tobacco smoke to testicular tissue and spermatogonia which are sensitive to radiation. This might damage the sperms and form abnormal sperms. 41 Conclusion: Cigarette smoking adversely affects sperm motility and morphology. The effect of the smoking are produced either by direct action of the toxic chemicals present in it or through its metabolites. 15 There is need of further studies to know what factors associated with smoking actually affects the quality of sperms. In the light of findings of present study, it will be advisable for the men with marginal seminal quality to quit smoking for improvement in the fertility status. Bibliography: 1 Chia SE, Ong CN, Tsakok FM. Effect of cigarette smoking on human semen quality. Arch Androl. 1994; 33(3): 163-168. 2 Dikshit RK, Buch JG, Mansuri SM. Effect of tobacco consumption on semen quality of a population of hypofertile males. Fertil Steril. 1987; 48(2): 334-336. 3 Evans HJ, Fletcher J, Torrance M, et al. Sperm abnormalities and cigarette smoking. Lancet. 1981 March 21: 627-629. 4 Gaur DS, Talekar M, Pathak VP. Effect of cigarette smoking on semen quality of infertile men. Singapore Med J. 2007; 48(2): 119-123. 5 Godfrey B. Sperm morphology in smokers. Lancet. 1981 April 25: 948. 6 Gopalkrishnan K, Hinduja IN, Mehta RH, Anand Kumar TC. Laboratory manual for human semen analysis. I C M R. Bombay, India. 1992: 1-34. 7 GraphPad Software, InStat guide to choosing and interpreting statistical tests, 1998, GraphPad Software, Inc., San Diego California USA, www.graphpad.com Copyright 1992-1998 GraphPad Software Inc. (131) 8 Hassa H, Yildirim A, Can C, et al. Effect of smoking on semen parameters of men attending an infertility clinic. Cli Exp Obstet Gynecol. 2006; 33(1): 19-22. 173 179

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27 Vogt HJ, Heller WD, Borelli S. Sperm quality of healthy smokers, ex-smokers, and never-smokers. Fertil Steril. 1986: 45(1): 106-110. 28 World Health Organization. WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. 4th Edition. Cambridge, England: Cambridge University Press, 1999. 29 Zavos PM, Correa JR, Karagounis CS, et al. An electron microscope study of the axonemal ultrastructure in human spermatozoa from male smokers and nonsmokers. Fertil Steril.1998; 69(3): 430-34. 171 181