Introduction:

The National Ribat University Faculty of Graduate Studies and Scientific Research The impact of cigarette smoking on semen parameters and sex hormones in infertile Sudanese male in Khartoum ( 2012 2015 ) تأثيز تذخين السجائز عل هعاييز السائل الونىي والهزهىنات الجنسيه عنذ هزض العقن السىدانيين الذكىر ف الخزطىم A Thesis Submitted in Fulfillment of the requirements PhD in Medical Laboratory Science (Clinical chemistry) By: Salah Eldin Omar Hussein B.S c - 2003 University of Science and Technology M.S c - 2008 Sudan University of Science and Technology Supervisor: Prof. Shamsoun Khamis Kafi Professor of Pathology The National Ribat University Co.Supervisor: Dr. Kamal Eldin Hussein Elhassan Associate Professor of Community Medicine The National Ribat University

DEDICATION To my: father, mother Who give me the meaning of the life To my wife, daughter and family members For their support and kindness To my colleagues' The persons whom I love, respect and appreciate

Acknowledgments I would like to express my profound thanks to my supervisor, Prof. Shamsoun Khamis Kafi, for his fruitful guidance, unlimited assistance, encouragement and sustained interest throughout the course of this work and also for Dr. Kamal Eldin Hussein Elhassan. I wish to extend my warmest thanks to the staff member of Dr. Alsir Abualhassan centers, for their continuous support and encouragement. Also I am grateful to all people from whom samples were taken. Abstract

This descriptive cross-sectional study was conducted during the period August 2012 to July 2015. The study included group I, 150 apparently infertile cigarette smoker volunteers (as a test group) and 150 apparently infertile non cigarette smoker volunteers (as a control group), group II included 150 apparently healthy cigarette smoker volunteers (as a test group) and 150 apparently healthy non cigarette smoker volunteers (as a control group). Semen parameters (volume/ml, count x 10 6, motility % and morphology %) and ( LH mlu/ml, FSH mlu/ml, Prolactin ng/ml, Testosterone ng/ml, TSH mlu/ml ) hormones levels were compared in the two groups. All study subjects were selected randomly from Dr. Alsir Abualhassan fertility center.the test group and the control group, were matched in terms of age and socioeconomic status. Group I: The sperm motility and Testosterone hormone levels were significantly reduced, while abnormal sperm morphology and prolactin hormone level were significantly raised. Semen volume, sperm count, LH, FSH and TSH were not significantly changed in the test group compared to the control group. Mean ± SD for infertile smokers versus controls show: Sperm motility (30.0±5.3)% versus (31.5±5.7), (P = 0.031 ),Testosterone (3.7±1.51) versus (3.9±1.01) ng/ml, ), (P = 0.039 ), abnormal sperm morphology (91.6±3.1) versus (89.1±4.3)%, (P = 0.001), Prolactin (6.8±3.41) versus (6.1±2.77) ng/ml, (P = 0.001),semen volume (2.3±0.55) versus (2.4±0.62) ml, (P = 0.183 ), sperm count (10.1±2.9) versus (11.8±3.0) x 10 6, (P = 0.140 ), LH (1.9±0.8) versus (2.1±0.9) mlu/ml, (P = 0.125 ), FSH (3.0±1.3) versus (3.1±1.5) mlu/ml, (P = 0.342 ), TSH (2.4±0.61) versus (2.4±0.67) mlu/ml (P = 0.725 ). Group II: The sperm motility, count and Testosterone hormone levels were significantly reduced while the number of sperms with abnormal morphology and prolactin hormone level were significantly raised. The semen volume, LH,

FSH and TSH hormones were not significantly change in the test group compared to the control group. Mean ± SD for fertile smokers versus controls show: Sperm motility (50.3±4.5)% versus (58.3±6.2), (P = 0.01 ), sperm count (31.9±4.2) versus (33.4±5.1) x 10 6, (P = 0.005 ), Testosterone (4.3±1.1) versus (4.5±1.4) ng/ml, (P = 0.013 ), abnormal sperm morphology (75.3±5.3) versus (54.8±4.1)%, (P = 0.015 ), Prolactin (7.3±2.8) versus (6.1±1.9) ng/ml, (P = 0.033 ), semen volume (2.4±0.7) versus (2.1±0.6) ml, (P = 0.560 ), LH (2.3±1.4) versus (2.4±1.5) mlu/ml, (P = 0.614 ), FSH (3.0±1.28) versus (3.1±1.39) mlu/ml, (P = 0.245 ), TSH (2.4±0.76) versus (2.4±0.83) mlu/ml (P = 0.518 ). From this study, it is concluded that; cigarette smoking is associated with reduced sperm count, motility and raised number of sperms with abnormal morphology while no significant change occurred on semen volume. Sperm count and volume correlate negatively with both the duration of smoking and the number of cigarettes smoked per day. Reduced testosterone hormone level correlates negatively with both the duration of smoking and the number of cigarettes smoked per day, while increase of prolactin hormone correlates positively with both the duration of smoking and the number of cigarettes smoked per day. In contrast LH, FSH, TSH hormones were not correlated with neither the duration of smoking nor the number of cigarettes smoked per day. هلخص الذراسة

أجريت هذه الدراسة المقطعية الوصفية خالل الفترة من أغسطس 2102 حتى يوليو 2105. حيث تمت مقارنة معايير السائل المنوى )الحجم, العدد,الشكل والحركة( والهرمونات الجنسيه ( الهرمون اللوتينى, الهرمون المنبه للجريب, هرمون البروالكتين, هرمون التيستيسرون والهرمون المنبه للدرق ) عند مجموعتين تضمنت المجموع األولى 051 من المدخنين المصابين بالعقم مع 051 من غير المدخنين المصابين بالعقم كمجموعة تحكم )مجموعة ضابطه( لتتضمن المجموعة الثانية 051 من المدخنين األصحاء مع 051 من غير المدخنين األصحاء كمجموعة تحكم )مجموعة ضابطه(. تم اختيار جميع المجموعات عشوائيا من داخل مركز دكتور السر ابوالحسن للخصوبة بحيث يتطابقون في العمر و الحالة االجتماعية و الجنس )ذكور(. المجموعة األولى: لوحظ انخفاض ذو داللة معنوية في حركة الحيوانات المنوية و هرمون التيستيسترون بينما كان هنالك ارتفاع فى نسبة الحيوانات المنوية المشوه و مستوى هرمون البروالكتين حيث كان االحتمال اإلحصائي للمقارنة اقل من 1.15. بينما لم يكن هنالك تأثير ملحوظ وذو داللة معنوية على حجم وعدد الحيوانات المنوية, الهرمون اللوتينى, الهرمون المنبه للجريب وهرمون البروالكتين حيث كان االحتمال اإلحصائي للمقارنة أكثر من. 1.15 وذلك عند مقارنة المستوى الوسطي للمدخنين المصابين بالعقم مقارنة بمجموعة التحكم و كانت النتائج كاآلتي 3 عدد الحيوانات. )00.1±0.1 )01.0±2.2 مقابل ( المنوية 3 ( حركة الحيوانات المنوية 3 )01.1±0.1 مقابل ( (,)00.5±5.5 الحيوانات المنويه المشوه 3 ( )20.9±0.0 مقابل (,)12.0±3.0 حجم الحيوانات المنوية 3 ( )2.0±1.55 مقابل ( )2.3±1.92, الهرمون اللوتينى 3 ( )2.0±0.3 مقابل (,)2.3±0.5 الهرمون المنبه للجريب ( 3 )0.1±0.21 مقابل (,)0.0±0.02 هرمون البروالكتين ( 3 )9.1±0.30 مقابل (,)9.0±2.55 هرمون التيستيسترون 3 ( )0.5±0.50 مقابل ( 0.2±0.10 (,الهرمون المنبه للدرق ( 3 )2.3±1.90 مقابل ( 1.95 )2.3±. ارتفاع المجموعة الثانية: لوحظ انخفاض ذو داللة معنوية في عدد و حركة الحيوانات المنوية و هرمون التيستيسترون بينما كان هنالك فى نسبة الحيوانات المنوية المشوه و هرمون البروالكتين حيث كان االحتمال اإلحصائي للمقارنة اقل من 1.15. بينما لم يكن هنالك تأثير ملحوظ وذو داللة معنوية على حجم الحيوانات المنوية, الهرمون اللوتينى, الهرمون المنبه للجريب وهرمون البروالكتين حيث كان االحتمال اإلحصائي للمقارنة أكثر من. 1.15 وذلك عند مقارنة المستوى الوسطي للمدخنين المصابين بالعقم مقارنة بمجموعة التحكم و كانت النتائج كاآلتي 3 عدد الحيوانات المنوية 3 (. )00.3±5.0 )00.2±3.2 مقابل ( حركة الحيوانات المنوية 3 ( )51.0±3.5 مقابل ( ( )55.0±5.0 مقابل,)53.1±3.0( ( )2.3±1.5 مقابل ( )2.0±1.9 الهرمون 0.0±0.02(, هرمون مقابل ( مقابل ( 3.5±0.3 (,الهرمون المنبه )0.1±0.21 2.3±0.5(, الهرمون المنبه للجريب ( 3 51.0±9.2( الحيوانات المنويه المشوه 3 )2.0±0.3 مقابل ( اللوتينى 3 ( البروالكتين ( 3 )5.0±2.1 مقابل (,)9.0±0.2 هرمون التيستيسترون 3 ( )3.0±0.0 للدرق ( 3 )2.3±1.59 مقابل ( 1.10 )2.3±. نخلص من هذه الدراسة لالتى : المنويه إن التدخين يؤدى إلى المشوه وهرمون البروالكتين انخفاض عدد وحركة الحيوانات المنوية وهرمون التيستيسترون وارتباط عدد وحجم الحيوانات المنويه مع ارتفاع نسبة الحيوانات ارتباطا سالبا مع مدة التدخين وعدد السجاير المدخنة فى اليوم بينما لم يحدث تغيير ذو داللة معنوية على حجم السائل المنوى, الهرمون اللوتينى, الهرمون المنبه للجريب والهرمون المنبه للدرق وقد تبين من خالل هذه الدراسة ارتفاع مستوى هرمون التيستيسترون والهرمون المنبه للدرق وارتباطهما ارتباطا موجبا بينما انخفض مستوى الهرمون اللوتينى, الهرمون المنبه للجريب وهرمون البروالكتين وارتباطهم ارتباطا سالبا مع مدة التدخين وعدد السجاير المدخنة فى اليوم. Contents

Subject Page Dedication. Acknowledgements. Abstract ( English ).. I ii iii Abstract (Arabic ). Contents... 7 v List of tables xii List of figures.. xiii List of abbreviation... xvi Chapter One Introduction. 1 1.2 Rationale 7 1.3 Objectives.. 7 1.3.1 General objective 7 1.3.2 Specific objectives. 7 Chapter Two 2. Literature review.. 9 2.1 Diagnosis of Infertility in Men... 9 2.1.1 Sexual or ejaculatory dysfunction... 10 2.1.2 Immunological cause... 10 2.1.3 Unexplained infertility. 11 2.1.4 Isolated seminal plasma abnormalities 11 2.1.5 Iatrogenic causes. 11 2.1.6 Congenital abnormalities. 12

2.1.7 Acquired testicular damage 13 2.1.8 Varicocele 14 2.1.9 Sexually transmitted diseases.. 15 2.1.10 Endocrine causes... 15 2.1.11 Seminal abnormalities.. 16 2.2 Risk factors of male infertility 17 2.2.1 Age. 17 2.2.2 Obesity. 18 2.2.3 Occupational exposure 19 2.2.4 Exercise... 20 2.2.5 Type of under trousers and position.. 20 2.2.6 Drinking alcohol and caffeinated beverages.. 21 2.2.7 Laptop and cell phones 21 2.2.8 War and stress. 21 2.2.9 Smoking... 22 2.2.9.1 Types of tobacco products 23 2.2.9.1.1 Smoking tobacco.. 23 2.2.9.1.1.1 Cigarettes 23 2.2.9.1.1.2 Bides.. 24 2.2.9.1.1.3 Cigars.. 24 2.2.9.1.1.4 Pipes... 24 2.2.9.1.2 Smokeless tobacco. 25 2.2.9.1.2.1 Chewing tobacco 25 2.2.9.1.2.2 Pan. 25 2.2.9.1.2.3 Snuff... 25 2.2.9.2 Smoking prevalence. 25 2.2.9.3 Tobacco and health.. 26 2.2.9.4 Cigarette smoking. 27

2..9.5 Other effect of smoking 29 2.2.9.6 Health benefit of smoking.. 30 2.3 Prevalence and causes of infertility... 30 2.4 Semen parameters.. 35 2.5 Sex Hormones 38 Chapter Three Materials and methods. 41 3.1Study approach 41 3.2 Study design.. 41 3.3 Study area. 41 3.4 Study period 41 3.5 Study population 41 3.5.1 Target population 41 3.5.2 Inclusion criteria. 41 3.5.3 Exclusion criteria 41 3.5.4 Ethical consideration.... 60 3.6 Sample size.. 42 3.6.1 Data collection 42 3.7 Clinical examination. 42 3.7.1 Specimen collection 42 3.7.2 Specimen processing.. 42 3.8 Data analysis.. 43 3.8.1 Semen collection and analysis.. 43 3.8.2 Blood sampling and processing. 43 3.8.3 Hormonal analysis 43 3.8.3.1 Testosterone hormone assay 43 3.8.3.1.1 Principle of the assay. 43 3.8.3.1.2 Kit components 44

3.8.3.1.3 Assay procedure 44 3.8.3.1.4 Validity of test 45 3.8.3.1.4.1 Calculation of results.. 45 3.8.3.1.4.2 Specificity. 46 3.8.3.1.4.3Sensitivity.. 45 3.8.3.1.4.4 Accuracy. 45 3.8.3.1.4.5 Precision. 46 3.8.3.1.4.6 Linearity. 46 3.8.3.1.5.7 Standard curve 46 3.8.3.2 Luteinizing hormone (LH) assay 46 3.8.3.2.1 Principle of the assay.. 47 3.8.3.2.2 Kit components 47 3.8.3.2.3 Assay procedure. 47 3.8.3.2.4 Validity of test 48 3.8.3.2.4.1 Calculation of results 48 3.8.3.2.4.2 Specificity. 48 3.8.3.2.4.3 Sensitivity.. 49 3.8.3.2.4.4 Accuracy 49 3.8.3.2.4.5 Precision. 49 3.8.3.2.4.6 Linearity 49 3.8.3.2.5.7 Standard curve.. 50 3.8.3.3 Follicle stimulating hormone (FSH) assay. 50 3.8.3.3.1 Principle of the assay 50 3.8.3.3.2 Kit components 51 3.8.3.3.3 Assay procedure... 51 3.8.3.3.4 Validity of test.. 52 3.8.3.3.4.1 Calculation of results... 52 3.8.3.3.4.2 Specificity.. 52

3.8.3.3.4.3 Sensitivity.. 52 3.8.3.3.4.4 Accuracy. 52 3.8.3.3.4.5 Precision 53 3.8.3.3.4.6 Linearity 53 3.8.3.3.5.7 Standard curve. 53 3.8.3.4 Prolactin hormone assay. 53 3.8.3.4.1 Principle of the assay 53 3.8.3.4.2 Kit components. 54 3.8.3.4.3Assay procedure 54 3.8.3.4.4 Validity of test 55 3.8.3.4.4.1 Calculation of results 55 3.8.3.4.4.2 Specificity.. 55 3.8.3.4.4.3 Sensitivity.. 56 3.8.3.4.4.4 Accuracy 56 3.8.3.4.4.5 Precision. 56 3.8.3.4.4.6 Linearity 56 3.8.3.4.5.7 Standard curve. 57 3.8.3.5 Thyroid Stimulating Hormone (TSH) assay.. 57 3.8.3.5.1 Principle of the assay 57 3.8.3.5.2 Kit components 58 3.8.3.5.3 Assay procedure. 58 3.8.3.5.4 Validity of test. 59 3.8.3.5.4.1 Calculation of results. 59 3.8.3.5.4.2 Specificity. 59 3.8.3.5.4.3 Sensitivity. 59 3.8.3.5.4.4 Accuracy 59 3.8.3.5.4.5 Precision. 60 3.8.3.5.4.6 Linearity. 60

3.8.3.5.5.7 Standard curve. 60 3.9 Quality Control.. 60 3.10 Statistical analysis 60 Chapter Four 4. Results 62 Chapter Five 5. Discussion. 82 Chapter Six 6. Conclusion and Recommendations. 85 6.1 Conclusion.. 85 6.2 Recommendations.. 85 References Appendices Questionnaire Luteinizing Hormone (LH) ELISA Follicle Stimulating Hormone (FSH) ELISA Prolactin ELISA Testosterone ELISA Thyroid Stimulating Hormone (TSH) ELISA List of tables Table Page

Table (4.1) Comparison of the means of semen parameters between the infertile smokers and non smokers groups... 64 Table (4.2) Comparison of the means of semen parameters between the fertile smokers and non smokers groups.. 65 Table (4.3) Comparison of the means of sex hormones between the infertile smokers and non smokers groups... 70 Table (4.4) Comparison of the means of sex hormones between the fertile smokers and non smokers groups 71 List of figures Figure Page Figure (4.1) A scatter plot shows the correlation between the

sperm count and the duration of cigarette smoking/ 66 years... Figure (4.2) A scatter plot shows the correlation between the sperm count and the number of cigarette smoking / 67 day. Figure (4.3) A scatter plot shows the correlation between the semen volume and the duration of cigarette smoking/ 68 years... Figure (4.4) A scatter plot shows the correlation between the semen volume and the number of cigarette smoking / 69 day.. Figure (4.5) A scatter plot shows the correlation between the LH and the duration of cigarette smoking/ 72 years... Figure (4.6) A scatter plot shows the correlation between the FSH and the duration of cigarette smoking/ 73 years..... Figure (4.7) A scatter plot shows the correlation between the TSH and the duration of cigarette smoking/ 74 years. Figure (4.8) A scatter plot shows the correlation between the Testosterone and the duration of cigarette smoking/ 75 years... Figure (4.9) A scatter plot shows the correlation between the LH and the number of cigarette smoking / 76 day.... Figure (4.10) A scatter plot shows the correlation between the FSH and the number of cigarette smoking / 77

day... Figure (4.11) A scatter plot shows the correlation between the TSH and the number of cigarette smoking / day.. Figure (4.12) A scatter plot shows the correlation between the Testosterone and the number of cigarette smoking /day. Figure (4.13) A scatter plot shows the correlation between the Prolactin and the duration of cigarette smoking/ years.... Figure (4.14) A scatter plot shows the correlation between the Prolactin and the number of cigarette smoking / day.. 78 79 80 81

List of abbreviations WHO SHBG HRT LH FSH TSH MAGI ASA TDS BMI DBCP EDB DDT CNS AVP ELISA QC World Health Organizat Sex Hormone B Hormone Repla Lute Follicle-Stim Thyroid Stim Male Accessory Anti S Testicular Dyg B Di-Bromo Ethy Dichloro-Diptenyl- Central Argin Enzyme-linked Immun

1. Introduction 1.1 Introduction: Male infertility is a problem of the reproductive system, and the word infertility itself means not fertile, and that would be equivalent to sterility. Sterility means that a man is totally unable to have a child. The World Health Organization (WHO) and the American Society for Reproduction Medicine Practice Committee defines infertility as no conception after at least 12 months of regular unprotected sexual intercourse. Infertility can be permanent (irreversible) or sub fertility which means the probability of spontaneous conception may be decreased. All men who are sterile would be considered infertile, but not all men who are infertile are sterile, because an infertile man can father a child with medical help or with simple change in his life style. (1, 2) A man is responsible in about 20% of infertility among couples, and contributes to infertility with a woman in another 30-40%. Infertility can either be primary or secondary; primary male infertility is when the man has never impregnated a woman, while secondary male infertility is when a man has impregnated a woman irrespective of the outcome of the pregnancy. Men with secondary infertility, in general, have better chance of future fertility. Duration of infertility is defined as the number of months during which the couple has been having sexual intercourse without the use of any contraceptive method. This indicator gives an important information about the couple's future fertility, if the duration of infertility of 3 years or less the couples have a better chance of future pregnancy, but if the duration has been longer, then there is a severe biological problem. But

in general couples tend to seek medical advice after a shorter duration of infertility. (2) Despite worldwide anti-smoking campaigns, cigarette smoking is very common. The highest prevalence of smoking is observed in young adult males during their reproductive period (46% smokers between 20 and 39 years. About 30% of the Austrian male population aged 15 and older are smokers. Smoking among men is increasing in Central and Eastern Europe. Overall 35% of European men smoke, with a prevalence of 44% or even higher in the Eastern parts (Bulgaria, Greece, Turkey) and 30% in the Western parts (UK, Sweden, Finland) of Europe. (3) Cigarette smoking may be associated with sub-fertility in males and may result in decreased sperm concentration, lower sperm motility, and a reduced percentage of morphologically normal sperm respectively. Nineteen studies evaluating the influence of smoking on semen parameters in infertile men and nine studies in fertile men have been published so far. The major shortcoming of these studies is a small overall patient number (only two studies included >500 men, and >200 smokers). In a recent meta-analysis, including 27 studies on the association between cigarette smoking and semen quality, a mean reduction in sperm concentration of 13%, a mean reduction of sperm motility of 10%, and a mean reduction of morphologically normal sperm of 3% was reported in smokers. Most of the studies, however, which reported a significant difference in semen quality, were performed in normal, non-infertility clinic men. Unfortunately, in 25 out of 27 studies in this meta-analysis, the number of smokers was <200 men. Another major shortcoming is the lack of accurate smoking dose information. Smoking may cause sub-fertility by influencing hormone levels. Testosterone levels may be unchanged, elevated, or decreased and estradiol levels are mainly found to be elevated in smokers. (3,4,5) Smoking may have impact on fertility, as reported in a recent study enrolling 200 men. In this study it was noted that cigarette smoking was

significantly associated with a decreased pregnancy rate and impaired semen parameters. Men with azoospermia were excluded and the authors did not report men with genital disease. In this study only 6% (n = 12) were smokers. Although there were only six smokers in both the pregnant and the non-pregnant group, a statistical significance (P = 0.02) was calculated. In order to overcome the shortcomings identified in other studies (i.e. low participant number, and lack of smoking dose data), we compared semen parameters and hormone levels of a large number of infertile smokers with non-smokers and ex-smokers and evaluated the smoking dose. It was recently concluded that men with marginal semen quality who wish to have children might benefit from stopping smoking. In addition, there are only limited data on whether men would stop smoking for the prospect of recovering from infertility. Therefore, they determined how many (3, 6) men would stop smoking if they thought it would increase their fertility. Infertility, defined as the inability to conceive after at least 1 year of unprotected intercourse, affects about 8-12% of couples in the world. Between countries and regions, infertility rates vary dramatically, corresponding to the incidence of preventable conditions, which can lead to infertility. In some areas, particularly in sub- Saharan Africa, up to one-third of couples are infertile and of them approximately 52% suffer from acquired infertility. On the contrary, the percentage of secondary infertility is lowest in Asia and in developed countries; 23% and 29%, respectively. Infertility could be caused by male factors such as Azoospermia, oligozoospermia, asthenozoospermia and /or teratozoospermia, or female factors such as tubal occlusion, ovulatory dysfunction, uterine abnormality, peritoneal factors and/or endometriosis. However, the problem could be from the male or the female partner alone or from both partners. Unfortunately only the woman is blamed for childlessness in our African society. This explains why woman are left on their own to find the solution to this problem from any source, ranging from spiritual to religious treatment. Therefore, this study aimed at determining the effect of smoking in infertility in

Sudanese males, based on clinical and laboratory findings in order to promote the involvement of males in reproductive health issues and in the prevention of infertility in particular. Infertility is a common problem affecting one in six couples. It can be defined as the incapacity to fulfill pregnancy after a reasonable time of sexual intercourse with no contraceptive measures taken. In 30% of infertile couples, the male factor, in the form of defective sperm quality, is a major cause. As a large number of men smoke worldwide, and the fact that cigarette smoke contains known mutagens and carcinogens, there has been much concern that smoking may have unfavorable effects on male reproduction. Several studies from different parts of the world have observed that cigarette smoking has an effect on the semen quality, especially in those who are heavy smokers or who have been smoking for many years. Measures of semen quality are used as surrogate measures of male fertility in andrology. Over years, undue importance has been given to sperm count, though it is meaningless without the required motility or normal sperm morphology. In fact, other parameters like seminal fluid volume, liquefaction time, sperm motility and viability can be of help in assessing the overall sperm quality and its fertility potential. The aim of our study determines the effect of cigarette smoking on quality of seminal fluid parameters. (7,8) Cigarette smoking has major effects on the reproductive potential of humans. It has an anti-oestrogenic effect in women. This is probably due to changes in hepatic oestrogen metabolism induced by smoking. Smoking has a powerful effect on the 2-hydroxylation pathway of oestradiol metabolism leading to increased production of 2-hydroxyestrogens. These compounds have minimal oestrogenic activity and are rapidly cleared from the circulation. Furthermore, in the circulation oestrogens bind avidly to sex hormone binding globulin (SHBG) (38%), loosely to albumin (60%) and the remainder is the free unbound fraction. In smokers, concentrations of SHBG are higher and lower concentrations of

biologically active oestrogens are thus seen. Animal data have also demonstrated (7, 8, 9) a direct toxic effect of cigarette smoke on ovarian follicles. Smoking also results in reduction in bone mineral density, making osteoporosis more common among female smokers. Though various mechanisms for this effect are described later in this review, part of the deleterious effect of smoking on bone is mediated through its oestrogen-lowering effect. It is important to take this into account when hormone replacement therapy (HRT) is considered for prevention of postmenopausal bone loss and osteoporotic fractures. The therapeutic efficacy of oral HRT, prescribed in conventional doses, is reduced in smokers. This occurs as a result of increased hepatic clearance, as described previously, and is seen with oral preparations only. Thus smoking can counteract the protective effect of oral HRT on bone. Increasing the dose of oral oestrogen is not recommended as it results in the production of toxic oestrogen conjugates, such as catechol oestrogens and 16α-hydroxyoestrone, which have been implicated in breast cancer. As transdermal administration of oestradiol by passes the liver and enables a lower dosage of oestrogen to be used, this route should be considered in women who continue to smoke despite all warnings. The parenteral route of HRT is another option. (10,11) Owing to its anti-oestrogenic action, certain diseases that depend on oestrogen for growth and development tend to be less common among smokers. The development of endometrial cancer is related to oestrogen levels and a lower prevalence of this cancer is seen among women who smoke. Similarly, hyperemesis gravidarum, uterine fibroids and endometriosis are common disorders in young women and are oestrogen dependent. Again smokers have a reduced risk of developing these conditions. Though breast tissue is oestrogen responsive, the association between smoking and breast cancer is less welldefined. In fact the inconsistent findings between smoking and breast cancer risk can be explained by the genetic susceptibility to carcinogens found in cigarette smoke and not the anti-oestrogenic effect. Ambrosone and coworkers found that

N-acetyl-transferase 2 genetic polymorphism plays an important role in breast cancer risk. (12) In males, the effect of smoking on androgen levels is important, given the recent interest in the association between low androgen levels and the metabolic syndrome, and coronary heart disease. Various studies examining the effects of smoking on serum testosterone levels have reported conflicting findings largely due to difficulties in the hormonal assays. Testosterone has a circadian rhythm with levels peaking between 0600 and 0800 h and reaching a nadir between 1800 and 2000 h. A significant proportion of the circulating total testosterone is inactive as it is tightly bound to Sex Hormone Binding Globulin (SHBG) (65 80%), whereas the biologically active fraction circulates either free (1 3%) in circulation or loosely bound to albumin (20 40%). The free plus the albuminbound testosterone is called the bio available testosterone. Thus levels of total testosterone can be affected by changes in the levels of SHBG and other plasma proteins. Significantly increased, decreased and unchanged levels of total testosterone in male smokers have been reported in various studies. Free testosterone levels have also been found to be higher among smokers. However, SHBG levels have been measured only in three studies [12, 13] and are reported to be higher amongst smokers. No significant differences in the levels of bio available testosterone have been demonstrated between smokers and nonsmokers. English and colleagues demonstrated that the increase in total testosterone observed in smokers is due to the raised SHBG levels. They also reported that SHBG levels and not testosterone correlated with serum nicotine levels, a measure of cigarette smoking. However, Svartberg found a positive association between testosterone and smoking even after adjusting for SHBG though other plasma proteins were not taken into account. It would seem likely that the effects of smoking on testosterone levels are due to changes in plasmabinding capacity rather than a direct effect of nicotine on androgens. (13,14) 1.2 Rationale:

Increased public awareness of a couple's infertility as a treatable condition and the availability of improved therapeutic options have resulted in a dramatically increased number of visits to fertility specialists in the last few years. Although diagnostic problems make it difficult to establish the extent of the male partner's contribution with certainty, a number of studies suggested that male problems represent the most common, single defined cause of infertility. Male-related disorders are probably present in up to 40% - 50% of childless couples, alone or in combination with female factors. This study was conducted to assess the effect of cigarette smoking on seminal and sexual hormones. 1.3 Objectives: 1.3.1 General objective: To evaluate the impact of cigarette smoking on semen parameters and sex hormones in infertile Sudanese males in Khartoum. 1.3.2 Specific objectives: 1- To identify the semen quality of cases in terms of the sperm count, motility, morphology, and semen volume. 2- To determine the serum level of sex hormones (LH, FSH, Prolactin, Testosterone, and TSH) in cigarette smokers with infertility compared to control subjects. 3- To identify correlation between the serum levels of sex hormones ( LH, FSH, Prolactin, Testosterone, and TSH), sperm count and semen volume with both; the duration of cigarette smoking per year and the number of cigarette smoking per day.

2.Literature review A man is responsible in about 20% of infertility among couples, and contribute to infertility with woman in another 30-40%. Infertility can either be primary or secondary; primary male infertility is when the man has never impregnated a woman, while secondary male infertility is when a man has impregnated a woman irrespective of the outcome of the pregnancy. Men with secondary infertility, in general, have better chance of future fertility. (15,16) Duration of infertility is defined as the number of months during which the couple has been having sexual intercourse without the use of any contraceptive method. This indicator gives an important information about the couple's future fertility, if the duration of infertility of 3 years or less the

couples have a better chance of future pregnancy, but if the duration has been longer, then there is a severe biological problem. But in general couples tend to seek medical advice after a shorter duration of infertility. (15) 2.1 Diagnosis of Infertility in Men: The most important steps in diagnosis of infertile men are a careful history taking and a physical examination. The past medical history of patients is very important because it contribute to the diagnosis in one-quarter of cases of infertility. Specific childhood illnesses may result in problems in the reproductive system like failing of testes to descend that result in cryptorchidism, post pubertal mumps orchitis (mumps accompanied with swelling of one or both testis), time of puberty, surgical history, therapeutic medications, and systemic diseases. (15,17) Physical examination is the second step in diagnosing abnormalities that causes infertility in men, measurement of height, weight, and blood pressure will give some information about systemic diseases. Body hair distribution gives an indication of androgen production, breasts should be inspected to detect gynaecomastia (breast enlargement), examination. (17) 2.1.1 Sexual or ejaculatory dysfunction: Difficulties with sexual intercourse or ejaculation are identified in about 2% of couples who have fertility problem. Sexual dysfunction can be as a result of either inadequate erection or inadequate frequency of sexual intercourse, if the average frequency of vaginal intercourse is twice or less per month it is inadequate. (15) Ejaculation to be considered adequate, it should occur intra vaginally, ejaculatory disturbance may results from ejaculation that occurs outside the vagina, no ejaculation takes place, or from retrograde ejaculation. Retrograde ejaculation is characterized by ejaculation into the bladder, because the bladder sphincter does not function properly. Normally, the sphincter of the bladder contracts before ejaculation forcing the semen to exit via the urethra.

Retrograde ejaculation could be occurred as a result of congenital absence of the bladder neck, nerve damage, diabetes, surgical procedures, and spinal cord injury, the diagnosis mainly based on founding spermatozoa in post-coital urine. (15,18) 2.1.2 Immunological cause: Sperm antibodies may be found in the semen of both fertile and infertile men,but it is diagnosed as immunological cause of male infertility when 50% or more of motile spermatozoa are found to be coated with antibodies. Sperm antibodies have been found in 3-7% of infertile men, and these antibodies may impair sperm function and may cause infertility in some men with impaired fertility like previous vasectomy, genital tract infection, and testicular injury or torsion. (15,18) 2.1.3 No demonstrable cause (Unexplained infertility): Unexplained infertility can describe 10 to 15% of infertile couples. Male is diagnosed as not having any demonstrable cause only if he has adequate sexual and ejaculatory function and the semen analysis is normal [3]. Normal semen parameters are of volume > 2cc, concentration >20 x 106/ml, morphology >30% normal, and motility > 50%. (19) 2.1.4 Isolated seminal plasma abnormalities: If the patient has normal spermatozoa but has abnormalities in the physical, or biochemical, or bacteriological composition of the seminal plasma, or increased number of white blood cells in semen then the patient is diagnosed with isolated seminal plasma abnormalities. (15) 2.1.5 Iatrogenic causes: When the abnormal spermatozoa are due to medical or surgical causes it called iatrogenic causes. There are some drugs that interfere with fertility like Sulphasalazine and Nitrofurantoin both may cause

impairment of sperm quality by direct toxicity, Colchicine and Niridazole can cause depression of fertility, Spironolactone may antagonize the action of androgen, Cimitidine may inhibit androgen effect. Hormonal treatments with high doses of corticosteroids, androgens, antiandrogens, progestogens, estrogens, and anabolic steroids that are taken by athletes can cause reduction in the gonadotropin secretion and lead to testicular atrophy. (15) Cancer therapy for some diseases can have a deleterious effect on fertility especially irradiation in the genital region, the degree of damage and suppression of spermatogenesis that occur as a result of treatment of malignancy depends on whether exposure occur before or after puberty, and the dosage and duration of exposure. If irradiation occurs during or after puberty the damage of germ cell is more severe. Cytotoxic drugs such as cyclophosphamide that used for cancer chemotherapy, when used in high dose or in combination regimens can cause severe germ cell damage [18]. Other drugs can cause erectile potency or ejaculation dysfunction include some anti hypertensives and tranquillizers. (15,18) Short term use of cocaine is associated with increase in the sexual performance, but chronic use is related to impotence in men. Marijuana also affects sexual function; chronic marijuana consumption can decrease sperm concentration. In heroin addicts and methadone treated patients there are abnormalities in their semen analysis especially sperm motility and morphology. (18) Several surgical procedures may influence the male fertility, testicular biopsy can result in a temporary suppression of spermatogenesis [15], bladder neck incision; treatment of urethral valves; and prostatectomy can results in retrograde ejaculation. Lumbar sympathectomy, hypospadius, epispadias, and vesicular exstrophy may cause ejaculatory disturbances. Hernia repair may cause damage to the vas deferens or leads to production of antisperm

antibodies, and that may also occur after hydrocelectomy or any other genital or inguinal surgery like vasectomy. (15) Many of systemic diseases can influence fertility in men, diabetes mellitus and neurological disease may cause erectile impotence and disorders of ejaculation. Chronic respiratory tract disease is associated with disorders of the sperm flagellum, tuberculosis can impair sperm transportation by causing epididymitis and prostatitis. Chronic liver and renal failure may result in infertility, hepatic cirrhosis can cause testicular damage and lead to testicular failure. Also fever exceeding 38.5c may cause suppression of spermatogenesis for a period of up to six months. (15,18) 2.1.6 Congenital abnormalities: Congenital abnormalities include a history of testicular maldescent, karyotype abnormalities, and azoospermia (sperm concentration is 0 x 106/ml) due to congenital agenesis of the vasa deferentia. Cryptorchidism (testicular maldescent) is the failing of the test is to descend normally from the abdomen in to the scrotum. Correction of testicular maldescent can be done surgically after puberty in men up to 32 years of age; however men over the age of 32 are at greater risk of death from surgery than from testicular malignancy. Karyotype abnormalities like in Klinefelter's syndrome that characterized by the presence of one or a number of extra X chromosomes, and in Down syndrome that associated with moderate to severe reduction in sperm production, also a number of rare complex genetic syndromes can affect fertility in men. In case of Y-chromosome gene deletion, micro deletion are more prevalent in infertile individuals, and deletions can cause severe spermatogenic defects ranging from non obstructive azoospermia to oligozoospermia. X-genes also affect male infertility in X-linked genetic disorders like Kultman's syndrome. Y-linked mutations can have adverse effects on spermatogenesis and normal sperm function, and it was found that men lacking expression of fertility genes of the Y chromosome are unable to make adequate function sperm. The prevalence of

these defects increases as the sperm count decreases. Congenital defects of the vas deferens, seminal vesicles, and epididymis may obstruct sperm transport and these include congenital absence of the vas and seminal vesicles, which is most commonly due to cystic fibrosis. (12,15,18,20) 2.1.7 Acquired testicular damage: Acquired testicular damage is recorded when the abnormal spermatozoa are caused by parotitis with orchitis. Mumps occurring before puberty and mumps not accompanied by orchitis do not affect fertility. The majority of men with previous bilateral mumps orchitis develop severe oligozoospermia or azoospermia, and therefore infertility that is irreversible. Testicular injury and testicular torsion can cause testicular damage. Testicular trauma as a cause of infertility is rare, but severe injury accompanied by tissue damage to the scrotum may cause disruption of the blood test is barrier and initiate antisperm antibody production. Testicular torsion is also infrequent cause of infertility, and fertility problems that results from a testicular torsion may be prevented by early treatment. (15) 2.1.8 Varicocele: Varicocele is a dilation of the testicular veins within pampiniform plexus of the spermatic cord that holds up a man's testicles Varicocele may cause infertility if it associated with abnormal semen analysis, but the mechanism is unclear. According to human report update (2001) varicocele is found in 15% of the general population including adolescents and adults, but the prevalence of varicocele among men attending the infertility clinics range between 30 to 40%. A study in 24 centers for the WHO found varicocele in 25.4% of men with abnormal semen compared with 11.7% of men with normal semen. So, not all men who have varicocele are infertile, but varicocele is more prevalent in infertile men. Varicocele occurs more frequently on the left side in about 90% of cases, and it is common in men with secondary infertility. (21,22)

The etiology of varicocele is multifactorial, the most common is the differences in the anatomy of the left and right spermatic vein, absence of valves in the spermatic vessels resulting in retrograde of the blood flow, and compression of the left renal vein causing a partial obstruction. Treatment of varicocele can be done by either surgery or embolisation. (23) In a review of literature in 2008 to evaluate the role of varicocele repair on male infertility it was found that varicocele repair is an effective treatment for selected patients and the most cost effective. But in 2009 another review to the effectiveness of varicocele treatment on restoring fertility in men the authors found that there is no evidence that treatment of varicocele will improve fertility. (24) 2.1.9 Sexually transmitted diseases: Sexually transmitted diseases and male accessory gland infection (MAGI) can impair male fertility by increasing the reactive oxygen species, or by causing inflammation lesions of the epididymis, or urithritis, or urethral strictures, or ejaculatory disturbance, or by stimulating anti sperm antibodies (ASA). It is hypothesized that infection with Chlamydia trachomatis, ureaplasma urealyticum, gram-negative bacilli, and mycobacterium tuberculosis results in accessory sex gland dysfunction and cause infertility. Infertile men may have a high incidence of herpes simplex and human papilloma virus in their semen, the presence of human papilloma virus in their semen may have an effect on sperm motility. (15,18) 2.1.10 Endocrine causes: The hypothalamus-pituitary endocrine system regulate the hormonal events that required to the normal testicular function. Hypothalamus stimulated the pituitary gonadotropins which are : Luteinizing Hormone (LH) stimulate the production of testosterone, and Follicle-Stimulating Hormone (FSH) which stimulate the production of seminiferous fluid. Normal levels of LH and FSH are necessary for maintenance of spermatogenesis,

disorders of the pituitary or hypothalamus will cause inadequate gonadotropin stimulation of the testis and that will lead to problems with fertility. (8,18) Disorders of sperm production may results from either diseases that affect the testis which called primary hypogonadism or from disorders of the pituitary or hypothalamus which called secondary hypogonadism. In men with primary hypogonadism the gonadotropin levels are increased (hypergonadotropic hypogonadism),while in men with secondary hypogonadism gonadotropin levels are low or low to normal (hypogonadotrophic hypogonadism). Measurement of FSH concentration is necessary to distinguish between hypergonadotropic and normo-or hypogonadotrophic hypogonadism. (18) Normal FSH concentration may indicate obstruction of sperm transport. Elevated FSH concentration may suggest severe defects in spermatogenesis, but in men with reduced testicular volume and signs of hypoandrogenism with the presence of high FSH level may indicate primary testicular failure, but if FSH is not elevated in these men that may due to failure of the hypothalamo-pituitary function or to pituitary tumor. Assessment of FSH level is not necessary in men with sperm concentration over 5Plasma testosterone level must be measured in men with signs of hypoandrogenism and in whom FSH is not elevated, and in men with sexual dysfunction. Prolactin is measured in men with sexual dysfunction or in men with signs of hypoandrogenism, some medication are responsible about increased prolactin concentration. Thyroid function must be assessed because hyperprolactinaemia may be associated with hypothyroidism, thyroid hormone assessment should be performed in men with suspected thyroid dysfunction. (15) 2.1.11 Seminal abnormalities: Idiopathic oligozoospermia is accepted if the sperm concentration is less than 20 x 10 6 /ml but more than 0 x 10 6 /ml and there is no other cause from

the causes mentioned above. Idiopathic asthenozoospermia in this case the sperm concentration is normal but there is a low proportion of spermatozoa with progressive motility and none of the other causes is applicable. Idiopathic teratozoospermia requires normal sperm concentration and motility but low morphology, and also none of the other causes is applicable. Idiopathic cryptozoospermia is diagnosed if no spermatozoa are found in the fresh semen sample, but few are found after centrifugation. (15) Obstructive azoospermia is diagnosed if the semen is azoospermia (no sperm are present in the semen) but the testicular biopsy reveals a full complement of spermatogenic in the seminiferous tubules. While patient's with idiopathic azoospermia has low or normal testicular volume million per ml and normal testicular volume. and spermatozoa are absent in any of the seminiferous tubules, the patient's is diagnosed with idiopathic azoospermia when the azoospermia is of unknown origin. (15) There is a strong evidence that most of the disorders of the male reproductive system such as testicular cancer; declining in semen quality; undescended testis; and hypospadius is of an antenatal origin as a results of disruption of embryonal programming and gonadal development during fetal life. All these are symptoms of one underlying concept the Testicular Dygenesis Syndrome (TDS), TDS can also be caused by either genetic or environmental factors. (24,25) 2.2 Risk factors of male infertility: 2.2.1 Age: Age is important risk factor for conception for both men and women. The peak rate of conception occurs at age 24 for both men and women and then after age 35 the rate begins to decline significantly. Studies have shown that blood testosterone level decline with age, and the risk of becoming infertile doubled in men who are over 35 years old compared with men who are under 25 years old, and five times longer to conceive at the age of 45.

Production of testosterone hormone begins to decrease around the age of 40, sperm quality changes with aging, also there is a decrease in the semen volume, motility, and normal morphology. (25,26) Studies showed that sperm concentration is stable, but the percentage of motility is the only parameter which decrease with age, and the fertilizing capacity does not seem to be decreased. However another study found that not only motility decreased with age but also sperm concentration, with normal sperm morphology decrease after the age of 45 years. In a study on a convenient sample of 55 healthy men ranging in age from 52 to 79 years old compared with a control group of men less than 52 years old found that older men had lower semen volume, with abnormal sperm morphology and reduce vitality. Another large retrospective study from a representative European database provided evidence that paternal age is an important risk factor for infertility. A study in Belgium by Mahmoud et al.2003 indicated that testicular volume of elderly males in their eighth decade was significantly less with 31% when compared with the young control group of 18 to 40 years old. (27,28) 2.2.2 Obesity: Several studies have shown that fertility decreased in overweight and obese women. Similarly, obesity may play a role in men fertility. A study in US investigating farmers and their wives showed that 10 kg increase in the body weight may reduce fertility by nearly 10%, and the great effect for men with a body mass index (BMI) of more than 32. A significant reduction in the number of normal motile sperm has been observed among men with BMI over 25, it also found that men with excess fat in the thigh and suprapubic area have poor semen quality. A Norwegian cohort study found that the risk of infertility is associated not only with high BMI but also with low BMI. (29)

2.2.3 Occupational exposure: Among the factors thought to affect male infertility is the occupational exposure, it was found that there is no significant association between infertility and occupational exposure. Another study conducted in Lebanon had demonstrated that occupational exposure to harmful physical and chemical agents is associated with increased risk of male infertility. Exposure to organic solvents at work associated with reduction in count of motile sperm, a number of solvents that are used Furthermore, welding may reduce the quality and quantity of semen, likewise, occupations in which the workers exposed to heat they have reduced sperm count. Also workers in agriculture or in a pesticide factory may experience a negative effect on reproduction Dibromochloropropane (DBCP) can cause testicular toxicity and reduce sperm production. In men who exposed to Ethylene Di-Bromide (EDB) had decreased sperm count and increase number of abnormal spermin industry may have an adverse effect on male reproductive function like carbon disulphide that had shown to affect semen quality but in low exposures had shown no effect. Previous exposure to glycol ethers in work place associated with decrease in the semen quality. (30,31) Furthermore, welding may reduce the quality and quantity of semen, likewise, occupations in which the workers exposed to heat they have reduced sperm count. Also workers in agriculture or in a pesticide factory may experience a negative effect on reproduction, Dibromochloropropane [DBCP] can cause testicular toxicity and reduce sperm production. In men who exposed to Ethylene Di-Bromide [EDB] had decreased sperm count and increase number of abnormal sperm, also insecticide have been found to have decreased sperm motility but there is no effect on fertility. Dichloro- Diptenyl-Trichloro-ethane [DDT] is a type of pesticides can lead to decreased fertility and altered sperm counts. (31)

Industrial and construction workers presents with an increase infertility rates because of greater exposure to stress, occupational stress was negatively correlated with the proportion of normal sperm. Heavy metals like cadmium and lead reduce the quality of semen, mercury can concentrate in the testes beside other organs, mercury poisoning leads to infertility. Furthermore, mercury and copper can interfere in spermatogenesis. (31,32) 2.2.4 Exercise: There are many health benefits of exercise, despite of that there are a conflict results about the effect of exercise on the male reproductive function. It was found that endurance training at highest level does not alter the male reproductive function, and there is no significant effect in hormonal profile and sperm parameters except for sperm motility in the cyclist (riding a bicycle) it was observed lower sperm motility but that may attributed to physical factors. The effect of vicious cycling was studied in another study and it was found that infertility was from the less common symptoms. But recent study suggesting that long term strenuous exercise have a deleterious effect on semen parameters, and also resistance exercise shows a significant decline in free and total testosterone. (33) 2.2.5 Type of under trousers and position: Types of under trousers affect the scrotal temperature, and semen quality. Wearing tight fitting under trousers is associated with increased scrotal temperature (as opposed to wearing loose under trousers or being naked). Note that left scrotal temperature is higher than right scrotal temperature. (33) Also the position or activity has its impact on increasing the scrotal temperature, walking is associated with significantly lower scrotal temperature than sitting, while driving for more than two hours continuously is associated with increasing the scrotal temperature. (34) 2.2.6 Drinking alcohol and caffeinated beverages:

Previous studies had found no association between alcohol consumption and male infertility. Whereas another study found that alcohol consumption affected the reproductive system at all levels. A recent study in Nigeria found a significant effect of alcohol consumption on infertility especially moderate to heavy alcohol intake. (35) Drinking caffeinated beverages may interfere with fertility in men; a study showed that men who consume more than three cups of tea daily is associated with decreased fertility. While another study found that there is no effect of caffeine on the semen quality and quantity. (36,37) 2.2.7 Laptop and cell phones: Exposure for a long time on a laptop will increase the scrotal temperature and have a negative impact on sperm parameters. Furthermore, using cell phones has been noted to have an adverse effect on male fertility due to decreased semen quality which paralleled of daily exposure to cell phones. Another study found that use of cell phones decreased the actual percentage of the live sperm and this correlated with the duration of using these phones. (38,39) 2.2.8 War and stress: For the relation between the war and infertility; a study on the French veterans found that only 9% of the gulf veterans have fertility problems and it was not in a high frequency, also in a review of literature on the reproductive health following the first gulf war it war found that there is some evidence of risk associated with service in the gulf war. However, when two studies were conducted in Lebanon it was found a strong relation between war and increased male infertility, that may due to exposure to toxins and stress. Men under stress their semen parameters are significantly decreased. (40) 2.2.9 Smoking:

The effect of smoking on male infertility and semen quality has been investigated in many studies on fertile and infertile men, their results are conflicting: several studies showed that smoking had an adverse influence on the semen quality specially among heavy smokers, a study in Singapore found that smoking increases the risk of infertility and there is no difference among the different smoking groups. (40,41) On the other hand, a mini review of studies on the effect of smoking on semen parameters showed that smoking had limited effect on semen quality, and another study found no significant effect of smoking cigarettes on semen quality. Whereas, another study found that there is no association between male smoking and delayed conception. (42) A study was done on young men from five European countries concluded that paternal smoking may reduce semen quality and testis size while current smoking had no effect on semen quality. So it is still not clear whether smoking or not affects semen quality and male infertility since human susceptibility may play an important role for this difference. (43) Cigarette smoking is a widely recognized health hazard, yet despite worldwide antismoking campaigns, some people continue to consume cigarettes on a regular basis, and the highest prevalence of smoking is observed in young adult males during their reproductive period. Reviewing the literature concerning the relationship between cigarette smoking and reproductive function, highlighted a strong body of evidence indicating the negative effect of cigarette smoking on male and female fertility. (44) A consistent number of studies have claimed that cigarette smoking is correlated with alterations in sperm quality such as semen volume, sperm concentration, motility, and morphology concomitant with a reduced concentration mainly of citrate and also of fructose. On the other hand, other studies did not find any alterations in conventional semen parameters. In infertile smoking men, the antioxidant level, particularly the superoxide

dismutase level, was recently found to be correlated with sperm concentration and negatively correlated with leukocytospermia. (45) Smoking is a practice where a substance, most commonly tobacco is burned and the smoke is tasted or inhaled, this is primarily done as a form of recreational drug use. Most common method today is through cigarette primarily industrially manufactured but also hand rolled from loose tobacco and rolling paper.other form through not as common are pipes, cigar, hookahs and bongs. (46) Tobacco smoking is today by far the most popular form of smoking and is practiced by over one billion people in the majority of all human societies, less common drugs that are smoked are considered to be addictive, some are hard as heroin and crack cocaine, but the use of the sears very limited and they are often not commercially available. (45,46) 2.2.9.1 Types of tobacco products: By the end of the twentieth century, manufactured cigarettes have come to be the predominant form in which tobacco is consumed around the world, it is also consumed in much other form, and some of the major forms of tobacco products currently in use are: 2.2.9.1.1 Smoking tobacco: 2.2.9.1.1.1 Cigarettes: Cigarette (French small cigar from cigar + ette ) is a product consumed through smoking and manufactured out cured and finally cut tobacco leaves and reconstituted tobacco often combined with other additives, A cigarette is distinguished from a cigar by its smaller size, use of processed leaf, and white paper wrapping. Cigars are typically composed entirely of whole-leaf tobacco. (47) Manufactured cigarettes are available in all countries, and filter-tipped cigarettes are usually more popular than plain-end cigarettes, handmade or "roll you- own cigarettes are also widely used in many countries. Other types of cigarettes include kreteks (clove- flavored cigarettes) sticks. (46,47)

2.2.9.1.1.2 Bides: Bid consists of a small amount of tobacco (0.2-0.3 g) wrapped in temburni leaf and tied with a small string. It is estimated that in the 1989And 1990s about 675.000 millions bides were smoked annually in India,50.000 million in Bangladesh and about 25.000 million in other countries. (46) 2.2.9.1.1.3 Cigars: Cigar are made of air cured and fermented tobaccos with a tobacco wrapped, and come in many shapes and size. (46) 2.2.9.1.1.4 Pipes: A pipe is a tool used for smoking. The designs of pipes vary considerably, but for the most part they are reusable and consist of a chamber, or bowl, in which the substance to be smoked is placed, a stem of some sort and a mouthpiece through which the smoke is sucked or inhaled,prior, slate and clay pipes have been used throughout Europe and the Americas. Water pipes are in common use in North Africa, the Mediterranean region and Asia. Having many names e.g. hoolah, gozahubble and shisha. (46) 2.2.9.1.2 Smokeless tobacco: 2.2.9.1.2.1 Chewing tobacco: Plug loose leaf and twist their use remains popular in certain sub populations. (46) 2.2.9.1.2.2 Pan: Pan chewing is widely practiced in south East Asia especially in India. (46) 2.2.9.1.2.3 Snuff: Dry snuff is powdered tobacco that is inhaled through the nasal passage or taken orally. Most snuff is taken orally. A small amount of ground tobacco is lied in the mouth between the cheek and the gum. Other smokeless tobacco

products are in use. One of these is khaini, mishri, zarda, kiwan and pills. These products, as well as others tobacco preparation known as gudakha. bajar and creamy snuff are often used for clearing teeth. (48) 2.2.9.2 Smoking prevalence: WHO estimate that there are about 1100 million smokers in the world representing about one third of the global population aged 15 years and over. The majority of the smokers are in developing countries (800million) and most of these are men (700 million). In china alone there are about 300 million smokers (90 % men, 10 % women ) about the same number as in all developed countries combined. About one third of regular smokers in developed countries are women compared with only about 1 in 8 in developing countries. Globally it is estimated that 47 % of men and 12 % of women smoke in developed countries, the corresponding figures are 42 % for men and 24 % for women. In developing countries, available data suggests that about 48 % of men and 7 % women smoke. Male smoking prevalence varies substantially among regions from less than 30 % in Africa region to 60 % in western pacific region. Even among developed countries, patterns of male smoking prevalence are not uniform in countries with established market economies, male smoking prevalence average 37% compared to 60 % in former socialist countries of central and Eastern Europe. (48) Smoking among women is most prevalent in former socialist, countries of central and Eastern Europe (29%), countries with established market economies (23%) and Latin American and Caribbean countries(21%) in most other countries fewer than 15 % of women smoke 17 old). (48) In many countries people begin to smoke at younger ages, with the median age of initiation under 15 yrs. starting to smoke at younger ages increase the risk of death. Young people who start smoking early in life will often find it difficult to quit smoking. Among those who continue to smoke throughout their lives, about one half can be expected to die from a smoking related issue, half of these

in the middle age (35 69)and the other half in old age (70 yrs and over).in countries, where more than 50%of young men are smokers aged 18-24 years and most of them began at a young age, a very heavy future death from tobacco use can be expected. (48) In general fewer cigarettes are smoked per day in developing countries than in developed countries, among developed countries the highest rate of daily consumption per smoker, 24 cigarettes per day: occur in countries with established market economies. (48,49) 2.2.9.3 Tobacco and health: Several decades of epidemiological research have identified cigarette smoking as a major cause of preventable mortality in developed countries. For smokers, the magnitude of the risk increase with increasing duration of smoking. Data from the mid of 1980s confirm that among smokers age 35 69, the death rate is three times that of non smoker. Among men in developed countries, smoking is estimated to be the cause of 40-45% of all cancer death, 90-95% of lung cancer death,75%of chronic obstructive lung disease death, just over 20%of vascular diseases deaths, and 35%of cardiovascular disease death in those aged 35-65 years. (50) Of all the diseases usually associated with smoking, lung cancer is the most well known, simply because in most populations almost all lung cancer deaths are due to smoking. However, smoking actually causes more deaths from diseases other than lung cancer. In 1995, therewere 541.000 smoking attributable lung cancer deaths in developed countries, compared to 625.000 smoking attributable deaths from heart and vascular disease in the same year. The use of smokeless tobacco (common among both men and women in areas such as south Asia) also poses serious heart risk, most notably elevated risks of cancer of the buccal cavity. The annual mortality from tobacco chewing in south Asia alone is of order of 50.000 deaths a year. (50)

In addition epidemiological evidence suggests that cigarette smoking is the major risk factor for chronic obstructive pulmonary diseases such as chronic bronchitis and emphysema for carcinogenesis and for cardiovascular disease, however, the precise mechanisms of these effects are incompletely understood. (50) Environmental tobacco smoke contains most of the toxic and carcinogenic compounds identified in mainstream smoke, absorption of smoke constituents from the environment has been documented in both infants and adults, and a number of epidemiological studies have demonstrated health effect in human. (50) 2.2.9.4 Cigarette smoking: Cigarette smoking is addictive because of the presence of alkaloid, nicotine, and its nicotine withdrawal that causes the unpleasant side effects after quitting smoking. Nicotine is an alkaloid found in night shade family of plant (solanaceose) which constitutes approximately 0.6 3.0 % of dry weight of tobacco. (50,51) As nicotine enters the body, it is distributed quickly through the blood stream and can cross the blood-brain barrier. On average it takes about seven seconds for the substance to reach the brain when inhaled. The half life of nicotine in the body is around two hours, the amount of nicotine inhaled with tobacco smoke is a fraction of the amount contained in the tobacco leaves. The amount of nicotine absorbed by the body from smoking depends on many factors, including the type of tobacco, whether the smoke is inhaled, and whether a filter is used. (52) For chewing tobacco, dipping tobacco and snuff, which are held in the mouth between the lip and gum, or taken in the nose, the amount released into the body tends to be much greater than smoked tobacco. Nicotine is metabolized in the liver by cytochrome P 450 enzymes (mostly CYP2A6, and also by CYP2B6). A major metabolite is cotinine. Nicotine easily crosses the blood-

brain barrier and there are nicotine receptors throughout the central and peripheral nervous system. (53) Nicotine acts on the nicotinic acetylcholine receptors, specifically the ganglion type nicotinic receptor and one CNS nicotinic receptor. The former is present in the adrenal medulla and elsewhere, while the latter is present in the central nervous system (CNS). In small concentrations, nicotine increases the activity of these receptors. Nicotine also has effects on a variety of other neurotransmitters through less direct mechanisms and nicotine has multiple neurological pathways, which control pleasure. (53) Nicotine's mood-altering effects are different by report. First causing a release of glucose from the liver and epinephrine(adrenaline) from the adrenal medulla, it causes stimulation. Users report feelings of relaxation, sharpness, calmness, and alertness. By reducing the appetite and raising the metabolism, some smokers may lose weight as a consequence. (54) Cigarette smoke also alters the structure and function of respiratory passages, capillaries and immune system of the lung. Nicotine has been found to damage the vascular endothelium, which could lead to the initiation of atherogenesis (capability of producing atheroma) and it is believed that smoking accelerates pre-existing athherogensis. (55) 2.2.9.5 Other effect of smoking: Cigarette smokers are ten times more likely to develop lung cancer than other smokers this risk is proportional to the number of cigarettes smoked per day, increasing to 20 to 25 times risk of nonsmokers in those who smoke two or more packs of cigarettes per day. This risk as also increased in those who inhale more deeply or began smoking at a younger age Cigarette smoking causes all of the major types of lung cancer. (56) Cigarette smokers have a great incidence of gastric and duodenal ulcers and delayed healing of theses ulcers. Several of these constituents of tobacco smoke are capable of inducing ghepatic microsomale systems which then alter

the metabolism of other drugs. Smokers have lower blood levels of vitamin c and vitamin B12, heamatocrit and hemoglobin level as well as carboxy heamoglobin level are elevated in smokers, and smoking is one cause of elevated red cell volume, smokers also have some small alteration in other diagnostic test, including a higher leukocyte count, but these differences are not usually significant for an individual patients. (56,57) Some of possible determinable effects of cigarette smoking on cardiovascular system increase in leukocyte count,platelet aggregation, fibrinogen level,plasma viscosity, and free ionized calcium total serum cholesterol and low density lipoprotein cholesterol. (58) 2.2.9.6 Health benefit of smoking: Studies suggest that smoking decreases appetite, but did not conclude that overweight people should smoke or that their health would improve by smoking. (58) Several types of "Smoker s Paradoxes (cases where smoking appears to have specific beneficial effects, the effect is eliminated if the individual stops smoking. Smoking appears to interfere with development of Kaposi's sarcoma breast cancer among women carrying the very high risk BRC Agene, and atopic disorders such as allergic asthma. A plausible mechanism of action in these cases may be the nicotine in tobacco smoke acting as an anti- inflammatory agent and interfering with the disease process. (59) Evidence suggests that non-smokers are up to twice as likely as smokers to develop Parkinson's disease or Alzheimer's disease. (60) 2.3 Prevalence and causes of infertility: Infertility becomes a public health problem when it's frequency exceeds 15% according to the WHO. Globally, it is expected that around 50-80 million people (8-12 % of couples) experience some form of infertility in their lives. There are large differences in prevalences of infertility among countries that might be due to differences in definitions and epidemiological designs. A

survey in 1992 in the United States reported that 8.5% of married couples had infertility, and this rate was similar to the rate among infertile couples in Northern Sweden (9%), of which 6% primary infertility and 3% secondary infertility. While In Western Siberia it was much higher, 16.7% of couples were considered to be infertile, 3.8% of all couples suffered from primary infertility and 12.9% had secondary infertility, and male infertility was 6.4% from the total prevalence. A population based study in United Kingdom found that the incidence of infertility among couples was 9%. In Spain, 257 males were studied for the cause of their infertility, endocrine cause was found in 3.5% of cases, 30% were idiopathic, 17.9% of cases had varicocele, 12.8% were associated to cryptorchidism, 8.9% to Klinfelter syndrome, and 6.6% were to exposure to toxic substances. (61,62,63) The aetiology of male infertility was studied in Kenya in 2005 on 43 men; 23% of the cases presented with signs of hypogonadism, 35% with signs of pain and swelling due to acute inflammation of the testes, 9% had prolactinaemia, 5% had signs of gonadotropin, another 5% had varicocele,and 23% of cases had idiopathic infertility. A similar study was conducted in Brazil on 822 men; 34.3% of the cases presented with varicocele, 31.6% idiopathic infertility, 10.3% seminal tract obstruction, 5.2% mumps, 4.5% pyospermia, 4.4% had systemic diseases, 4.1% testicular failure, 1.7% cryptorchidism, and 1.3% had ejaculatory dysfunction. In Northern Tanzania, 112 couples were evaluated for the causes of their infertility; 37.1% of them had primary infertility and 62.9% had secondary infertility, and male factor alone was found in 6.8% of cases. In southeastern Nigeria also, 314 couples were evaluated for the causes of their infertility; of them 65% had primary infertility while 35% had secondary infertility and male factor alone was found in 42.4% of the cases. A retrospective study in Northern Nigeria was conducted on 537 infertile men; primary male infertility was seen in 96% and secondary male infertility in 4%, about half (48.7%) of the cases had genitourinary tract infection. In addition

azoospermia from testicular pathology was seen in 3.4% and obstruction to the vas or epididymis was seen in 14%, 45% had oligospermia resulting from testicular insufficiency while 11.4% had oligospermia due to seminal tract obstruction. (64,65,66) The causes of infertility among 430 infertile couples were studied in Mongolia, about one quarter (25.6%) of the couples infertility was due to male factor and 18.8% of the couples infertility was diagnosed in both partners. The most common causes of male infertility were a history of sexual transmitted diseases (44.2%), previous testicular damage (33.5%), obstructive azoospermia (8.4%), MAGI (6.7%), and acquired testicular damage (5.4%). In China 7872 newly married couples were followed up to assess the prevalence of infertility among those couples, the prevalence of infertility was found to be 5.1% (after 24 months of unprotected sexual intercourse). A retrospective study was performed in Thailand between 1999-2004 on 172 infertile couples. That study revealed 61.8% of primary infertility and 35.6% of secondary infertility. The causes of infertility in this study were found in 55.6% in both partners, 19.4% in male partner and 17.5% in female partner, whereas 4.7% of couples had unexplained infertility. In Bangladesh two recent studies were conducted on infertile couples. The first study estimated the prevalence of infertility among couples to be 10%; of which 40% male, 50% female, and 10% involve both sexes. The second study was conducted to find out the causes of couples infertility among them; primary infertility was present in 61.9% and secondary infertility in 38%, a positive male factor alone was found in 13% of couples and oligospermia was the most common cause of male infertility. A study about primary infertility in Kashmir region in India on 250 couples found that primary infertility account for 15%, and male infertility alone was 22.4%. In Iran, three studies were conducted to determine the prevalence of infertility among couples; in 2004 in Tabriz city the prevalence of infertility was 3.3%; 2% as primary infertility and 1.2% as secondary infertility. (67,68,69)

From 2004-2005 a study was conducted in all provinces of Iran to assess the prevalence of primary infertility, and it was found to be 3.4%. In 2007 another study was conducted to explore the prevalence and the risk factors of infertility (after 24 months of unprotected sexual intercourse), the overall prevalence of infertility was 8% ; 4.6 % as primary infertility and 3.4% as secondary infertility. In Turkey most studies focused on genetics and its relation to male infertility. A study was carried out on 208 patients who had either non obstructive azoospermia or severe oliozoospermia compared with 20 fertile men, the rate of genetic abnormalities among infertile men was 12.5%. A survey in 2009 was conducted on 1935 males with male factor infertility to assess the genetic abnormalities among those men. The researcher found that the genetic abnormality rate increased with the severity of infertility. In Arab countries, a retrospective study in Saudi Arabia was performed on 230 testicular biopsies, 31.3% showed normal spermatogenesis, 39.1% of cases had germinal cell aplasia, 13% showed hypospermatogenesis, 10.9% showed maturation arrest, 5.2% tubular sclerosis, and only 0.5% had karyotypic abnormalities. (70,71) Sixty four infertile men were evaluated between the period 2001-2005 in Kuwait; 38% of them were azoospermic while 62% were oligospermic, 50% of oligospermic cases had varicocele, the most common cytopathology was sertoli cell-only-syndrome, and varicocele imbolization resulted in a significant rise in the sperm count in oligospermic patients. Another study was held in Kuwait on the genetics of primary male infertility on 289 patients, the study showed that chromosomal anomalities and Y microdeletions were found in 10.4% of the infertile men in the study. A cross-sectional study in Qatar on married men revealed that there was a strong association between male infertility and diabetes mellitus, the prevalence of male infertility in men with type 2 diabetes mellitus was 35.1%. Causes of infertility were studied for 250 couples in Iraq, 77.2% of whom had primary infertility and 22.8% had secondary infertility, male factor

alone was found in 36.8% of the cases. In a study was carried out in a center for IVF in Yemen on 485 testicular biopsies, 33.8 % of the cases showed germ cell aplasia, 19.2% showed fibrosis, 20.4% had obstructive azoospermia, and normal spermatogenesis was found in 27.5% of cases. In a study done to understand the medical causes of infertility among 710 Sudanese couples, primary infertility was 62.4% and secondary infertility was 37.6%; male factor alone was found in 36.2% of couples, female factor in 49.3%, 1.5% had a combination of male and female factor, and the cause was unexplained in 13% of couples. In Cairo, 1488 infertile couples were followed up between the period 1980-1989, primary infertility was found the majority of couples (70.7%) and secondary infertility about 29.3% of couples, 20.6% of couples had male factor alone and in 12.2% of couples both the man and his wife suffered of infertility. Another study in Egypt found that there was a decrease in sperm quality and increase in Anti-Sperm Antibody ASA in patients with varicocele compared with men without varicocele. (72,73,74,75) Also a study in Syria was done to assess the association between ASA and unexplained infertility, found a strong association between ASA and unexplained infertility. Many studies were conducted in Jordan on reproductive health. One of these studies was about the prevalence of some abnormalities in male reproductive system, the study found that the prevalence of inguinal hernia and undescended testis were 3% and 0.5% respectively. Another case control study compared men who were exposed to x-ray with another group that was not exposed. A significant association between exposure to radiation and male infertility was found. In Lebanon, five studies investigated male infertility, three of them about war and its effect on male infertility, the study found a strong relation between war and increased male infertility, this may be due to exposure to toxins and stress. Another study about the occupational and environmental exposure to heavy metals as a risk factors for male infertility found that exposure to harmful physical and chemical agents is associated with

increased risk of male infertility. The last study regarded consanguinity and family clustering and its effect on male factor, this study demonstrated a significant association between consanguinity and family clustering on male infertility. A study in Israel investigated the prevalence of genital Chlamydia and mycoplasma infection in 135 infertile couples attending a male infertility clinic compared with 88 fertile couples, found that the prevalence of Chlamydia and mycoplasma was higher in infertile couples. Two other studies dealt with risk factors of male infertility; the first being the effect of vicious cycling on urogenital disorders, and infertility was from the less common symptoms. The other study investigated the association between male infertility and occupational psychological stress found that male infertility is associated with industry and construction jobs. (76,77) Many other studies that covered several topics about infertility Israel including gene variation, in vitro fertilization, and assisted reproductive technology, from these; a study on genotyping of idiopathic oligospermic and azoospermic men, resulted in that Y chromosome microdeletion contributed to male infertility. In Palestine there are no studies available about the causes of male infertility or even for the prevalence of infertility in men or infertility in general at the national level. Whereas, there is only one study about time to pregnancy that was conducted in agricultural villages in Hebron on newly married couples, the researcher found that prolonged time to pregnancy associated with oldest age category for both genders. (78,79) 2.4 Semen parameters: Recent studies about semen quality have provided conflicting evidence, some studies suggested that there is strong evidence that semen quality is declined over the years. In Paris from 1973 through 1992 the seminal volume, the sperm concentration, and the percentages of motility and morphology of normal spermatozoa were measured in 1351 healthy fertile men, during that period there had been a decline in the concentration, motility, and

morphology but there was no changed in semen volume. Another study on 577 men in Scotland provided that sperm concentration and motility decreased with age. The same results were found in a study conducted in India on 7770 subjects in the period between 1993 to 2005 providing that the sperm concentration, motility, and morphology were lower during 2004-2005 compared with 1993-1994. Also a study in Jerusalem conducted to investigate the changes in semen quality among men involved in infertile relationships between 1990 and 2000 found that sperm count and motility declined significantly among men treated by intrauterine insemination. While other studies have found no evidence of any changes, a retrospective review of semen analysis in USA in 1996 for 1283 men over 25 years, concluded that there was no decline in sperm concentration. The same results found in an Indian study in 2003 that have analyzed semen analysis for the last eleven years. In Israel, a retrospective study of semen parameters among healthy sperm donors in Jerusalem for over fifteen years found that there were no significant changes in semen concentration and motility, but there was an increase in the semen volume during the study period. (80,81,82) Furthermore, seasonal variation affected the semen concentration. A study in Europe found that the highest sperm concentration observed during the winter season and the lowest counts observed at the summer season, while no seasonal variation was detected for sperm morphology. Although another study showed that sperm concentration is highest in winter and lowest in fall, and greater percentage of sperm with normal morphology in winter also and the lowest in summer. Geographical regions can affect semen quality. (83) A cross sectional study in four European countries (Denmark, France, Scotland, and Finland) found a significant differences in semen quality between the four European cities. Another study was conducted in France to investigate if there is a difference in semen within state found that there is difference between the north and the south in the total number of sperm. The north had

higher number of sperm compared the south. Evaluation for semen analysis in USA for male partners of women presenting for an infertility consultation, 52% of subjects had at least one sperm abnormality, of them 51% had abnormality in sperm motility, 18% in sperm concentration,14% in sperm morphology, and 4% were azoospermic. A retrospective study in Spain based on reviewing the seminogram forms from 571 clinical files for couples that seekedconsultation for infertility from 1993 until 2001, of the 571seminogram forms; 65%had alteration in the seminal parameters,24 were azoospermic,11.9%had asthenonecro-zoospermia,11.6% had hypospermia, asthenozoospermia in 8.9%, oligoastheno zoospermia in 8.4%,hypo asthenozoospermia in 4.9%,cryptospermia in 2.8%, and hypooligoasthenoteratozoospermia in 1.9%.In 1985 classification for semen, samples were done on 500 Nigerian male partners of infertile couples, of them 74.2% were normozoospermic while 16.2% were azoospermic, 5.6% were necrospermic, and 4.1% were asthenozoospermic. Moreover the degree of oligospermia was mild in 35.9%, severe in 23.2%, and very severe in 40.9%. Another evaluation for semen parameters were done in Ibadan region in Nigeria between 1990 and 1999 on 824 male partners of infertile couples, 27.3% of these subjects had abnormal semen analysis with;27.8% had asthenozoospermia, 6.7% had azoospermia, 25.5% had oligoastheno- zoospermia, and 13.1% of the study subjects had oligoasthenoterato-zoospermia. In 2006 another semen evaluation for 348 Nigerian men, showed that 68% had semen abnormalities, 30% had single factor abnormalities while 38% had combined factor anomalities. (84,85,86) 2.5 Sex Hormones: Various conditions such as alcohol or tobacco smoking can influence serum levels of sex steroid hormones leading to impairment of male reproductive system. Cigarette smoking has major effects on the reproductive potential of humans. Cigarette smoking stimulates the release of several anterior and posterior pituitary hormones. Smoking acutely increases the plasma levels of

prolactin, arginine vasopressin (AVP) without significant changes in TSH, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). (87) These effects are directly proportional to the nicotine content of cigarettes, with greater hormonal responses observed in high content cigarettes. Though all these studies were done in males, similar acute hormonal changes with smoking in women would be expected.. The neurochemical events that occur with nausea are coordinated by the brain stem emetic centre and nicotine is known to stimulate the emetic centre and could thus contribute to smoking-induced nausea. Another possible mechanism is via nicotine-stimulated cyclic AMP production as demonstrated in rats. Stress per se could also cause the release of these hormones. A direct effect of nicotine or neurotransmitters released by nicotine, acting on the anterior pituitary or hypothalamus, could be another possibility. In chronic smokers, however, inhibition of prolactin secretion occurs. (88) The inhibitory effects of chronic nicotine exposure on prolactin secretion are probably produced via an activation of nicotinic receptors of the tuberoinfundibular dopamine neurones releasing dopamine as a prolactininhibitory factor. Besides this, in a study using the GH3 rat pituitary cell line, nicotine was shown to downregulate prolactin gene expression. Baseline prolactin levels are thus lower in chronic smokers than non-smokers. This may contribute to the reduced fertility in smokers. Importantly, pregnant women who smoke have lower prolactin levels towards the end of pregnancy. (89) Furthermore, significantly lower prolactin levels are found to occur in breast-feeding smokers, though suckling-induced acute increases in serum prolactin and oxytocin-linked neurophysin were not influenced by smoking. In this study, smokers were found to wean their babies significantly earlier than non-smokers. Thus smoking women may have a shorter period of lactation due to the lower prolactin levels.. However, in habitual smokers both active and passive smoking is associated with elevated FSH concentrations in perimenopausal women. This results in a shorter duration of the transitional

period to menopause. In men, the levels of gonadotrophins have been reported to be unchanged though others have found increased or decreased LH levels in smokers. Smoking acutely increases vasopressin levels. This could account for the acute hypertensive responses after smoking. As nicotine given intravenously does not affect vasopressin levels, an airway-specific mechanism, through irritation of the sensory nerve terminals in the respiratory epithelium by cigarette smoke, could be responsible for vasopressin release. Maternal smoking also causes disturbances in the endocrine equilibrium of the foetus. Increased levels of prolactin, GH and IGF-I are observed which are more pronounced between 30 and 37 weeks of gestation than at term. The most pausible explanation of hormonal abnormalities in neonates of smoking mothers is foetal distress due to underperfusion of the foetoplacental unit and acute decreases of placental blood flow associated with smoking. However, a direct effect of nicotine is another possibility. The high hormone levels observed in the above study persisted for at least the first 3 days of life likely due to the additional stress caused by adaptation to the extrauterine environment. (89,90) In males, the effect of smoking on androgen levels is important, given the recent interest in the association between low androgen levels and the metabolic syndrome, and coronary heart disease. Various studies examining the effects of smoking on serum testosterone levels have reported conflicting findings largely due to difficulties in the hormonal assays. (90,91)

3.Materials and methods 3.1Study approach: Quantitative approach. 3.2 Study design: This is a descriptive cross-sectional, and analytical case-control study. 3.3 Study area: The study was done in, Dr. Alsir Abualhassan and Nile fertility centers, Khartoum State. 3.4 Study period: This study was conducted during the period from August 2012 to January 2015. 3.5 Study population: 3.5.1 Target population: The target population was infertile Sudanese smokers diagnosis by consultant, aged 20-50 years attending the study areas for medical care. 3.5.2 Inclusion criteria:

Long standing cigarette smokers (5 years and more)were included as a test group in this study. 3.5.3 Exclusion criteria: Men with known causes of infertility, using contraceptive methods, newly married men ( less than one year married ) and men with chronic diseases or use chronic medications. 3.5.4 Ethical consideration: Permission of this study was obtained from the local authorities in the area of the study. The objectives of the study were explained to all individuals participating in this study. An informed consent was obtained from all participants in the study. Health education about the hazards of cigarette smoking. 3.6 Sample size: A total of 600 Sudanese men were included in this study ( 300 infertile smokers and 300 healthy fertile men as control group ). The sample size was calculated based on the formula for unmatched casecontrol studies. Open EPI-INFO statistical package version 7 was used with 99 % two sided confidence level, 80% power and 40 % of controls exposed, 57 % of cases exposed and odds ratio > 2. The ratio of controls to cases is 1:1. 3.6.1 Data collection: * 150 infertile smokers (test group). * 150 infertile non smokers (control group). * 150 fertile smokers (test group). * 150 fertile non smokers (control group). 3.7 Clinical examination: 3.7.1 Specimen collection:

Interviews with the cigarette smokers and the controls were done to obtain the clinical data. A questionnaire was specifically designed to obtain information which helps in either including or excluding certain individuals in or from the study. 3.7.2 Specimen processing: Clinical history & diagnosis of the test group and the control group were checked by a physician. 3.8 Data analysis: 3.8.1 Semen collection and analysis: Semen samples were collected from smokers and non smokers by masturbation in sterile polypropylene containers after sexual abstinence of 3.5 days. Semen volume was measured. Routine semen analysis was carried out by light microscopy. The concentration, motility and morphology of spermatozoa were assessed according to WHO criteria (WHO, 1992). 3.8.2 Blood sampling and processing: Venous blood sample (5 ml) was drawn by a well trained medical technologist into plain vacutainer tubes from smokers and non smokers. The Blood was left for a while allowed to clot. Then serum samples were obtained by centrifugation at room temperature at 3000 rpm/10 minutes then used for hormonal analysis. 3.8.3 Hormonal analysis: 3.8.3.1 Testosterone hormone assay: Testosterone hormone level was determined according to the method of Tiez, 1986 using ELISA TECO kit for testosterone. 3.8.3.1.1 Principle of the assay: The testosterone ELISA Kit is a solid phase enzyme-linked immune sorbent assay (ELISA), based on the principle of competitive binding. The micro titer wells are coated with an antibody directed towards an unique antigenic site

on the testosterone molecule. Endogenous testosterone of a patient sample competes with a testosterone horseradish peroxidase conjugate for binding to the coated antibody. After incubation the unbound conjugate is washed off. The amount of bound peroxidase conjugate is inversely proportional to the concentration of testosterone in the sample. After addition of the substrate solution, the intensity of color developed is inversely proportional to the concentration of testosterone in the patient sample. 3.8.3.1.2 Kit components: 1. Microtiter wells, 12x8 (break apart) strips, 96 wells coated with mouse monoclonal anti-testosterone antibody 2. Standard (Standard 0-6), 7 vials, 1 ml, ready to use concentrations: 0-0.2-0.5. 1. 2. 6-16 ng/ml Conversion: 1 ng/ml = 3.467 nmol/l 3. Enzyme conjugate, 1 vial, 25 ml, ready to use testosterone conjugated to horseradish peroxidase 4. Substrate solution, 1 vial, 25 ml, 5. Stop solution, 1 vial, 14 ml, ready to use contains 0.5M H2SO4. 6. Wash solution, 1 vial, 30 ml (40X concentrated) Note: Additional standard 0 for sample dilution is available on request. 3.8.3.1.3 Assay procedure: All samples and reagents were allowed to reach room temperature (~25 C).Reagents mixed by gentle inversion before use. Standards, controls and samples assayed in duplicate. 1. Microtitration strip was marked to be used. 2. Twenty-five µl of the standards, controls and samples were added into each corresponding well. 3. Two hundred µl of conjugate reagent were added into each well using a precision pipette. 4. The wells were mixed for 10 seconds. 5. The wells were incubated for 60 minute at room temperature (~25 C).

6. Each well was aspirated and washed 3 times by adding 400 µl of working wash solution. 7. Two hundred µl of substrate solution were added into each well using a precision pipette and gently mixed for 10 seconds. 8. The wells were incubated in the dark for 15 minute at room temperature (~25 C). 9. One hundred µl of stop solution were added into each well using a precision pipette and mixed for 10-20 seconds. 10.The absorbances of the solution in each well were read at 450 nm. 3.8.3.1.4 Validity of test: 3.8.3.1.4.1 Calculation of results: The absorbance for each standard, control, or samples were obtained (Absorbance at 450 nm), and then the standard curve prepared by plotting the absorbance readings for each of the standards along the Y-axis versus standard concentrations in ng/ml along the X-axis, the mean absorbance values for each sample were determined the corresponding concentration of testosterone in ng/ml from the standard curve. Normal reference values of testosterone for adult male between 2.0-7.0 ng/ml. 3.8.3.1.4.2 Specificity: Haemoglobin (up to 4 mg/ml), Bilirubin (up to 0.25 mg/ml) and Triglyceride (up to 7.5 mg/ml) have no influence on the assay results. 3.8.3.1.4.3 Sensitivity: The analytical sensitivity was calculated from the mean plus two standard deviations of twenty (20) replicate analyses of Standard 0 and was found to be 0.083 ng/ml. 3.8.3.1.4.4 Accuracy: Use controls at both normal and pathological levels. The controls and the corresponding results of the QC-Laboratory are stated in the QC certificate added to the kit. The values and ranges stated on the QC sheet always refer to the

current kit lot and should be used for direct comparison of the results. It is also recommended to make use of national or international Quality Assessment programs in order to ensure the accuracy of the results. Employ appropriate statistical methods for analyzing control values and trends. If the results of the assay do not fit to the established acceptable ranges of control materials patient results should be considered invalid. In this case, please check the following technical areas: Pipetting and timing devices; photometer, expiration dates of reagents, storage and incubation conditions, aspiration and washing methods. 3.8.3.1.4.5 Precision: Intra-assay reproducibility was determined by measurement of 20 replicates of serum pool in a single run. Mean (ng/ml): 4.88 SD (ng/ml): 0.51 CV (%): 3.28 3.8.3.1.4.6 Linearity: A study was performed diluting a standard (1:2,1:4,1:8,1:16) containing an elevated level of Testosterone with the 0 ng/ml calibrator to determine the linearity of the MICRO-ELISA Testosterone test. Table 3.1 Standard curve: standard concentrations in ng/ml Optical Units (450 nm) Standard 0 (0 ng/ml) 0.00 Standard 1 (0.2 ng/ml) 0.15 Standard 2 (0.5 ng/ml) 0.33 Standard 3 (1.0 ng/ml) 0.45 Standard 4 (2.0 ng/ml) 0.69 Standard 5 (6.0 ng/ml) 0.77 Standard 6 (16.0 ng/ml) 0.91 3.8.3.2 Luteinizing hormone (LH) assay: Luteinizing hormone level was determined according to (Lenton in et al, 1982) method using ELISA TECO kit for LH.

3.8.3.2.1 Principle of the assay: The essential reagents required for an immunoenzymometric assay include excess amount of antibodies (both enzyme conjugated and immobilized) with high affinity, high specificity and contain different epitopes with distinct recognition and native antigen. In this assay procedure, the immobilization takes place at the surface of a microplate well through the interaction of streptavidin coated on the well and exogenously added biotinylated monoclonal anti-lh antibody. Upon mixing, a reaction results between the native antigen contained in serum, the monoclonal biotinylated antibody and the enzymelabeled antibody, without competition or steric hindrance, to form a soluble sandwich complex. Simultaneously, the complex is deposited to the well through the high affinity reaction of streptavidin and biotinylated antibody. After equilibrium is attained, the antibody-bound fraction is separated from unbound antigen by decantation or aspiration. The enzyme activity in the antibody-bound fraction is directly proportional to the native antigen concentration. By utilizing several different serum references of known antigen value, a dose response curve can be generated from which the antigen concentration of an unknown can be ascertained. 3.8.3.2.2 Kit components: One stripholder containing 96 microtitration wells coated with streptavidin, six LH reference standards with concentrations of approximately (0, 5.0, 25, 50, 100 and 200 miu/ml). Enzyme conjugate, TMB chromogen solution, stop solution and wash solution concentrate. 3.8.3.2.3 Assay procedure: All samples and reagents were allowed to reach room temperature (~25 C). Reagents mixed by gentle inversion before use. Standards, controls and samples assayed in duplicate. 1. Microtitration strip was marked to be used.

2. Fifty µl of the standards, controls and samples were added into each appropriate well. 3. One hundred µl of conjugate reagent were added into each well using a precision pipette and then mixed for 30 seconds. 4. The wells were incubated for 60 minutes at room temperature (~25 C). 5. Each well was aspirated and washed 3 times by added 300 µl of working wash solution. 6. One hundred µl of TMB reagent were added into each well and gently mixed for 10 seconds. 7. The wells were incubated in the dark for 15 minutes at room temperature (~25 C) without shaking. 8. Fifty µl of stop solution were added into each well and gently mixed for 10-20 second. 9. The absorbance for each well was read at 450 nm. 3.8.3.2.4 Validity of test: 3.8.3.2.4.1 Calculation of results: The absorbance for each standard, control, or samples were obtained (Absorbance at 450), and then the standard curve prepared by plotting the absorbance readings for each of the standards along the Y-axis versus standard concentrations in miu/ml along the X-axis, the mean absorbance values for each sample were determined the corresponding concentration of LH in miu/ml from the standard curve Normal reference values of LH for adult male between 2.0-13.0 miu/ml. 3.8.3.2.4.2 Specificity: Haemoglobin (up to 4 mg/ml), Bilirubin (up to 0.5 mg/ml) and Triglyceride (up to 30 mg/ml) have no influence on the assay results. 3.8.3.2.4.3 Sensitivity:

The analytical sensitivity was calculated from the mean plus two standard deviations of twenty (20) replicate analyses of Standard 0 and was found to be 1.27 miu/ml. 3.8.3.2.4.4 Accuracy: Use controls at both normal and pathological levels. The controls and the corresponding results of the QC-Laboratory are stated in the QC certificate added to the kit. The values and ranges stated on the QC sheet always refer to the current kit lot and should be used for direct comparison of the results. It is also recommended to make use of national or international Quality Assessment programs in order to ensure the accuracy of the results. Employ appropriate statistical methods for analyzing control values and trends. If the results of the assay do not fit to the established acceptable ranges of control materials patient results should be considered invalid. In this case, please check the following technical areas: Pipetting and timing devices; photometer, expiration dates of reagents, storage and incubation conditions, aspiration and washing methods. 3.8.3.2.4.5 Precision: Intra-assay reproducibility was determined by measurement of 20 replicates of serum pool in a single run. Mean (miu/ml):15.72 SD (miu/ml): 0.71 CV (%): 4.50 3.8.3.2.4.6 Linearity: A study was performed diluting a standard (1:2,1:4,1:8,1:16) containing an elevated level of LH with the 0 miu/ml calibrator to determine the linearity of the MICRO-ELISA LH test. 3.8.3.2.5.1 Standard curve: Standard concentrations in miu/ml Optical Units (450 nm)

Standard 0 (0 miu/ml) 0.00 Standard 1 (10 miu/ml) 0.10 Standard 2 (20 miu/ml) 0.30 Standard 3 (40 miu/ml) 0.60 Standard 4 (100 miu/ml) 1.40 Standard 5 (200 miu/ml) 2.70 3.8.3.3 Follicle stimulating hormone (FSH) assay: Follicle stimulating hormone level was determined according to (Vitt et al., 1998)method, using ELISA TECO kit for FSH. 3.8.3.3.1 Principle of the assay: The essential reagents required for an immunoenzymometric assay include excess amount of antibodies (both enzyme conjugated and immobilized) with high affinity, high specificity and contain different epitopes with distinct recognition and native antigen. In this assay procedure, the immobilization takes place at the surface of a microplate well through the interaction of streptavidin coated on the well and exogenously added biotinylated monoclonal anti-fsh antibody. Upon mixing, a reaction results between the native antigen contained in serum, the monoclonal biotinylated antibody and the enzymelabeled antibody, without competition or steric hindrance, to form a soluble sandwich complex. Simultaneously, the complex is deposited to the well through the high affinity reaction of streptavidin and biotinylated antibody. After equilibrium is attained, the antibody-bound fraction is separated from unbound antigen by decantation or aspiration. The enzyme activity in the antibody-bound fraction is directly proportional to the native antigen concentration. By utilizing several different serum references of known antigen value, a dose response curve can be generated from which the antigen concentration of an unknown can be ascertained. 3.8.3.3.2 Kit components:

One stripholder containing 96 microtitration wells coated with streptavidin, six FSH reference standards with concentrations of approximately (0, 5.0, 25, 50, 100 and 200 miu/ml). Enzyme conjugate, TMB chromogen solution, stop solution and wash solution concentrate. 3.8.3.3.3 Assay procedure: All samples and reagents were allowed to reach room temperature (~25 C). Reagents mixed by gentle inversion before use. Standards, controls and samples assayed in duplicate. 1. Microtitration strip was marked to be used. 2. Fifty µl of the standards, controls and samples were added into each appropriate well. 3. One hundred µl of conjugate reagent were added into each well using a precision pipette and then mixed for 30 seconds. 4. The wells were incubated for 60 minutes at room temperature (~25 C). 5. Each well was aspirated and washed 3 times by added 300 µl of working wash solution. 6. One hundred µl of TMB reagent were added into each well and gently mixed for 10 seconds. 7. The wells were incubated in the dark for 15 minutes at room temperature (~25 C) without shaking. 8. Fifty µl of stop solution were added into each well and gently mixed for 10-20 second. 9. The absorbance for each well was read at 450 nm. 3.8.3.3.4 Validity of test: 3.8.3.3.4.1 Calculation of results: The absorbance for each standard, control, or samples were obtained (Absorbance at 450), and then the stander curve prepared by plotted the absorbance readings for each of the standards along the Y-axis versus standard

concentrations in miu/ml along the X-axis, the mean absorbance values for each sample were determined the corresponding concentration of FSH in miu/ml from the standard curve Normal reference values of FSH for adult male between 2.5-10.0 miu/ml. 3.8.3.3.4.2 Specificity: Haemoglobin (up to 4 mg/ml), Bilirubin (up to 0.5 mg/ml) and Triglyceride (up to 30 mg/ml) have no influence on the assay results. 3.8.3.3.4.3 Sensitivity: The analytical sensitivity was calculated from the mean plus two standard deviations of twenty (20) replicate analyses of Standard 0 and was found to be 0.856 miu/ml. 3.8.3.3.4.4 Accuracy: Use controls at both normal and pathological levels. The controls and the corresponding results of the QC-Laboratory are stated in the QC certificate added to the kit. The values and ranges stated on the QC sheet always refer to the current kit lot and should be used for direct comparison of the results. It is also recommended to make use of national or international Quality Assessment programs in order to ensure the accuracy of the results. Employ appropriate statistical methods for analyzing control values and trends. If the results of the assay do not fit to the established acceptable ranges of control materials patient results should be considered invalid. In this case, please check the following technical areas: Pipetting and timing devices; photometer, expiration dates of reagents, storage and incubation conditions, aspiration and washing methods. 3.8.3.3.4.5 Precision: Intra-assay reproducibility was determined by measurement of 20 replicates of serum pool in a single run. Mean (miu/ml): 14.24 SD (miu/ml): 0.64 CV (%): 4.54 3.8.3.3.4.6 Linearity:

A study was performed diluting a standard (1:2,1:4,1:8,1:16) containing an elevated level of FSH with the 0 miu/ml calibrator to determine the linearity of the MICRO-ELISA FSH test. 3.8.3.3.5.1 Standard curve: Standard concentrations in miu/ml Optical Units (450 nm) Standard 0 (0 miu/ml) 0.00 Standard 1 (5 miu/ml) 0.20 Standard 2 (10 miu/ml) 0.40 Standard 3 (20 miu/ml) 0.80 Standard 4 (50 miu/ml) 1.50 Standard 5 (100 miu/ml) 2.90 3.8.3.4 Prolactin hormone assay: Prolactin hormone level was determined according to (Tietz, 1995) method using ELISA TECO kit for prolactin. 3.8.3.4.1 Principle of the assay: The essential reagents required for sandwich enzyme-linked immunoassay include excess amount of antibodies (both enzyme conjugated and immobilized) with high affinity, high specificity and contain different epitopes with distinct recognition and native antigen. In this assay, a certain amount of prolactin hormone calibrator, patient specimen or control is first added to a microplate well. Biotinylated monoclonal and enzyme labeled antibodies (directed against distinct and different epitopes of PRL) are added and the reactants mixed. Immobilization of the tagged PRL occurs through the interaction of streptavidin coated on the well and the added biotinylated monoclonal anti-prl antibody. Upon mixing a reaction results between the native antigen contained in serum, the monoclonal biotinylated antibody and the enzyme-labeled antibody, without competition or steric hindrance, to from a soluble sandwich complex bound to the surface of microplate through the streptavidin-biotin system. After equilibrium is attained the antibody-bound fraction is separated from unbound

antigen by decantation or aspiration. The enzyme activity in the antibody-bound fraction is directly proportional to the native antigen concentration. By several different serum references of known antigen value, a dose response curve can be generated from which the antigen concentration of an unknown can be ascertained. 3.8.3.4.2 Kit components: One stripholder containing 96 microtitration wells coated with streptavidin, six prolactin reference standards with concentrations of approximately (0, 5.0, 10, 25, 50 and 100 ng/ml). Enzyme conjugate, TMB chromogen solution, stop solution and wash solution concentrate. 3.8.3.4.3Assay procedure: All samples and reagents were allowed to reach at room temperature (~25 C). Reagents mixed by gentle inversion before use. Standards, controls and samples assayed in duplicate. 1. Microtitration strip was marked to be used. 2. Twenty-five µl of the standards, controls and samples were added into each appropriate well. 3. One hundred µl of conjugate reagent were added into each well using a precision pipette. 4. The wells were mixed for 30 seconds. 5. The wells were incubated for 60 minute at room temperature (~25 C). 6. Each well was aspirated and washed 3 times by added 300 µl of working wash solution. 7. One hundred µl of TMB reagent were added into each well using a precision pipette and gently mixed for 10 seconds. 8. The wells were incubated in the dark for 15 minute at room temperature (~25 C). 9. Fifty µl of stop solution were added into each well using a precision pipette and mixed for 10-20 seconds.

10. The absorbance's of the solution in each well were read at 450 nm. 3.8.3.4.4 Validity of test: 3.8.3.4.4.1 Calculation of results: The absorbance for each standard, control, or samples were obtained, and then the standard curve prepared by plotted the absorbance readings for each of the standards along the Y-axis versus standard concentrations in ng/ml along the X-axis, The mean absorbance values for each sample were determined the corresponding concentration of prolactin in ng/ml from the stander curve. Normal reference values of prolactin for adult male between 2.0-12.0 ng/ml. 3.8.3.4.4.2 Specificity: Any improper handling of samples or modification of this test might influence the results. Haemoglobin (up to 4 mg/ml), Bilirubin (up to 0.5 mg/ml) and Triglyceride (up to 0.9 mg/ml) have no influence on the assay results. 3.8.3.4.4.3 Sensitivity: The analytical sensitivity was calculated from the mean plus two standard deviations of twenty (20) replicate analyses of Standard 0 and was found to be 0.13 ng/ml. 3.8.3.4.4.4 Accuracy: Use controls at both normal and pathological levels. The controls and the corresponding results of the QC-Laboratory are stated in the QC certificate added to the kit. The values and ranges stated on the QC sheet always refer to the current kit lot and should be used for direct comparison of the results. It is also recommended to make use of national or international Quality Assessment programs in order to ensure the accuracy of the results. Employ appropriate statistical methods for analyzing control values and trends. If the results of the

assay do not fit to the established acceptable ranges of control materials patient results should be considered invalid. In this case, please check the following technical areas: Pipetting and timing devices; photometer, expiration dates of reagents, storage and incubation conditions, aspiration and washing methods. 3.8.3.4.4.5 Precision: Intra-assay reproducibility was determined by measurement of 20 replicates of serum pool in a single run. Mean (ng/ml): 15.72 SD (ng/ml): 0.71 CV (%): 4.50 3.8.3.4.4.6 Linearity: A study was performed diluting a standard (1:2,1:4,1:8,1:16) containing an elevated level of Prolactin with the 0 ng/ml calibrator to determine the linearity of the MICRO-ELISA Prolactin test. 3.8.3.4.5.1 Standard curve: standard concentrations in ng/ml Optical Units (450 nm) Standard 0 (0 ng/ml) 0.00 Standard 1 (0.2 ng/ml) 0.30 Standard 2 (0.5 ng/ml) 0.50 Standard 3 (1.0 ng/ml) 0.90 Standard 4 (2.0 ng/ml) 1.30 Standard 5 (6.0 ng/ml) 1.70 Standard 6 (16.0 ng/ml) 2.10 3.8.3.5 Thyroid Stimulating Hormone (TSH) assay: thyroid stimulating hormone level was determined according to (Vitt et al., 1998)method, using ELISA TECO kit for TSH. 3.8.3.5.1 Principle of the assay:

The essential reagents required for an immunoenzymometric assay include excess amount of antibodies (both enzyme conjugated and immobilized) with high affinity, high specificity and contain different epitopes with distinct recognition and native antigen. In this assay procedure, the immobilization takes place at the surface of a microplate well through the interaction of streptavidin coated on the well and exogenously added biotinylated monoclonal anti-tsh antibody. Upon mixing, a reaction results between the native antigen contained in serum, the monoclonal biotinylated antibody and the enzymelabeled antibody, without competition or steric hindrance, to form a soluble sandwich complex. Simultaneously, the complex is deposited to the well through the high affinity reaction of streptavidin and biotinylated antibody. After equilibrium is attained, the antibody-bound fraction is separated from unbound antigen by decantation or aspiration. The enzyme activity in the antibody-bound fraction is directly proportional to the native antigen concentration. By utilizing several different serum references of known antigen value, a dose response curve can be generated from which the antigen concentration of an unknown can be ascertained. 3.8.3.5.2 Kit components: One stripholder containing 96 microtitration wells coated with streptavidin, six TSH reference standards with concentrations of approximately (0, 5.0, 25, 50, 100 and 200 miu/ml). Enzyme conjugate, TMB chromogen solution, stop solution and wash solution concentrate. 3.8.3.5.3 Assay procedure: All samples and reagents were allowed to reach room temperature (~25 C). Reagents mixed by gentle inversion before use. Standards, controls and samples assayed in duplicate. 1. Microtitration strip was marked to be used. 2. Fifty µl of the standards, controls and samples were added into each appropriate well.

3. One hundred µl of conjugate reagent were added into each well using a precision pipette and then mixed for 30 seconds. 4. The wells were incubated for 60 minutes at room temperature (~25 C). 5. Each well was aspirated and washed 3 times by added 300 µl of working wash solution. 6. One hundred µl of TMB reagent were added into each well and gently mixed for 10 seconds. 7. The wells were incubated in the dark for 15 minutes at room temperature (~25 C) without shaking. 8. Fifty µl of stop solution were added into each well and gently mixed for 10-20 second. 9. The absorbance for each well was read at 450 nm. 3.8.3.5.4 Validity of test: 3.8.3.5.4.1 Calculation of results: The absorbance for each standard, control, or samples were obtained (Absorbance at 450), and then the stander curve prepared by plotted the absorbance readings for each of the standards along the Y-axis versus standard concentrations in miu/ml along the X-axis, the mean absorbance values for each sample were determined the corresponding concentration of TSH in miu/ml from the standard curve Normal reference values of TSH for adult male between 0.27-4.2 miu/ml. 3.8.3.5.4.2 Specificity: Haemoglobin (up to 4 mg/ml), Bilirubin (up to 0.5 mg/ml) and Triglyceride (up to 30 mg/ml) have no influence on the assay results. 3.8.3.5.4.3 Sensitivity: The analytical sensitivity was calculated from the mean plus two standard deviations of twenty (20) replicate analyses of Standard 0 and was found to be 0.041 miu/l.

3.8.3.5.4.4 Accuracy: Use controls at both normal and pathological levels. The controls and the corresponding results of the QC-Laboratory are stated in the QC certificate added to the kit. The values and ranges stated on the QC sheet always refer to the current kit lot and should be used for direct comparison of the results. It is also recommended to make use of national or international Quality Assessment programs in order to ensure the accuracy of the results. Employ appropriate statistical methods for analyzing control values and trends. If the results of the assay do not fit to the established acceptable ranges of control materials patient results should be considered invalid. In this case, please check the following technical areas: Pipetting and timing devices; photometer, expiration dates of reagents, storage and incubation conditions, aspiration and washing methods. 3.8.3.5.4.5 Precision: Intra-assay reproducibility was determined by measurement of 20 replicates of serum pool in a single run. Mean (miu/l): 15.23 SD (miu/l): 0.48 CV (%): 5.21 3.8.3.5.4.6 Linearity: A study was performed diluting a standard (1:2,1:4,1:8,1:16) containing an elevated level of TSH with the 0 miu/l calibrator to determine the linearity of the MICRO-ELISA TSH test. 3.8.3.5.5.1 Standard curve: Standard concentrations in miu/ml Optical Units (450 nm) Standard 0 (0 miu/ml) 0.00 Standard 1 (0.25 miu/ml) 0.40 Standard 2 (0.75 miu/ml) 0.90 Standard 3 (2.0 miu/ml) 1.20 Standard 4 (5.0 miu/ml) 1.70 Standard 5 (15.0 miu/ml) 2.10 3.9 Quality Control:

The precision and accuracy of all methods used in this study were checked each time a batch was analyzed by including commercially prepared control sera. 3.10 Statistical analysis: Finally the result were analyzed by SPSS version 19. The mean and SD were obtained and t test used for comparison. Linear regression was also use for correlation. P. value was obtained to assess the significance of the results ( p value of < 0.05 was considered to be significant ).

4. Results A total 600 Sudanese males were recruited to participate in this study (300 infertile and 300 fertile). The infertile included 150 smokers and 150 nonsmokers as control and fertile group included 150 smokers and 150 non-smokers as another control group. The mean age of participants was 42.5 ± 7.6 year for range 20 50 year the infertile and fertile smokers and 41.0 ± 6.3 year range 35 52 year for the infertile and fertile non-smokers. However the variation in the age between the smokers and non-smokers was not statistically significant (P value 0.31). In the infertile smokers the percentage sperm motility was found to be statistically lower compared to the infertile non-smokers (P values 0.031). On the other hand the percentage of sperm with abnormal morphology was significantly high in the group of infertile smokers (P values 0.001). However there were statistically insignificant differences between the two groups regarding the mean sperm count and semen volume (P values 0.140 &0.183 respectively). (Table 4.1). The percentage of sperm motility and sperm count were found to be significantly lower in the fertile smokers than fertile non-smokers (P values 0.01 & 0.005 respectively). While the percentage of sperm with abnormal morphology was significantly higher in fertile smokers compared to the non smokers (P values 0.015). The results of this study showed statistically insignificant difference in semen volume between fertile smokers and nonsmokers (P values 0.560). (Table 4.2).

In infertile smokers negative correlation was observed between the sperm count with; the duration of cigarette smoking / years and the number of cigarette smoked / day (Figure 4.1, 4.2). No correlation was found between the levels of semen volume with; the duration of cigarette smoking / years and the number of cigarette smoked / day (Figure 4.3, 4.4). Cigarette smoking was found to significantly increase prolactin level and reduced the mean testosterone level in the infertile smokers compared to the nonsmokers (P values 0.001 & 0.039 respectively). In contrast insignificant differences were obtained between the two groups regarding the means LH, FSH and TSH levels (P values 0.125, 0.342 & 0.725 respectively). (Table 4.3). The means prolactin level was significantly higher in fertile smokers compared to the non-smokers (P values 0.033). While mean testosterone level was significantly lower in the fertile smokers than the non-smokers (P values 0.013). The results of this study showed statistically insignificant differences in LH, FSH and TSH levels between fertile smokers and non-smokers (P values 0.614, 0.245 & 0.518 respectively). (Table 4.4). In infertile smokers negative correlations between the levels of LH, FSH, TSH and Testosterone with both ; the duration of cigarette smoking in years and the number of cigarette smoked / day (Figure 4.5, 4.6, 4.7, 4.8, 4.9, 4.10, 4.11, 4.12). Significant positive correlation was found between the level of Prolactin with both; the duration of cigarette smoking in years and the number of cigarette smoked / day (Figure 4.13, 4.14). Table (4.1) Comparison of the means of sperm count, sperm motility, sperm morphology, semen volume between the infertile smokers and non smokers groups:

Test group Control group Variable (Infertile smokers) )mean + SD ) (Infertile non smokers) )mean + SD ) P. value n = 150 n = 150 Sperm count x 10 6 (10.1 + 2.9) (11.8 + 3.0) 0.140 Sperm motility % (30.0 + 5.3) (31.5 + 5.7) 0.031 Abnormal sperm morphology % (91.6 + 3.1) (89.1 + 4.3) 0.001 Semen volume ml (2.3 + 0.55) (2.4 + 0.62) 0.183 Th e table shows the mean + SD, range in brackets ( ) and probability (P). T- test was used for comparison. Table (4.2) Comparison of the means of sperm count, sperm motility, sperm morphology, semen volume between the fertile smokers and non smokers groups: Test group Control group Variable (Fertile smokers) )mean + SD ) (Fertile non smokers) )mean + SD ) P. value n = 150 n = 150

Sperm count x 10 6 (31.9 + 4.2) (33.4 + 5.1) 0.005 Sperm motility % (50.3 + 4.5) (58.3 + 6.2) 0.01 (75.3 + 5.3) (54.8 + 4.1) 0.015 Abnormal sperm morphology % (2.4 + 0.7) (2.1 + 0.6) 0.560 Semen volume ml Th e table shows the mean + SD, range in brackets ( ) and probability (P) T- test was used for comparison

Sperm count x 10 6 0.00 3.00 6.00 9.00 12.00 15.00 18.00 Duration of smoking (years) Figure (4.1):- Shows moderate negative correlation between the sperm count and the duration of cigarette smoking / years in the infertile smokers ( r = -0.54, P = 0.047 ).

Sperm count x 10 6 Number of cigarettes smoked/day Figure (4.2):- shows low negative correlation between the sperm count and the number of cigarette smoking / day in the infertile smokers ( r = -0.31, P = 0.051 ).

Semen volume ml 2.80 2.40 2.00 1.60 1.20 0.80 0.4 0 0.00 0.00 3.00 6.00 9.00 12.00 15.00 18.00 Duration of smoking (years) Figure (4.3):- Shows no correlation between the semen volume and the duration of cigarette smoking / years in the infertile smokers ( r = 0.012, P = 0.79 ).

Semen volume ml 2.80 2.40 2.00 1.60 1.20 0.80 0.40 0.00 Number of cigarettes smoked/day Figure (4.4):- Shows no correlation between the semen volume and the number of cigarette smoking / day in the infertile smokers ( r = -0.023, P = 0.98 ). Table (4.3) Comparison of the means of LH, FSH, Prolactin, Testosterone, TSH between the infertile smokers and non smokers groups: Test group Control group Variable (Infertile smokers) )mean + SD ) (Infertile non smokers) )mean + SD ) P. value n = 150 n = 150

LH mlu/ml (1.9 + 0.8) (2.1 + 0.9) 0.125 FSH mlu/ml (3.0 + 1.3) (3.1 + 1.5) 0.342 Prolactin ng/ml (6.8 + 3.41) (6.1 + 2.77) 0.001 Testosterone ng/ml ( 3.7 (2.4 + + 1.51 ) 0.61) (3.9 (2.4 + + 1.01) 0.67) 0.039 0.725 TSH mlu/ml Th e table shows the mean + SD, range in brackets ( ) and probability (P). T- test was used for comparison. Table (4.4) Comparison of the means of LH, FSH, Prolactin, Testosterone, TSH between the fertile smokers and non smokers groups: Test group Control group Variable (Fertile smokers) )mean + SD ) (Fertile non smokers) )mean + SD ) P. value n = 150 n = 150

LH mlu/ml (2.3 + 1.4) (2.4 + 1.5) 0.614 FSH mlu/ml (3.0 + 1.28) (3.1 + 1.39) 0.245 Prolactin ng/ml (7.3 + 2.8) (6.1 + 1.9) 0.033 Testosterone ng/ml (4.3 + (2.4 + 1.1) 0.76) (4.5 + (2.4 + 1.4) 0.83) 0.013 0.518 TSH mlu/ml Th e table shows the mean + SD, range in brackets ( ) and probability (P). T- test was used for comparison.

Serum LH mlu/ml 0.00 3.00 6.00 9.00 12.00 15.00 18.00 Duration of smoking (years) Figure (4.5):- Shows weak negative correlation between the level of LH and the duration of cigarette smoking / years in the infertile smokers ( r = -0.13, P = 0.61 ).

Serum FSH mlu/ml 0.00 3.00 6.00 9.00 12.00 15.00 18.00 Duration of smoking (years) Figure (4.6):- Shows weak negative correlation between the level of FSH and the duration of cigarette smoking / years in the infertile smokers ( r = -0.19, P = 0.73 ).

Serum TSH ng/ml 0.00 3.00 6.00 9.00 12.00 15.00 18.00 Duration of smoking (years) Figure (4.7):- Shows weak negative correlation between the level of TSH and the duration of cigarette smoking / years in the infertile smokers ( r = -0.23, P = 0.71).

Serum Testosterone ng/ml 0.00 3.00 6.00 9.00 12.00 15.00 18.00 Duration of smoking (years) Figure (4.8):- Shows moderate negative correlation between the level of Testosterone and the duration of cigarette smoking / years in the infertile smokers ( r = -0.69, P = 0.039 ).

Serum LH mlu/ml Number of cigarettes smoked/day Figure (4.9):- Shows weak negative correlation between the level of LH and the number of cigarette smoking / day in the infertile smokers ( r = -0.15, P = 0.57 ).

Serum FSH mlu/ml Number of cigarettes smoked/day Figure (4.10):- Shows weak negative correlation between the level of FSH and the number cigarette smoking / day in the infertile smokers ( r = -0.32, P = 0.12 ). of

Serum TSH mlu/ml Number of cigarettes smoked/day Figure (4.11):- Shows weak negative correlation between the level of TSH and the number cigarette smoking / day in the infertile smokers ( r = -0.19, P = 0. 947 ). of

Serum Testosterone mlu/ml Number of cigarettes smoked/day Figure (4.12):- Shows moderate negative correlation between the level of Testosterone and the number of cigarette smoking / day in the infertile smokers ( r = -0.54, P = 0.049 ).

Serum Prolactin ng/ml 0.00 3.00 6.00 9.00 12.00 15.00 18.00 Duration of smoking (years) Figure (4.13):- Shows strong positive correlation between the level of Prolactin and the duration of cigarette smoking / years in the infertile smokers ( r = 0.91, P = 0.017 ).

Serum Prolactin mlu/ml Number of cigarettes smoked/day Figure (4.14):- shows strong positive correlation between the level of Prolactin and the number of cigarette smoking / day in the infertile smokers ( r = 0.93, P = 0.016 ). 6. Discussion Several studies have examined the effect of cigarette smoking on male fertility and showed a negative effect on sperm production, motility and morphology. The results of this study indicated that cigarette smoking affects sperm motility and sperm morphology, causing reduced motility and increased abnormal sperm morphology and these findings confirm the earlier studies examining the relationship between cigarette smoking and sperm motility and abnormal sperm