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: