Natural course of idiopathic oligozoospermia: Comparison of mild, moderate and severe forms

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1 International Journal of Urology (2010) 17, doi: /j x Original Article: Clinical Investigationiju_ Natural course of idiopathic oligozoospermia: Comparison of mild, moderate and severe forms Chong Won Bak, 1 Seung-Hun Song, 1 Tae Ki Yoon, 2 Jung Jin Lim, 3 Tai Eun Shin 3 and Suye Sung 3 Departments of 1 Urology and 2 Obstetrics and Gynecology, and 3 Andrology Lab, Fertility Center, CHA Gangnam Medical Center, CHA University, Seoul, Korea Objectives: To investigate the natural courses of mild, moderate and severe idiopathic oligozoospermia, and which factors or semen variables were of utmost importance in predicting the courses. Methods: A total of 208 men (age years) who were diagnosed with mild, moderate and severe idiopathic oligozoospermia in a 9-year-period between January 2000 and December 2008 were followed up for more than 6 months. Results: Overall, 16 (24.6%) of 65 patients with severe oligozoospermia developed azoospermia, whereas two (3.1%) patients with moderate oligozoospermia developed azoospermia and none of the patients with mild oligozoospermia developed azoospermia. Initial follicle stimulating hormone level and testicular volume between the subgroups were significantly different (P = and , respectively). The subgroup of patients who became azoospermic (n = 18) showed statistically significant differences in terms of body mass index and the level of prolactin (PRL) from the subgroup that maintained the initial lingering sperm count (n = 190; P = and , respectively). As the vitality of semen variables increased 1%, the risk of progression to azoospermia diminished by fold, according to Cox s proportional hazards model analysis. A receiver operating characteristic curve analysis showed that the area under the curve was and the sperm concentration value with the highest sensitivity and specificity was the reference value of 3 5 million/ml, with a sensitivity of and specificity of (P = 0.01). Conclusions: Patients with severe oligozoospermia should be warned of the possibility of becoming azoospermic and hence sperm freezing should be encouraged as early as possible. Key words: male infertility, moderate oligozoospermia, natural course, severe oligozoospermia, sperm cryopreservation. Introduction Infertility affects approximately 15% of couples attempting pregnancy, with male factor infertility identified in approximately 50% of the cases. 1 3 Reproductive fecundity depends on coordinated functions of various organs along the hypothalamo pituitary gonadal reproductive tract axis. Intracytoplasmic sperm injection (ICSI), since its introduction in 1992, has revolutionized the treatment of male infertility. However, complete loss of paternity casts a devastating effect on a man, and the preservation of paternity for the future is of utmost importance. To our knowledge, this is the first report on the natural fate of different degrees of oligozoospermia. Correspondence: Seung-Hun Song MD, Department of Urology, Fertility Center, CHA Gangnam Medical Center, CHA University, Seoul , Korea. shsong02@cha.ac.kr Received 31 May 2010; accepted 11 August Online publication 10 September 2010 Methods Patients A total of 208 eligible men were identified from 1201 men with fertility problems of severe, moderate and mild degrees of oligozoospermia who sought evaluation for infertility in the fertility center of our university hospital during a 9-year period between January 2000 and December The present study was approved by the local hospital ethics committee. Inclusion and exclusion criteria Other than the 208 men who represented the final study cohort, the remaining 993 patients decided to undergo assisted reproductive technology (ART) within 6 months of their initial visit, were not eligible for the study due to exclusion criteria or were lost to follow up mainly owing to visiting other centers. Men were considered to have male factor fertility problems based on a clinical presentation with abnormal semen analysis variables as defined by World 2010 The Japanese Urological Association 937

2 CW BAK ET AL. Health Organization criteria (fourth edition, 1999). Inclusion criteria were severe oligozoospermia (less than 5 million sperm/ml), moderate oligozoospermia (between 5 and 10 million sperm/ml) and mild oligozoospermia (between 10 and 20 million sperm/ml) using a centrifuged specimen at the initial visit and follow up of more than 6 months. To completely rule out transient worsening of semen parameters, strict exclusion criteria including previous history of infection, varicocele, cryptorchidism, exposure to gonadotoxin, surgery of the genitourinary tract, insufficient sexual abstinence period and more than 3 months of any medical therapy, including oriental or herbal medicine as well as genetic abnormalities, such as chromosome anomaly and Y-chromosome microdeletion, was applied. Measurements All semen samples were obtained by masturbation into a wide-mouthed plastic container in a separate room. Sexual abstinence of a minimum of 48 h was requested and the reported abstinence time was recorded. The semen characteristics evaluated were sperm concentration, percentage of total motile spermatozoa, strict morphology and percentage of living spermatozoa by the vital stain. Testicular volume was measured using a Prado orchidometer. Complete personal information, including demographics as well as previous history of infection or surgery regarding the reproductive tract, and hormonal evaluations, such as follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL) and testosterone were carried out. Evaluation of chromosome anomalies and Y-chromosome microdeletion were also carried out. Statistical analysis All statistical analyses were carried out using a commercially available software program (SAS Enterprise Guide 4.1; SAS Institute, Cary, NC, USA). The Kaplan Meier method was used to calculate unadjusted estimates of azoospermia. Odds ratios and the corresponding 95% confidence intervals were used to measure the strength of the association. The c 2 -test, Kruskal Wallis test and t-tests (or Wilcoxon rank sum test) were used to compare parameters between groups, and multiple logistic regression analysis was carried out to assess the independent contributions of variables on the development of azoospermia. Cox s proportional hazards model analysis was used to estimate hazard ratio, and logistic regression analysis was applied to discover the risk of becoming azoospermic with every 1 million decrease in sperm count. One-way analysis of variance (ANOVA) was utilized to evaluate vitality, morphology and motility of each subgroup. A receiver operating characteristic (ROC) curve analysis was carried out to determine the threshold sperm concentration. Statistical significance was defined as a P-value <0.05 and all statistical tests were two-sided. Results Table 1 presents the characteristics of the study population. Of 208 eligible participants, a total number of 18 cases of azoospermia developed. In detail, 16 (23.5%) of the severe oligozoospermic patients developed into azoospermia, whereas just two (3.0%) of the moderate degree developed into azoospermia. Not a single patient in the subgroup of mild oligozoospermia became azoospermic. Table 1 also clearly summarizes different characteristics of the three different degrees of oligozoospermia. Initial severe oligozoospermia recorded a declining tendency of sperm concentration, as time went by, from the initial concentration of *10 6 /ml (mean SD) to *10 6 /ml after the interval of days and again reduced to *10 6 /ml after days. In contrast, initial mild oligozoospermia showed a slight improvement of sperm concentration from the initial concentration of *10 6 /ml to *10 6 /ml after days then *10 6 /ml after days. A total of 13 men in the mild oligozoospermia subgroup achieved fatherhood before undergoing a third semen analysis. Not a single man with a mild degree of oligozoospermia developed azoospermia on their natural course, whereas six men in the subgroup showed transient worsening to the level of moderate oligozoospermia that eventually recovered back to the mild degree and even to the normal ranges. As shown in Figure 1, the Kaplan Meier method was used to calculate unadjusted estimates of progression to azoospermia (P = ). The median duration to developing azoospermia in the present study group was 1193 days, with a standard error of 94.6 days. The subgroup of severe oligozoospermia had statistically smaller testicles than the other two groups (11.9 vs 14.3 ml vs 14.5 ml, P = ). Severe oligozoospermic men also had a statistically higher level of FSH ( miu/ml) than the moderate oligozoospermic men ( miu/ml), and men with mild oligozoospermia ( miu/ml; P = ). Other study parameters, such as age, body mass index (BMI) and smoking, as well as other hormonal levels, were also not statistically related to progression to azoospermia in the present study groups. The study population of 208 was divided into the two subgroups in terms of progression to azoospermia, that is, the subgroup that progressed to azoospermia (n = 18) and the subgroup that maintained semen parameters (n = 190), as shown in Table 2. Student s t-test and Wilcoxon rank sum test showed that there were statistically significant differences in BMI and PRL between the two groups (P = The Japanese Urological Association

3 Different fates of oligozoospermia Table 1 Characteristics of study participants Severe oligozoospermia Moderate oligozoospermia Mild oligozoospermia P-value No. patients N/A No. patients developed azoospermia * Initial sperm count, *10 6 /ml (median;range) N/A (0.5; 0.05 to 5.0) (10; 5.2 to 10.0) (16; 11 to 19) Second sperm count, N/A (0.2; 0 to 4.8) (11; 0 to 46.0) (18; 11 to 25) Interval between 1 st and 2 nd semen analysis (days) N/A (97.5; 23 to 1033) (171; 14 to 2041) (153; 14 to 895) Third sperm count, *10 6 /ml (median; range) N/A (0.05; 0 to 3.5) (16.5; 0.5 to 46.0) (18.5; 11 to 39) Interval between 2 nd and 3 rd semen analysis (days) N/A (160; 19 to 1417) (178; 14 to 1290) (198; 14 to 1814) Fourth sperm count, *10 6 /ml (median; range) N/A (0.075; 0 to 1.8) (12; 0.5 to 58) (20; 11 to 57) Age (y) *** BMI (kg/m 2 ) ** Mean follow-up duration (days) *** Smoker 43(66.15%) 43(66.15%) 39(55.7%) * Testis volume (ml) ** FSH (miu/ml) *** LH (miu/ml) *** PRL (ng/ml) *** Testosterone (ng/ml) *** *(c 2 -test. **ANOVA test. ***Kruskal Wallis test. Values are means SD. BMI, body mass index; FSH, follicle stimulating hormone; LH, luteinizing hormone; N/A, not applicable; PRL, prolactin. Cumulative survival Follow - up (days) Fig. 1 Different fates of severe and moderate idiopathic oligozoospermia., Mild oligozoospermia;, censored group of mild oligozoospermia;, moderate oligozoospermia;, censored group of moderate oligozoospermia;, severe oligozoospermia;, censored group of severe oligozoospermia. and , respectively). Table 3 shows the characteristics of the two subgroups of severe oligozoospermia; one group that diminished to azoospermia (n = 16) and the other group that maintained already impaired semen variables (n = 49). Wilcoxon rank sum test found that the two groups showed statistically significant differences with regard to BMI and PRL (P = and , respectively). Vitality was found to be the most important factor for predicting the natural course of severe oligozoospermia, that as the vitality of semen parameter increased 1%, the risk of progression to azoospermia diminished by fold according to Cox s proportional hazards model analysis that also produced a hazard ratio (HR) of motility of 1.012, HR of morphology of 0.936, HR of BMI of and HR of testicular volume of Cox s PH model analysis of various hormones showed HR of FSH of 0.888, HR of LH of 1.307, HR of PRL of and HR of testosterone of Logistic regression analysis of the data found that FSH has no statistical significance with the progression to azoospermia. However, the analysis showed that there was an increased risk of becoming azoospermic with every decrease of 1 million in sperm count (OR = 1.411). ROC curve showed 2010 The Japanese Urological Association 939

4 CW BAK ET AL. Table 2 Characteristics of the patients classified according to progression to azoospermia Diminish to azoospermia No decline to azoospermia P-value No. patients N/A Age (years) NS Sperm count (*10 6 /ml) at initial visit * BMI (kg/m 2 ) ** Smoker 10 (55.56%) 86 (66.15%) NS Testis volume (ml) NS FSH (miu/ml) NS LH (miu/ml) NS PRL (ng/ml) * Testosterone (ng/ml) NS *Wilcoxon rank sum test. **t-test. Values are means SD. BMI, body mass index; FSH, follicle stimulating hormone; LH, luteinizing hormone; N/A, not applicable; NS, not significant; PRL, prolactin. Table 3 Characteristics of the severe oligozoospermics classified according to progression to azoospermia Diminish to azoospermia Not decline to azoospermia P-value No. patients N/A Age (years) NS Smoker 10 (55.56%) 32 (65.31%) NS BMI (kg/m 2 ) * Testis volume (ml) NS FSH (miu/ml) NS LH (miu/ml) NS PRL (ng/ml) * Testosterone (ng/ml) NS Sperm count (*10 6 /ml ) NS *Wilcoxon rank sum test. Values are means SD. BMI, body mass index; FSH, follicle stimulating hormone; LH, luteinizing hormone; N/A, not applicable; NS, not significant; PRL, prolactin. that the area under the curve was and the sperm concentration value with the highest sensitivity and specificity was the reference value of 3 to /ml, with a sensitivity of and specificity of 0.711(P = 0.01) (Fig. 2). Discussion Several studies suggest that sperm concentration has decreased over time. 4 6 Subfertility is defined as the failure to conceive after 1 year of regular, unprotected intercourse with the same partner. Approximately 10 17% of all couples experience primary or secondary subfertility at some time during their reproductive life, 7,8 with male factor infertility identified in approximately 50% of the cases. 1 Subfertile couples try to conceive with all possible techniques, such as ART, which do not actually treat the cause of the subfertility. Sensitivity Specificity Fig. 2 Receiver operating characteristic curve of sperm concentration The Japanese Urological Association

5 Different fates of oligozoospermia Advances in in vitro fertilization (IVF) techniques, namely ICSI, have allowed the use of very few sperm to achieve fertilization, which leads to the possibility of cryopreservation of semen, even from patients with very low sperm counts. 9 However, there has been no large scale study on the fate of patients with severely impaired semen status. Among several definitions of severe oligozoospermia, we consented to the idea of less than 5 million sperm/ml implying underlying impaired spermatogenesis. 10 It is well known that neuroendocrine regulation of mammalian fertility is governed by the episodic release of the hypothalamic peptide gonadotropin-releasing hormone (GnRH), which acts in a hypophysiotropic manner to drive the anterior pituitary gonadotropes to secrete FSH and LH in a pulsatile fashion. 11 One of the key findings in the present study is that FSH in the cohort of severe oligozoospermia shows a statistically higher level than the group of moderate oligozoospermia (P = ). FSH, a dimeric glycoprotein hormone of the pituitary, stimulates the Sertoli cells to produce androgen-binding protein, inhibin and a variety of growth factors. 12 FSH plays a crucial role in males as the most important tropic hormone regulating Sertoli cell function. The hormone interacts with its signaling repertoire in the gonads, stimulates spermatogenesis by activating the specific receptor, FSH receptor (FSHR), that is a member of the G protein-coupled receptor family, including the classical Gs-linked systems. 13 In males, the FSHR system is highly specific, as mrna is expressed only in testicular Sertoli cells. However, FSHR haplotype is not associated with different serum FSH levels, but it is differently distributed in normal and azoospermic men. 14 We believe that, as shown in Table 1 and Figure 1, severe, moderate and mild oligozoospermia are different disease entities. There is plenty of room for men with mild oligozoospermia to improve their semen quality by changing their lifestyle, avoiding harmful environments, treating undetected infections and hence adopting preventive measures. However, men with severe oligozoospermia, especially those with a sperm concentration of approximately 1 million/ml, are facing a great danger of developing azoospermia, as shown in the present study (23/65 patients). However, the remaining 42 men in the present study barely maintained their initial lingering sperm concentration until they opted to undergo ART. Nine subjects in the subgroup of mild oligozoospermia showed an improvement, though slow and time consuming, of semen quality. However, 61 patients maintained an initial sperm concentration of a mild degree of oligozoospermia after a period of days with a median of 153 ranging from days. A third semen analysis of the subgroup was carried out in 52 patients, showing an improvement in the sperm concentration of 21 patients to a level above 20*10 6 /ml, whereas the level of mild oligozoospermia was maintained in the remaining of 31 men. Men with moderate oligozoospermia are not in a safe zone, but in gray zone, because two out of the 65 patients in the present study eventually developed azoospermia, whereas the remaining 63 men either maintained the initial lingering sperm concentration or the semen quality improved during a longer period of observation than for men with severe oligozoospemia. The underlying genetic and epigenetic causes of male infertility are not fully understood, but have just begun to unfold and, hence, be further elucidated. One of the surprising findings in the present study is the statistically different level of PRL between the subgroup that diminished to azoospermia (n = 18) and the subgroup that maintained already impaired semen variables (n = 190; P = ), whereas a similar difference presents between patients whose sperm count decreased to azoospermia (n = 16) and those who maintained the lingering sperm count (n = 49; P = 0.015) among the men with severe oligozoospermia. The physiological role of PRL in male sexual behavior remains to be elucidated. However, a recent study of hypoprolactinemia documented that PRL in the lowest quartile levels (<113 mu/l or 5 ng/ml) is associated with metabolic syndrome and arteriogenic erectile dysfunction, as well as with premature ejaculation and anxiety symptoms. 15 The present results showed that men who developed azoospermia recorded statistically lower levels of PRL ( ng/ml) than others who did not develop azoospermia ( ng/ml). According to previous studies, low PRL levels in a group of infertile men might be one of the primary causes of their infertility. 16,17 LH binds to a G protein-coupled reception in the Leydig cells. Germ cells do not have androgen receptors, so androgens secreted by Leydig cells act through receptors on the Sertoli cells. 18 Testosterone secreted from Leydig cells, inhibin secreted from Sertoli cells and estradiol formed from aromatization of testosterone act on the hypothalamus and pituitary to regulate gonadotropin secretion by negative inhibition. There were significant associations between decreased levels of testosterone and increased severity of erectile dysfunction, longer duration and poor metabolic control of diabetes, ischemic heart disease, hyperprolactinemia and low desire. 19 Although estrogen receptor-alpha (ERa) has traditionally been considered an important regulator of female development, its crucial role in the male reproductive tract was recently shown. 20 The study shows that the BMI of the subgroup that diminished to azoospermia (n = 18) and not-declined-to-azoospermia (n = 190) presents statistically significant differences (P = ), whereas a similar difference presents between those who decreased to azoospermia (n = 16) and those who maintained the lingering semen count (n = 49; P = ) among the men with severe oligozoospermia. Male factor infertility has been considered to be associated with a higher incidence of obesity. Serum leptin is also known to mediate a link between obesity and male infertility. 21 Obesity was 2010 The Japanese Urological Association 941

6 CW BAK ET AL. also been shown to affect the GnRH LH/FSH pulse that might impair Leydig and Sertoli cell functions and interfere with the release of sex hormones with a consequent effect on sperm maturation. 22 However, the present study produced the result that not only extreme levels of obesity, but also a BMI in the normal range is involved in the development of azoospermia; as seen in men who became azoospermic an recorded a statistically lower BMI than men who did not develop azoospermia (P = ). According to a recent report, BMI was unrelated to sperm concentration, motility or morphology despite the fact that only extreme levels of obesity might negatively influence male reproductive potential. 23 It remains an enigma why patients in the severe oligozoospermia group developed azoospermia when they had such unique characteristics in terms of BMI and PRL, despite recent evidence showing a role for PRL in adipogenesis. 24,25 Fertility depends on coordinated functions of organs along the hypothalamo pituitary gonadal reproductive tract axis. In the present study, severe oligozoospermic men showed statistically smaller testicles than the moderate group (11.9 vs 14.3 ml, P = ). Testicular volumes are known to be significantly correlated with sperm density, total sperm count and total motile sperm count. 26 It is also well known that a decrease in size of one or both testes is an important symptom of spermatogenic defects. 27 We consent to the idea that a variety of environmental hazards related to testicular dysgenesis syndrome affects sperm quality of men with an already severely impaired sperm count. 28 In the present study, sperm vitality turns out to be the most important factor for predicting the natural course of severe oligozoospermia. The risk of progression to azoospermia declines fold as the vitality of semen variable increases 1%. The percentage of sperm vitality has been known to be affected by smoking as well as fever, though not significantly. 29,30 It has also been found that the percentage of non-vital spermatozoa increases with the degree of oligozoospermia. 31 Sperm vitality is also known to bear a significant relationship to pregnancy; 32 meanwhile, sperm morphology assessment is very useful for the selection of patients for ICSI. 33 The reason why patients with genetic abnormalities, such as chromosome anomaly or Y chromosome microdeletion, were excluded from the present study was that few microdeleted patients were reported to have a decline in sperm output over time A case report documented that a father of four sons was found to be azoospermic with Y chromosomal microdeletions at the age of 63 years, suggesting that spermatogenic defects with Y chromosomal microdeletions might worsen with age. 37 Despite the fact that the present study has several limitations, including the nature of the retrospective design, lack of a control group, missing data and individual variation of semen variables, the result clearly showed the diminishing tendency of sperm count over time in the cohort of severe oligozoospermia, especially in comparison with the group of moderate oligozoospermia. The present study findings show that idiopathic severe oligozoospermia poses quite a risk of progression to azoospermia, whereas moderate oligozoospermia is less associated with the risk. To our knowledge, these are the first large scale data to report such a relationship. Thus, it is important that our results are confirmed in additional studies. It should be emphasized that patients with lower BMI and lower PRL level among initial severe oligozoospermics are more likely to worsen to azoospermia and, hence, require more attention. We trust that it is of interest for subfertile men as well as clinicians to be familiar with the importance of the differential diagnosis of and outcome of mild, moderate and severe oligozoospermia. Sperm cryobanking has been mainly available to men facing gonadotoxic treatment or awaiting ART, but not available on the day of ART. The present study, however, definitely suggests that patients with severe oligozoospermia, especially those with a sperm concentration of less than 5 million/ml, should be given enough information about the possibility of loss of fatherhood on the course and the option of cryopreservation from the very beginning of evaluation. And if a trend in the decline of semen variables is shown, they should be encouraged to cryopreserve sperm without any further delay. Although men with idiopathic severe oligozoospermia hoping to conceive should be reminded of the relentless declining quality of their sperm as time passes, those with moderate oligozoospermia should also be informed of the possibility of becoming azoospermia, though the risk is less. Acknowledgments We thank Ms Yon-Wan Han for her helpful comments and excellent editing assistance. The present study was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry of Health, Welfare & Family affairs, Republic of Korea (A084923). The sponsor had no role in the design and conduct of the study, in the collection, analysis or interpretation of the data, or in the preparation, review or approval of the manuscript. References 1 Bhasin S, de Krester DM, Baker HW. Clinical review 64: pathophysiology and natural history of male infertility. J. Clin. Endocrinol. Metab. 1994; 79: Brugh VM III, Matschke HM, Lipshultz LI. Male factor infertility. Med. Clin. North Am. 2004; 88: Sokol RZ. Endocrinology of male infertility: evaluation and treatment. Semin. Reprod. Med. 2009; 27: Carlsen E, Giwercman A, Keiding N, Skakkebaek NE. Evidence for decreasing quality of semen during past 50 years. BMJ 1992; 305: The Japanese Urological Association

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