Soluble Fas and gonadal hormones in infertile men with varicocele

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1 Soluble Fas and gonadal hormones in infertile men with varicocele Hatem Zedan, M.D., a Abdel Wasea M. M. El-Mekhlafi, M.D., a Amira M. El-Noweihi, M.D., b Nagwa E. Abd El-Azim, M.D., a and Taymour Mostafa, M.D. c a Department of Dermatology and Andrology and; b Department of Medical Biochemistry, Faculty of Medicine, Assiut University, Assiut, Egypt; and c Department of Andrology and Sexology, Faculty of Medicine, Cairo University, Cairo, Egypt Objective: To assess gonadal hormones in serum and semen as well as seminal antiapoptotic factor; soluble fibroblast associated (sfas) in infertile men associated with scrotal varicocele. Design: Prospective. Setting: Academic setting. Patients: Eighty-eight males: fertile healthy controls (Gr1, n ¼ 12), fertile normozoospermia with varicocele (Gr2, n ¼ 31), and infertile oligoasthenozoospermia with varicocele (Gr3, n ¼ 45). Main Outcome Measure(s): Serum and seminal gonadal hormones: follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), and testosterone (T), in addition to seminal sfas. Results: There were significant higher mean levels of serum FSH, serum, and seminal LH with significant lower seminal T levels in cases of Gr3 compared with Gr2. Mean seminal sfas in Gr3 were significantly higher than its levels in Gr1 and 2 (mean SE vs and ng/ml, respectively). Nonsignificant differences between serum and seminal gonadal hormones were elicited between Gr2 and controls. Seminal sfas in various varicocele grades demonstrated nonsignificant differences. There were significant positive correlations between seminal sfas with serum FSH, serum LH, semen FSH, sperm abnormal forms percentage, and significant negative correlations with sperm concentration and sperm motility. Conclusion(s): sfas could play a role in germ cell apoptosis in varicocele-associated cases. (Fertil Steril Ò 2009; 91: Ó2009 by American Society for Reproductive Medicine.) Key Words: Male infertility, semen, varicocele, apoptosis, Fas, sfas Apoptosis has been observed in testicular germ cells, and is thought to be one of the important factors in regulating the production of spermatozoa. Regulation of apoptosis in various cell systems is mediated through specific factors including hormones, the presence or withdrawal of which activates the apoptotic death pathway (1 3). Furthermore, the physiologic regulation of germ cell apoptosis was found to involve signaling pathways triggered by Fas ligand (FasL). The Fas system has been implicated as a key regulator of germ cell apoptosis in the mammalian testis (4 6). Although the incidence of varicocele in the male general population is roughly 15%, it has been implicated as a factor responsible for infertility in as much as one-third of the infertile population (7, 8). Scrotal varicocele could be associated in infertile cases with impaired semen parameters: decreased sperm count, sperm motility, and morphologically normal spermatozoa (8 10). It is suggested that the spermatogenic dysfunction in varicocele testis may be related partly to an abnormal control of apoptosis (11, 12). Regarding the reproductive hormones, FSH and LH are essential for preventing cellular death in the seminiferous tubules and interstitial cells (13). Furthermore, a decrease in T concentration induces a significant increase in the number of apoptotic germ cells in most stages of the cycle of the spermatogenesis process (14). The Fas system in the testis has been identified as a paracrine signaling system by which Sertoli cells, expressing FasL, can initiate killing of Fas expressing germ cells. In addition, soluble forms of cell surface receptors such as soluble Fas (sfas) can be produced either by proteolysis cleavage of membrane-bound receptors or by alternative splicing (15), and it is believed to inhibit Fas-Fas L binding and thereby block Fas-mediated apoptosis (16). Fujisawa et al. (17) indicated that apoptosis was decreased in the testes of patients with varicocele compared with those of controls. Contrarily, Fujisawa and Ishikawa (18) demonstrated increased apoptosis in oligozoospermic patients with varicocele having lower seminal sfas than patients without varicocele or fertile men, suggesting that hyperthermia in these patients may inhibit its production. Celik-Ozenci et al. (12) detected that expression of FasL in spermatids was significantly down-regulated after induction of experimental varicocele. This study aimed to assess seminal sfas in relation to gonadal hormones in infertile men with varicocele. Received September 24, 2007; revised and accepted November 28, Reprint requests: Taymour Mostafa, M.D., Department of Andrology and Sexology, Faculty of Medicine, Cairo University, Cairo 11562, Egypt ( taymour1155@link.net). PATIENTS AND METHODS This study was conducted on 88 male cases after institute review board approval and informed consent. They were 420 Fertility and Sterility â Vol. 91, No. 2, February /09/$36.00 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 divided into group 1 (n ¼ 12), fertile healthy men without varicocele as controls; group 2 (n ¼ 31), fertile normozoospermia with scrotal varicocele; group 3 (n ¼ 45), infertile oligoasthenozoospermia with varicocele. Exclusion criteria included associated hydrocele, Klinefelter syndrome, chronic medical illness, or azoospermia. Diagnosis of varicocele was based on local examination of both testicles and spermatic cords in addition to Doppler ultrasonography. Scrotal varicocele was classified according to Hudson et al. (19) into grade I (n ¼ 12), grade II (n ¼ 30), and grade III (n ¼ 34). They were subjected to the following: history taking, clinical examination, and semen analysis. Serum and seminal FSH, LH, T, and PRL hormones were estimated in addition to seminal sfas. Fasting blood samples were collected from all patients and controls in addition to two semen samples collected after 4 days of sexual abstinence. The first sample was incubated at 37 C for semen analysis within 30 minutes according to the World Health Organization guidelines (20). To estimate sfas and gonadal hormones level, the second semen samples were centrifuged at 3,000 rpm for 10 minutes to pellet spermatozoa. Seminal plasma and sera were separated and stored at 70 C until the assay. Reproductive hormones were estimated in both serum and seminal plasma by the enzyme immunoassay kit method (BioSource Europe SA, Nivelles, Belgium). FSH assay had a sensitivity of 0.15 miu/ml, intraassay variations of 6.4%, interassay variations of 3.8%, and an assay range of miu/ml. LH assay had a sensitivity of 0.1 miu/ml, intraassay variations of 4.6%, interassay variations of 6.7%, and an assay range of miu/ml. T assay had a sensitivity of 0.05 ng/ml, intraassay variations of 4.9%, interassay variations of 6.9%, and an assay range of ng/ml. PRL assay had a sensitivity of 11 uiu/ml, intraassay variations of 5.4%, interassay variations of 6.5%, and an assay range of 100 6,000 uiu/ml. Seminal plasma sfas levels were determined by an ELISA (R&D Systems Inc., Minneapolis, MN). Its minimum detectable concentration was 0.02 ng/ ml, intraassay variations of 3.8%, and interassay variations of 4.5%. Data were presented as mean SE. Comparisons were performed using a two-tailed unpaired Student t-test. Correlations between different parameters were performed using the Spearman s rank correlation coefficient. P<.05 was considered statistically significant. RESULTS Mean semen volume and sperm concentration, sperm motility, and sperm normal forms percentage in cases with varicocele were significantly lower than in fertile controls. Mean seminal and serum FSH and PRL levels were significantly higher in varicocele-associated cases compared with fertile controls. Infertile oligoasthenozoospermic cases with varicocele showed significantly higher levels of serum FSH, serum, and seminal LH, and significantly lower seminal T levels compared with normozoospermic cases with varicocele. Mean seminal sfas in infertile oligoasthenozoospermic cases with varicocele was significantly higher than normozoospermic cases with or without varicocele (Table 1). Seminal sfas in various varicocele grades demonstrated nonsignificant differences (Table 2). In varicocele-associated cases, significant positive correlations were found between seminal sfas and serum FSH (r ¼ 0.485, P¼.001), serum LH (r ¼ 0.378, P¼.01), seminal FSH levels (r ¼ 0.509, P¼.04), and sperm abnormal forms percent (r ¼ 0.329, P¼.01). Seminal sfas levels showed significant negative correlations with sperm concentration (r ¼ 0.384, P¼.001) and sperm motility (r ¼ 0.353, P¼.01) (Figs. 1A and B). DISCUSSION Several hypotheses have been raised to explain the mechanisms by which scrotal varicocele may exert a deleterious effect on spermatogenesis and male fertility. They include renal and adrenal reflux, hypoxia, hormonal dysfunction, hyperthermia, and apoptosis of germ cells (17, 21, 22). In the present study, sperm concentration, sperm motility and normal form percentages in varicocele-associated patients were significantly lower than fertile controls obtained from numerous reports (23 25). Circulating gonadotropins and T have been shown to regulate testicular germ cell apoptosis in a stage-specific manner (26, 27). T is able to effectively inhibit in vitro-induced apoptosis of human spermatocytes and spermatids, and is thus a germ cell survival factor (1). In the present study, there was nonsignificant difference between serum T levels of varicocele-associated cases and fertile controls obtained from results yielded by different investigators showing reduced serum T levels in males suffering from varicocele that increased after varicocelectomy (28, 29). Seminal T levels were significantly decreased in infertile oligoasthenozoospermic cases with varicocele compared with normozoospermic cases with varicocele obtained from similar studies (30, 31). Mean levels of serum and seminal FSH in varicocele-associated cases were significantly higher than fertile controls. In addition, serum FSH levels were significantly increased in oligoasthenozoospermic cases with varicocele compared with normozoospermic cases with varicocele in agreement with different investigators (25, 32, 33). Adamopoulos et al. (34) suggested that raised serum FSH concentration in these cases signified damaged tubular epithelium probably related to the varicocele. In addition, varicocele-associated cases had significant higher mean serum PRL levels than fertile controls, a finding similar to that reported by Arowojolu et al. (35). Odell and Larsen (36) pointed that high serum PRL may inhibit LH action on interstitial cells and alter gonadal reaction to gonadotrophin. Although hormonal factors are essential for successful spermatogenesis, growth factors and cytokines are also Fertility and Sterility â 421

3 TABLE 1 Comparison between data in different studied groups (mean ± SE). Group 1 (n [ 12) Group 2 (n [ 31) Group 3 (n [ 45) Semen parameters Semen volume (ml) a b Sperm conc. (mill/ml) a a,b Sperm motility a,b (a þ b) (%) Sperm abn a,b forms (%) Serum hormones FSH (miu/ml) a,b LH (miu/ml) b PRL (miu/ml) a a,b T (ng/ml) Semen hormones FSH (miu/ml) a,b LH (miu/ml) a,b PRL (miu/ml) T (ng/ml) b Seminal sfas (ng/ml) a,b Note: SE ¼ standard error. a Significant difference compared to group 1. b Significant difference compared to group 2. involved in the local control mechanisms of testicular function (37). Pentik ainen et al. (38) suggested that when the testicular environment could no longer support spermatogenesis, a specific pathway leading to germ cell apoptosis is activated. Apoptosis has been observed in testicular germ cells, and this is thought to be one of the important factors in regulating the production of spermatozoa (4, 5). Simsxek (39) revealed that mean percentage of apoptotic cells was quite rare in testicular tissue of controls compared with varicocele associated cases (2% vs. 14.7%). They concluded that apoptosis may have a significant role in spermatogenetic dysfunction associated with varicocele. The Fas system has been implicated as a key regulator of germ cell apoptosis in the mammalian testis. In the human testis, FasL constitutively expressed by Sertoli cells is suggested to bind to Fas of germ cells causing death of these Fas-bearing germ cells (38, 40, 41). In addition, soluble forms of cell surface receptors such as sfas can be produced either by proteolytic cleavage of membrane-bound receptors or by alternative splicing, and was believed to inhibit Fas FasL binding and thereby block Fas-mediated apoptosis (16, 42). We showed that seminal levels of sfas in oligoasthenozoospermic varicocele-associated cases were significantly higher than in normozoospermic cases with varicocele and fertile controls. In accordance with these results, Fujisawa et al. (17) reported that apoptosis is decreased in the testes of patients with varicocele compared with those of fertile men. However, Fujisawa and Ishikawa (18) reported that oligozoospermic patients with varicocele had lower seminal levels of sfas than patients without varicocele or normal men, suggesting that the Fas FasL system may be one of the factors in the mechanism responsible for germ cell TABLE 2 Seminal sfas levels in patients with different grades of varicocele (mean ± SE). Grade I (n [ 12) Grade II (n [ 30) Grade III (n [ 34) P sfas (ng/ml) NS Note: NS ¼ nonsignificant (P>.05). 422 Zedan et al. Seminal sfas in men with varicocele Vol. 91, No. 2, February 2009

4 FIGURE 1 (A) Correlation of sfas and sperm concentration in cases with varicocele. (B) Correlation of sfas and sperm motility in cases with varicocele. ASperm motility ( ) Sperm concentration (mil./ml) B r = p < sfas (ng/ml) sfas (ng/ml) r = p < 0.01 apoptosis. Furuya et al. (43) showed that patients with oligozoospermia had a lower seminal plasma sfas than those with normal sperm concentration. Seminal sfas levels were shown to be inversely correlated with sperm concentration, sperm motility, and sperm normal morphology in varicocele-associated cases that could be attributed to reduced clearance of spermatozoa (abortive apoptosis) by increased sfas having an antiapoptotic effect (16, 44, 45). Sakkas et al. (46) showed that percentage of Fas-positive spermatozoa was small in normozoospermic semen samples, but this percentage can be as high as 50% in men with abnormal semen parameters. Immunohistochemical studies among these men showed that Fas protein was mostly expressed in germ cells with degenerative features such as chromatin condensation and margination typical of apoptosis (47 49). Therefore, a link can exist between expression of Fas in germ cells and a compensatory increase in sfas production. Celik-Ozenci et al. (12) reported significant down-regulation of FasL in experimentally induced varicocele cases without a significant increase in its number in apoptotic germ cells. They concluded that the Fas system may have another role in male reproduction instead of, or in addition to, apoptosis. Pentikainen et al. (50) demonstrated that the expression of the FasL was down-regulated by tumor necrosis factor alpha controlling physiologic germ cell apoptosis. It is concluded that sfas could play a role in the germ cell apoptosis process in varicocele-associated cases, and that the concomitant changes in gonadal hormones may be related to this activity. REFERENCES 1. Erkkila K, Henriksen K, Hirvonen V, Rannikko S, Salo J, Parvinen M, et al. Testosterone regulates apoptosis in adult human seminiferous tubule in vitro. J Clin Endocrinol Metab 1997;82: Shaha C. Modulators of spermatogenic cell survival. Soc Reprod Fertil Suppl 2007;63: O Neill DA, McVicar CM, McClure N, Maxwell P, Cooke I, Pogue KM, et al. Reduced sperm yield from testicular biopsies of vasectomized men is due to increased apoptosis. Fertil Steril 2007;87: Allan DJ, Harmon BV, Roberts SA. Spermatogonial apoptosis has three morphologicaly recognizable phases and shows no circadian rhythm during normal spermatogenesis in the rat. Cell Prolif 1992;25: Troiano L, Fustini MF, Lovato E, Frasoldati A, Malorni W, Capri M, et al. Apoptosis and spermatogenesis: evidence from an in vivo model of testosterone withdrawal in the adult rat. Biochem Biophys Res Commun 1994;202: Kim SK, Yoon YD, Park YS, Seo JT, Kim JH. Involvement of the Fas Fas ligand system and active caspase-3 in abnormal apoptosis in human testes with maturation arrest and Sertoli cell-only syndrome. Fertil Steril 2007;87: Kamal KM, Jarvi K, Zini A. Microsurgical varicocelectomy in the era of assisted reproductive technology: influence of initial semen quality on pregnancy rates. Fertil Steril 2001;75: Jarow JP, Sharlip ID, Belker AM, Lipshultz LI, Sigman M, Thomas AJ, et al Best practice policies for male infertility. J Urol 167: Schatte EC, Hirshberg SJ, Fallick ML, Lipschultz LI, Kim ED. Varicocelectomy improves sperm strict morphology and motility. J Urol 1998;160: Mostafa T, Anis TH, El-Nashar A, Imam H, Othman IA. Varicocelectomy reduces reactive oxygen species levels and increases antioxidant activity of seminal plasma from infertile men with varicocele. Int J Androl 2001;24: Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995;267: Celik-Ozenci C, Sahin Z, Ustunel I, et al. The Fas system may have a role in male reproduction. Fertil Steril 2006;85: Tapanainen JS, Tilly JL, Vihko KK, Hsueh AJ. Hormonal control of apoptotic cell death in the testes: gonadotropins and androgens as testicular cell survival factors. Mol Endocrinol 1993;7: Henriksen K, Hakovirta H, Parvinen M. Testosterone inhibits and induces apoptosis in rat seminiferous tubules in a stage-specific manner: in situ quantification in squash preparations after administration of ethane dimethane sulfonate. Endocrinology 1995;136: Riccioli A, Starace D, D Alessio A, Starace G, Padula F, De Cesaris P, et al. TNF-alpha and IFN-gamma regulate expression and function of the Fas system in the seminiferous epithelium. J Immunol 2000;165: Fertility and Sterility â 423

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