ORIGINAL ARTICLE Follicle-stimulating hormone receptor polymorphism and seminal anti-Mu¨ llerian hormone in fertile and infertile men A. A. Zalata1, A. H. Hassan2, H. A. Nada1, F. M. Bragais3, A. Agarwal3 & T. Mostafa4 1 Medical Biochemistry Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt; 2 Dermatology & Andrology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt; 3 Center for Advanced Research in Human Reproduction, Cleveland Clinic Foundation, Cleveland, OH, USA; 4 Andrology & Sexology Department, Faculty of Medicine, Cairo University, Cairo, Egypt Keywords Summary Anti-Mu¨ llerian hormone—FSH receptors—male infertility— Follicle-stimulating hormone (FSH) is fundamental for Sertoli cell function polymorphism—semen stimulating spermatogenesis and follicular growth by a specific receptor (FSHR). This work aimed to investigate the occurrence of Asn and Ser FSHR Correspondence gene variants and its relationship with seminal anti-Mu¨llerian hormone Dr Taymour Mostafa, Department of (AMH) among normozoospermic and infertile oligoasthenozoospermic (OAT) Andrology & Sexology, Faculty of Medicine, males. Eighty-two Caucasian males grouped into normozoospermic healthy Cairo University, Cairo 11562, Egypt. Tel.: +20 10 515 0297; controls (n = 30) and infertile OAT males (n = 52). FSHR gene variants were E-mail: [email protected] determined by DNA from anti-coagulated blood and underwent polymerase chain reaction (PCR) amplification and electrophoresis in detecting amplifica- Accepted: July 16, 2008 tion products. AMH in seminal plasma was determined by ELISA. The results showed that the frequency of FSHR gene variants among fertile men was 46.7% Asn/Asn (N680S), 33.3% Asn/Ser, and 20% Ser/Ser, whereas among OAT men were 34.6%, 38.5% and 26.9% respectively with nonsignificant differences. Seminal AMH was significantly higher in fertile than infertile OAT men. There was significant increase in seminal AMH with Asn/Asn variant of FSHR gene than those with Asn/Ser or Ser/Ser. It is concluded that FSH gene variants showed no difference in distribution between fertile or infertile OAT men. However, when correlated with seminal AMH values, there was an increase in Asn/Asn in men with high seminal AMH. more than 1234 bp (Gromoll et al., 1996; Simoni et al., Introduction 2002). Although exon 10 is fundamental for signal trans- Follicle-stimulating hormone (FSH) stimulates spermato- duction, it is not necessary for ligand binding. The trans- genesis and follicular growth by a specific receptor membrane domain, however, might contact hormone (FSHR) which is a member of the G protein-coupled bound to the extracellular domain (Simoni et al., 1997). receptor family (Le´cureuil et al., 2007; Meduri et al., The two most common SNPs in the coding region of 2008). FSHR gene is a single-copy gene spanning a region FSHR occur at nucleotides 919 (919A>G, T307A, rs6165) of 54 kbp in the humans consisting of 10 exons and nine and 2039 (2039A>G, N680S, rs6166) in exon 10 corre- introns. The single nucleotide polymorphisms (SNP) in sponding to amino acid positions 307 and 680 of the exon 10, influences serum FSH levels in women, but not mature protein (Simoni et al., 1999; Gromoll & Simoni, in men. The extracellular domain of the human receptor 2005). One of the polymorphisms in exon 10 is Asn/Ser is encoded by nine exons ranging 69–251 bp. The C-ter- 680 present in 60% of the population (Simoni et al., minal part of the extra cellular domain, transmembrane 2002; Gromoll & Simoni, 2005) with the Ser 680 variant and the intracellular domain is encoded by exon 10 with occurring in 40% (Gromoll & Simoni, 2001). ª 2008 The Authors 392 Journal Compilation ª 2008 Blackwell Publishing Ltd Æ Andrologia 40, 392–397 A. A. Zalata et al. FSHR polymorphism and seminal AMH As regards the effects of exon 10 SNPs on testicular Materials and methods function, it has no effect on serum levels of FSH or other clinical parameters in men with either normal or This study included 82 Caucasian men attending the impaired spermatogenesis (Simoni et al., 1999; Asatiani andrology outpatient clinic at the university hospital after et al., 2002). There were no differences in FSH levels in institutional review board (IRB) approval. They were men with different FSHR genotypes. Although the SNPs divided into group (gp)1 (n = 30) normozoospermic, in exon 10 might have physiological effects in the testis, fertile and healthy volunteers who had achieved concep- there is no reliable parameter to measure this. By per- tion within 1 year, and gp2 (n = 52) infertile OAT males. forming variant screening, it was reported that the FSHR Normozoospermia means sperm concentration >20 mil- ) variants, Asn 680 and Ser 680, are similarly distributed in lion ml 1, sperm motility >50% (A + B) and normal infertile men and fertile controls, thereby excluding the sperm morphology >30%. OAT means sperm concentra- ) possibility that one of the two isoforms is involved in the tion <20 million ml 1, sperm motility <50% (A + B) and pathogenesis of idiopathic male infertility (Simoni et al., sperm normal morphology <30%. Exclusion criteria were 1999; Song et al., 2001). However, the two allelic variants, varicocele, cryptorchidism, karyotype anomalies, Y chro- at position )29 and exon 10, showed a significant differ- mosome microdeletions and leucocytospermia. Semen ent distribution between controls and men with non- samples were obtained by masturbation after 4 days of sex- obstructive azoospermia (NOA) (Ahda et al., 2005). ual abstinence. Computer-assisted semen analyses (Auto- Anti-Mu¨llerian hormone (AMH) has long been known sperm) (Fertipro NV, Beerneme, Belgium) were carried for causing regression of the Mu¨llerian ducts as a require- out according to guidelines of the World Health Organiza- ment for normal male reproductive tract development tion (1999). Sperm morphology was evaluated by phase (Jost, 1947; Teixeira et al., 2001). AMH is a member of the contrast microscope and sperm Mac stain (Fertipro NV). tissue growth factors superfamily of growth and differenti- Peroxidase-positive white blood cells were detected by ation factors (Massague´ & Chen, 2000; Gruijters et al., Endtz stain (Shekarriz et al., 1995). 2003). Visser et al. (1998) showed that AMH expressed by Seminal plasma was obtained by centrifugation of the testis during male sex differentiation is continued to be semen samples for 15 min and the supernatant seminal secreted into adult life. Josso et al. (2001) indicated that plasma was analysed for alpha-glucosidase activity and AMH is a marker of both Sertoli cell proliferation and pro- AMH. Ten milliliters of overnight fasting blood samples tein synthesis activity in response to FSH before puberty were withdrawn from men included in the study. One ml and also a useful marker of FSH action in the assessment was taken on EDTA and stored at )30 °C until the subse- of testicular function in pre-pubertal boys. quent use for genomic DNA extraction. The rest of the Al-Qahtani et al. (2005) found that seminal AMH con- blood sample was used to separate sera for FSH, lutenis- centrations in male factor infertility were not significantly ing hormone (LH), testosterone (T) and prolactin (PRL) different from fertile men. Fujisawa et al. (2002) corre- assessment by enzyme immunosorbant (ELISA) assay. lated seminal AMH significantly with sperm concentra- tion, testicular volume, serum LH but not with serum Genotyping of FSH receptor gene using PCR FSH, testosterone or oestradiol. Fallat et al. (1996) sug- gested that AMH may have a function in modulating DNA was extracted from EDTA anti-coagulated blood sperm motility. Seminal AMH was undetectable in using genomic DNA purification kit (Puregene; Gentra patients with obstructive azoospermia (Fujisawa et al., Systems, Minneapolis, MN, USA) for DNA purification 2002; Mostafa et al., 2007). from whole blood (Sudo et al., 2002). Conventional PCR Anti-Mu¨llerian hormone promoter activity was shown amplification was used for FSHR Gene (Asn-Ser 680) to be enhanced by the classical FSH-regulated signaling polymorphism. The region of nucleotide number 1624 to cascade involving a G protein, adenylate cyclase and PKA 2143 in the FSHR gene was amplified. The amplification being capable of enhancing AMH gene transcription. The product of 520- base pairs was indicative of the presence positive effect of FSH on testicular AMH production was of gene. found to be due to both the proliferation of Sertoli cells and to the increase in AMH transcriptional activity per Restriction analysis Sertoli cell (Rey, 1998; Lukas-Croisier et al., 2003). Al-Attar et al. (1997) demonstrated that testicular AMH Codon 680 of FSHR gene expresses asparagine (Asn) output could be stimulated by FSH. (AAT) (contain A at position 2039 nucleotide of the This study aimed to demonstrate the relation between receptor gene). If A at position 2039 becomes G then Asn the Asn and Ser FSHR gene variants and seminal AMH at position 680 will be Serine (Ser) (AGT). This A to G levels in infertile OAT males. transition creates recognition site for BsrI restriction ª 2008 The Authors Journal Compilation ª 2008 Blackwell Publishing Ltd Æ Andrologia 40, 392–397 393 FSHR polymorphism and seminal AMH A. A. Zalata et al. endonuclease. The Asn 680 allele gives an undigested Table 1 Data of studied groups (median, range) fragment of 520 bp, whereas the Ser 680 allele gives two Normozoospermia OAT fragments of 413 and 107 bp. For heterozygous (Asn/Ser), (n = 30) (n = 52) agarose gel electrophoresis allows visualisation of three bands 520 bp, 413 bp and 107 bp. Semen parameters a Seminal plasma AMH was carried out using an ELISA Testicular volume (ml) 21 (18–25) 16 (10–22) Semen volume (ml) 4 (2–7) 4 (1–9.2) kit (DSL, Diagnostic System Laboratories Inc., Webster, a Sperm concentration 70.4 (44.8–96) 8.2 (0.85–18.8) TX, USA), an enzymatically amplified two-site immuno- (106 ml)1) assay.