International Journal of

Introduction
 

T

he number of couples which meet the definition of infertility at reproductive ages is increasing worldwide. Infertility has different causes and conditions, which may root from congenital disorders or morbidity in males/females (1). One of the most known conditions of infertility in male individuals is azoospermia, defined as the complete absence of spermatozoa in the semen (2). Azoospermia does not mean complete sterility, because many azoospermic men may have varying levels of sperm within their testes. Azoospermia presents in two forms, named obsteructive azoospermia (OA) and non-obstructive azoospermia (NOA). While in OA form the spermatogenesis is normal, due to the absence of vasa deferentia or a mechanical blockage sperm cells are not present in the ejaculatory fluid (3). In contrast, in NOA form spermatogenesis is impaired (2). Several factors may predispose men to the OA or NOA syndromes, including congenital abnormalities, infection, medication, trauma, prostatic cysts, surgical procedure, and endocrine disorders (2, 4). Follicle stimulating hormone (FSH) is essential for normal reproductive function in males and females. FSH is fundamental for Sertoli and granulosa cells function and also for a normal gametogenesis. In the male, FSH is generally considered essential for the pubertal initiation of spermatogenesis and maintenance of quantitative normal sperm production in adults (5). FSH acts through its specific receptor named FSHR. FSHR as a member of G protein-coupled receptor family, after binding to its ligand outside the cell, promotes a signal transduction pathway within the target cell that finally leads to cellular response (6). FSH acts through the FSHR, which is expressed only in Sertoli cells in humans. The FSHR gene consists of 10 exons and 9 introns (7). Mutation screening of the FSHR gene revealed various single nucleotide polymorphisms (SNPs) both in the core promoter and in the coding region, which may affect receptor function (8). Among several SNPs within the FSHR gene, A919G and A2039G have been reported that may affect the receptor function (8).
There are several studies regarding FSHR SNPs and its relationship with infertility. However, there are only a few publications regarding the relationship between FSHR SNP and azoospermia.
Thus, the present study aimed to investigate the correlation between FSHR gene polymorphism (A919G and A2039G) and susceptibility to azoospermia in a group of Iranian infertile men from Fars province (Iran) in comparison to a healthy control group. The association between FSH levels within the sera and A919G and A2039G alleles and genotypes were also investigated.
 
Materials and methods
 
Subjects
Due to the ethical concern of the research, all the participants were ascertained that their blood samples would be used for research purposes and a written consent were obtained from them. In addition, the study was approved by the ethics committee, Shiraz University of Medical Sciences.
This case control study was performed on 212 primary azoospermic patients (126 non-obstructive, mean age 36.2 ± 3.7 years, and 86 obstructive, mean age 35.4 ± 4.2 years) and 200 healthy men (mean age 35.3 ±4. 6 years) who were referred to the Infertility Research Center, Mother and Child Hospital, Shiraz University of Medical Sciences between May 2011 and May 2013. The cases and controls were selected based on age and ethnic was matched from the same geographic region (Fars province, southwest of Iran). The clinical condition of the patients and the type of azoospermia were obtained from their medical records, checked and confirmed by a urologist. At first, azoospermia was confirmed based on two separate semen analysis. Inclusion criteria for NOA were, having no history of genital infections and existence of bilateral vas deferens and the exclusion criteria were, having history of surgery or vasectomy. All OA cases were selected among men with primary idiopathic epididymis obstruction. Excluding criteria for OA cases were azoospermia due vas deferens or ejaculatory duct. Moreover, patients with genital infections, vasectomy, or other iatrogenic injuries to the male reproductive tract were excluded from the study.
 
Genotyping
DNA was extracted from the peripheral blood using PrimePrepTM Genomic DNA Isolation Kit according to the manufacturer’s protocol (Genet Bio, Korea). Locus specific primers were selected in order to amplify the FSHR gene polymorphisms at locus’s A919G and A2039Gby polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Primer sequences and PCR conditions are shown in Table I. For genotyping of the FSHR gene at position A919G, a 305bp product was amplified and subjected to digestion with AhdI (Fermentas, Lithuania) in order to create 217 and 88 bp fragments (Figure 1). The amplified PCR product for A2039G was 577 bp. After digestion with BsrI restriction enzyme (Fermentas, Lithuania), several bandssizing403, 174, and143 bp was created based on the genotypes. Finally, the digested bands were monitored by electrophoresis in 2.5% agarose gel (Figure 2).
 
FSH measurement
Sera were separated from 2 ml of peripheral blood after centrifuging at 1000 x g for 10 minutes. All sera were stored at -70^°C until assay. The level of FSH was measured using FSH ELISA kit (Vidas, BioMetrieux, France) according to the manufacturer's guideline and was compared to the results of genotyping of the FSHR.
 
Statistical analysis
Prior to the statistical analysis, Hardy-Weinberg equilibrium of expected and observed frequencies was evaluated using Alrequin 3.1 software algorithm. Subsequently, SPSS.16 (Statistical Package for the Social Sciences, version 16.0, SPSS Inc, Chicago, Illinois, USA) was used to perform the statistical analyses. Between-group comparisons were carried out using chi-square test. In order to determine which genotypes accounted for statistical differences, Bonferroni method was performed using the following formula p_c=p×n, where the p_c is the corrected value, p is the uncorrected p-value, and n is the number of tests. P-value less than 0.05 was considered as statistically significant.
 
Results
 
In this study, two SNPs of FSHR gene at positions A919G and A2039G were genotyped and the relationship with azoospermia was investigated in 212 azoospermic patients (126 non-obstructive and 86 obstructive) and 200 healthy Iranian men. The genotype frequencies in azoospermic patients and healthy controls were in concurrence with Hardy-Weinberg equilibrium. The results of the statistical analysis for A919G and A2039G genotypes and allele frequencies have presented in Tables II-V.
Accordingly, AA genotype and A allele at A919G position were more frequent in OA cases in comparison with the healthy controls (P<0.001 for both comparisons, Tables II and III), although no significant difference was observed between NOA men and healthy controls regarding the distribution of genotypes and allele frequencies, AA genotypes and A allele were more frequent in OA compared to NOA patients (p<0.001 for both comparisons, Table II and Table III).
Considering A2039G polymorphism, no significant difference was found between OA and NOA patients as well as between the patients and the healthy controls regarding both genotype and allele frequencies (Table IV and Table V).
The findings of the current study indicated that the mean level of serum FSH was higher in the NOA men compared to the OA patients (23.8 versus 13.8, respectively, p=0.04). Additionally, analysis of the correlation between the serum FSH concentration and FSHR genotypes at positions A919G and A2039G indicated that OA patients with the AA genotype at position A919G had higher levels of FSH in comparison with those with genotypes GG and AG. However, no relationship was found between A2039G genotypes and FSH level in OA patients (P=0.04, Table VI). In addition, no significant association was observed between the levels of FSH and A919G or A2039G genotypes in NOA patients (Table VI).