The susceptibility of FSHB -211G > T and FSHR G-29A, 919A > G, 2039A > G polymorphisms to men infertility: an association study and meta-analysis

Background Male infertility is a complex disorder caused by genetic, developmental, endocrine, or environmental factors as well as unknown etiology. Polymorphisms in the follicle stimulating hormone beta subunit (FSHB) (rs10835638, c.-211G > T) and follicle stimulating hormone receptor (FSHR) (rs1394205, c.-29G > A; rs6165, c.919A > G; rs6166, c.2039 A > G) genes might disturb normal spermatogenesis and affect male reproductive ability. Methods To further ascertain the aforementioned effects, we conducted a case-control study of 255 infertile men and 340 fertile controls from South China using the Mass ARRAY method, which was analyzed by the t-tests and logistic regression analysis using SPSS for Windows 14.0. In addition, a meta-analysis was performed by combining our results with previous reports using STATA 12.0. Results In the FSHB or FSHR gene single nucleotide polymorphism (SNP) evaluation, no statistically-significant difference was found in the frequency of allelic variants or in genotype distribution between cases and controls. However, a significant association for the comparison of GAA (P: 0.022, OR: 0.63, 95%CI: 0.43–0.94) was seen between the oligozoospermia and controls in haplotype analysis of rs1394205/rs6165/rs6166. In the meta-analysis, rs6165G allele and rs6166 GG genotype were associated with increased risk of the male infertility. Conclusions This study suggested that FSHR GAA haplotype would exert protective effects against male sterility, which indicated that the combination of three SNP genotypes of FSHR was predicted to have a much stronger impact than either one alone. Then in the meta-analysis, a significant association was seen between FSHR rs6165, rs6166 polymorphisms and male infertility. In terms of male infertility with multifactorial etiology, further studies with larger sample sizes and different ethnic backgrounds or other risk factors are warranted to clarify the potential role of FSHB and FSHR polymorphisms in the pathogenesis of male infertility.


Background
Worldwide, approximately 15% of couples cannot have a child, and in half of the cases the reason is the result of male infertility [1], which is a complex disorder caused by genetic, developmental, endocrine, or environmental factors or still unknown etiology [2]. It has been demonstrated that approximately 30% of infertility cases could be attributed to genetic defects, such as Klinefelter syndrome (KS), disorders of sexual development (DSD), or congenital absence of the vas deferens. Therefore, it is necessary to prevent and treat male infertility from the genetic viewpoint [3].
FSH acts as a spermatogonial survival factor in the adult testis and is also a necessary signal for Sertoli cell proliferation, a process that occurs during fetal, neonatal, and prepubertal life [16]. FSH is a double-stranded glycoprotein consisting of two chains, including 92 amino acids forming the α chain and 111 amino acids comprising the β chain, which are coupled by a noncovalent bond [17]. Most clinical studies reported so far only focused on the single nucleotide polymorphism (SNP) rs10835638 (c.-211G > T), which is located in an element of the FSHB promoter influencing gene transcription by affecting the binding of the LHX3 homeodomain transcription factor [15,18,19]. In order to exert its stimulatory effect, FSH binds to its receptor, the FSHR expressed on the membrane of granulosa cells (GCs) in the ovary and Sertoli cells in the testis to bring about folliculogenesis [20] and spermatogenesis [21], respectively. The FSHR gene is located on chromosome 2p21 and consists of 9 introns and 10 exons [22]. Given the significant role of FSH in fertility, genetic abnormalities of the FSHR could cause infertility. FSHR SNP rs6166 exchanges asparagine (Asn) for serine (Ser) in the intracellular domain of the receptor, introducing a potential phosphorylationsite; while rs6165 replaces threonine (Thr) with alanine (Ala), resulting in a change from a polar (T) to a nonpolar, hydrophobic (A) amino acid and removing a potential O-linked glycosylation site [22]. A previous studies in human granulosa-lutein cells (hGLC) has revealed that the two FSHR polymorphisms, blunted ERK1/2 (extracellular regulated protein kinases 1/2) activation, which indicated that Ala307-Ser680 FSHR was indeed less "active" in vitro, providing a molecular explanation for the clinical data [23]. Clinical studies suggested that those men with both mutated FSHR variants may have significantly higher basal serum FSH levels. SNP (rs1394205) exchanges a nucleotide in the promoter region of the FSHR gene (c.-29G > A), resulting in a significant 56% decrease in the transcriptional promoter activity of A allele [18]. In summary, SNPs involved either in signal transduction (FSHR exon 10) or in transcriptional activity (FSHR and FSHB promoter) in vitro could result in an overall change of FSH action.
Among these candidates, the linked SNPs at positions 307 and 680 in exon 10 of the FSHR gene have been extensively investigated as the potential cause of male infertility in various ethnic populations [14,22,24,25], and the results largely failed to demonstrate significant associations. Polymorphism rs1394205 is located in the 5′-untranslated region (5′-UTR) of FSHR (c.-29G/A), which has been reported to affect the serum level of FSH [22]. For the FSHB, rs10835638 is a -211G > T polymorphism located in the element of the FSHB promoter, which has been reported to be associated with lower testicular volume, lower sperm count, lower testosterone, and higher luteinizing hormone (LH) serum levels [18,22]. However, the associations between these SNPs in the FSHR gene and FSHB and male infertility remain uncertain.
To further verify the effects of the polymorphisms (FSHB rs10835638, FSHR rs6165, rs6166, rs1394205) on the risk of male infertility and to quantify potential heterogeneity between various studies, we conducted a case-control study of 255 infertile men and 340 healthy controls from South China, as well as performing a meta-analysis of the results of previous reports and this study.

Study population
Only men of Han-Chinese ethnicity were recruited between April 2013 and July 2015 among the participants in the Institute of Laboratory Medicine, Jinling Hospital, Nanjing University School of Medicine. This study population was consisted of 340 fertile men as healthy control, who had at least 1 child in the last year by direct survey and lacked any history of requiring assisted reproduction technology, and 255 infertile men, including 166 with azoospermia or severe oligozoospermia (sperm concentration < 5 ×106/ml), and 89 with oligozoospermia (sperm concentration5-15×106/ml), with at least 1 year of infertility. Individuals with known causes of infertility including genetic factors (chromosome anomalies), AZF microdeletions, clinical factors (varicocele, crytorchidism), obstructive azoospermia and infections were excluded from this study. All controls and cases were ethnic Han-Chinese.

Evaluations
All the enrolled patients were examined at least two semen analyses according to the World Health Organization guidelines (WHO, 2010). In brief, after ejaculation, the semen was incubated at 37°C for 30-40 min for liquefaction. Semen volume was estimated by weighing the collection tube with the semen sample and subsequently subtracting the predetermined weight of the empty tube assuming 1 g = 1 mL. For assessment of the sperm concentration, the samples were diluted in a solution of 0.6 mol/L NaHCO 3 and 0.4% (v/v) formaldehyde in distilled water. The sperm concentration was assessed using the improved Neubauer haemocytometers.
In addition, the participants underwent medical and andrological examination including medical history, hormonal analysis for the measurement of serum LH, FSH, total testosterone (T), Estradiol (E 2 ), Prolactin, karyotype, and Y chromosome microdeletion screening. Karyotype and Y chromosome microdeletion were determined by G-banding in lymphocytes and multiplex polymerase chain reaction (PCR) using primers (sY84, sY86, sY127, sY134, sY254, sY255, SRY, ZFX/ZFY) specific for the diagnosis of microdeletion of the AZFa, AZFb, and AZFc regions, respectively. Then serum hormone levels were detected by chemiluminescent microparticle immunoassay on an Abott-ARCHITECT Immunoanalyser (Abbott Laboratories Abbott Park, IL, USA). The intraand interassay coefficients of variation (CV) for measurement of both FSH and LH were 3 and 4.5%, for total testosterone <8% and <5%, for estradiol 7.5% and 13%, respectively.

Meta-analysis Study selection
To identify the related articles, a comprehensive systematic searching was performed in the PubMed, Web of Science and the Chinese National Knowledge Infrastructure (CNKI) database, using the search words "FSHR rs6165", "FSHR rs6166", "FSHR rs1394205", "polymorphism" and "male infertility". Included studies had to meet the following criteria: (1) evaluation of the FSHR rs1394205, rs6165, rs6166 and male infertility; (2) involving in human beings; (3) a case-control study; (4) with detailed genotype frequency of cases and controls or obtained the article text. Statistical analysis and meta-analysis Analysis of genetic data T-test was used to measure the differences in the distributions of clinical characteristics, including age, hormone and sperm parameters between groups and cases. The difference of FSHR rs1394205, rs6165 and rs6166 polymorphism and FSHB rs10835638 polymorphism between the infertile and fertile groups was calculated using a logistic regression model, SNPs coded as three categories: wild-type homozygote (WW, reference), the heterozygous (WR) and rare allele homozygote (RR), which yielded a p value and odds ratio (OR) with the corresponding 95% confidence interval (CI), using SPSS for Windows 14.0 (SPSS, Inc., Chicago, Illinois). And P < 0.05 was considered statistically significant. The linkage disequilibria and haplotypes were analyzed with SHEsis software (http://analysis.bio-x.cn/SHEsisMain.htm). Because only the data of Age was collected completely, genetic association tests have been adjusted for Age effects.

Statistics for meta-analysis
This study and other related case-control studies were combined for the meta-analysis, which was performed using STATA 12.0 (STATA Corporation LP, College Station, TX, USA). Odds ratio (OR) and 95% confidence interval (CI) were calculated to estimate the associations of FSHR rs6165、rs6166、rs1394205 polymorphisms with male infertility susceptibility based on three genetic models, including co-dominant model [the rare allele homozygote (RR) vs. wild-type homozygote (WW), the heterozygous (WR) vs. WW], dominant model (RR + WR vs. WW), recessive model (RR vs. WW + WR). In addition, stratified analyses were performed by HWE (>0.05 and <0.05), race (Caucasian, Asian and Brazilian population) and case counts (>200 and <200). HWE < 0.05 indicates unbalanced distribution in the frequency of the population. So studies with SNPs with HWE < 0.05 were removed from metaanalysis. Heterogeneity across the studies was evaluated by t-test test based on Q test and was considered significant if P-value for heterogeneity (P h ) was <0.05. A fixed-effect model with no heterogeneity (P h > 0.05 or I 2 < 50%) using the Mantel-Haenszel method and a random effects model with a high heterogeneity (P h < 0.05 or I 2 > 50%) using the DerSimonian and Laird method were used to pool the results. Moreover, a sensitivity analysis, by which a single study in the meta-analysis was deleted each time to determine the influence of the individual data set to the overall pooled OR, was performed to assess the stability of the results. To test the publication bias, Begg's Funnel plots and Egger's linear regression test were applied. HWE in the controls of each study was calculated using a web-based program (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl).

Clinical characteristics of the study population
A total of 255 infertile men and 340 infertile men were collected in this case-control study. The clinical charactereristics of the participants were presented in Table 1. The observed frequencies of all tested genotypes in controls were in agreement with the HWE (P: 0.538 for FSHB rs10835638, P: 0.975 for FSHR rs1394205, P: 0.884 for rs6165, P: 0.391 for rs6166, respectively). The serum FSH and LH concentrations in fertile men were 4.72 ± 2.51 IU/L and 3.37 ± 1.46 IU/L, whereas the FSH and LH values in infertile patients were 15.73 ± 16.15 IU/L and 6.46 ± 4.98 IU/L, respectively. The FSH and LH levels in the infertile patients were significantly higher than that in the fertile men (P < 0.05). And fertile men had higher sperm concentration and sperm motility compared with the infertile men. No statistical difference was observed in the other indicators.

Case-control study of FSH and FSHR gene polymorphisms
Logistic regression analysis revealed that, when the FSHR SNPs at nucleotide −211, −29, codon 307 and codon 680 was separately analyzed, no statistically significant difference was found in the frequency of allelic variants or in genotype distribution between cases and controls, as showed in Table 2.
For rs1394205 polymorphism, no significant association was showed in overall analyses and subgroup analyses.

Test of heterogeneity and sensitivity analysis
Sensitivity analyses on the FSHR SNPs rs6165, rs6166 and rs1394205 under four models were conducted. Among rs6165 polymorphism, we found slight heterogeneity for the comparison of dominant model (GA + GG vs. AA: P h : 0.042) in overall analysis, homozygote model (GG vs. AA: P h : 0.038), dominant model (GA + GG vs. AA: P h : 0.044) in subgroup analysis of case count > 200. The heterogeneity was decreased respectively to 0.120, 0.266 and 0.201 when omitting the paper reported by Gharesi-Fard et al. [13]. Among rs1394205 polymorphisms, a significant heterogeneity was apparent in the overall analyses under the homozygote model, heterozygous model and dominant model. Consistently, subgroup analyses by HWE, race and case count for rs1394205 also showed a significant heterogeneity, which indicated in Table 5. Sensitivity analysis revealed that the study reported by Li Y et al. [28], was the origin of substantial heterogeneity, and this was decreased when it was removed.

Publication bias
Begg's Funnel plots and Egger' test were performed to assess publication bias. For the FSHR rs6165, rs6166 and rs1394205 funnel plot shape did not show any evidence of obvious asymmetry in all comparison models. And the Egger's test was used to provide statistical evidence for funnel plot symmetry, which showed no obvious evidence of publication bias.

Discussion
In the present study, we included 340 fertile males and 255 infertile males, consisting of 166 with azoospermia or severe oligozoospermia, and 89 with oligospermia, and we investigated the association between FSHB, FSHR gene polymorphisms and male infertility. Compared with the fertile controls, the infertile patients had higher FSH and LH levels, and lower sperm concentration and sperm motility. Then the most common FSHR allelic variants in the core promoter and exon 10, and the FSHB variants in the core promoter were genotyped with respect to male infertility status using the Mass ARRAY platform. However, the distributions of FSHB and FSHR allele, genotype frequencies among azoospermic, severe oligozoospermic, or oligozoospermic men and fertile men based on age-adjusted estimates were similar. However a more precise analysis should be conducted if all individual raw data were available, to allow    Inclusion criteria were as follows: (i) a minimum of 1 year of infertility (ii) sperm count below 20 × 106/ml as determined by at least two semen analyses. Patients were excluded if they had: (i) hypogonadotrophic hypogonadism or abuse of androgenic (anabolic) steroids (ii) obstructive azoospermia (iii) undergone treatment with chemotherapeutic agents or radiotherapy. Azoospermic and severely oligozoospermic men with karyotype abnormalities and Y chromosome long arm microdeletions were excluded.
for the adjustment according to hormone, sperm parameters and other lifestyle factors. Consistent with previous reportes [31,34], we also identified a significant association for the comparison of GAA (P: 0.022, OR: 0.63, 95%CI: 0.43-0.94) among the oligozoospermic men in haplotype analysis, which indicated that the GAA haplotype would exert protective effects against male sterility. Previous studies examining potential associations between FSHR polymorphisms and male infertility parameters have produced contradictory results. Variants of FSHR have been shown to affect the serum FSH, inhibin B, anti-Mullerian hormone (AMH) and total testes volume [5,14,25,33]. However, the majority of studies have failed to detect any link between common FSHR isoforms and male reproductive parameters. It is regrettable that not all clinical fertility parameters in this study were collected, as we could not find any association between the clinical fertility parameters and the FSHB and FSHR genotypes.
FSH secreted by the anterior pituitary, together with other endocrine factors, plays a central role in establishing and maintaining human fertility. In males, circulating FSH stimulates gametogenesis and steroidogenesis in the gonads by binding to its receptor (FSHR). Tuttelmann et al. reported that the FSHB -211G > T T-allele showed significant dosage effects on FSH, LH and bilateral testicular volume. Moreover, FSHR 2029A > G significantly modulated the more dominant effect of FSHB -211G > T on serum FSH and testicular volume among the T-allele carriers [18], suggesting that the interplay between polymorphisms in hormone and receptor is of relevance under physiological conditions. We searched available databases, such as GWAS Central, National Human Genome Research Institute GWAS Catalog and PUBMED, but failed to find a relevant genome-wide association study (GWAS) on all SNPs. To date, no clear consensus appears to have been reached in the literature on the relationship between FSHR polymorphisms and male infertility risk.
To resolve the conflicting results, we carried out a meta-analysis to obtain a more precise estimation of the associations. Contrary to previous meta-analyses that found no significant association between FSHR rs1394205 (G-29A), rs6165 (Thr307Ala), or rs6166 (Asn680Ser) polymorphisms and the risk of male Table 4 Main characteristics of all studies on the genotype of FSHR rs6165, rs6166 and rs1394205 included in the meta-analysis (Continued) Galan [2,30,34], this study indicated that the rs6165G allele was associated with increased risk of male infertility, particularly in the subgroup analysis of HWE > 0.05. In addition the rs6166 GG genotype was also observed to be a risk factor for infertility in the subgroup analysis of HWE > 0.05. One possible reason for these discrepant findings is specific selection of the publications. However, the FSHR rs1394205 polymorphism was not associated with male infertility, which was similar to the findings of other studies. In addition, identifying the source of heterogeneity is one of the most important goals of the meta-analysis. In each case, the heterogeneity could be a result of differences in ethnicity, sources of controls, methods used and so on. In this study, we found that significant heterogeneity existed in overall analysis and sub-group analysis for the comparison of FSHR rs1394205 polymorphisms and male infertility. Sensitivity analysis revealed that the study reported by Li et al. [28], contained substantial heterogeneity, and this was decreased when it was removed. The subjects of the casecontrol study by Li et al. [28], comprised 364 idiopathic infertile patients (97 with non-obstructive azoospermia, 79 with oligozoospermia and 188 with normozoospermia) and 285 fertile men were collected [28]. However, previouslyreported meta-analyses including our meta-analysis considered 188 men with normozoospermia and 285 fertile men as controls, which brought out different associations between the FSHR rs1394205A allele and male infertility, resulting in the significant heterogeneity.
It should be pointed out that there are some limitations in this meta-analysis. Firstly, inadequate sample size and inappropriate control subjects resulted in limited power for exploring the real association, and subgroup analyses by HWE, ethnicity, and case count involved relatively small groups, which were more likely to reveal greater beneficial effects than a large-scale trial. Secondly, owing to lack of the original data, we could not further evaluate the potential interactions between genes, which might affect male infertility. Thirdly, much detailed information, including body mass index, age, work, smoking or alcohol habits, environmental exposure and other lifestyle factors, is not available, so that our results were based on unadjusted estimates. A more precise analysis should be conducted through adjustment by other co-variants. In fact, some other genes as well as environmental exposure could also play an important role in spermatogenesis.
Owing to the critical role of FSH in spermatogenesis, polymorphisms in the FSHB and FSHR genes might disturb normal spermatogenesis and affect male reproductive ability. Because of this, FSH treatment has always been tempting and is actively prescribed by many doctors even though the efficacy of FSH therapy remains a contentious issue. One study showed that patients with at least one FSHR c.2039A > G G allele had a significant increase in total sperm count after 3 months of treatment with recombinant FSH (rFSH, 150 IU/three times per week). Another study considered the FSHB-211G > T genotype and showed that TT homozygote, representing 25% of men with oligozoospermia and low FSH levels, could significantly benefit from FSH treatment [23]. However, there have been other studies showing that the outcome of FSH treatment was poor. Possible reasons include that the doses used were too low, with a high dose of recombinant rFSH necessary for treatment to be effective. Another explanation is that FSH therapy might require stratification of men according to genotype into FSH-responsive and non-responsive individuals, which depending on the FSHB and FSHR genotypes [36].

Conclusions
This study suggested that FSHR GAA haplotype would exert protective effects against male sterility, which indicated that the combination of three SNP genotypes of FSHR was predicted to have a much stronger impact than either one alone. Then in the meta-analysis, a significant association was seen between FSHR rs6165, rs6166 polymorphisms and male infertility. In terms of male infertility with multifactorial etiology, further studies with larger sample sizes and different ethnic backgrounds or other risk factors are warranted to clarify the potential role of FSHB and FSHR polymorphisms in the pathogenesis of male infertility. had been involved in drafting the manuscript or revising it critically for important intellectual content. MCZ, WWL and QZ agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. XYX and YXC given final approval of the version to be published. Each author should have participated sufficiently in the work to take public responsibility for appropriate portions of the content. All authors read and approved the final manuscript.
Ethics approval and consent to participate The manuscript has been read and approved by all the authors. The research adhered to the tenets of The Declaration of Helsinki. The Ethics Committee of Jinling Hospital approved the protocol. The patients gave written informed consent.

Consent for publication
Written informed consent about the patients for publication was obtained.