Association study of novel single nucleotide polymorphisms of androgen receptor and estrogen receptor-α genes with male infertility in Northwest of Iran: A case-control study

Abstract Background: Observational evidence on the association of novel single nucleotide polymorphisms (SNPs) of androgen receptor (AR) and estrogen receptor-α (ER- α ) genes with odds of male infertility are rare. This is particularly relevant in the Iranian population where male infertility is relatively high. Objective: This study was designed to investigate the relationship between different SNPs of these genes and male infertility among the Iranian population. Materials and Methods: The present project was a population-based, case-control study conducted on 120 idiopathic azoospermia or severe oligospermia infertile cases alongside 120 age-matched subjects enrolled as controls. Overall, 3 variants from the AR gene and 2 variants from ER- α were genotyped - ARrs137852568, ARrs137852599 and AR rs137852563, and ER- α rs796065354 and ER- α rs104893956- using amplification refractory mutation system methods. Results: The obtained results indicated a significant association between AR rs1378525568 TT genotype as well as AR rs137852599 C allele with odds of male infertility (OR: 0.433, CI: 0.197-0.951 and OR: 0.545, CI: 0.304-0.978, respectively). Other variants of AR were not related to male infertility. A significant association was noted between predisposition polymorphism ER- α rs796065354 genotypes with male infertility. This significant association was not seen between ER- α rs104893956 and the risk of idiopathic azoospermia or severe oligospermia. Heterozygote overdominance was also observed in ESR rs796065354 but not in the other variants studied. Conclusion: Pieces of evidence were found on the association of novel polymorphisms of AR and ER- α with male infertility among the Iranian population. However, larger studies are warranted to confirm our findings.Key words: Male infertility, Androgen receptor, Estrogen receptor- α .


Introduction
Infertility is a universally common issue defined as a failure to achieve pregnancy after a year of unprotected intercourse (1). According to the World Health Organization report, around 60-80 million couples of reproductive age suffer from infertility worldwide (2).
Although a complex network of environmental factors and genetic aberrations play a crucial role in the etiology of male infertility, the main cause of impaired spermatogenesis is unknown (3). In the case of spermatogenesis, androgen receptor (AR) mediates the androgen signaling in the testis.
It has been pinpointed that disruption of the AR-signaling pathway results in a disturbance of maintenance of spermatogonial numbers, bloodtestis barrier integrity, completion of meiosis, adhesion of spermatids, and spermiation (4,5). Due to the fundamental effects of AR in spermatogenesis, various gene polymorphisms of AR concerning male infertility have been studied. Several meta-analyses have reported the association between AR gene CAG repeat length polymorphisms and male infertility (6)(7)(8)(9).
Despite these conclusions, some populationbased primary studies did not find any significant associations between AR gene CAG as well as GGN repeat length polymorphisms and infertility among their male population (8,10,11).
However, a recent local survey in southwest Iran has revealed that GGN repeat length polymorphisms are one important polymorphism leading to male infertility among the Iranian population (12). These findings have led to the hypothesis that other polymorphisms in the AR gene should be examined which might be considered a risk factor for male infertility. Another hormonal receptor that was studied is estrogen receptor alpha (ER-). Female hormones and their receptor gene polymorphisms, in particular estrogen receptors, have been challenged in elucidating their role in male infertility (13). It should be borne in mind that similar to AR, ERs also contribute to several steps of spermatogenesis such as modulated sperm metabolism, reduced testicular size, and severe oligospermia. Besides, gene expression of ER is significantly reduced in Sertoli cell-only tests (14). Evidence has added new significant findings on the association between ER gene polymorphisms, especially p and x alleles, and increased male infertility (10,15

Participants
This population-based, case-control study was carried out in 120 infertile men aged 25-

Genotyping
The allele-specific PCR or amplification Samples were re-genotyped to confirm the results.

AR single nucleotide polymorphisms (SNPs)
The ARA>T (rs137852568) was amplified by using a common reverse primer

ER-α SNPs
To amplify the ER-A>G (rs796065354), 4 primers were used as follows: forward inner primer

Statistical analysis
The frequency of genotypes in the case and control groups were evaluated for Hardy-

Results
The  There were no significant associations between AR rs137852563 genotypes and infertility risk among men compared to the normal homozygote.   The results of this population-based, casecontrol study suggested that AR rs1378525568

Discussion
TT genotype and AR rs137852599 C allele have a protective effect on fertility and may reduce idiopathic azoospermia or severe oligospermia risk. We also observed an association between ER-rs796065354 genotypes (AG/AA, GG, GA+GG, and GA/GG+A) and odds of infertility among men. However, the results failed to find any significant association between other polymorphisms of AR and ER-genes with the risk of infertility among our study population. To the best of our knowledge, this study is among the first case-control studies examining the association between specific polymorphisms of AR and ER-genes with the risk of male infertility among the Iranian population.
Most meta-analysis studies have concluded that the increase in CAG repeat length in AR is associated with male infertility (6)(7)(8)(9). However, the findings of an up-to-date meta-analysis which were classified by region and sub-types of male infertility were inconsistent. It seems that Caucasian populations are more susceptible to this polymorphism due to AR dysfunctions (7-9).
We found for the first time, an inverse association between AR rs1378525568 TT genotype and AR rs137852599 C allele with male infertility. Most previous studies have focused on CAG repeat length polymorphisms, and their results are in contrast to our findings (12,18,19). Besides, some studies failed to find any significant relationship between AR polymorphisms and the odds of various sub-types of male infertility (20). However,  However, based on recent studies, they are regarded as a "survival factor". According to this concept, the absence of ER-results in reducing the epididymis sperm content, sperm motility, and fertilizing ability (21,22). It seems that ERconfers a stronger estrogen effect. We found, as the first case-control study, ER-rs796065354 genotypes (AG/AA, GG, GA+GG and GA/GG+A) might increase the odds of male infertility. Similar to AR, ER-gene polymorphisms investigated in previous observational studies were different from the polymorphisms we studied. However, it has been reported that the (TA) repeat polymorphism may negatively influence spermatogenesis (22).
Some studies have demonstrated that XbaI and PvuII polymorphisms of the ER-gene are associated with the risk of male infertility (15,23). ER-gene polymorphisms might affect sperm quality in men. ER-397T/T and ER-351A/A genotypes were associated with lower sperm motility in men with oligozoospermia (24).
Semen variables including sperm count, motility, velocity, and morphology as well as sperm acrosin activity were significantly higher among infertile oligoasthenoteratozoospermia men who had pp and xx genotypes (15). Nevertheless, we found no significant relationship between ER-rs104893956 genotype and idiopathic azoospermia or severe oligospermia among our study population. It should be noted that our studied polymorphisms were completely different from other studies; therefore, this may lead to some inconsistency. Also, some controversial findings might be a reflection of the difference in study design and population. Similar to AR, it is Being the first report on the association of novel polymorphisms of AR and ER-with odds of male reproductive function, as well as the age-matched design that led to control of potential agedependent confounders, are the strengths of the present study. However, some limitations should be considered in the interpretation of our findings.
Control genotypes should be in HWE, as long as the population they are selected from is random and large in size. A significant result showing that controls are not in HWE could arise because of 1) random chance (one of every 20 markers tested will give a p-value < 0.05 by chance); 2) incorrect genotyping; and 3) heterogeneous population.
Provided the controls are in HWE, the cases may then be tested. If the SNP has a true genetic effect that is not controlled by a multiplicative model, the cases will not be in HWE. The test has little power to detect small departures from HWE (26).
Environmental factors, smoking, high-risk jobs, residence in high-risk areas, etc. are some confounders that were not included in this study.
Therefore, the generalizability of the findings should be done cautiously.

Conclusion
In conclusion, we found evidence of an association between novel polymorphisms of AR and ER-and male infertility among the Iranian population. Based on the results, AR rs1378525568 TT genotype and AR rs137852599 C allele may have a protective effect on male fertility. In contrast, there was a significant association between predisposition polymorphism ESR-rs796065354 and male infertility that may be considered as a biomarker for male infertility. However, further studies with different ethnic populations and larger samples are needed to validate the findings.