Promoter methylation status of methylenetetrahydrofolate reductase (MTHFR) gene in spermatozoa of Iraqi infertile men

Abstract

In the vast majority of instances, the causes of male infertility are unknown, although scientists have lately attempted to correlate epigenetics to infertility mechanisms. DNA methylation is an important biological modification that gives phenotypic diversity and adaptability. The aim of this study was whether the methylation status of the methylenetetrahydrofolate reductase (MTHFR) gene promoter in Iraqi men’s spermatozoa is correlated with male infertility. Semen samples were collected from 100 infertile and healthy fertile men. Divided into 4 groups included 25 subjects of healthy fertile men as a control group(C) and infertile groups, which divided into (3) subgroups: Asthenozoospermia (A) (n=25), Oligoasthenozoospermia (OA) (n = 25) and severe Oligoasthenoteratozoospermia (SOA) (n =25). Sperm DNAs and RNAs from all samples were isolated; followed by sperm-specific methylation detection using the EpiJET DNA Methylation Analysis Kit, which is simple digestion was performed with two restriction enzymes (MspI/HpaII). The methylation percentage (mean ± SD) of the MTHFR gene promoter was quantified in each studied group as follow:[ (C) ( 1.76±0.46) (A) (3.59±0.89), (OA) (26.10±4.14) and (SOA)( 29.13±4.88)]. The percentage of MTHFR promoter methylation infertile men was significantly higher than that in the healthy control group (p = 0.00). Methylation level was determined via receiver operator characteristic (ROC) analysis as the cut-off values of each infertile subgroup were as follows: [(A) (2.43%), (OA) (7.59%) and (SOA) (10.96%)]. According to WHO guidelines, sperm concentration, progressive motility and morphologically normal sperm were determining (WHO, 2010). The qRT-PCR was used to evaluate MTHFR gene expression in all sperm samples. The results revealed a substantial reduction in fold expression (2–∆∆Ct) in all infertile groups when compared to the control g roup, with a P-value (0.002). In this research findings suggest that epigenetic impairment (hypermethylation) of the MTHFR promoter region in sperm DNA from OA and SOA patients may increase male infertility risk.