Idiopathic male infertility is strongly associated with aberrant promoter methylation of methylenetetrahydrofolate reductase (MTHFR)

Abstract

Background: Abnormal germline DNA methylation in males has been proposed as a possible mechanism compromising spermatogenesis of some men currently diagnosed with idiopathic infertility. Previous studies have been focused on imprinted genes with DNA methylation in poor quality human sperms. However, recent but limited data have revealed that sperm methylation abnormalities may involve large numbers of genes or shown that genes that are not imprinted are also affected.

Methodology/principal findings: Using the methylation-specific polymerase chain reaction and bisulfite sequencing method, we examined methylation patterns of the promoter of methylenetetrahydrofolate reductase (MTHFR) gene (NG_013351: 1538-1719) in sperm DNA obtained from 94 idiopathic infertile men and 54 normal fertile controls. Subjects with idiopathic infertility were further divided into groups of normozoospermia and oligozoospermia. Overall, 45% (41/94) of idiopathic infertile males had MTHFR hypermethylation (both hemimethylation and full methylation), compared with 15% of fertile controls (P<0.05). Subjects with higher methylation level of MTHFR were more likely to have idiopathic male infertility (P-value for trend = 0.0007). Comparing the two groups of idiopathic infertile subjects with different sperm concentrations, a higher methylation pattern was found in the group with oligozoospermia.

Conclusions: Hypermethylation of the promoter of MTHFR gene in sperms is associated with idiopathic male infertility. The functional relevance of hypermathylation of MTHFR to male fertility warrants further investigation.

Figure 1. The CpG island promoter region of the MTHFR gene.

The CpG Island Searcher Program identified a CpG island within the MTHFR gene promoter region, downstream of the transcriptional start site. Arrow represents the transcriptional start site, blue line depicts the CpG island and the vertical bars represent a CpG site. Black lines and yellow lines represent the location of the MSP primers and BSP primers, respectively.

Figure 2. Frequencies of MTHFR hypermethylation (both hemimethylation and full methylation) in case and control groups.

Significant differences were marked with *P<0.05 compared with the control.

Figure 3. Methylation status of the promoter of MTHFR in genomic DNA prepared from ejaculated human sperm.

(A) Representative results of the MSP analysis of MTHFR. DNA obtained from the sperms was amplified with primers specific to the unmethylated (U) or the methylated (M) of MTHFR after treatment with sodium bisulfite. (a) Fertile controls a1–a13 showed only the unmethylated allele. Others showed both methylated and unmethylated alleles. (b) Idiopathic infertile males with normozoospermia (Case 1) b1–b9 showed only the unmethylated allele. Idiopathic infertile males with normozoospermia b10–b14 showed both methylated and unmethylated alleles. Other showed only the methylated allele. (c) Idiopathic infertile males with oligozoospermia (Case 2) c1–c8 showed only the unmethylated allele. Idiopathic infertile males with oligozoospermia c9–c13 showed both methylated and unmethylated alleles. Others showed only the methylated allele. (B) Representative results of bisulfite-PCR sequencing of MTHFR. Filled and open circles represent methylated and unmethylated CpGs, respectively. L: molecular weight markers; P: positive control (universal methylated DNA).