Association of the MTHFR C677T (rs1801133) polymorphism with idiopathic male infertility in a local Pakistani population

Infertility is a state of failure to conceive in a year of unprotected intercourse with the same partner [1]. The male factors are responsible of infertility in 20.0 to 25.0% of couples [2]. In men, the main causes of infertility are oligospermia, asthenospermia, teratozoospermia and azoospermia [3,4]. Like other nutritional factors, availability of bioactive folate has recently been found to be related to male fertility [5].

Folates are an inter-convertible group of enzymes that metabolize amino acid, synthesize and methylate deoxy-ribonuleotides (dNTPs) [6,7]. Methyltetrahy-drofolate (MethylTHF) is the biologically active form of folate in plasma. An important enzyme, methylene-tetrahydrofolate reductase (MTHFR), reduces 5,10 methylenetetrahydrofolate into 5, MethylTHF and during this reduction, a homocysteine is converted into methionine by attaining a methyl group from methylenetetrahydrofolate [8].

Methionine is converted to S-adenosyl methionine that acts as a ‘methyl’ donor for DNA methylation. The 5,10 methylenetetrahydrofolate converts uracil into thymine for DNA synthesis [9]. Therefore, deficiency in folate intake or polymorphism(s) in the enzymes of the folate pathway may result in aberrant DNA synthesis and methylation, especially in rapidly dividing cells such as bone marrow and spermatogonia [10].

The MTHFR gene is composed of 11 exons and is located on the short arm of chromosome 1 at 1p36.22 [8,11]. Being an important enzyme of folate metabolism, the MTHFR gene product has been studied and found in a total of 65 polymorphisms. The C677T (rs1801133) polymorphism in the MTHFR gene reduces the enzyme activity by 35.0% [11] resulting in impairment of nucleic acid metabolic pathways. However, the condition can be managed by folic acid supplementation for determining the frequency of the MTHFR C677T mutation is a prerequisite.

The results of many of the molecular epidemiological studies on the association of the MTHFR C677T polymorphism and male infertility remain controversial due to small sample size, ambiguously defining infertility and confounding factors including ethnicity. In the present study, we determined an association between idiopathic sperm disorders and the MTHFR 677 CT polymorphism.

Genotype distribution of the MTHFR 677C>T polymorphism of the 655 subjects analyzed: 472 (72.06%) subjects were homozygous for the C allele (CC), 166 (25.34%) subjects were heterozygous (CT), and 17 (2.60%) subjects were homozygous for the T allele (TT). The minor allele (T) frequency of the MTHFR 677C>T polymorphism was 0.153, and the genotype distributions did not deviate from the Hardy-Weinberg equilibrium (p >0.05) (Tables 1 and 2).

Spermatogenesis is a complex process involving about 2000 genes and various non genetic factors. The gene mutations and disrupted expression may distort maturation of spermatocytes. DNA methylation ensures the regulated expression of genes. However, DNA methylation is done only in the presence of methionine. A homocysteine is converted into methionine by MTHFR through transferring a methyl group from methylenetetrahyfrofolate [8]. Therefore, a mutation in the MTHFR such as C677T may reduce methylation of DNA [11]. In addition, a reduced conversion of homocysteine into methionine may lead to hyper-homocysteinemia, and cause DNA and cell membrane damages, testicular arterial sclerosis, and impairment of spermatogenesis [11,15-17].

However, results of most of the molecular epidemiological studies on association of MTHFR 677CT polymorphism and male infertility remain controversial. A total of 20 of 41 studies showed association of the polymorphism with male infertility. Out of four [18-21] studies carried out on African populations, only one ([18] showed a significant association of the 677T with infertility in Egyptian men. Furthermore, a study on Moroccan infertile men showed a protective role of the MTHFR 677T polymorphism [22]. Comparatively, more than half the number of Asian studies (14 out of 22) [22-32,36-38,41-42] expressed this association, while only a few Caucasian studies (six out of 15) [44-47,50-51] exhibited it.

Similarly, all the studies [22-28] except one [29] reported a significant association of the 677T anomaly with male infertility. Seven studies on Indian populations reported contrasting observations to each other [30-36]. Four Indian studies [30-33] showed a significant association of mutant genotypes with infertility, while three studies [34-36] showed no statistically significant difference of 677 C>T variants between infertile and fertile males. Dhillon et al. [35] explained the variations in results that their study included a majority of OAT patients in comparison to the majority of azoospermic cases in the study by Singh et al. [29]. Gupta et al. [30] included both azoospermic and OAT individuals, showing a significant association of mutant alleles and genotypes with infertility. It was reported that the three Indian populations studied were not ethnically different [30]. Three [36-38] of five [36-40] other Asian studies showed significantly higher mutant genotypes in infertile groups than fertile groups of Iranian men. Similarly, studies carried out on Korean infertile men also had contrasting results [41-43].

Moreover, in Caucasian populations, few studies reported that the polymorphism in MTHFR was a risk of male infertility [44-48]. However, other studies inferred that the mutant genotype is not such a risk [49-58]. Moreover, few studies gave contrasting conclusions for the same population [47,48].

Therefore, the differences in the results could be attributed to other factors such as variations in recruitment of subjects, sample size, ethnicity and geographic factors (Tables 3 and 4). The results are also dependent on the reproductive health (testicular, hormonal, and epididymal, etc.), general health (infections, surgeries, fever and antibodies, etc.) and lifestyle factors (clothing, occupation, smoking, caffeine intake, etc.), which were not addressed while selecting subjects in many of the previous studies. There were eight studies [18,22,23,26,27,31,37,49] that showed association of 677T with male infertility and seven studies [20,39,40,50-52,58] that lack association and did not mention criteria of selection of infertile men. Furthermore, nine studies [23,24,26-28,32,37,38,47] showing association and eight studies [19,28,34,39,40,48,50,57] that showed no association, did not mention selection criteria of fertile men as a control group (data not shown).

In addition, the MTHFR C677T association with male infertility was also affected by folate supplementation, usually prescribed in infertility. The status of folate and supplementation is not mentioned in the previous studies except four [34,36,42,55].

Although a single study that took folate levels as a factor of infertility in Caucasian men also found no association of 677T with male infertility, we can assume, depending on the socioeconomic and health facilities that the Caucasians may have comparatively more chances of folate supplementation than Asians, thus lowering the association of 677T with infertility [42,59,60]. The previous studies described that 35.0% of the Pakistani population is folate deficient (61) and no organized folate supplementation program is currently working for the general population.

The present study of the association between idiopathic sperm disorders and the MTHFR 677T polymorphism suggests that the polymorphism might be a factor of male infertility in Pakistan. The results showed a significantly (p <0.05) higher T allele and gene frequencies in the infertile men as compared to the fertile men. Furthermore, the carriers of the T allele were more in all semen groups of infertile men as compared to the control group. Association of male infertility with MTHFR C677T polymorphisms provokes investigation of the effects of folic acid supplementation in infertile men on sperm parameters. However, the present study is about only one ethnic group in Pakistan, which was self-defined by the subjects, while, MTHFR 677T frequencies in the Pakistani populations have been reported to be greatly variable and may influence the frequencies of SNPs. Therefore, studies must be conducted to investigate the association of SNPs also at the ethnic level. Secondly, the screening of infertile men must include karyotyping that was not done in the present study. We suggest that further studies must consider folate levels and folate supplementation while selecting subjects in genetic studies. Similarly, the studies must consider other well defined factors of reproductive health, general health and lifestyle factors in exclusion criteria. Further research, analyzing multiple fertility regions/loci in idiopathic infertile men is also required through high throughput analyzers, i.e., microarray, etc.