Among non-users of vitamins, the infertile men in our study had significantly lower serum folate concentrations than fertile controls. RCF concentrations were not significantly lower in cases after adjusting for education, suggesting that lower socioeconomic status-related dietary factors could explain the difference. Cases and controls did not differ in B12 and tHcy concentrations. No metabolite concentrations were correlated significantly with any semen parameters. Of the folate/B12/homocysteine-related genes that we studied, variants for two: the transcobalamin receptor gene, TCblR, and the choline pathway gene, PEMT, were related to infertility before correction for multiple comparisons.
Only a few folate-, B12- or homocysteine-related polymorphisms have been investigated as risk factors for idiopathic male infertility. We attempted to confirm many of them. A recent meta-analysis of MTHFR
677C>T reported a significant association.12
We found no association even when we limited our analysis to subjects with folate concentrations below the median; however, the upper bound of our 95% CI is within the range of the reported effect. A few studies have looked for an association between male infertility and variants in two key enzyme genes involved in methylation and homocysteine metabolism, MTR
, with mixed results.8, 11
We did not find SNPs in either MTR
to be associated with infertility; we also confirmed that RFC1
(also known as SLC19A1
) R27H was not associated with infertility.18
The other eight genes we investigated in the areas of folate, B12 and homocysteine metabolism have not, to our knowledge, been investigated previously. Variants in these enzyme genes are potentially important because they affect homocysteine and choline metabolism (BHMT, PEMT), folate transport and action (FOLH1, MTHFD1, SHMT1, PCFT) and B12 uptake (TCblR) and transport (TCN2).
Of the 20 SNPs that we studied, PEMT
M175V and TCblR
rs173665 showed associations with infertility. The PEMT
variant that was significantly more common in cases is noteworthy because it is a loss of function mutation that has been shown to cause increased tHcy when folate status is marginal.19
This result suggests that choline metabolism may play a role in idiopathic male infertility. The TCblR
variant is also of interest because the minor A allele was also found to be protective against neural tube defects in a recent population-based study.20
None of the variants that we found to be associated with infertility was associated with significantly poorer semen quality suggesting that these findings require additional investigation.
Previous studies have produced mixed results regarding the role of folate in male infertility. A randomized controlled trial performed by Landau et al.21
reported that folic acid supplementation does not improve overall semen quality. Another randomized controlled trial of folic acid and zinc found a 74% increase in the sperm count in the men who took folic acid supplements.22
An uncontrolled study of folinic acid, 15 mg daily for 3 months, showed a significant improvement in spermatozoa number and motility.23
Somewhat surprisingly, blood folate, B12 and tHcy concentrations in infertile men have not been studied often and the results have been conflicting. One study reported that serum folate and B12 were lower and tHcy was higher in infertile subjects, but a P
value was reported only for tHcy.10
In contrast, a trial to determine whether folic acid could improve sperm parameters reported that fertile and subfertile men had almost identical folate concentrations (18.0 nmol l−1
) prior to treatment.24
A third study reported no difference in blood folate, B12 and tHcy values between their fertile and infertile groups.25
Our data do not show an association between B12 or tHcy and semen parameters. After adjusting for education, only serum folate, not RCF, was significantly lower in infertile men not taking folic acid supplements. The inclusion of RCF is a major advantage over most previous studies because it provides a long-term (90–120 days) measure of folate status.
Several limitations of our study should be noted. The infertile men were undergoing fertility evaluations and provided semen samples on a different day than the blood samples. Fertile men provided semen and blood samples on the same day. The lifetime of an erythrocyte is 90–120 days and spermatogenesis takes 72 days, so our RCF measure would reflect the period of spermatogenesis. Abstinence time was not available for 45% of controls. Strengths of our study include the extensive genotyping and biochemical data on a relatively large sample of men carefully screened for known causes of infertility.
In conclusion, this study provides little support for the importance of low folate or B12 in the pathogenesis of idiopathic male infertility. The role of genetic variants (PEMT and TCblR) in choline and B12 metabolism merits further investigation.