High-dose folic acid supplementation alters the human sperm methylome and is influenced by the MTHFR C677T polymorphism

Hum Mol Genet. 2015 Nov 15;24(22):6301-13. doi: 10.1093/hmg/ddv338. Epub 2015 Aug 24.


Dietary folate is a major source of methyl groups required for DNA methylation, an epigenetic modification that is actively maintained and remodeled during spermatogenesis. While high-dose folic acid supplementation (up to 10 times the daily recommended dose) has been shown to improve sperm parameters in infertile men, the effects of supplementation on the sperm epigenome are unknown. To assess the impact of 6 months of high-dose folic acid supplementation on the sperm epigenome, we studied 30 men with idiopathic infertility. Blood folate concentrations increased significantly after supplementation with no significant improvements in sperm parameters. Methylation levels of the differentially methylated regions of several imprinted loci (H19, DLK1/GTL2, MEST, SNRPN, PLAGL1, KCNQ1OT1) were normal both before and after supplementation. Reduced representation bisulfite sequencing (RRBS) revealed a significant global loss of methylation across different regions of the sperm genome. The most marked loss of DNA methylation was found in sperm from patients homozygous for the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, a common polymorphism in a key enzyme required for folate metabolism. RRBS analysis also showed that most of the differentially methylated tiles were located in DNA repeats, low CpG-density and intergenic regions. Ingenuity Pathway Analysis revealed that methylation of promoter regions was altered in several genes involved in cancer and neurobehavioral disorders including CBFA2T3, PTPN6, COL18A1, ALDH2, UBE4B, ERBB2, GABRB3, CNTNAP4 and NIPA1. Our data reveal alterations of the human sperm epigenome associated with high-dose folic acid supplementation, effects that were exacerbated by a common polymorphism in MTHFR.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • DNA / genetics
  • DNA / metabolism
  • DNA Methylation
  • Dietary Supplements*
  • Epigenesis, Genetic / drug effects
  • Folic Acid / administration & dosage*
  • Folic Acid / adverse effects
  • Folic Acid / blood
  • Genes, Regulator
  • Genotype
  • Humans
  • Male
  • Methylenetetrahydrofolate Reductase (NADPH2) / genetics*
  • Polymorphism, Genetic
  • Spermatozoa / drug effects*
  • Spermatozoa / enzymology
  • Spermatozoa / physiology*
  • snRNP Core Proteins / genetics


  • snRNP Core Proteins
  • DNA
  • Folic Acid
  • MTHFR protein, human
  • Methylenetetrahydrofolate Reductase (NADPH2)