Abstract
Unexplained male factor (MF) infertility accounts for a significant proportion of in vitro fertilization (IVF) failures, with increasing evidence supporting an association between the quality of the male gamete and live birth. Recent studies have linked MF infertility and poor embryo development with altered DNA methylation profiles. In this study we hypothesized that changes to the methylome in normozoospermic samples are associated with poor developmental outcomes, reflecting a subset of unexplained MF infertility. Research study. Normozoospermia sperm from infertile couples undergoing IVF using fertile donor oocytes (eliminating all female factor infertility) was donated with patient consent. Sperm samples (n=12) underwent methylome analyses using the Human Methylation450 BeadChip (Illumina). Beta value and statistical calculations were determined using Illumina GenomeStudio software and subject to pathway analyses (DAVID 6.7) where p<0.05 was considered significant. Sperm samples were segregated into 2 groups: good quality embryogenesis (D5>20% “AA”; >60% total blastocysts) and poor quality embryogenesis (D5=0% “AA”; <13% total blastocysts). Validation on additional normozoospermic samples (n=6) was performed by targeted bisulfite sequencing (Bio Basic Inc). Sperm methylation array data showed 17,987 CpG sites that were statistically significant between the two embryo developmental groups (p<0.05), 8,349 sites were enriched at key developmental loci that may significantly impact embryogenesis. Three distinct classes were identified: A) hypermethylated loci, B) hypomethylated loci, and C) variably methylated loci. Validation confirmed methylation array differences, including hypermethylated genes ARID3A (DNA binding protein) and KLF6 (transcriptional activator), hypomethylated genes HOXA11 (DNA-binding transcription factor) and SMARCD3 (transcription regulator by chromatin remodeling) and variably methylated genes GATA3 (negative regulator of transcription) and RASA3 (cellular proliferation and differentiation). The results of this study confirm the presence of tightly regulated genes, both hyper- and hypomethylated, as well as loosely regulated genes with variable methylation in normozoospermic samples. Having eliminated female factor infertility, significantly altered methylation profiles enriched at developmental loci were observed in normozoospermic samples resulting in poor embryogenesis, reflecting a subset of unexplained MF infertility. Our data suggests that moderate epigenetic changes throughout the genome may have a cumulative effect on fertility and embryonic developmental capacity.
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