Higher sperm H3K4me3 levels are associated with idiopathic recurrent pregnancy loss

Background: During fertilization, spermatozoa contribute genetic and epigenetic factors such as chromatin packaged with protamines and histones; DNA methylome, micro RNAs etc. Human sperm chromatin retains 5–15% nucleosomes which can play a key role in embryonic development. Recurrent Pregnancy loss (RPL) is a condition mainly attributed to defects in embryo and placenta development. Majority of the known RPL factors are of maternal contribution while ~ 50% RPL cases are termed idiopathic (iRPL). Besides paternal genetic factors, epigenetic factors via sperm could also be responsible for iRPL. Hence, we investigated alterations in retained nucleosome content of iRPL sperm (n = 46) as compared to fertile male population (n = 40). Results: We measured the relative abundance of core histone H4 and Protamine-2 content along with the modified histones H4Ac, H3K4me3, H3K27me3 and H3K9me3 by flow cytometry. Enrichment of these modified histones at regulatory loci have either transcription activating or repressing roles and Protamine-2 contributes to sperm chromatin compaction. H4 and Protamine-2 levels were comparable in both groups and showed significant negative correlation. The iRPL group was found to have significantly higher levels of sperm H3K4me3 as compared to the fertile control group. The other modified histones and protamine levels showed no alterations among the two groups. Furthermore, we analyzed possible correlation of sperm DNA fragmentation index (DFI) and 5-mC content with the relative sperm core histone H4 levels. Sperm DFI was found to be significantly positively correlated with H4 MFI in both control and iRPL sperm. Conclusion: A fraction of the H3K4me3 enrichment is now known to resist embryonic epigenetic reprogramming; and hence, such elevated levels in the sperm would question its developmental competence requiring its implications to be explored further in RPL pathology. Also, incidence of sperm DNA fragmentation is associated with increased histone retention in both fertile and iRPL cases.