Sperm chromatin immaturity is associated with DNA methylation profiles related to male reproduction and development in a general population cross-sectional study

Successful spermatogenesis requires marked DNA condensation, chromatin remodeling via the histone-to-protamine transition, and maintenance of specific DNA methylation (DNAm) patterns. DNA fragmentation index (DFI), a marker of sperm DNA damage, and high DNA stainability (HDS), a marker of incomplete protamination, have been linked to poor reproductive outcomes. However, their relationship with sperm DNAm patterns remains largely unknown. The objective of this study was to investigate sperm DNAm profiles that are associated with sperm chromatin structure, assessed by DFI and HDS. This nested cross-sectional study assessed sperm DNAm using the EPIC V1 Infinium Methylation Beadchip from 376 participants from the general population enrolled in the prospective preconception pregnancy cohort: Longitudinal Investigation of Fertility and the Environment (LIFE) Study. DFI and HDS were measured with the sperm chromatin structure assay (SCSA). Linear regression ( CpGassoc ) and generalized estimating equations ( Aclust2.0 ) algorithms, adjusted for male age and methylation at DLK1 and corrected for genomic inflation, were used to identify differentially methylated CpGs (DMCs) and regions (DMRs), respectively. Enrichment analysis of DMCs and DMRs across genic features, transcription factor (TF) binding motifs, and regions of histone retention was performed, as well as functional enrichment to elucidate biological relevance. DFI was not associated with sperm DNAm. HDS was associated with 18,187 DMCs and 1,241 DMRs (q<0.05), which were predominantly hypermethylated. Functional enrichment analysis revealed HDS-associated DNAm at genes involved in male reproduction and development-related pathways, such as male gamete generation, DNAm in gamete generation, male meiotic nuclear division, and epithelial tube morphogenesis. DMCs and DMRs were enriched in 39 TF binding sites (q<0.05), including those for master regulators of the cell cycle and pluripotency. HDS-associated DMCs and DMRs were also enriched in regions with retained testis-specific histone-variant-TH2B (absolute fold change >2; p <0.05). This is the first study, to our knowledge, to report associations between HDS and sperm DNAm profiles enriched in genomic regions important for spermatogenesis and male reproduction, as well as regions with retained TH2B. Despite the cross-sectional nature of the study, these findings suggest potential epigenetic communication between DNAm and chromatin remodeling during spermatogenesis, which may also involve intermediate histone proteins such as TH2B.