Sperm AluY Epimutations in Male Partners of Couples with Unexplained Recurrent Pregnancy Loss

Recurrent pregnancy loss (RPL) affects 5% of couples worldwide. Over half of cases remain unexplained (uRPL), in part because paternal factors are rarely evaluated beyond paternal karyotype. Sperm DNA methylation is a putative paternal contributor to RPL. Using ONT direct whole methylome sequencing, we generated the first and largest cohort of locus-resolved, single-molecule sperm methylome maps from men with uRPL to date (n=39). We identified 294 differentially methylated regions (DMRs), of which 158 (53.7%) overlapped AluY repetitive elements, representing significant enrichment (z=40.9); most were hypomethylated in uRPL sperm and spanned the full length of individual AluY insertions. Because of single-molecule resolution, we show that the methylation differences found in uRPL sperm occur in a subpopulation of sperm rather than a uniform DNA methylation change across all sperm. Affected loci were underrepresented for imprinting-associated chromatin marks and instead overrepresented for H2A.Z. Although DMR-associated genes were enriched for neuronal GO terms, many converge on reported roles in implantation, placentation, and early embryonic development. AluY epimutation represents a previously unrecognized, predominant signature of paternal epigenetic perturbation in uRPL, establishing repetitive elements as a new axis of sperm epigenetic risk.