Sex-specific differences in liver DNA methylation patterns and epigenetic aging in mice

Biological sex has been shown to influence aging outcomes, contributing to distinct trajectories in disease susceptibility and lifespan. DNA methylation patterns provide a quantitative measure of biological aging. This study investigated whether aged male and female mice display distinct liver DNA methylation patterns and differences in epigenetic aging. Liver samples were collected from 17 aged c57BL/6 mice (6 males, 11 females). Genomic DNA was extracted and bisulfite-converted before targeted enrichment of 2,045 murine age-associated CpG loci. Biological age (DNAge) was estimated using a previously developed DNA methylation-based predictor generated through elastic net regression. The difference (ΔDNAge) between DNAge and chronological age was computed. Sex-specific differences were assessed by comparing site-specific methylation ratios, ΔDNAge values, and through principal component analysis (PCA) and multiple linear regression. Twelve CpG sites across six genes (<i>Fam84b</i>, <i>Zswim6</i>, <i>Hsf4</i>, <i>Mn1</i>, <i>Qprt</i>, and <i>Rapgefl1</i>) showed significant sex-associated differences in methylation. <i>Fam84b</i> demonstrated the largest and most consistent sex-associated effect, with all three associated CpG sites showing higher methylation in males (regression coefficients: -0.204, -0.281, and -0.294). <i>Zswim6</i> exhibited consistent lower methylation ratios in females, whereas the other genes showed higher methylation in females. There were no sex differences in biological age or ΔDNAge (<i>P</i> = 0.596). Although the epigenetic clock did not reveal differences between sexes in aging, aged mice did exhibit sex-specific liver methylation patterns different from those reported in younger mice, suggesting that sex-dependent epigenetic changes may emerge later in life and may reflect sexual dimorphism in liver function with age.<b>NEW & NOTEWORTHY</b> Males and females are known to age differently and develop certain diseases at different rates. Here, we examined the livers of aged male and female mice to see if they show different DNA methylation patterns. We found that aged male and female mice had distinct DNA methylation patterns at specific genes. Interestingly, most of these methylation differences were not present in younger mice, suggesting that sex differences in the genome may change with age.