Sex-Specific Differences in Liver DNA Methylation Patterns and Epigenetic Aging in Mice

Background: 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. Methods: Liver samples were collected from 17 aged c57BL/6 mice (6 males, 11 females). Genomic DNA was extracted and bisulfite-converted prior to 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. Results: Twelve CpG sites across six genes ( Fam84b, Zswim6, Hsf4, Mn1, Qprt, and Rapgefl1) showed significant sex-associated differences in methylation. Fam84b 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, -0.294). Zswim6 exhibited consistent lower methylation ratios in females, while the other genes showed higher methylation in females. There were no sex differences in biological age or ΔDNAge® (p = 0.596). Conclusions: 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.