Immune cell-associated DNA methylation responses to exercise in women: A bioinformatics analysis comparing pre- and postmenopausal stages

INTRODUCTION: Menopause is associated with immunosenescence and altered immune profiles, potentially reducing immune competence. Physical exercise may counteract these changes by modulating DNA methylation in fitness-related genes. METHODS: This observational bioinformatics study analyzed genome-wide DNA methylation profiles derived from whole blood using two publicly available datasets from Brazilian cohorts (Illumina MethylationEPIC 850K). The analysis included pre- (PreM, n = 13; 34 ± 4.7 years) and postmenopausal (PostM, n = 49; 59.8 ± 4.7 years) women who completed supervised combined exercise training. Four linear models were applied to examine associations between DNA methylation, cardiorespiratory fitness (VO₂ peak), and exercise intervention (pre- vs. post-training), while adjusting for age, fat percentage, and estimated immune-cell proportions (EpiDISH). Interaction terms were tested to assess whether immune cell composition or menopausal status modulated the relationship between VO₂ peak, exercise response, and DNA methylation. RESULTS: After adjusting for baseline values using analysis of covariance (ANCOVA), no statistically significant between-group differences were observed in the estimated immune cell proportions (FDR > 0.05). In PostM, exercise-induced DNA methylation changes were significantly associated with baseline VO₂ peak and were modulated by B cells, CD4⁺ T cells, NK cells, and monocytes. Functional enrichment highlighted pathways involved in lipid kinase regulation, nucleobase metabolism, and membrane organization. CONCLUSION: Postmenopausal women showed distinct epigenetic patterns in response to exercise, although these differences must be interpreted cautiously given the small premenopausal sample size. These results suggest potential immune cell-associated DNA methylation markers to inform personalized exercise strategies supporting healthy aging in women.