Menopause timing, hormone therapy, epigenetic aging, and breast cancer outcomes: a secondary analysis of the PLCO Cancer Screening Trial

Background: Current recommendations limit the use of menopausal hormone therapy (MHT) principally to the management of menopausal symptoms, largely because of concerns regarding increased breast cancer (BC) risk. The impact of MHT use on systemic biological aging and blood methylome and its overlap with that of menopause timing remains incompletely understood. Methods: In females (N=78,209) of the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial, we used Cox models to assess the associations of menopause timing (ordinal age at menopause; binary early-M [<45 years] vs non-early-M [[&ge;]45 years]) and MHT use with BC incidence, mortality, and survival. In a nested case-control subset with blood DNA methylome (DNAm; 758 cases, 745 controls), we related both exposures to epigenetic clocks and DNAm-based telomere length (DNAmTL) and used polygenic instruments to test both causal directions. Results: Ever MHT use was associated with higher BC incidence (aHR 1.17, 95% CI 1.10-1.24), particularly for hormone-receptor positive (HR+) subtypes (1.19, 1.11-1.27), whereas early-M was associated with lower HR+ incidence (0.88, 0.81-0.96) but not with overall incidence after adjustment (0.94, 0.88-1.01). However, ev-er MHT use was associated with lower BC mortality (0.72, 0.62-0.83) and better overall survival (0.87, 0.81-0.95). In the DNAm subset, non-early-M was associated with slower epigenetic aging and longer DNAmTL par-ticularly in cases. Higher cumulative aging and pace-of-aging markers predicted worse survival and partially mediated the early-M associations. Genetic analyses supported shared polygenic architecture between meno-pause timing and DNAm aging biomarkers rather than simple causation. MHT showed no broad epigenetic age acceleration and its profound methylome and immune-marker signatures largely did not overlap with those of menopause timing. Conclusions: Ever MHT use was associated with lower BC mortality and better overall survival despite higher BC incidence. Systemic aging detected more than a decade after menopausal transition correlates with early menopause. MHT use shows molecular signatures distinct to those seen for menopause timing.