Early to Mid-Gestational Testosterone Excess Leads to Epigenetic Modulation of Fetal Sheep Heart

Excess testosterone (T) exposure from early to mid-gestation days (GD) 30-90 leads to intrauterine growth retardation and sexually dimorphic adverse cardiac programming in mid-gestation persisting into late gestational fetuses, culminating in cardiometabolic dysfunction in adult female offspring. We hypothesized that sex-specific disruption in the cardiac epigenome induced by early to midgestational T treatment persisting into late gestation underlies the adverse cardiac programming observed in offspring. Pregnant ewes received 100 mg T propionate intramuscularly 2x weekly from GD 30 to 90 (term GD 147). At GD90±5 (end of T treatment) and GD120±5 ( 30 days post cessation of T treatment) fetal hearts were harvested, and cardiac left ventricle (LV) from control (C) female (CF, n=5-6), T female (TF, n=6-7); C male (CM, n=5-6), T male (TM, n=6-7) were processed for global methylation assay (% 5-methylcytosine, %5-mC) and real-time qPCR gene expression analysis. Data were analyzed using a two-way ANOVA, and p< 0.05 was considered significant. Cohen’s effect size (d) > 0.8 represented a large effect size. At GD90, T excess induced cardiac epigenetic modification only in TF, not TM, manifested as 1) a non-significant, large effect size increase in %5-mC 2) significant decrease in expression of various epigenetic modulators, including DNA methyltransferase (DNMT 1 and 3B), Histone deacetylase (HDAC) 1, Sirtuin 1 (SIRT1), Lysine (K)-specific demethylase 1A (KDM1A), Enhancer of zeste homolog 2 (EZH2) and Histone-lysine N-methyltransferase (SUV39H1). Interestingly, disruption in gene expression of certain epigenetic modulators seen at GD90 in TF were also noted at GD120 including 1) non-significant large magnitude decreases in DNMT3B, significant decrease in HDAC1 expression and non-significant, large magnitude decrease in EZH2. In contrast, at GD120, cardiac epigenetic modifications were more pronounced in TM including 1) non-significant, large magnitude decrease in %5-mC 2) non-significant large magnitude decrease in DNMT 3B, non-significant, large magnitude increase in SIRT1 and significant decrease in SUV39H1. Overall, these findings are indicative of differential timing in the disruption of the cardiac epigenome in males vs females by prenatal T excess, and such disruption likely underlies the sex specific adverse cardiac programming persisting into adulthood. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.