Abstract
Disclosure: M. Agarwal: None. K.M. Halloran: None. R.C. Cardoso: None. V. Padmanabhan: None. Polycystic ovary syndrome (PCOS), a common hyperandrogenic disorder in reproductive aged women, is linked to metabolic dysfunction including dyslipidemia, insulin resistance, oxidative stress, and adipocyte defects. Prenatal testosterone (T) excess from gestational days (GD) 30-90 (term 147 days) induces similar dysfunctions in sheep, a precocial model of translational relevance. Considering obese PCOS women exhibit a more severe phenotype and visceral adipose tissue (VAT) influences insulin sensitivity, we used GD 60-90 T treatment to replicate subclinical metabolic perturbations and assess impact of postnatal obesity. We hypothesized prenatal T excess from GD 60-90 induces defects in VAT and postnatal obesity amplifies these defects. Pregnant sheep received bi-weekly im injections of T propionate or vehicle from GD 60-90. Offspring were weaned at 5 months and fed a maintenance (M) diet or overfed (O) to induce obesity. At ∼3 years, ewes were weighed and VAT collected to assess oxidative stress and triglyceride content as an index of functional compromise and subjected to RNA sequencing to identify differentially expressed genes (DEGs) between control (C), T maintenance (TM), and T overfed (TO) ewes (n = 6/group). Commercial kits were used to measure triglyceride content and malondialdehyde (MDA) as a marker for oxidative stress in VAT. Data were analyzed with ANOVA with post-hoc Tukey’s tests and Cohen’s d effect size (d≥0.8 is large effect size). RNA sequencing was analyzed with DESeq2 R package (Adj P<0.1) to identify DEGs. Genes passing threshold of log2 fold change >1 and FDR <0.05 were analyzed with DAVID Gene Ontology (GO). Body weights were higher in TM vs C (d=1.4), TO vs C (d=2.3, P=0.002), and TO vs TM (d=1, P=0.07). In VAT, MDA was higher in TM vs C (d=0.9) and TO vs C (d=2.2, P=0.02). Total triglyceride content was higher in TO vs C (d=2.4, P=0.01) and TO vs TM (d=1.18). On the transcriptional level, there were only 2 DEGs (1 up, 1 down) in TM vs C, 421 DEGs (86 up, 335 down) in TO vs C, and 1929 DEGs (1110 up, 819 down) in TO vs TM. GO analysis suggested alterations in pathways involving lipid metabolism, carboxylic acid metabolism, fatty acid biosynthesis, and metabolic processes in VAT of TO vs both TM and C ewes. TO vs TM ewes had enrichment of immune response in signaling pathways, inflammatory response and cellular signaling pathways. Collectively, a trend for increased oxidative stress and triglyceride content in TM ewes, both of which are significantly increased in TO ewes, coupled with transcriptional changes in lipid metabolic pathways in TO vs C and TM, and activation of immune pathways in addition in TO vs TM, suggests adipose defects induced by prenatal T exposure that may remain masked is unmasked or worsened by obesity. These results provide support for the role played by obesity in amplifying the severity of metabolic disease in a translationally relevant PCOS model. Funding NIH R01HD099096 Presentation: Monday, July 14, 2025