Antioxidant intervention restores oocyte quality and somatic-germline communication compromised by lipotoxicity during early folliculogenesis

Elevated circulating non-esterified fatty acids (NEFA), hallmarks of metatabolic stress and negative energy balance, are increasingly associated with reduced female fertility (1,2). While lipotoxicity is well-characterized during late oocyte maturation (2,3), its impact during early folliculogenesis, a critical window for establishing oocyte developmental competence, remains unclear. This study investigated whether sustained, physiologically buffered NEFA elevation during this stage compromises the somatic-germline axis and oocyte quality. Prepubertal ovine preantral follicles were cultured for 18 days in a 3D in-vitro folliculogenesis system under physiological (70 µM) or moderately elevated (140 µM) NEFA. Follicular integrity and development, developmental competence, redox-related molecular mechanisms, and transcriptomic landscapes were evaluated to assess lipotoxic damage and the protective effects of Trolox. To determine how extensively lipotoxicity affected the follicular unit, signals from exhausted culture media, somatic compartments (follicular walls and cumulus cells; FW and CC), and oocytes were characterized. Despite preserved follicular growth and morphology, elevated NEFA induced a pro-oxidant microenvironment characterized by ROS and DAMP accumulation, including increased 8-OHdG and mtDNA D-loop oxidation in culture medium. Within the somatic compartment, these conditions progressively impaired follicular wall integrity and disrupted somatic-oocyte communication, evidenced by dysregulated HAS2 expression in cumulus cells. RNA sequencing revealed compartment-specific transcriptomic reprogramming: while the follicular wall showed few differentially expressed genes (DEGs), cumulus cells displayed marked dysregulation under lipotoxic and antioxidant rescue conditions. Altered pathways involved epigenetic regulation, extracellular matrix remodeling, and cytoskeletal organization. At the oocyte level, lipotoxicity reduced mtDNA copy number and mitochondrial membrane potential. Trolox restored mitochondrial activity, normalized cumulus cell function, and rescued redox imbalance across compartments. Overall, moderate NEFA elevation during early folliculogenesis induces lipotoxicity impairing oocyte quality despite apparently normal follicular development. Our findings identify redox imbalance, mitochondrial depletion, and disruption of the somatic-germline transcriptomic landscape as early determinants of metabolic infertility, highlighting antioxidant intervention as a promising strategy to preserve reproductive competence.