Moderate NEFA Reprogram Early Follicular Development and Oocyte Competence: Evidence for a Targetable Redox Mechanism

Elevated circulating non-esterified fatty acids (NEFA), a hallmark of metabolic stress and negative energy balance, are increasingly associated with reduced female fertility. While most studies focus on late oocyte maturation, metabolic disturbances during follicular growth—particularly during the preantral–early antral transition, a critical window for establishing oocyte developmental competence—may already impair oocyte quality. Here, we investigated whether sustained, physiologically buffered NEFA elevation during this stage affects oocyte developmental competence. Preantral follicles were cultured for 18 days in a three-dimensional ovine in vitro folliculogenesis system under physiological (70 µM) or moderately elevated (140 µM) NEFA conditions, with defined fatty acid composition and albumin buffering. The cytoprotective effects of the antioxidants Trolox and resveratrol were also evaluated. Chronic exposure to moderately elevated NEFA induced a pro-oxidant follicular microenvironment, characterized by reactive oxygen species (ROS) accumulation and oxidative DNA damage, including increased 8-OHdG and mtDNA D-loop oxidation. This stress impaired cumulus cell function and somatic–oocyte communication, reduced oocyte mtDNA copy number and mitochondrial activity, and compromised meiotic and developmental competence despite preserved follicular growth. Antioxidant treatment with Trolox restored mitochondrial function, normalized cumulus activity, and rescued blastocyst development, partially reversing the NEFA-induced phenotype. Prolonged moderate NEFA elevation during early folliculogenesis impairs oocyte competence despite preserved follicular morphology, identifying chronic lipotoxicity and redox imbalance as early and clinically relevant determinants of impaired fertility.