Lipotoxicity Impairs Early Follicle Development: Mechanistic Insights and Trolox-Mediated Protection

Purpose Female fertility is strongly affected by metabolic imbalance, particularly by elevated non-esterified fatty acids (NEFAs) associated with obesity and high-fat diets. Excess NEFAs induce oxidative stress and distrupt key ovarian physiological processes. However, their specific impact on early follicular growth and oocyte competence remains insufficiently understood. This study aims to clarify how high NEFA concentrations affect early folliculogenesis reproduced in vitro using the ovine model and to evaluate whether antioxidant supplementation can counteract NEFA- induced damage. Method: Lamb preantral follicles were mechanically isolated and cultured on 3D PCL- based “artificial ovary” platform. Follicles were cultured for 18 days under four conditions: NFAT (70 µM NEFAs), HFAT (140 µM NEFAs), NFAT + antioxidant (70 µM NEFAs + 150 µM Trolox), and HFAT + antioxidant (140 µM NEFAs + 150 µM Trolox). Follicular survival, growth dynamics, and antrum formation were monitored throughout culture. At the end of culture, meiotic and developmental competence were assessed. NEFA-induced molecular mechanisms were investigated by quantifying reactive oxygen species (ROS), evaluating mitochondrial integrity—including mtDNA D-loop damage and 8-OH-dG levels—measuring lipid accumulation, and analyzing expression of key antioxidant (SOD2, SOD3) and detoxification genes (EPHX1, GSTA4). Results HFAT exposure accelerated follicle growth but compromised developmental quality by increasing degeneration (23.3% vs 9.4%, p<0.05) and profoundly impairing meiotic competence (11.3% vs 66.5%, p<0.0001) and developmental competence (0% cleavage after parthenogenesis, p<0.0001). ROS levels were greatly elevated by HFAT (p<0.05–0.0001) by mediating the increase of mitochondrial stress markers (8-OH-dG), and mtDNA D-loop damage. At the same time HFAT altered antioxidant gene expression (downregulation of SOD2/SOD3 and upregulation of GSTA4/EPHX1) in somatic follicular compartment. Trolox co-treatment mitigated degeneration, normalized antioxidant gene profiles, reduced ROS and mtDNA damage (p<0.001), and fully rescued meiotic and developmental competence (p>0.05). Discussion These findings demonstrate that high NEFA concentrations compromise early follicular health by disrupting redox homeostasis and triggering mitochondrial dysfunction. The strong protective effect of Trolox integrates molecular, cellular, and developmental readouts, indicating that oxidative stress is the primary driver of NEFA- induced lipotoxicity. This underscores the relevance of antioxidant-based diet approaches to safeguard female fertility under metabolically challenging conditions. Conclusion High NEFA levels severely impair early follicular development and oocyte competence by inducing oxidative and mitochondrial stress. Trolox supplementation effectively protects follicles and oocytes from NEFA-mediated lipotoxicity, positioning it as a promising antioxidant strategy to mitigate the metabolic impact