Primed Avian Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Restore Granulosa Cell Homeostasis in a CTX-Induced POI-like Dysfunction Model Under Human Menopausal Gonadotropin Stimulation

Premature ovarian insufficiency (POI) is a heterogeneous disorder, and cyclophosphamide (CTX)-induced ovarian injury represents an acquired POI-like condition rather than the full clinical spectrum of POI. In this study, CTX was used to establish a granulosa cell dysfunction model to evaluate whether primed avian mesenchymal stem cell-derived small extracellular vesicles (primed AMSC-sEVs) could restore granulosa cell homeostasis under human menopausal gonadotropin (hMG) stimulation, a clinically relevant gonadotropin context used in ovarian stimulation. Human granulosa cells were exposed to CTX and subsequently treated with hMG, naïve AMSC-sEVs, or primed AMSC-sEVs. Cell viability, mitochondrial membrane potential, AMH and FSHR expression, and hormone secretion were examined. CTX reduced cell viability, mitochondrial membrane potential, AMH and FSHR expression, and AMH/estradiol secretion, confirming the establishment of a POI-like granulosa cell dysfunction state. hMG partially restored selected functional markers but showed limited effects on cell viability and mitochondrial recovery. In contrast, primed AMSC-sEVs markedly restored granulosa cell viability, mitochondrial membrane potential, AMH and FSHR expression, and endocrine output, with stronger effects than naïve AMSC-sEVs. Co-treatment with hMG did not consistently enhance the restorative effects of primed AMSC-sEVs beyond those achieved by primed AMSC-sEVs alone. These findings indicate that primed AMSC-sEVs primarily act by restoring granulosa cell and mitochondrial homeostasis, thereby supporting the cellular competence required for gonadotropin responsiveness in an acquired POI-like condition.