Cyclophosphamide-induced iron homeostasis imbalance triggers ovarian toxicity through ferroptosis of ovarian granulosa cells in a mitophagy crosstalk manner.

Chemotherapy, while effective in targeting cancerous cells, often results in collateral damage to normal cells with high proliferative capacity, leading to iatrogenic ovarian injury. Ferroptosis has been implicated as a key driver of ovarian injury caused by chemotherapy drugs. However, the underlying molecular mechanisms remain incompletely understood. Therefore, this study was designed to elucidate the mechanism by which chemotherapy drugs induce ovarian injury through ferroptosis. Our findings demonstrate that the chemotherapeutic drug cyclophosphamide (CTX) impairs ovarian function and disrupts follicular development. Further analysis revealed characteristic features of ferroptosis within CTX-induced atretic follicular granulosa cells (GCs), including elevated Fe2+ accumulation, increased lipid peroxidation, and dysregulation of key ferroptosis-related molecules. Consistently, in vitro experiments demonstrated that treatment with the ferroptosis inhibitor Ferrostatin-1 (Fer-1) significantly alleviated CTX-induced cytotoxicity in KGN cells. Moreover, using the iron chelator deferoxamine (DFO) and the iron supplement ferric ammonium citrate (FAC), we demonstrated that modulating iron homeostasis could attenuate or aggravate CTX-induced ferroptosis in GCs, thereby confirming that disrupted iron homeostasis is a primary trigger for CTX-induced ferroptosis in GCs. Notably, we observed that Fe2+ accumulation occurred within mitochondria and subsequently triggered excessive mitophagy. This heightened mitophagy response exacerbated mitochondrial damage, thereby establishing a vicious cycle of persistent mitochondrial impairment that ultimately sensitized GCs to ferroptosis. Conversely, treatment with the mitophagy inhibitor cyclosporin A (CSA) alleviated CTX-induced ferroptosis in GCs. In conclusion, our study reveals that mitophagy-mediated ferroptosis is a critical mechanism underlying CTX-induced ovarian injury. These findings suggest that therapeutic strategies targeting mitophagy or ferroptosis hold promise for the treatment and prevention of chemotherapy-associated ovarian toxicity.