Protective effect of a novel hydrogel loaded with CM-UCMSCs on vitrified-thawed ovaries during in vitro culture

Ovarian tissue cryopreservation and transplantation (OTCT) is an important fertility preservation method for female cancer patients; however, its efficacy is limited by post-transplantation ischemia-reperfusion injury, leading to oxidative stress, apoptosis, and fibrosis that impair ovarian reserve and graft function. Mesenchymal stem cell-conditioned medium shows therapeutic potential through paracrine actions, but clinical use is restricted by relatively limited antioxidant capacity and delivery challenges. To address this, an antioxidant-enriched hydrogel (PG-gel) was developed from N-acetylcysteine-modified gelatin and poly (ethylene glycol) succinimidyl succinate, loaded with conditioned medium from umbilical cord mesenchymal stem cells (CM-UCMSCs). This study evaluated the efficacy of PG-gel in protecting vitrified-thawed ovarian tissue during in vitro culture. The CM-UCMSCs-loaded PG-gel significantly suppressed intracellular reactive oxygen species generation. The PG + CM-UCMSCs group showed markedly reduced follicle loss, improved follicle morphology, decreased collagen deposition, lower apoptosis (fewer TUNEL-positive cells and reduced caspase-3 expression), diminished oxidative damage (lower 8-OHdG), and enhanced glucose consumption compared with the other culture groups. Transcriptomic analysis revealed downregulation of apoptosis-related genes (e.g., Ddit3, Trib3 and Hmox1) and upregulation of mitochondrial metabolism genes (e.g., Mt-atp8, Mt-nd1 and Mt-cyb). In conclusion, the PG + CM-UCMSCs system provided comprehensive protection to cryopreserved ovarian tissue by mitigating oxidative stress, fibrosis, and apoptosis, likely through regulation of apoptotic signaling and enhancement of mitochondrial energy metabolism, thereby offering a promising strategy to improve OTCT outcomes.