Abstract: Objective  To investigate the mechanisms underlying follicle loss after ovarian tissue transplantation and evaluate the protective effects of N-acetylcysteine (NAC) on primordial follicles.Methods  Frozen-thawed human ovarian tissue was transplanted onto the chorioallantoic membrane (CAM) of chick embryos. Four groups were established: fresh ovarian tissue (fresh group), standardized cryopreservation and transplantation (control group), PI3K inhibitor treatment (PI3Ki group), and NAC treatment (NAC group). Follicle counts were assessed by hematoxylin-eosin staining (HE) staining, mitochondrial membrane potential (ΔΨm) was measured by JC-1 staining using flow cytometry, and the expression of phosphorylated protein kinase B (p-Akt) and phospholated forhead box o3a (p-Foxo3a) in the PI3K-Akt-Foxo3a pathway was detected by Western blotting.Results  Compared with the fresh group, all transplantation groups showed significantly reduced follicle counts (P<0.008 3) and increased mitochondrial depolarization (P<0.001). Compared with PI3Ki group, the control group exhibited activation of the PI3K-Akt-Foxo3a pathway, as indicated by elevated p-Akt and p-Foxo3a levels. This activation was suppressed in the PI3Ki group. In the NAC group, the expression of p-Akt and p-Foxo3a was significantly lower than in the control group (P<0.008 3), and the mitochondrial depolarization was the lowest among all transplanted groups(P<0.008 3).Conclusion  NAC protects primordial follicles during ovarian tissue transplantation by downregulating the PI3K-Akt-Foxo3a signaling pathway, thereby inhibiting excessive follicle activation and reducing mitochondrial damage.

Key words: ovarian tissue cryopreservation and transplantation, follicle loss, N-acetylcysteine, PI3K-Akt-Foxo3a signaling pathway, chick embryo chorioallantoic membrane model, follicle activation