Improving Follicle Development After Cryopreservation of Prepubertal Ovarian Tissue Through In Vitro Activation

Background: Fertility preservation in pediatric patients requires effective cryopreservation of ovarian tissue, yet the relative performance of slow freezing and vitrification in this age group remains unclear. This study compared the two cryopreservation methods in prepubertal ovarian tissue and examined their functional consequences through in vitro activation and short-term culture. Methods: Ovarian tissues from forty-six girls aged one to fourteen years were processed fresh, slow-frozen, or vitrified. Follicular morphology and developmental stage assessed by hematoxylin and eosin staining; apoptosis assessed by TUNEL assay; follicular activation assessed by Western blotting and immunohistochemistry; follicular growth after IVA culture. Appropriate statistical analyses were applied based on data distribution, including t-tests, non-parametric tests, and contingency table analyses. Results: Slow freezing preserved follicular morphology better than vitrification, with significantly less oocyte damage and stromal cell apoptosis. IVA significantly increased pFOXO3a/FOXO3a ratios at day 2 in both groups and promoted nuclear translocation of pFOXO3a. By day 6, IVA-treated tissues showed a higher increase in primary follicle numbers compared to untreated controls, particularly in the slow freezing group. Conclusions: Slow freezing is more effective than vitrification for pediatric ovarian tissue cryopreservation. IVA further enhances follicular activation after thawing and may provide a promising strategy for optimizing pediatric fertility preservation.