Vitrification versus slow freezing for prepubertal ovarian tissue cryopreservation: insights from a porcine xenotransplantation model

Ovarian cryopreservation is crucial for preserving fertility in prepubertal patients requiring gonadotoxic therapies. However, the optimal cryopreservation strategy remains unresolved. This study was conducted to compare vitrification and slow freezing techniques for prepubertal ovarian tissue cryopreservation, focusing on post-thaw viability and post-transplant functional recovery using a porcine xenotransplantation model. Ovarian cortical tissues from prepubertal gilts were cryopreserved via vitrification (V group) or slow freezing (S group), with fresh tissue (F group) as control. Post-thaw apoptosis (TUNEL, caspase-3), ultrastructure (TEM), and follicular integrity were evaluated. Tissues were xenografted into ovariectomized nude mice and harvested at days 9 and 30 post-transplantation. Outcome measures included follicular morphology, apoptosis, neovascularization (CD31, VEGFA immunohistochemistry), and endocrine function (AMH, estradiol, progesterone levels). Upon thawing assessment, TUNEL fluorescence staining revealed comparable apoptotic rates between the two cryopreservation protocols. In contrast, ultrastructural analysis by transmission electron microscopy demonstrated inferior stromal cell integrity preservation in the vitrified group relative to the slow-freezing group. At 9 days post-transplantation, no significant differences were observed in apoptosis, angiogenic marker (CD31 and VEGFA), or hormone profiles among the groups. By 30 days post-transplantation, the S group exhibited a significantly lower proportion of morphologically intact follicles (p < 0.05), yet demonstrated enhanced angiogenic capacity, as evidenced by increased CD31 staining intensity and expanded VEGFA-positive area (both p < 0.05), compared to the V group. Hormonal profiles remained comparable between the two groups at this later time point. Both vitrification and slow freezing maintain short-term follicular viability post-transplantation. However, slow freezing appears to promote superior neovascularization and morphological preservation of stromal architecture, whereas vitrification better preserves follicular morphology in the longer term. These findings suggest a differential susceptibility of stromal versus follicular compartments to cryoinjury. Future optimization strategies—such as stromal preconditioning or VEGF supplementation—may improve graft outcomes. Translation to clinical protocols for pediatric fertility preservation warrants further investigation.