Impact of post-thaw incubation time on viability and neovascularization of cryopreserved human ovarian tissue

Background This study aimed to investigate the effects of delayed transplantation after thawing on the quality of cryopreserved human ovarian cortex tissue. Methods Cryopreserved human ovarian cortex tissue samples from five oncological patients (mean age 26 ± 3.6 years; AMH > 3 µg/L; mean: 7.64 ± 2.24 µg/L; oncological entities: brain tumor, breast cancer ( n = 2), Hodgkin lymphoma, non-Hodgkin lymphoma) undergoing ovarian tissue cryopreservation were analyzed immediately after thawing, following different culture periods (1 h, 2 h, 4 h, 24 h) at room temperature, and after grafting onto the chorioallantoic membrane (CAM) of fertilized chicken eggs for short-term culture. Follicle viability and relative differences in viable follicle counts were assessed by Neutral Red staining. Tissue morphology, proliferation, apoptosis, angiogenesis and molecular expression of selected ovarian tissue markers were evaluated by histology (hematoxylin and eosin), immunohistochemistry (proliferation: Ki-67, apoptosis: cleaved Caspase-3), blood vessel quantification on CAM, and quantitative polymerase chain reactions (RT-qPCRs; apoptosis: BCL-2, BAX, hypoxia: HIF1A, angiogenesis: VEGFA, proliferation: mTOR, YAP, and follicle activation: GDF9, BMP15). Culture medium quality was examined by pH and microbiological analyses. Results Ovarian cortex samples incubated for less than 4 h after thawing displayed preserved tissue integrity and viability, with minimal follicle activation and stable medium quality. Samples subjected to delays of more than 4 h showed structural damage and reduced tissue viability as well as relative difference in viable follicle counts of up to 67% after 24 h and markedly impaired neovascularization with a mean blood vessel count difference of 52.35%. Conclusions Human ovarian cortex tissue maintained viability and structural integrity when transplantation was performed within 4 h after thawing. Delays exceeding 4 h were associated with reduced neovascularization, impaired tissue integrity, and increased relative difference in viable follicle counts. The data support minimizing avoidable delay and using four hours only as a provisional handling target requiring confirmation. Trial registration Not applicable. Clinical trial number Not applicable.