Fertility preservation in women: enhanced neovascularization, follicle viability and tissue integrity in cryopreserved human ovarian cortex by remaining medulla tissue.

backgroundOvarian tissue cryopreservation is a critical fertility preservation method for women undergoing gonadotoxic treatments. Current protocols typically discard medullary tissue, although preclinical evidence suggests the medulla may enhance neovascularization and follicle survival after transplantation. This study investigates the impact of medullary tissue retention on tissue integrity and viability in human ovarian cortex.

methodsOvarian tissue was obtained from five women with a mean age of 27 years (20–33 years), different tumor entities (breast carcinoma [n = 2], B-cell lymphoma, melanoma, medulloblastoma) and AMH levels above 2 ng/mL (4.28 ng/mL ± 1.89). Cryopreserved and thawed human ovarian tissue fragments, either cortex-only, medulla-containing cortex or medulla-only, were transplanted onto the chorioallantoic membrane (CAM) of fertilized chicken eggs. Neovascularization was assessed using the CAM model by counting blood vessels converging towards the grafts and expression of angiogenic related genes (VEGFA and HIF1alpha) by qPCR. Tissue quality was evaluated by histology and immunohistochemistry for Caspase-3 (apoptosis) and Ki-67 (proliferation). Follicle viability was determined after enzymatic digestion and Calcein-AM staining.

resultsMedulla-containing cortex grafts exhibited significantly higher neovascularization after grafting on the CAM compared to cortex-only grafts, with a greater number of blood vessels converging towards the graft, higher VEGFA expression and reduced HIF1A expression by qPCR measurements. The proliferation rate, measured by Ki-67 positivity, was also increased in medulla-containing cortex tissue. After CAM cultivation, the number of viable follicles was markedly greater in medulla-containing cortex than in cortex-only tissue. All tissue types demonstrated excellent post-thaw integrity and high tissue quality. Histological analysis revealed well-preserved tissue architecture and intact follicle morphology. Apoptosis was minimal, as evidenced by Caspase-3 staining with rates below 4% across all groups, indicating that the cryopreservation method for different types of tissues is unproblematic.

conclusionThis experimental study demonstrates that retaining medulla tissue in cryopreserved human ovarian cortex leads to improved neovascularization, higher cellular proliferation, reduced expression of hypoxic genes and increased follicle viability compared to cortex-only tissue after short-term CAM culture. These findings suggest that clinical protocols for ovarian tissue cryopreservation and retransplantation should consider preserving the medulla-cortex connection to optimize transplantation outcomes and functionality for fertility preservation.