Assessment of viability of cryopreserved ovarian tissue

Objective. To evaluate the effectiveness of culturing intact pieces of ovarian tissue for follicular maturation over a three week culture period. The effect of cryopreservation on ovarian tissue viability was evaluated by histology, by live/dead cell viability assays, and by immunohistochemical staining for PCNA, Ki-67, and CPP32.; Design. Previously cryopreserved ovarian tissue fragments from seven patients were cultured in vitro over three weeks. Tissue was retrieved weekly for histological evaluation and immunohistochemical staining with PCNA, Ki-67, and CPP32.; Results. No histological differences were identified microscopically in either follicular or stromal cells in ovarian tissue samples before and after cryopreservation. The percentage of atretic follicles increased greatly after the first week in culture. Immunohistochemical staining for Ki-67 demonstrated minimal cell proliferation while many follicles stained positive for CPP32, a marker for apoptosis.; Conclusion. Immunohistochemical staining with Ki-67 and CPP32 are good indicators of cell viability. The culture system used in this study did not promote follicle growth to the MII stage.