EXTRACORPOREAL DEVELOPMENT AND ULTRARAPID FREEZING OF HUMAN FETAL OVA

Purpose: The present study was performed to culture human fetal ova to determine whether they can be matured and cryopreserved using ultrara pid freezing. Methods: Thirty-three pairs of fetal ovaries were obtain ed from fetuses of 16-20 weeks' gestation following elective abortion. Ovarian tissues were minced into approximately I-mm sizes and culture d in Waymouth media either before or after ultra rapid freezing. The W aymouth medium was supplemented with 15% (v/v) fetal bovine serum, 0.0 3 IU/ml FSH and 35 ng/ml insulin. The tissue was cultured at 37 degree s C in 5% CO2 in air for 5-25 days in Falcon dishes and 30-40 days in Costar Transwell-COL membranes prior to induction of final maturation in the presence of LH and human follicular fluid. Minced tissues were also frozen by ultra rapid freezing in M199 with 4.2 M dimethylsulfoxi de (DMSO) and 0.35 M sucrose and plunged directly into liquid nitrogen . For thawing, the straws were plunged into a 37 degrees C water bath for 5 s. The contents were then expelled and diluted 1:5 with thawing medium containing 0.42 M sucrose. After washing the thawed tissues wer e cultured as described for the fresh tissues. Results: Patches of mon olayer consisting of fibroblasts had formed within 2-3 days of culture of fresh tissues. After 1 week of culture, follicles separated out fr om the ovarian tissue bur remained attached to the monolayer. The maxi mal number of follicles separating out from the tissue appeared about I week after initiating the culture (154 follicles per 10 fields at Da y 5 and 61 and Day 25). After 40 days of culture in Costar dishes, 34% of the ova reached a diameter of more than 80 mu m, which was signifi cantly higher than at the beginning of culture (6%; P < 0.05). Among t hese ova, 34% were found to be surr ounded by the zona pellucida, whic h was not observed at the beginning of culture. Following induction of final maturation, extrusion of the first polar body was noted in 25% of ova grown in Costal dishes for 40 days. Twelve percent of the oocyt es show,ed the first polar body wizen they were grown in Costar dishes for less than 30 days. For frozen-thawed tissues, 14% of minced ovari an tissues displayed central necrosis immediately after thawing. Follo wing digestion and Trypan blue staining, 75% of ova and 85% of somatic cells survived ultrarapid freezing. Nineteen percent of the ova which have been cultured as described for fresh tissues displayed extrusion of the first polar body, comparing favorably with the 25% maturation rate observed with the fresh tissue (P > 0.05). Conclusion: This study demonstrates that morphologically normal, mature human ova can be obt ained from primordial follicles in vitro development. Using a simple, quick ultrarapid freezing method, human fetal ova can be cryopreserved in the form of minced tissue without significantly compromising their ability to grow in vitro.