Vascularised Chorioallantoic Membrane (CAM) Culture System for Cryopreserved Human Ovarian Tissue as an Alternative to Xenotransplantation

Purpose: Previously there were only two effective ways to determine the quality of cryopreservation procedures for ovarian tissue after thawing: xenotransplantation and in vitro culture in a big volume of medium with permanent mechanical agitation. The Belgian group of J. Donnez has shown that the chorioallantoic membrane (CAM) culture system offers a new approach to study human ovarian tissue transplantation in its first ischemic stages, yielding information on the timing of tissue changes before neovascularization is established. The aim of this study was to compare the effectiveness after thawing of human ovarian tissue cultured in vitro in a big volume of medium with agitation with a CAM culture system. Material and Methods: Ovarian tissue fragments from 5 patients were transported within 20 min at 32-34 degrees C to the laboratory. The fragments were divided into smaller pieces (1-2 x 0.7-1 mm), frozen, thawed and randomly divided into the following two groups: Group 1: tissue cultured in vitro for 7 days in a big volume of medium with mechanical agitation; Group 2: tissue cultured in a CAM system for 5 days. The viability of the tissue from the respective method of cultivation was evaluated by immunohistochemistry (cytokeratin and Ki-67) and assessed according to the development of follicles and follicular cell proliferation. Results: 85 and 87% of the follicles were morphologically normal in group 1 and group 2, respectively. Immunohistochemical analysis showed that the proliferative characteristics of follicular cells after culture in the CAM system were significantly increased. Conclusion: Both the CAM system and in vitro culturing in a big volume of medium with permanent mechanical agitation are suitable for culturing human ovarian tissue. However, the CAM system provides more information