Study of follicular growth and differentiation after transplantation of cryopreserved human ovarian tissue
(2012)
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- (en) Ovarian tissue cryopreservation and transplantation is a well-developed procedure for preservation of fertility in young cancer patients. Although 17 live births have been achieved so far, this procedure has some limitations (difference in follicular size compared to follicles in situ, recovery of empty follicles from a transplanted ovary and low recovery rate of oocytes). It is unclear, however, if these alterations are associated with ovarian tissue freezing or transplantation procedures. The aim of this research project was to compare follicular growth and the expression of key regulators of folliculogenesis after grafting of fresh and frozen-thawed ovarian tissue in order to understand the processes affecting follicular quality following cryopreservation and transplantation. For this purpose, human ovarian tissue was frozen, thawed and transplanted intraperitoneally (under conditions mimicking those applied in the clinics) in a murine xenotransplantation model. Immunostaining and morphometric analyses were carried out on grafts recovered after 3 weeks (follicular development up to the secondary stage) and 28 weeks (follicular development up to the antral stage). After 3 weeks’ xenotransplantation, activation of follicle growth occurred in both fresh and frozen-thawed grafts, as indicated by the higher proportion of growing follicles compared to ungrafted controls. There was no significant difference in the follicular population between fresh and frozen-thawed grafts, indicating that freezing does not have a major impact on initial growth, structural features, or survival of preantral follicles. Our study evidences, for the first time, the expression of c-kit, KL and GDF-9 in pre-antral follicles after freezing and transplantation. After long-term xenotransplantation, follicular activation and follicular growth to the antral stage were observed. Although there was no significant difference in oocyte or follicle diameter in fresh or frozen-thawed grafts, lower expression of KL was observed in primordial and primary follicles after transplantation of both fresh and frozen-thawed tissue. On the other hand, consistent expression of AMH, an essential inhibitor of follicle growth, was found in most growing follicles. Interestingly, the present study suggests that most primordial follicles remaining in 28-week-old grafts are quiescent. In antral follicles, although no difference was observed in antrum area or zona pellucida thickness, the theca layer was found to be significantly thinner in frozen-thawed grafts compared to fresh grafts. Our studies demonstrate that both freezing and grafting may have an impact on follicular quality. Follicular expression of KL, a key activator of follicle growth and differentiation, was affected after grafting possibly because of the massive activation of follicular growth in grafted ovarian tissue. However, although follicular recruitment and KL expression were altered after grafting, resting primordial follicles were found in grafts. Their maintenance could be due to factors produced by growing follicles such as AMH. These findings suggest that cross-talk between oocyte, granulosa and stroma cells could be affected following grafting causing differences in follicular characteristics. Freezing appears to be responsible for alteration in antral follicles theca cell layer which may be explained by cryoinjuries to stromal cells, acting as theca cell precursors and granulosa cells, inducing their recruitment. Since theca cells play a key role in the synthesis of androgens, a change in their population (number and/or function) may lead to failure in final follicular development and function.
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David, A. (2012). Study of follicular growth and differentiation after transplantation of cryopreserved human ovarian tissue. https://hdl.handle.net/2078.5/160678