Regulation of human ovarian folliculogenesis in vitro

Abstract: Cryopreservation of ovarian tissue containing immature oocytes is one approach to preserving the potential fertility of young women who risk losing their oocytes as a consequence of treatment with anti-cancer agents, or genetic disorders. This cryopreserved tissue can then be transplanted back into the ovary when the woman wants to have children. Our research group has also been developing an alternative procedure, namely maturation of ovarian follicles and their oocytes in vitro, for cancer patients that risk retransmission of the disease after transplantation. Live offspring can already be produced in mice from small antral follicles that have matured and been fertilized in vitro. In the case of women, it is much more challenging to obtain mature oocytes from ovarian tissue in vitro, due to the much longer period required for maturation and the dense structure of this tissue. Employing ovarian biopsies obtained from volunteers, our research group has been actively optimizing conditions for culturing human ovarian tissue and we have shown that if structural and biochemical systems are kept intact (i.e., by culturing pieces of tissue instead of isolated follicles), primordial human follicles can develop into primary and even secondary follicles in vitro. However, fully mature oocytes have not yet been obtained and further optimization of this system is required. The mechanisms controlling the initiation of the growth of small ovarian follicles are not yet known in detail, although a number of factors produced in the ovary itself are known to be involved. These include the family of transforming growth factor beta, as well as other growth factors. Our present findings can be summarized as follows: Ovarian cortical tissue should be cultured in the form of cubes on diluted Matrigel matrix and the composition of the extra cellular matrix (ECM) should be chosen on the basis of the goals of the study in question (Article I). Kit ligand (KL) mRNA and c-Kit mRNA and protein are expressed in follicles during all stages of development, from primary to antral stage, and the reduction in the survival of follicles in long-term culture caused by an antibody that blocks the c-Kit receptor indicates that signaling via KL/c-Kit plays an important role in the early development of human ovarian follicles (Article II). Moreover, anti-müllerian hormone (AMH) plays a key role in suppressing the entry of follicles into the growing pool, i.e., is one of the hormones involved in inhibiting the recruitment of primordial follicles (Article III). Finally, endogenous growth differentiation factor-9 (GDF-9) is an important regulator of the transition from primary to secondary follicles; BMPRII-Fc can suppress this transition; and, furthermore, the rhGDF-9 protein promotes the early development and growth of follicles, an effect which could be of clinical value. Thus, we report here important new information concerning the early maturation of human oocytes and follicles in this culture system. This system provides a valuable tool for the identification of factors that promote or inhibit the recruitment of ovarian follicles and will aid in the improvement of procedures for assisted reproduction.