Encapsulated three-dimensional (3-D) culture of ovarian follicles for female fertility preservation: A decade of experience and future directions

摘要

Introduction: The combination of successful ovarian tissue cryopreservation with in vitro maturation of follicles to produce competent oocytes is emerging as a potential clinical option to preserve female fertility following cancer treatment, especially for patients for whom ovarian tissue transplantation poses a risk for reintroduction of malignant cells. Three-dimensional (3-D) approaches designed to maintain the cell-cell and cell-matrix interactions known to be important in regulating follicle development, were successfully applied to mouse follicles. Alginate encapsulation supported normal murine follicle development and oocyte maturation with production of live offspring. This biomaterials approach was applied to nonhuman primate follicles to determine whether the difficulty of maintaining follicle architecture during culture in non-rodent species could be overcome for maturation of follicles and their enclosed oocytes. Materials and Methods: Individual primary or secondary follicles were manually dissected from ovarian cortex of rhesus macaques, encapsulated in alginate alone or with fibrin, and cultured under chemically defined conditions that included gonadotropin hormones. Follicles achieving antral stages were exposed to a bolus of human chorionic gonadotropin to induce oocyte meiosis, and punctured to obtain mature oocytes for in vitro insemination and embryo culture. Results: The survival of cultured follicles was dependent upon animal age, phase of the menstrual cycle at collection and the presence of gonadotropins in the media. Surviving follicles were heterogeneous with regard to their growth activity, and categorized as non-growing, slow-growing and fast-growing follicles. These follicle categories differed in their potential to reach the antral stage, and their production of steroids and paracrine factors (anti-Mullerian hormone, vascular endothelial growth factor) was stage-dependent. More primary follicles developed within fibrin-alginate than alginate alone. Secondary follicles achieved antral stages (1mm diameter) by 5 weeks, but primary follicles required 13 weeks. When antral follicles enclosed oocytes of 110 micron diameter, reinitiation of meiotic maturation, in vitro fertilization, and early zygotic cleavage was possible. Discussion: Secondary follicles can grow and mature autonomous of the surrounding ovarian stroma if sufficient physical support is provided to them by a bioengineered environment. Primary follicles can also develop in the absence of ovarian stroma, but require a more rigid environment in vitro. This culture system offers a novel opportunity to utilize primary and secondary follicles for in vitro follicle maturation, with the major goal of producing live, normal offspring. This system also allows important studies on the basic biology of primate follicles at various stages of development. Conclusion: The development of a culture system for human follicles has been perceived as problematic because of the prolonged period of follicle development required. However, a rationally designed tissue engineering approach to follicle maturation in the nonhuman primate is providing new insights into the physiology of the follicle with translational significance for cancer patients. Continued studies are needed to understand the role of hormones in follicle growth and the integration of hormones with the physical environment. Whether primordial follicles can develop and mature independent of their cellular environment or would benefit from tissue engineering is an important question of current interest. Future challenges include production of competent oocytes from preantral follicles derived from cryopreserved ovarian tissue or individually cryopreserved follicles. These fundamental studies will impact development of a robust system for human follicle development and oocyte maturation for cancer patients who cryopreserve ovarian tissue prior to gametotoxic cancer therapies.