PROJECT SUMMARY/ABSTRACT Ovarian tissue cryopreservation (OTC), a fertility preservation option for cancer patients and others at increased risk of developing infertility that is ideally performed prior to the gonadotoxic treatment, has enabled >140 live births. However, fertility restoration is currently limited to transplantation of OTC tissue, and some patients may not be able to use this option due to the risk of reintroducing their disease. An alternative that is not yet developed for the clinic, would be to use the primordial follicles, the oocyte and support cell units that are cryopreserved in OTC, and perform in vitro growth and maturation (IVGM) to produce eggs. Current assisted reproductive technologies require 20 – 30 eggs to offer a good chance of producing a child. However, current IVGM protocols performed in the research lab are not efficient and yield only a few eggs for every 10 or more patients. Importantly, the success of current methods for isolating primordial follicles from ovarian tissue drastically differs between patients and, at best, only a small percentage are obtained intact. Secondary follicles more easily remain intact, but few are cryopreserved during OTC. Therefore, primordial follicles must be activated to grow to secondary follicles if OTC tissue is used to make eggs. The rate limiting steps for advancing IVGM are: (1) efficiently and reliably isolating healthy primordial follicles that are activated to grow, and (2) efficiently and reliably growing and maturing secondary follicles into good quality eggs. 3D-printed bioscaffolds of specific architectures can support the viability and growth of secondary follicles through egg maturation in vitro, as well as primordial follicle growth and maturation through egg maturation, ovulation, and live birth in mice. These promising results lay the foundation to further explore the use of specifically designed bioscaffolds to address current limitations of IVGM. This application tests the hypothesis that a dynamic synthetic microenvironment will provide the necessary mechanical and architectural cues to induce stromal cell migration out of ovarian tissue, release of the embedded follicles, and support follicle growth and oocyte maturation into an egg. 3D-printed bioscaffolds of varying mechanical and remodeling properties using tunable highly porous biomaterials will be utilized to induce follicle migration from cortical tissue and provide a dynamic environment that remodels over time as the follicles grow. Bovine ovaries as used to mimic human ovaries in size, cortical density, follicle growth and maturation for the following aims: (1) to define the bioscaffolds that support stromal cell migration and subsequent release, activation, and growth of primordial follicles; and (2) to define the bioscaffolds that induce growth and maturation of isolated secondary follicles in vitro. These studies will identify specific properties of synthetic microenvironments that can enhance follicle isolation from ovarian tissue, as well as those that support the growth and maturation of follicles into eggs. If successful, this work would enable researchers to study folliculogenesis from primordial follicle activation to an egg in a controlled environment, would uncover a completely novel approach to IVGM and support the development of future technologies for IVGM in humans.

PROJECT NARRATIVE The development of eggs from ovarian tissue cryopreserved for future fertility would significantly expand fertility restoration options for patients, including childhood cancer survivors. Current processes for isolating, growing and maturing ovarian follicles (oocyte-containing units) that produce eggs in a dish, however, are not efficient or reliable. The goals of this study are to develop new tools and more efficient strategies using 3D-printed bioscaffolds to enhance the isolation of follicles from ovarian tissue, as well as support their growth and maturation to produce eggs.