The long-term goal of this work is to establish a broad fertility preservation option for women facing infertility as a result of gonadotoxic treatments. The overall objective of this proposal in working towards the long term goal and mitigating the risks associated with autotransplantation is to create a biomimetic environment that promotes in vitro growth of immature follicles. The low success rates of follicle development, or folliculogenesis, are attributed to the complex and poorly understood paracrine, autocrine and endocrine signaling between the cells in a follicle, neighboring follicles and their microenvironment. The central hypothesis is that recreating the ovarian microenvironment through co-encapsulation of follicles with adipose-derived stem cells (ADSCs) in a hydrogel which retains cell-secreted extracellular matrix (ECM) will supply the necessary support for primary follicle growth in vitro. The rationale for the proposed work is that by recapitulating the natural ovarian microenvironment and deciphering transcription factor and cytokine networks, the culture system can be further developed to promote folliculogenesis of human follicles. In the first aim, ECM-sequestering peptides will be incorporated in a biomimetic poly(ethylene glycol) (PEG) hydrogel system using Michael-type addition chemistry to facilitate deposition of ECM components and mimic the native ovarian tissue. The follicle basement membrane is composed of ECM proteins and it functions as a structural support for the cells, a selective barrier for materials entering the follicle, and a scaffold for retaining growth factors. It is continuously remodeled as the follicle grows, but cell-secreted ECM molecules are unable to adhere to unmodified PEG for self-assembly. By integrating ECM-sequestering peptides in the PEG hydrogels, the structural and biological roles of ECM can be restored for in vitro folliculogenesis. In the second aim, Transcriptional Activity Cellular Array (TRACER) will be used to determine the dynamic transcription factor (TF) activity in granulosa cells of growing follicles when cultured in a biomimetic hydrogel which includes paracrine and ECM support. This information will give insight to the internal cell processes which lead to follicle growth and survival. The contribution of this work is expected to be a novel in vitro follicle culture that supports primary follicle growth, and a better understanding of the underlying mechanisms which drive folliculogenesis. The contribution of this work will be significant because it will guide the development of a standardized in vitro culture for maturation of human follicles and a safe fertility preservation option for patients unable to produce mature eggs as a result of gonadotoxic treatments. The proposed work is innovative in that it will be the first instance of follicle culture in a synthetic ECM-sequestering matrix, and the first time TRACER will be used to study follicles in PEG.