144 A decellularized extracellular matrix scaffold for creating an in vitro ovary

Species conservation and human fertility restoration share a common goal: preserving the ability to reproduce. In both endangered species and humans with conditions such as premature ovarian insufficiency (POI), maintaining fertility is crucial. With a global prevalence of POI at 3.5%, over 138 million women experience subfertility or complete infertility along with hormonal disruptions. Additionally, oncofertility patients with metastatic ovarian disease cannot transplant native ovarian tissue, leaving many patients without the possibility of having biological children or hormones. This underscores the need for fertility preservation techniques that can benefit both wildlife and humans. Such approaches require a deeper understanding of the ovarian microenvironment to engineer optimal conditions for follicle growth and maturation. Thus, we have created a biomimetic extracellular matrix (ECM) hydrogel from cortex (Ctx) and medulla (Med) to develop an ovary three-dimensional model, supplying isolated cells and follicles with an in vivo-like environment. Bovine ovarian tissues were decellularized; prepared into hydrogels; checked for biocompatibility by culturing stromal cells (n = 3); and proteomics (n = 5), rheological properties (nanoindentation; n = 4), and porosity (n = 4) analysis were performed to characterize their biomechanical properties. Bovine primordial follicles were isolated and cultured in CTx-dECM, and follicle viability was evaluated at Day 3 (n = 3). Data analyses were carried out in R, using a generalized linear model. Both core and affiliated ECM proteins (regulators, affiliated, and secreted factors) were detected in the Ctx and Med dECMs (119 and 91, and 130 and 120; respectively). A total of 185 matrisome proteins were common to both dECMs: Ctx-dECM had 10 unique proteins (such as ZP3, ZP4, and WNT4), while 63 proteins were only detected in Med-dECM (including MST1, FST, and SMOC1), showing that there are differentially expressed matrisome proteins across these regions. Overall, collagens were more abundant in the cortex (86% vs. 78%; P < 0.05), while medulla had higher proteoglycans content (14% vs. 8%; P < 0.05). Core ECM components known to have roles in folliculogenesis, such as laminin, nidogen, and versican, were found in both dECMs. Estrus stage had a minimal effect on the dECM composition. Porosity of Ctx-dECM and Med-dECM were lower than the native tissues (P < 0.05). Stromal cell culture promoted ECM degradation, but the addition of 1% alginate reduced this loss significantly, all while preserving high cell viability (>95%, n = 3). Ctx dECMs had reduced stiffness compared with the native tissue (1.74 vs. 7.45 kPa; P < 0.05), and Med-dECM had similar stiffness (3.2 vs. 4.45 kPa; P > 0.05). All dECMs had similar elastic and visco-elastic properties to the tissue. Primordial follicle survival was higher in Ctx-dECM than in two-dimensional cultures (70.8% vs. 50%, respectively). By validating the dECM, we aim to create a biomimetic ovary-on-a-chip platform that better supports follicle growth and maturation, laying the foundation for advanced biomaterials to enhance fertility restoration for a diverse range of individuals.