Assessment of Survival and Expansion of Mouse Pre‐Antral Follicles in 3D System Within Hyaluronic Acid‐Alginate Hydrogel Cocultured With Human Theca Stem and Progenitor Cells

Cancer treatments can impair fertility in young women, and existing fertility preservation methods are often unsuitable for prepubertal patients. Replicating the natural follicular microenvironment in a three-dimensional (3D) in vitro culture of ovarian follicles presents a promising alternative, and co-culturing follicles with ovarian cells may enhance oocyte development. This study used a 3D culture system of hyaluronic acid and alginate to grow mouse preantral follicles and human theca stem and progenitor cells (hTSCs and hTPCs). Ovarian tissue was obtained from five transsexual women; hTSCs were isolated, expanded, confirmed, and differentiated into hTPCs. Preantral mouse follicles were collected and co-cultured with hTSCs and hTPCs in 0.5% hyaluronic acid-alginate (HA-Alg) hydrogel for 14 days. hTSCs were identified by spindle-shaped fibroblast morphology, positive alkaline phosphatase activity, expressing mesenchymal markers like CD29, CD44, and CD90, and differentiation potential to adipocytes and osteocytes-like cells. hTSCs differentiation into hTPCs causes an increase in steroidogenic gene expression, hormone release, and follicular recruitment. Coculturing of mouse preantral follicles with hTSCs and hTPCs in HA-Alg hydrogel induced follicle survival, hormone release, and oocyte maturation. Human theca cells in the stem and progenitor cells stage may support mouse follicle growth, survival, and steroidogenesis.