The significance of in vitro culture medium in directing human ovarian preantral follicle development and ECM secretion

Ovarian tissue cryopreservation and transplantation is an alternative to preserve fertility in female cancer patients who cannot undergo oocyte or embryo cryopreservation. On the other hand, transplantation of cryopreserved ovarian tissue from patients at high risk of ovarian metastasis, such as those with leukemia, neuroblastoma, and Burkitt lymphoma, is not recommended due to the potential reintroduction of malignant cells. Ovarian tissue engineering offers a promising alternative by encapsulating human ovarian follicles to support folliculogenesis. Studies have explored both transplantation and in vitro culture of isolated human preantral follicles within hydrogels. However, in vitro follicle culture remains challenging due to stage-specific culture requirements and the mechanical properties of the hydrogel. Notably, most studies have relied on high fetal bovine serum (FBS) concentrations in the culture medium. In this study, we aim to evaluate in vitro culture medium effects on the development and extracellular matrix (ECM) secretion of human preantral follicles encapsulated in a PEGylated fibrin hydrogel. Isolated follicles were encapsulated in a tailored PEGylated fibrin hidrogel, which mechanically mimic ovarian tissue at reproductive age 1, and cultured in vitro using a fetal bovine serum (FBS)-based culture medium supplemented with follicle-stimulating hormone (FSH) and/or growth differentiation factor-9 (GDF-9). The viability, growth, and ECM production of follicles were then analyzed. Additionally, the different concentrations of FBS have been analyzed to assess their impact on follicle growth, ECM retention, and production. Peripheral mean fluorescent intensity of a follicle was calculated on a rectangular of 10×4 µm2, with its center located on the edge of the follicle. Whole follicle mean fluorescent intensity was also calculated by drawing the freeform shape of spline contour around a follicle. In conclusion, our findings recommend in vitro culture of early-stage human preantral follicles in a medium containing less serum components, 1% FBS, to provide support for both ECM secreting and follicular growth. Our results revealed the effects of FSH, GDF-9, and different levels of serum on human preantral follicle development and ECM deposition, advancing ovarian tissue engineering for fertility preservation.