Assessing the effect of adipose-tissue-derived stem cell conditioned medium on follicles and stromal cells in bovine ovarian tissue culture.

RESEARCH QUESTION: Does adipose-tissue-derived stem cell conditioned medium (ASC-CM) supplementation enhance follicle and stromal cell outcomes in vitro? DESIGN: Bovine ovaries (n = 8) were sectioned and cultured in vitro for 8 days in two different groups: (i) standard culture (OT Ctrl D8); and (ii) culture with ASC-CM supplementation (OT + CM D8). Half of the culture medium was replaced every other day, and stored to measure the production of oestradiol. Follicle classification was established using haematoxylin and eosin staining. Follicle and stromal cell DNA fragmentation was assessed by TUNEL assays, while growth differentiation factor-9 (GDF-9) staining served as a marker of follicle quality. Additionally, three factors, namely vascular endothelial growth factor (VEGF), interleukin 6 (IL-6) and transforming growth factor beta 1 (TGF-ß1), were evaluated in ASC-CM in order to appraise the potential underlying mechanisms of action of ASC. RESULTS: The OT + CM D8 group showed a significantly higher proportion of secondary follicles (P = 0.02) compared with the OT Ctrl D8 group. The OT + CM D8 group also demonstrated significantly lower percentages of TUNEL-positive follicles (P = 0.014) and stromal cells (P = 0.001) compared with the OT Ctrl D8 group. Furthermore, follicles in the OT + CM D8 group exhibited a significant increase (P = 0.002) in expression of GDF-9 compared with those in the OT Ctrl D8 group, and oestradiol production was significantly higher (P = 0.04) in the OT + CM D8 group. All studied factors were found to be present in ASC-CM. VEGF and IL-6 were the most widely expressed factors, while TGF-ß1 showed the lowest expression. CONCLUSIONS: Addition of ASC-CM to culture medium enhances follicle survival, development and oestradiol production, and promotes the viability of stromal cells. VEGF, IL-6 and TGF-ß1 could be paracrine mediators underlying the beneficial effects.

Comprehensive Review of In Vitro Human Follicle Development for Fertility Restoration: Recent Achievements, Current Challenges, and Future Optimization Strategies.

Ovarian tissue cryopreservation (OTC) and subsequent transplantation (OTT) is a fertility preservation technique widely offered to prepubertal girls and young fertile women who need to undergo oncological treatment but are at a high risk of infertility. However, OTT is not considered safe in patients with certain diseases like leukemia, Burkitt's lymphoma, and ovarian cancer because of the associated risk of malignant cell reintroduction. In vitro follicle development has therefore emerged as a promising means of obtaining mature metaphase II (MII) oocytes from the primordial follicle (PMF) pool contained within cryopreserved ovarian tissue, without the need for transplantation. Despite its significant potential, this novel approach remains highly challenging, as it requires replication of the intricate process of intraovarian folliculogenesis. Recent advances in multi-step in vitro culture (IVC) systems, tailored to the specific needs of each follicle stage, have demonstrated the feasibility of generating mature oocytes (MII) from early-stage human follicles. While significant progress has been made, there is still room for improvement in terms of efficiency and productivity, and a long way to go before this IVC approach can be implemented in a clinical setting. This comprehensive review outlines the most significant improvements in recent years, current limitations, and future optimization strategies.