In vitro mouse preantral follicle development in 2D and suspension culture: α-MEM vs SAGE 1-step.

abstractThe in vitro culture of immature follicles remains a challenge in reproductive biology, ART, and fertility preservation. This study investigated the impact of α-MEM and SAGE 1-Step media on mouse preantral follicle development and oocyte maturation using two-dimensional (2D) and suspension culture systems. Preantral follicles (∼130 μm) from 14-day-old NMRI mice were cultured for 13 days in either medium under 2D or suspension conditions. On day 13, hCG was added to induce meiotic resumption. We evaluated follicular growth, antrum formation, oocyte maturation, meiotic spindle organization, and expression of genes related to maturation (Bmp15, Gdf9), cumulus expansion (Has2, Ptgs2, Lhr, Adamts1), and apoptosis (Bax, Bcl2). Antral formation was higher in α-MEM in both culture systems (P > 0.05). However, SAGE 1-Step significantly improved oocyte maturation across both systems (P = 0.01 in 2D vs P = 0.006 in suspension compared to α-MEM). Spindle staining demonstrated that suspension culture significantly increased the percentage of oocytes with normal meiotic spindle organization (P = 0.002). Gene expression analysis revealed a significant upregulation of Adamts1 (P = 0.008) and downregulation of Bax (P = 0.01) in suspension culture. Furthermore, the apoptotic ratio of Bax/Bcl2 was significantly decreased in the suspension system (P = 0.01). In conclusion, the SAGE 1-Step medium significantly enhances oocyte maturation, while the suspension system superiorly preserves meiotic spindle architecture and reduces apoptosis. The combination of SAGE 1-Step and suspension culture provides an optimized microenvironment for producing high-quality oocytes. LAY SUMMARY: Growing immature egg outside the body, a process known as in vitro follicle culture, is a promising but challenging technique for preserving fertility. This study investigated novel approaches to improve this process, aiming to create healthier, more viable eggs for future use. The researchers found that using a specific medium significantly improved egg development and maturation. Importantly, they found that culturing these immature eggs in suspension, where the follicles remained floating in the medium, rather than in a traditional two-dimensional system where follicles inevitably flatten and partially lose their three-dimensional structure, resulted in significantly better outcomes. Eggs grown in suspension showed more organized cellular structures and reduced cell death compared to those cultured on a flat surface. These findings represent an important step toward reliably producing healthy, developmentally competent eggs outside the body, offering new hope for fertility preservation and assisted reproduction.