FSH and GDF-9 modulate extracellular matrix remodeling in human preantral follicles in a 3D hydrogel culture

Human preantral follicle development requires finely tuned signaling between oocytes and granulosa cells, supported by a structured extracellular matrix (ECM). This study investigates how follicle-stimulating hormone (FSH) and growth differentiation factor 9 (GDF-9), alone or combined, influence follicular growth and ECM organization in a 3D cell culture system. All culture conditions maintained high follicle viability on Days 1 and 7 with no significant intergroup differences; Day-7 viability ranged from ~ 69–76% across conditions. On Day 1, follicle diameters were comparable among groups. By Day 7, follicles cultured without added FSH or GDF-9 reached a significantly larger mean diameter (171.98 ± 61.05 μm) than those cultured with both supplements (130.22 ± 56.75 μm; p < 0.05); diameters in single-supplement groups were intermediate and not significantly different from the unsupplemented group. Immunofluorescence revealed that laminin expression at the follicle peripheral and across the whole follicle was significantly higher in the FSH-only and GDF-9-only groups than in the unsupplemented and combined-supplement groups. In contrast, collagen IV exhibited a distinct pattern: total collagen IV was lowest in the GDF-9-only group, whereas combined-supplemented group resulted in the higher collagen IV fluorescent intensity in both peripheral and total compared to GDF-9-only group, highlighting the importance of synchronized proliferative and organizational cues. These findings underscore the critical need to evaluate follicle quality not only by growth but also by ECM integrity. They also reinforce the translational value of serum-free systems enriched with physiological regulators to support oocyte-somatic coordination and preserve follicular architecture for reproductive engineering and fertility preservation applications.