Enhanced maturation of mouse immature oocytes using a novel three-dimensional culture system

The effective utilization of immature oocytes holds promise for improving conception rates in women with low ovarian reserve. The oocyte microenvironment, comprising the extracellular matrix and intercellular communication with somatic cells, is crucial for supporting oocyte maturation. This study hypothesizes that a three-dimensional (3D) culture system, constructed with hydrogels modified by laminin-mimetic peptides-a major component of the extracellular matrix-and granulosa cells (GCs) from mature mouse oocytes, can replicate the biomechanical microenvironment required for oocyte maturation, thereby promoting the development of immature mouse oocytes. To validate this hypothesis, we initially assessed the physical properties of hydrogels and determined the optimal concentration for in vitro oocyte culture. A total of 304 germinal vesicle (GV) phase mouse oocytes were retrieved and randomly allocated to two-dimensional (2D), 3D, and 3D-GCs culture groups. The 3D-GCs group exhibited the highest rate of first polar body extrusion (83.1 ± 5.0 %). Oxidative stress levels and mitochondrial membrane potential were evaluated using DCFH-DA and JC-1 staining, confirming that 3D culture significantly reduced oxidative stress and enhanced mitochondrial activity. Cytoplasmic maturation, assessed by cortical granule distribution and endoplasmic reticulum organization, further highlighted the superiority of the 3D-GCs system. The 3D-GCs group demonstrated 87.0 % of oocytes with grade III cortical granule distribution and 84.6 % with mature endoplasmic reticulum, significantly surpassing the other groups (p < 0.05). In conclusion, the 3D-GCs culture system effectively supports synchronized nuclear and cytoplasmic maturation in immature oocytes. This approach provides a promising platform for improving the developmental competence of oocytes, potentially benefiting women with diminished ovarian reserve.