Engineering ovarian asymmetric mechanical niches in a dual-layer microcavity array enhances mouse secondary follicle...

With global population aging and declining fertility rates, the preservation of reproductive health has become an important biomedical challenge. In vitro follicle culture, especially secondary follicle culture, represents a promising strategy for advancing fertility preservation research; however, current systems often fail to recapitulate the spatially heterogeneous ovarian mechanics, particularly the mechanical asymmetry between cortex and medulla, limiting follicular development and culture scalability. Here, we engineered a dual-layer biomimetic microcavity array (MCA) for high-throughput three-dimensional culture of mouse secondary follicles by integrating a mechanically supportive agarose outer scaffold with softer alginate hydrogel compartments that generate a softer medulla-like microenvironment. This platform enables standardized and parallel culture of isolated follicles with spatially organized mechanical regulation. Compared with conventional alginate microsphere culture, the MCA system significantly enhanced secondary follicle growth, survival, estradiol secretion, and oocyte maturation. Furthermore, oocytes derived from MCA-cultured follicles exhibited improved developmental competence relative to microsphere controls, with higher fertilization and blastocyst formation rates. Blastocyst formation increased from 7.7% in the microsphere group to 34.5% in the MCA group, although it remained below that of the superovulated in vivo control. Mechanistically, the dual-layer architecture generated distinct solid mechanical stress distributions, including altered shear components arising from follicular expansion against the surrounding matrix, which were associated with changes in mechanotransduction-related signaling pathways, including PI3K/Akt-related signaling, in follicular cells. Collectively, this study establishes a biomimetic platform for investigating the role of mechanical microenvironments in follicular development and provides a research framework for future advances in ovarian tissue engineering and fertility preservation.