Recent advancements in 3-dimensional (3D) in vitro culture systems for follicles, oocytes, and embryos represent a paradigm shift in assisted reproductive technologies and reproductive biology. By mimicking the native physiological microenvironment more faithfully than conventional 2-dimensional systems, these innovative platforms directly address critical limitations of current in vitro embryo production protocols, including low blastocyst development rates, compromised embryo quality, and concerning epigenetic aberrations. This comprehensive review examines a spectrum of emerging 3D strategies, microfluidic devices, dynamic culture conditions, spheroid/organoid models, and advanced co-culture systems with supportive somatic and stem cells. Accumulating evidence demonstrates that these technologies (by preserving 3D follicular and embryonic architecture, enhancing nutrient and gas exchange, and restoring vital cell-cell and cell-matrix interactions) significantly improve oocyte maturation rates, embryo developmental competence, and overall in vitro fertilization outcomes. Nevertheless, challenges such as species-specific optimization, scalability, high costs, and the imperative for clinical standardization remain significant hurdles. Future directions to overcome these obstacles involve the development of smart, physiologically responsive biomaterials, the integration of cutting-edge technologies, such as artificial intelligence and organ-on-a-chip platforms, and the creation of cost-effective systems for widespread application. This review underscores the transformative potential of 3D culture systems as a foundational new approach in reproductive research and fertility treatments.
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