Beyond Histology: A Validated CUBIC-Based Workflow for Volumetric Analysis of Follicles and Cortical Vasculature in Human Ovarian Tissue

Research question: Can tissue clearing, combined with volumetric imaging, enable reliable, quantitative three-dimensional analysis of follicles and vasculature in intact human ovarian tissue? Design: A CUBIC-based clearing protocol was adapted for human ovarian medulla and cryopreserved cortex. Tissue from reproductive-aged donors was cleared, fluorescently labeled, and imaged using confocal and light sheet microscopy. Tissue expansion, imaging depth, and vascular morphometrics were quantified and follicle density was compared to conventional histology. Results: , and volume fractions of 1.9-2.3%. Volumetric imaging further illustrated heterogeneous spatial relationships between follicles and surrounding vessels. Conclusion: Tissue clearing and volumetric imaging complement routine histology and enable quantitative three-dimensional investigation of follicle-vascular interactions in intact human ovarian tissue, providing a framework for advancing fertility preservation and ovarian tissue transplantation research.