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

Research questionCan tissue clearing, combined with volumetric imaging, enable reliable, quantitative three-dimensional analysis of follicles and vasculature in intact human ovarian tissue? DesignA 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. ResultsClearing produced optically transparent tissue with a linear expansion factor of 1.2 across cortex and medulla. Imaging depth increased 6.5-11-fold in cortex and 6-8-fold in medulla. Follicle density measurements in immunolabeled cleared cortex were comparable to histology, supporting the validity of volumetric follicle quantification. Light sheet microscopy of lectin-labeled cortex revealed no significant donor-to-donor differences in vascular morphometrics, including mean vessel diameters of 12-14 {micro}m, branch point densities of 632-965 points/mm3, vessel length densities of 117-175 mm/mm3, and volume fractions of 1.9-2.3%. Volumetric imaging further illustrated heterogeneous spatial relationships between follicles and surrounding vessels. ConclusionTissue 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.