Three‐Dimensional Architecture of Ectopic Epithelium and Vasculature in Ovarian Endometriosis Revealed by Tissue‐Clearing Imaging

Ovarian endometriosis (OEM) is characterised by ectopic endometrial tissue growth within the ovary. In these ectopic lesions, the ectopic epithelium plays a crucial role in OEM progression and has been associated with malignant transformation in a subset of cases. However, conventional histology limits understanding of ectopic epithelial distribution, structure, and its perivascular microenvironment, thus impeding pathogenesis studies. To address this, we employed a modified tissue-clearing method and three-dimensional (3D) imaging to systematically characterise OEM, revealing key, previously unreported spatial characteristics. We found significantly higher densities of ectopic epithelium and vasculature in the outer cystic wall versus the inner. Furthermore, our method improved the detection rate of ectopic epithelium and revealed its morphological polymorphism at both tissue and cellular levels. Besides, we demonstrated that vessels preferentially cluster around ectopic epithelium, with their distribution pattern strongly linked to the location of ectopic epithelium. Strikingly, we observed endometrial-like structures in lesional vasculature in 3 of 49 cases, representing a novel morphological observation that warrants further investigation. This study significantly advances our understanding of OEM histopathology, offering insights for clinical diagnosis and treatment.