In vitro modeling of myometrial disorders microenvironment- challenges and methodological advances to unveil pathogenetic mechanism and therapeutic targets

Three-dimensional culture systems provide more physiologically relevant models of uterine tissues than conventional two-dimensional monolayers, as they better preserve cell–cell and cell–matrix interactions and allow extracellular matrix (ECM) deposition within tissue-like architectures. Uterine smooth muscle tumors, including benign leiomyomas and malignant leiomyosarcomas, arise from the myometrium and pose a major clinical challenge, from highly prevalent fibroids to rare but aggressive sarcomas. This thesis reports the development and characterization of 3D spheroid models of human myometrium, leiomyoma, and leiomyosarcoma generated by an agarose-based manual method and by extrusion-base bioprinting in commercial and in-house hydrogels. Phase-contrast and fluorescence microscopy, histological and histochemical staining, and Field Emission-Scanning Electron Microscopy demonstrated that these scaffolds support viable, proliferative, and structurally organized spheroids suitable for quantitative morphometric, ultrastructural and molecular analyses. These platforms were then used to investigate the endocrine disruptor bisphenol A (BPA) in uterine smooth muscle pathophysiology by assessing cell viability in 2D and 3D systems (MTT and PrestoBlue assays) and evaluating fibrogenic responses through COL1A1, fibronectin and activin A mRNA expression by RT-PCR and for fibronectin and COL1A1 protein expression by immunohistochemistry after exposure to biologically relevant BPA concentrations. Overall, the data indicate that bioprinted 3D uterine spheroids constitute robust and versatile models to dissect ECM remodeling and endocrine disruption in myometrial tumors. In addition, preliminary findings suggest that low-dose BPA may contribute to the pathogenesis of myometrial neoplasms, particularly leiomyomas, reinforcing the need to integrate environmental endocrine disruptors into uterine tumor biology and risk assessment frameworks.