Ovulatory follicular fluid promotes the clonogenicity and invasion of ectopic and eutopic endometrial cells

A BSTRACT Objectives: Endometriosis is a chronic, estrogen-dependent disorder characterized by the ectopic growth of endometrial-like tissue. Despite its significant impact on fertility and quality of life, the pathogenesis of endometriosis remains poorly understood. This study investigates the potential role of ovulatory follicular fluid (FF) in driving disease progression. Based on the “double engines theory,” which posits that retrograde menstruation provides the “seeds” for ectopic lesions while ovulation fuels their growth, we hypothesize that FF enhances endometrial cell proliferation, clonogenicity, migration, and invasion. Materials and Methods: Primary eutopic and ectopic endometrial cells were isolated from infertile women and treated with 10% diluted FF collected from in vitro fertilization patients. Cellular morphology, proliferation, clonogenicity, migration, and invasion were evaluated using colony formation, transwell, and Matrigel assays. Statistical analyses were performed to compare cellular responses between eutopic and ectopic cells. Results: Morphological analysis revealed distinct adaptive changes, with ectopic cells predominantly adopting a more fibroblast-like phenotype. Ectopic cells exhibited higher clonogenicity (29.8-fold) capacities, lower proliferation (0.49-fold) and migration (0.11-fold) capacities, and similar invasion capabilities compared to eutopic cells. FF significantly enhanced proliferation (1.7-fold in eutopic and 1.3-fold in ectopic cells) and clonogenic capacity, with eutopic cells forming 31.3 times more colonies and ectopic cells showing a 1.3-fold increase. The clonogenic area expanded dramatically, increasing 261-fold in eutopic and 4.9-fold in ectopic cells. In addition, FF promoted migration (1.8-fold in eutopic and 2.9-fold in ectopic cells) and invasion (9.1-fold in eutopic and 4.8-fold in ectopic cells). These findings suggest that FF may play a pivotal role in the early establishment and progression of ectopic lesions. Conclusion: The results highlight the critical role of FF in enhancing endometrial cell survival, proliferation, and dissemination. This supports the “double engines theory” of endometriosis, emphasizing the significant contribution of ovulation to the pathogenesis of the disease.