Role of macrophages in the pathogenesis of uterine adenomyosis

Uterine adenomyosis is a common benign gynecological disorder affecting reproductive-age women and causing symptoms such as pelvic pain, abnormal uterine bleeding, and infertility. Despite its high prevalence and severity of symptoms, its pathogenesis has not yet been elucidated, thus, to date, no drug can definitively cure it. Even though surgical approaches and off-label medical treatments exist, they are not suitable for patients who wish to conceive. Histologically, adenomyosis is characterized by the presence of ectopic endometrial tissue within the myometrium, which is widely believed to occur due to invagination of the endometrium into the uterine muscle. The mechanism of endometrial invasiveness remains unknown but several cell invasion processes have been proposed, including epithelialmesenchymal transition (EMT) and collective cell migration (CCM). As our laboratory previously demonstrated an increase in M2-like macrophage accumulation in adenomyosis compared to healthy endometrium and given the macrophages’ ability to induce tumour progression, our work aims to elucidate the involvement of M2 macrophages in the pathogenesis of adenomyosis, and more specifically, in endometrial invasiveness. Therefore, in the first instance, we developed an in vitro co-culture model, which allows the interaction between primary endometrial cells and THP-1-derived M2 macrophages. Endometrial epithelial and stromal cells were then analysed in terms of their invasion ability as well as expression of genes involved in EMT. Our invasion assay demonstrated that endometrial cells from adenomyosis and healthy patients do acquire a more invasive phenotype in the presence of M2 macrophages, but this does not occur through EMT, according to our gene expression analysis. Next, we performed immunohistochemical analysis to investigate the specific mechanism of invasiveness of adenomyotic endometrium with the following markers : E- and N-cadherin, which are markers of cell-cell junctions, and matrix metalloproteinase-9 (MMP9), an indicator of cell invasion activity. Results indicate that, in adenomyosis, both E- and N-cadherin are strongly expressed in the invasive glands of the endometrium, along with an increase of MMP9 expression in eutopic endometrium. Thus, our results suggest that CCM underlies the invasion of endometrial cells into the myometrium observed in adenomyosis. Finally, we set up gland-derived organoid and endometrial assembloid cultures to examine their potential as a more advanced in vitro model of disease pathogenesis. Future research will focus on using these culture models to investigate CCM and the role of macrophages in this process. Understanding the pathogenic mechanisms responsible for adenomyosis onset, progression and related symptoms is crucial for development of effective and safe treatments that would offer patients a better quality of life and even effectively cure their disease.