SINGLE CELL ANALYSIS REVEALS HETEROGENEITY IN THE DISEASE RELEVANT CELL TYPES OF ADENOMYOSIS.

To identify cellular composition and gene expression profiles of the cell types in adenomyotic tissue, eutopic endometrium, and normal myometrium of the women with diffuse adenomyosis. Adenomyotic tissue (n=3), eutopic endometrium (n=3), and myometrium (n=3) were obtained from premenopausal women who underwent hysterectomy for diffuse adenomyosis and confirmed by frozen section histologic evaluation. Following digestion, the stromal and smooth muscle cells were collected, and the epithelial cell pellets were further digested. After checking the viability, which was at least 75%, samples subjected the 10x Genomics® Single Cell Protocol. Processed data from the Cell Ranger pipeline was analyzed using the R package Seurat (v3.2). Uniform Manifold Approximation and Projection was generated to visualize cell clusters. The Wilcoxon rank sum test was used to compare the gene expression profile for each cluster which were annotated by marker genes. We identified ciliated and unciliated epithelial cells, fibroblast-like cells, smooth muscle cells (SMC), endothelial cells, pericytes, vascular SMCs, progenitor cells, T cells, NK cells, B cells, MQ, and monocytes, neurons and myeloid progenitors in the adenomyotic uterus. Notably, comparative transcriptome analysis of epithelial cells in eutopic endometrium and adenomyotic tissue revealed a small subset of differentially expressed genes, indicating the stability of the transcriptional program of these cells. On the other hand, we identified a high degree of heterogeneity among the fibroblast populations. Intriguingly, fibroblast-like cells in adenomyotic vs eutopic endometrial tissue showed higher expression of progesterone-responsive genes. In contrast, eutopic endometrial epithelial cells displayed higher expression of progesterone-responsive genes. Pseudo-time analysis in eutopic endometrium have demonstrated evidence of both epithelial-mesenchymal and mesenchymal-epithelial transition originating from a progenitor cell population and pericytes, respectively. Our analyses revealed 15 distinct cell populations in adenomyotic tissue. Fibroblast-like cells constituted the majority of all cell types which showed a high degree of heterogeneity, which may be due to different transcriptional states linked to the intense inflammatory processes in adenomyosis. Our results suggest that progesterone response may vary between cell types in adenomyotic tissue. The lack of a substantial difference between epithelial cells in adenomyosis and adjacent endometrium supports the postulated mechanism whereby invagination of eutopic endometrial tissue is responsible for adenomyosis.