Single-cell transcriptomic analysis of eutopic endometrium and ectopic lesions of adenomyosis

Abstract

Background Adenomyosis (AM) is a common benign chronic gynaecological disorder; however, the precise pathogenesis of adenomyosis is still poorly understood. Single-cell RNA sequencing (scRNA-seq) can uncover rare subpopulations, explore genetic and functional heterogeneity, and reveal the uniqueness of each cell. It provides us a new approach to reveal biological issues from a more detailed and microscopic perspective. Here, we utilize this revolutionary technology to identify the changes of gene expression patterns between ectopic lesions and the eutopic endometrium at the single-cell level and explore a potential novel pathogenesis of AM. Methods A control endometrium (sample with leiomyoma excluding endometrial disorders, n = 1), eutopic endometrium and ectopic lesion (from a patient with adenomyosis, n = 1) samples were analysed by scRNA-seq, and additional leiomyoma (n = 3) and adenomyosis (n = 3) samples were used to confirm colocalization and vasculogenic mimicry (VM) formation. Protein colocalization was visualized by immunofluorescence, and CD34-periodic acid-Schiff (PAS) double staining was used to assess the formation of VM. Results The scRNA-seq results suggest that cancer-, cell motility- and inflammation- (CMI) associated terms, cell proliferation and angiogenesis play important roles in the progression of AM. Moreover, the colocalization of EPCAM and PECAM1 increased significantly in the ectopic endometrium group (P < 0.05), cell subpopulation with high copy number variation (CNV) levels possessing tumour-like features existed in the ectopic lesion sample, and VNN1- and EPCAM-positive cell subcluster displayed active cell motility in endometrial epithelial cells. Furthermore, during the transformation of epithelial cells to endothelial cells, we observed the significant accumulation of VM formation (positively stained with PAS but not CD34, P < 0.05) in ectopic lesions. Conclusions In the present study, our results support the theory of adenomyosis derived from the invasion and migration of the endometrium. Moreover, cell subcluster with high CNV level and tumour-associated characteristics is identified. Furthermore, epithelial-endothelial transition (EET) and the formation of VM in tumours, the latter of which facilitates the blood supply and plays an important role in maintaining cell growth, were also confirmed to occur in AM. These results indicated that the inhibition of EET and VM formation may be a potential strategy for AM management.

Single-cell transcriptomic analysis of endometriosis provides insights into fibroblast fates and immune cell heterogeneity

“…The Cell Ranger software pipeline (version 3.1.0) provided by 10 × Genomics was used to demultiplex cellular barcodes, map reads to the genome and transcriptome using the STAR aligner, and down-sample reads as required to generate normalised aggregate data across samples, producing a matrix of gene counts versus cells [ 59 , 60 ]. We processed the UMI count matrix using the R package Seurat (version 3.0.0) to remove low-quality cells and likely multiples.…”

“…We then annotated the clusters based on the average expression of curated gene sets of the following major cell types: epithelial cells ( EPCAM, KRT19, KRT18, KRT5, KRT15 ), macrophages/monocytes ( CD68, MS4A4A, MS4A7 ), T cells ( CD2, CD3D/E/G ), MAST cells ( CD117, TPSB2, TPSAB1 ), NK cells ( TRDC, KLRC1 ), neutrophils ( CD16, CD66 ), ECs ( AQP1, MYCT1, CDH5, PECAM1 ), and FBs ( COL1A1, COL3A1, COL1A2 )(representative marker reference from: http://biocc.hrbmu.edu.cn/CellMarker/ and https://panglaodb.se/search.html ) [ 60 ].…”

Adenomyosis as a Risk Factor for Myometrial or Endometrial Neoplasms—Review

“…Liu et al proved the theory about the migration and invasion of the endometrium into the myometrium. Their results indicated that the inhibition of EET (epithelial–endothelial transition) and VM formation (vascular-mimicry formation) may be a potential strategy for adenomyosis management [ 42 ].…”

Uterine Adenomyosis: From Disease Pathogenesis to a New Medical Approach Using GnRH Antagonists

“…Further studies indeed corroborated the hypothesis of invasiveness, as adenomyotic glands appear to resemble those of eutopic endometrium and most likely originate from them [ 18 ]. Moreover, single-cell transcriptomic data detected a clear upturn in genes related to cell motility and cancer-like features in adenomyosis [ 19 ]. It has also been hypothesized that estrogen itself drives EMT in adenomyosis, although other studies have proposed inflammation-associated factors as mediators of this process [ 16 , 20 , 21 ].…”