Single-Cell Profiling Uncovers the Roles of Endometrial Fibrosis and Microenvironmental Changes in Adenomyosis

J Inflamm Res. 2023 May 5:16:1949-1965. doi: 10.2147/JIR.S402734. eCollection 2023.

Abstract

Purpose: Adenomyosis (AM) is a common benign uterine disorder that has deleterious effects on women's health. However, the pathogenesis of AM is not clearly understood. We aimed to investigate the pathophysiological changes and molecular mechanism in AM.

Methods: Single-cell RNA sequencing (scRNA-seq) was employed to construct a transcriptomic atlas of various cell subsets from the ectopic endometrium (EC) and eutopic endometrium (EM) of one AM patient and evaluate differential expression. The Cell Ranger software pipeline (version 4.0.0) was applied to conduct sample demultiplexing, barcode processing and mapping reads to the reference genome (human GRCh38). Different cell types were classified with markers with the "FindAllMarkers" function, and differential gene expression analysis was performed with Seurat software in R. The findings were confirmed by Reverse Transcription Real-Time PCR using samples from three AM patients.

Results: We identified nine cell types: endothelial cells, epithelial cells, myoepithelial cells, smooth muscle cells, fibroblasts, lymphocytes, mast cells, macrophages and unknown cells. A number of differentially expressed genes, including CLO4A1, MMP1, TPM2 and CXCL8, were identified from all cell types. Functional enrichment showed that aberrant gene expression in fibroblasts and immune cells was related to fibrosis-associated terms, such as extracellular matrix dysregulation, focal adhesion and the PI3K-Akt signaling pathway. We also identified fibroblast subtypes and determined a potential developmental trajectory related to AM. In addition, we identified increased cell-cell communication patterns in EC, highlighting the imbalanced microenvironment in AM progression.

Conclusion: Our results support the theory of endometrial-myometrial interface disruption for AM, and repeated tissue injury and repair could lead to increased fibrosis in the endometrium. Therefore, the present study reveals the association between fibrosis, the microenvironment, and AM pathogenesis. This study provides insight into the molecular mechanisms regulating AM progression.

Keywords: AM; ectopic endometrium; eutopic endometrium; extracellular matrix; fibrosis; scRNA-seq.

Grants and funding

The research was supported by the National Natural Science Foundation of China (grant no. 81873330, 82104916), the Natural Science Foundation of Shandong Province (grant no. ZR2020QH337, ZR2021MH198) and the Science and Technology Plan Project of Jinan (grant no. 201907009), the Shandong Province: Taishan Scholars Project (grant no. tsqn201909185), The Traditional Chinese Medicine Science and Technology Development Plan Project of Shandong Province (Grant no. 2021M186) and the China Postdoctoral Science Foundation (Grant no. 2022M722000).