Fibroblast heterogeneity and abnormal phenotype transition in vaginal wall prolapse at single-nucleus transcriptional...

Pelvic organ prolapse significantly impacts women's physical and mental health. Although current research primarily focuses on the analysis of extracellular matrix components, the underlying pathologic imbalances of pelvic organ prolapse remain incompletely understood. The functional roles and phenotypic changes of fibroblasts within the complex microenvironment of prolapsed tissue warrant further investigation.

To decipher the pathological basis of pelvic organ prolapse at single-cell resolution through analyzing the heterogeneity of fibroblasts, exploring prolapse-specific fibroblast subtypes and their functional characteristics.

A total of 6 full-thickness prolapsed vaginal wall tissue samples were collected from patients with stage III or IV pelvic organ prolapse (pelvic organ prolapse group), along with 3 control tissue samples from patients with stage 0-I prolapse who underwent total hysterectomy for benign gynecologic diseases (CTRL group). Nuclei were isolated from frozen tissues and processed into single-nucleus suspensions for single-nucleus RNA sequencing. The sequencing data were subjected to dimensionality reduction, clustering, cell type annotation, and subpopulation identification to construct a single-nucleus transcriptome atlas of the anterior vaginal wall, with a focused exploration of fibroblast heterogeneity. Gene Ontology biological process enrichment analysis was performed to investigate dysregulated cellular functions and the functional characteristics of fibroblast subpopulations. Pseudotime trajectory analysis was performed to construct fibroblast differentiation trajectories and investigate fibroblast subpopulation specific differentiation aberrations. The main findings of bioinformatics analysis were validated by immunofluorescence staining and in vitro functional assays.

The transcriptomes of 96,622 vaginal wall cell nuclei isolated from pelvic organ prolapse and CTRL group were profiled. Fibroblasts, endothelial cells, and epithelial cells were the major cell populations in the anterior vaginal wall. Fibroblasts exhibited significantly higher enrichment scores for extracellular matrix and related gene sets compared to other cell types, underscoring their critical role in regulating extracellular matrix homeostasis in pelvic floor tissues. In the pelvic organ prolapse group, we observed an increased proportion of proinflammatory (IL6ST⁺_FIB) and matrix-degrading (MMP2⁺_FIB) fibroblast subpopulations, alongside a decreased proportion of extracellular matrix-synthesis (COL1A1⁺_FIB) and mesenchymal (POSTN⁺_FIB) fibroblast subpopulations. Pseudotime trajectory analysis further revealed that fibroblast differentiation in pelvic organ prolapse samples shifted away from an extracellular matrix-synthesis phenotype toward pro-inflammatory and matrix-degradating phenotypes. Functional enrichment analysis of fibroblasts showed that biological processes related to extracellular matrix remodeling and negative regulation of cell migration and growth were upregulated. Consistently, fibroblasts isolated from prolapsed tissue exhibited impaired proliferation and migration capabilities.

Fibroblasts in prolapsed vaginal wall exhibited marked heterogeneity compared to nonprolapsed tissue, comprising 6 functionally distinct subpopulations, including those involved in extracellular matrix synthesis, pro-inflammatory, and matrix degradation. In the prolapse microenvironment, an aberrant phenotypic shift was observed that fibroblasts transitioned from an extracellular matrix-stable phenotype predominantly characterized by COL1A1⁺_FIB toward proinflammatory (IL6ST⁺_FIB) and matrix-degrading (MMP2⁺_FIB) phenotypes, which contributes to extracellular matrix disorganization and impaired tissue repair capacity. These findings further enhance the understanding of the fibroblast phenotype imbalance underlying pelvic organ prolapse and provide novel therapeutic strategies targeted at modulating fibroblast function to restore pelvic floor.