Single-cell Transcriptome Profiling of the Murine Vagina with Age

Objectives: Risk of vaginal and pelvic floor injury with fetal delivery and the long-term risk of pelvic floor disorders increases with maternal age. With more women having their first child later in life, there is an intensifying need to identify and understand vaginal changes with age. Age-related decreases in tissue elasticity and the inability to accommodate large tissue stretches during vaginal delivery may drive increased risk of obstetric injury and pelvic organ prolapse (POP). Our prior work focused on mechanical testing of the murine vagina showed a diminished contribution of elastic fibers and decreased smooth muscle cell (SMC) contractility with age. However, the cell populations and cell-specific signaling contributing to changes in the vaginal extracellular matrix (ECM) and SMC behavior with age remains unknown. The objective of this study was to use single cell RNA sequencing (scRNAseq) to identify changes in cell populations and gene expression with age in the murine vagina. We hypothesized that cells will show age-related differences in genes associated with ECM components specific to elastic fibers, contractile proteins, and immunologic mediators.

Methods: Non-pregnant CD-1 mice aged 2-3 and 7-9 months (n=3/group; IACUC approved) were sacrificed, and the vagina was immediately isolated. Single cell suspensions were created by mechanical dissociation, followed by repeated digestion in media containing Collagenase I, Dispase II, Elastase and DNase I. Cells were collected through a cell strainer and resuspended in PBS with BSA before confirming viability and concentration. Single cell barcoding and library preparation was performed (Chromium platform, 10x Genomics; Illumina NextSeq 2000) and CellRanger was used for barcode filtering and read alignment. R packages (Seurat, clustifyr and clusterProfiler) were used for QC, clustering, vaginal cell identification, labeling, and differential expression (DE) analysis.

Results: Our dissociation protocol consistently provided single cells in suspension with high yield and viability (1,270,000 cells with 84.9% viability, on average). Analysis of scRNAseq data allowed for identification of multiple vaginal cell types and subtypes (Figure 1a). Expression of Eln (elastin) was primarily localized to fibroblasts (Figure 1b); additional canonical gene markers are shown: Myh11 (smooth muscle), Col1a1 (fibroblast), and Ptprc (immune). DE analysis revealed higher relative expression of Eln in fibroblasts from young mice. Age-dependent changes in contractile gene expression were found in SMCs; multiple genes were altered in immune and epithelial cells which may suggest altered function with age (Figure 2; top 20 DE genes). Notably, the identified proportion of adaptive immune cells in the vagina was elevated (16% young vs. 27% old of total immune population) suggesting an altered immunological environment with age.

Conclusions: Understanding the distribution of cells and gene expression profiles that contribute to vaginal structural integrity with age can help identify therapeutic targets for obstetric injury and POP.