Mechanosensing in Human Vaginal Cells Relating to Pelvic Organ Prolapse

Objectives: Vaginal stromal cells play an important role in extracellular matrix (ECM) deposition which is responsible for maintenance of tissue integrity. Disordered ECM is associated with the development of pelvic organ prolapse (POP), but it is unknown if this is due to an intrinsic defect in stromal cell mechanosensing ability as tissue changes over time. In this study we aimed to determine the response of vaginal stromal cells to changes in matrix stiffness. We hypothesized that there will be a differential response between cells obtained from pre- and post-menopausal women with and without POP.

Methods: Vaginal tissue was obtained from pre-menopausal women with and without prolapse and post-menopausal women with prolapse (n=4, age range 34-70years). Stromal cells were isolated and seeded on 1kPA or 10kPA polyacrylamide gel matrix plates. RNA was isolated and quantitative polymerase chain reaction (qPCR) was performed to quantify gene expression of vimentin, alpha smooth muscle actin (ACTA2), nuclear protein Ki67, and matrix metalloproteinases (MMPs) 2, 7, and 9. Statistical analysis was performed using two-way ANOVA followed by a Tukey post-hoc test.

Results: Gene expression of ACTA2, MMP-7, and MMP-9 was higher in the post-menopausal prolapse group, compared to the pre-menopausal prolapse group on both the 1kPA (p < 0.0001) and 10kPA (p < 0.01) gel matrix plates. Ki67 expression was higher in the post-menopausal group compared to the pre-menopausal group on the 1kPa gel matrix plates (p=0.006). There was no difference in Vimentin gene expression between the pre-menopausal and post-menopausal prolapse patients on both the 1kPA and 10kPA gel matrix plates. Vaginal stromal cells of pre-menopausal women with prolapse exhibit an increase in Ki67 and MMP-2 expression when grown on the higher stiffness substrate, compared to post-menopausal women with prolapse. In contrast, MMP-9 expression decreased at higher stiffness in the post-menopausal prolapse group (Figure). There were no significant differences in relative expression of ACTA2 and Vimentin between the 1kPA and 10kPA gel matrix plates in any of the groups.

Figure. Quantitative gene expression for (a) Ki67 (b) MMP-2 and (c) MMP-9 when fibroblasts grown on low 1kPa versus high 10kPa gel matrix plates in pre- and post-menopausal women with prolapse. *p < 0.05

Conclusions: In this in-vitro study we found that vaginal stromal cells of post-menopausal women with prolapse have a higher expression of pro inflammatory (MMP-7 and MMP-9) genes compared to those of pre-menopausal women with prolapse, regardless of matrix stiffness. The relative increase in Ki67 and MMP2 expression in the pre-menopausal group with prolapse compared to the post-menopausal group with prolapse suggests that the mechanosensing ability of the pre-menopausal group with prolapse may remain intact. Our findings provide insights into the potential differences in POP pathophysiology in pre- vs post-menopausal women.