Pelvic organ prolapse (POP) is a prevalent and debilitating condition that affects up to 30-50% of woman, causing symptoms in nearly 25% of patients. Although prosthetic materials used in pelvic floor reconstruction have improved outcomes, there are concerns about high rates of symptomatic recurrence and associated complications. The underlying mechanisms driving these complications remain poorly elucidated, but there is growing interest in studying the nature of mesh interaction with pelvic floor support tissues mechanically compromised and biologically dysfunctional.  
Fibroblasts are crucial for maintaining the vaginal wall’s integrity, including tissue structure, damage repair, and extracellular matrix (ECM) regulation. In the context of POP, vaginal fibroblasts undergo notable alterations compared to healthy counterparts, such as reduced proliferation, morphological changes, differential ECM expression, and impaired adaptability to substrate stiffness [1,2]. These fibroblast dysfunctions collectively contribute to POP pathogenesis and complications from mesh-based pelvic floor reconstruction. Understanding the distinctive responses of these dysfunctional fibroblasts is essential for characterizing the role of the biological microenvironment in POP and developing more effective, personalized treatments.
The aim of the study was to identify novel molecular pathways involved in POP pathogenesis by dissecting the transcriptional landscape of vaginal wall fibroblasts, focusing on the differential expression of genes encoding ECM and cell adhesion-related molecules in POP and control non-POP fibroblasts. Given the central role of vaginal tissue biomechanics, especially stiffness, in POP onset and subsequent repair using biomaterials [3], the study also assessed and compared the impact of the microenvironment stiffness on the fibroblasts’ transcriptional profiles.

Full thickness biopsies (>1 cm2) from the anterior vaginal wall were surgically excised from women suffering POP and healthy controls operated on for benign gynecological pathology (n=5 both groups, based on information from previous studies). Tissue collection was approved by the Clinical Research Ethics Committee of our hospital and all participants signed a written informed consent. Biopsies were collected and immediately processed to isolate primary human vaginal fibroblasts by a double trypsin digestion. Experiments were carried out with fibroblast-derived cell lines within 3-8 passages. 
To examine the impact of matrix stiffness on fibroblasts, we cultured 2 × 105 cells in collagen coated polydimethylsiloxane (PDMS) substrates with various stiffness covering a physiological range that mimicked the vaginal wall (2 - 32 kPa) [1], and a standard cell culture plastic (approximately 1 GPa). After 48 hours under standard culture conditions, fibroblasts were processed for total RNA extraction. Two samples from pooled fibroblasts were used for each condition. The cDNA was reverse-transcribed and analyzed using a SYBR green-based quantitative real-time qPCR array (Qiagen; Hilden, Germany) to profile 84 genes coding for ECM proteins and proteoglycans, as well as ECM-cell adhesion molecules. Results were normalized against 3 reference genes. Genes with Ct values above 30 were considered negative and excluded from the analysis. Normalized gene expression was calculated relative to calibrators using the 2−ΔΔCt method. Whole analysis included principal component analysis (PCA) and hierarchical clustering to classify genes into different co-expression modules according to their similarity in the expression profiles based on Pearson’s correlation. Analysis of functional enrichment and interaction among the genes (PPI networks) were performed using Metascape (www.metascape.org) for each co-expression module.

Among the initial 84 genes examined, 58 genes met the criteria for inclusion in the analysis based on Ct values. Principal component analysis effectively distinguished POP and non-POP fibroblasts, indicating notable disparities in the expression of ECM and cell adhesion genes in response to substrate stiffness variations. Non-supervised heatmap analysis unveiled four distinct co-expression modules (Figure 1): 1) a cluster primarily associated with ECM organization, with higher expression in POP fibroblasts irrespective of substrate stiffness, where genes like CD44 and ITGB1 were central in the PPI network, and co-expressed alongside TGFBI and TIMP1; 2) a smaller cluster enriched in cell adhesion and membrane-ECM interaction genes, up-regulated in both POP and non-POP fibroblasts on the stiffest substrate (polystyrene plastic, PS); 3) another cluster enriched in integrin-related and collagen formation genes, also up-regulated in POP cells on the stiffest substrates, including FN1 and COL1A1, particularly prominent and central within the PPI network; and 4) a minor group comprising collagenases MMP1 and MMP3, and MMP16, significantly down-regulated in POP fibroblasts compared to non-POP, regardless of experimental conditions.

Results were able to distinguish POP fibroblasts from those of healthy non-POP tissues, supporting the distinctive phenotype of POP fibroblasts. Notably, POP fibroblasts showed robust upregulation of transforming growth factor beta 1 (TGB1), various fibrillar and non-fibrillar collagens, and ECM glycoproteins, indicative of a fibrotic signature. Moreover, our findings showed a concomitant reduction in extracellular proteolytic activity, attributed to alterations in the expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), further underscoring the dysregulated ECM remodeling characteristic of POP (Figure 2). In addition to elucidating the ECM remodeling dynamics, our study revealed that POP fibroblasts displayed divergent expression patterns of adhesion molecules, particularly integrins, which mediate cell-ECM interactions, aligning with a myofibroblast phenotype associated with fibrosis [1, 3]. Furthermore, our results demonstrated a significant effect of substrate stiffness on the transcriptional profile of POP fibroblasts, with the highest stiffness exacerbating the fibrotic phenotype, characterized by increased expression of ECM structural proteins, together with decreased MMP and TIMP gene expression.

Our study unveils molecular mechanisms driving the pathogenesis of POP, shedding new light on the intricate interplay between extracellular stiffness, gene expression patterns, and the fibrotic phenotype of vaginal fibroblasts. Stiffness exacerbated the fibrotic phenotype of POP fibroblasts, potentially leading to the deposition of a stiffer ECM. These findings suggest the existence of a profibrotic positive feedback loop, where the fibrotic ECM secreted by POP fibroblasts perpetuates through chemical and mechanical signaling pathways. This differential response of POP fibroblasts to stiffness underlies the weakening process of the vaginal wall characteristic of POP, and may contribute to the complications associated with pelvic floor reconstruction. The identified molecules and associated pathways represent promising targets for future investigations aimed at elucidating novel preventive or therapeutic interventions for POP. These findings may pave the way for development of personalized biomaterials-based treatments tailored to address the multifactorial and complex nature of this condition, ultimately improving patient outcomes and quality of life.

Ruiz-Zapata, A.M.; Heinz, A.; Kerkhof, M.H.; van de Westerlo-van Rijt, C.; Schmelzer, C.E.H.; Stoop, R.; Kluivers, K.B.; Oosterwijk, E. Extracellular Matrix Stiffness and Composition Regulate the Myofibroblast Differentiation of Vaginal Fibroblasts. Int. J. Mol. Sci. 2020, 21, 4762. https://doi.org/10.3390/ijms21134762
Quiles, M.; Sabadell, J.; Rodriguez-Contreras, A.; Manero, J.M.; Salicru, S.; Montero, A.; Poza, J.; Gil-Moreno, A.; Armengol, M.; Arbós, M. Differential interaction between vaginal fibroblasts and polypropylene from prolapse and healthy controls and the effect of material’s surface modifications. Continence 2022, 2, Suppl. 2, 100244. https://doi.org/10.1016/j.cont.2022.100244.
Knight, K. M.; King, G. E.; Palcsey, S. L.; Suda, A.; Liang, R.; Moalli, P. A. (2022). Mesh deformation: A mechanism underlying polypropylene prolapse mesh complications in vivo. Acta Biomater. 2022, 148, 323. https://doi.org/10.1016/j.actbio.2022.05.051.