According to a recent cross-sectional study, the prevalence of symptomatic pelvic organ prolapse in Chinese women is 9.6%[1]. One of the risk factors is transvaginal delivery[16], which can produce a huge stretch on the vaginal tissues by increasing intra-abdominal pressure, thus affecting the function of fibroblasts leading to changes in collagen metabolism, altering the strength of the tissues and eventually prolapse. Another important factor is menopause, which rapidly decreases estrogen production in post-menopausal women, resulting in atrophy, thinning and inflammation of the vagina, which deprives the pelvic floor of the ability to compensate for the trauma associated with childbirth, there by weakening the support network[13]. In addition, it has been shown that estrogen can influence the level of LOXL1[17] and thus the occurrence of POP. The connective tissue at the site of prolapse is stiffer and exhibits a higher modulus of elasticity compared to healthy tissue, similar to the characteristics of sarcoid tissue[18]. Several studies have suggested that the abnormal extracellular matrix in prolapsed tissue may maintain POP by regulating the differentiation of vaginal fibroblasts to myofibroblasts[19–21]. Fibroblasts can sense an increase in environmental stiffness and differentiate into myofibroblasts, a process known as fibroblast myofibroblast transformation[22]. The marker for myofibroblasts is α-SMA, which accelerates tissue repair during the repair phase of tissue damage, but sustained activation can cause pathological deposition of extracellular matrix and loss of tissue elasticity[23]. It has been shown that α-SMA levels in the anterior vaginal wall are significantly higher in patients with POP [18], suggesting that fibroblasts are relatively quiescent under physiological conditions and that myofibroblasts have a higher abundance in POP patients, but the mystery in between remains to be explored.
The main treatment for POP is currently surgery. Natural (allograft, autograft and xenograft) and synthetic implants (permanent and biodegradable) have been introduced to surgically correct POP[24, 25]. Synthetic permanent polypropylene (PP) mesh has been used for the surgical repair of POP, which provides mechanical support to the pelvic floor by inducing a foreign body response. However, PP mesh has been associated with clinical complications, such as the use of polypropylene mesh that is stiffer than native tissue for vaginal reconstruction, which can further compromise the mechanical properties of the vagina[7, 26]. In animal models it has been shown that the mechanical properties of the vagina deteriorate after implantation of stiffer patches, suggesting that not only the vaginal wall but also the stiffness of the mesh can affect the biological properties of the vagina[7]. In addition, it has been suggested that the increased risk of pelvic floor disorders after menopause may be related to estrogen deficiency and changes in estrogen receptor expression[15]. Women with persistently low estrogen levels show delayed recovery due to impaired wound healing[27]. Therefore, the addition of estrogen to the surgical site may promote fibroblast proliferation and collagen synthesis, thereby promoting pelvic floor tissue repair[28].
In summary, abnormal stiffness in the anterior vaginal wall may maintain POP by regulating fibroblast to myofibroblast differentiation, and estrogen has an irreplaceable role in the treatment of POP. It is therefore clinically important to investigate the fate regulation of fibroblasts by extracellular matrix stiffness and the therapeutic mechanisms of estrogen in POP.
Our results found that the anterior vaginal wall of POP patients had a significantly higher elastic modulus than controls, i.e. increased extracellular matrix stiffness, and a significant increase in α-SMA expression in vaginal wall tissue, suggesting a higher abundance of vaginal wall myofibroblasts, but the relationship between extracellular matrix stiffness and fibroblast differentiation remains to be explored. In recent years, there has been evidence that epigenetic mechanisms in vascular smooth muscle cells play a key role in mediating environmental stiffness signalling to induce cell phenotype regulation. However, understanding of the epigenetic regulation triggered by mechanical stimulation of fibroblasts is limited. Here, we report the effects of microenvironmental stiffness on fibroblast differentiation mediated by DNMT1. DNMT1 is a mechanosensitive protein and our study identifies a fundamental role for DNMT1 as a downstream component of mechanosensors in how fibroblasts perceive their physical microenvironment. When the matrix stiffens in elasticity, cells produce less DNMT1, leading to a decrease in DNA methylation levels. The detailed mechanotransduction mechanism by which ECM stiffness regulates DNMT1 awaits further characterisation, but our experimental results suggest that the process involves the polymerisation of microtubules. These findings have potential implications for the study of mechanotransduction and highlight the importance of DNMT1 in mediating the interaction between fibroblast and stromal stiffness. In addition, postmenopausal estrogen reduction is an important risk factor for the development of POP, and it has been shown that estrogen enhances the efficacy of vaginal pelvic floor electrical stimulation (PFS) for pelvic organ dysfunction and delays its recurrence[29]. We therefore investigated the possibility that estrogen inhibits fibroblast differentiation. We were encouraged to find that estrogen treatment increased DNMT1 expression and decreased α-SMA and CTGF expression in fibroblasts. However, there are some limitations to this study. Firstly, we have not yet determined how physical signals are translated into biological signals through cellular microtubule aggregation. Our preliminary study found that the integrin family and Hippo family may be involved in this process. Second, how estrogen regulates DNMT1 expression remains to be explored, and preliminary studies suggest that estrogen may affect DNMT1 expression through interaction with estrogen receptor2(ER2). Our study suggests a potential mechanism for fibroblast differentiation by showing that when mechanosensitive proteins (in our case DNMT1) receive signals from the mechanical microenvironment, the cells respond by changes in their stiffness, and thus offers new ideas and possibilities for the treatment of patients with POP and the selection of stiffness-related properties of implant materials. This article may contribute to a better understanding of the role of fibroblasts in pelvic organ prolapse and how to optimise surgical approaches, such as the choice of implant material stiffness.