BACKGROUND: Fibrosis of the connective tissue in the vaginal wall predominates in pelvic organ prolapse (POP), which is characterized by excessive fibroblast-to-myofibroblast differentiation and abnormal deposition of the extracellular matrix (ECM). Our study aimed to investigate the effect of ECM stiffness on vaginal fibroblasts and to explore the role of methyltransferase 3 (METTL3) in the development of POP. METHODS: Polyacrylamide hydrogels were applied to create an ECM microenvironment with variable stiffness to evaluate the effects of ECM stiffness on the proliferation, differentiation, and expression of ECM components in vaginal fibroblasts. METTL3 small interfering RNA and an overexpression vector were transfected into vaginal fibroblasts to evaluate the effects of METTL3 silencing and overexpression on matrix stiffness-induced vaginal fibroblast-to-myofibroblast differentiation and abnormal modulation of the ECM. Both procedures were detected by 5-ethynyl-2'-deoxyuridine (EdU) staining, Western blotting (WB), quantitative real-time polymerase chain reaction (RT-qPCR), and immunofluorescence (IF). RESULTS: Vaginal fibroblasts from POP patients exhibited increased proliferation ability, increased expression of α-smooth muscle actin (α-SMA), decreased expression of collagen I/III, and significantly decreased expression of tissue inhibitors of matrix metalloproteinases (TIMPs) in the stiff matrix ( P <0.05). Compared with those from non-POP patients, vaginal wall tissues from POP patients demonstrated a significant increase in METTL3 content ( P <0.05). However, silencing METTL3 expression in vaginal fibroblasts with high ECM stiffness resulted in decreased proliferation ability, decreased α-SMA expression, an increased ratio of collagen I/III, and increased TIMP1 and TIMP2 expression. Conversely, METTL3 overexpression significantly promoted the process of increased proliferation ability, increased α-SMA expression, decreased ratio of collagen I/III and decreased TIMP1 and TIMP2 expression in the soft matrix ( P <0.05). CONCLUSIONS Elevated ECM stiffness can promote excessive proliferation, differentiation, and abnormal ECM modulation, and the expression of METTL3 plays an important role in alleviating or aggravating matrix stiffness-induced vaginal fibroblast-to-myofibroblast differentiation and abnormal ECM modulation.
Humans ; Female ; Extracellular Matrix/metabolism* ; Cell Differentiation/genetics* ; Methyltransferases/metabolism* ; Pelvic Organ Prolapse/pathology* ; Fibroblasts/metabolism* ; Myofibroblasts/metabolism* ; Vagina/metabolism* ; Cell Proliferation/physiology* ; Cells, Cultured ; Middle Aged

OBJECTIVES: Pelvic organ prolapse (POP) is a common condition in postmenopausal women, with an increasing prevalence due to aging. Some women experience POP recurrence after surgical treatment, significantly affecting their physical and mental health. The uterosacral ligament is a critical pelvic support structure. This study aims to investigate the molecular pathological changes in the uterosacral ligament of postmenopausal women with recurrent POP using transcriptomic analysis. METHODS: Transcriptomic data of uterosacral ligament tissues were obtained from the public dataset GSE28660, which includes samples from 4 postmenopausal women with recurrent POP, 4 with primary POP, and 4 without POP. Differentially expressed genes (DEGs) were identified between recurrent POP and both primary and non-POP groups. Further analysis included intersection analysis of DEGs, gene ontology enrichment, protein-protein interaction (PPI) network construction, gene set enrichment analysis (GSEA), single-sample GSEA, and xCell immune cell infiltration analysis to explore molecular pathological changes in recurrent POP. Additionally, histological and molecular differences in the uterosacral ligament were compared between simulated vaginal delivery (SVD) rat models with and without ovariectomy. RESULTS: Compared with primary POP and non-POP groups, recurrent POP exhibited activation of adipogenesis and inflammation-related pathways, while pathways related to muscle proliferation and contraction were downregulated in the uterosacral ligament. Nine key DEGs (ADIPOQ, FABP4, IL-6, LIPE, LPL, PCK1, PLIN1, PPARG, and CD36) were identified, with most enriched in the peroxisome proliferator-activated receptor (PPAR) signaling pathway. These genes were significantly correlated with lipid accumulation, monocyte infiltration, and neutrophil infiltration in the uterosacral ligament. Urodynamic testing revealed that the bladder leak point pressure was significantly higher in ovariectomized SVD rats, both of which had higher values than the sham group. Masson staining showed pronounced adipogenesis in the uterosacral ligament of ovariectomized SVD rats, along with reduced collagen and muscle fibers compared to the sham and non-ovariectomized SVD groups. Furthermore, real-time RT-PCR confirmed significantly elevated expression of key DEGs, including ADIPOQ, IL-6, PCK1, and PLIN1, in the uterosacral ligaments of ovariectomized SVD rats. CONCLUSIONS Adipogenesis and inflammation in the uterosacral ligament may contribute to its reduced supportive function, potentially leading to recurrence POP in postmenopausal women.
Female ; Humans ; Ligaments/pathology* ; Pelvic Organ Prolapse/metabolism* ; Postmenopause ; Animals ; Rats ; Adipogenesis/genetics* ; Recurrence ; Gene Expression Profiling ; Transcriptome ; Middle Aged ; Ovariectomy ; Protein Interaction Maps ; Aged ; Rats, Sprague-Dawley ; Uterus

Humans ; Matrix Metalloproteinase 9 ; Collagen Type I ; Pelvic Organ Prolapse/metabolism*

BACKGROUNDPelvic organ prolapse (POP) is a major health problem in adult women that involves many factors. No proteomic analysis has been conducted exclusively in POP patients. This study aimed to identify the differential expression of proteins that may be involved in POP by proteomic analysis.

METHODSSamples of the uterosacral ligament (USL) were collected from five POP patients and five non-POP patients matched according to age, parity, and menopausal status and analyzed using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the mRNA expression of proteins that showed differential expression in the proteomic analyses.

RESULTSProteins differentially expressed between POP and non-POP patients were detected. Eight proteins that were down-regulated in the POP group were identified by MALDI-TOF-MS. These proteins included electron transfer flavoprotein, apolipoprotein A-I, actin, transgelin, cofilin-1, cyclophilin A, myosin, and galectin-1, and their expression was verified by qRT-PCR.

CONCLUSIONUsing comparative proteomics, we identified eight differentially expressed proteins (including four cytoskeleton proteins and three proteins related to apoptosis) in the USL that may be involved in apoptosis associated with the tissue effects in POP pathophysiology.

Actins ; metabolism ; Aged ; Apolipoprotein A-I ; metabolism ; Cyclophilin A ; metabolism ; Cytoskeleton ; metabolism ; Female ; Flavoproteins ; metabolism ; Galectin 1 ; metabolism ; Humans ; Ligaments ; metabolism ; Microfilament Proteins ; metabolism ; Middle Aged ; Muscle Proteins ; metabolism ; Myosins ; metabolism ; Pelvic Organ Prolapse ; metabolism ; Postmenopause ; metabolism ; Proteomics ; methods ; Reverse Transcriptase Polymerase Chain Reaction ; Sacrum ; metabolism ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Uterus ; metabolism

PURPOSE: To evaluate the possible influence of G-->T substitution at the Sp1-binding site of the COLIA1 gene on the risk of pelvic organ prolapse (POP). MATERIALS AND METHODS: The study group consisted of 15 women with advanced stage POP. Fifteen control subjects with uterine myomas among the postmenopausal women were matched for age and parity. DNA was obtained from peripheral blood leukocytes. The fragments of the first intron of the COLIA1 gene were amplified by real time polymerase chain reaction. The polymorphism was identified using LightCycler Technology with hybridization probes. Sequencing reactions were performed on each template using commercial primer. RESULTS: Two groups had no significant difference in medical history, surgical, and smoking history. The homozygous peaks in two groups were noted at 57degrees C on melting curve analysis. Sequencing reactions confirmed the G/G alleles in the 30 specimens tested. We could not find any polymorphism at the Sp1-binding site in COLIA1 gene with advanced stage POP. Statistical significance was considered to be p < .05. CONCLUSION: The polymorphism of the Sp1-binding site in the COLIA1 gene did not contribute to the development of POP in Korea.
Aged ; Asian Continental Ancestry Group/genetics ; Binding Sites/genetics ; Collagen Type I/*genetics ; Female ; Genetic Predisposition to Disease ; Humans ; Middle Aged ; Pelvic Organ Prolapse/*genetics ; Polymerase Chain Reaction ; Polymorphism, Genetic/*genetics ; Sp1 Transcription Factor/*metabolism

OBJECTIVE: To identify 1) whether the endogenous steroid hormone metabolism in patients with pelvic organ prolapse was different from that of normal women, 2) the relationship between endogenous steroid hormone metabolites and the stage of the pelvic organ prolapse. METHODS: Twenty postmenopausal women who were clinically diagnosed as having pelvic organ prolapse and 20 volunteer postmenopausal women not having pelvic organ prolapse were included in the study. We compared the urinary profiles of endogenous steroids between the two groups and investigated the relationship between urinary profiles of the endogenous steroids and the degree of pelvic organ prolapse. Urinary profiles of the endogenous steroids were assayed by gas chromatography-mass spectrometry. RESULTS: The ages of the patients and control group were 64.6 +/- 6.5 and 63.5 +/- 3.9 years, and the Body Mass Index (BMI) was 23.96 +/- 3.14 and 24.11 +/- 2.73 kg/m2 in patients and in normal subjects, respectively. The number of patients in each stage were 4 in stage I, 4 in stage II, 6 in stage III and 6 in stage IV. 5-androstene-3beta, 16beta, 17beta-triol (5-AT), 11beta-hydroxy androstenedione (An) and 17beta-estradiol were significantly increased in patients with pelvic organ prolapse over that of the control group (0.76 +/- 0.67 vs 0.06 +/- 0.03 micro mole/g creatinine; p=0.002, 1.16 +/- 0.83 vs 0.65 +/- 0.23 micro mole/g creatinine; p=0.04, 15.08 +/- 9.81 vs 8.53 +/- 6.19 micro mole/g creatinine; p=0.04). However, tetrahydrocortisone (THE) was significantly increased in the control group over that in patients having pelvic organ prolapse (9.80 +/- 6.21 vs 5.22 +/- 4.89 micro mole/g creatinine; p=0.04). The androgen metabolites, 5-AT and THE significantly correlated with the POP-Q stage (R=0.418; p=0.027, R=0.46; p=0.016). Among the estrogen metabolites, 17beta-estradiol was correlated to the POP-Q stage but not mathematically significantly (R=0.38; p=0.05) and the 17beta-estradiol/estrone ratio weakly correlated to pelvic organ prolapse stage (R=0.14; p=0.49), by showing a low correlation coefficiency. CONCLUSION: The urinary concentrations of 17beta-estradiol, 5-AT and 11beta-hydroxy An increased in patients with pelvic organ prolapse over that of the control group and 5-AT, THE and 17beta-estradiol showed a relationship to the progression of pelvic organ prolapse in Korean women. The metabolites of endogenous steroid hormones could be contributing factors in the pathogenesis of pelvic organ prolapse.
Androstenedione ; Body Mass Index ; Creatinine ; Estrogens ; Female ; Gas Chromatography-Mass Spectrometry ; Humans ; Metabolism* ; Pelvic Organ Prolapse* ; Steroids ; Tetrahydrocortisone ; Volunteers