Regional Variation in Tissue Composition and Biomechanical Properties of Postmenopausal Ovine and Human Vagina

Objective

There are increasing numbers of reports describing human vaginal tissue composition in women with and without pelvic organ prolapse with conflicting results. The aim of this study was to compare ovine and human posterior vaginal tissue in terms of histological and biochemical tissue composition and to assess passive biomechanical properties of ovine vagina to further characterise this animal model for pelvic organ prolapse research.

Study Design

Vaginal tissue was collected from ovariectomised sheep (n = 6) and from postmenopausal women (n = 7) from the proximal, middle and distal thirds. Tissue histology was analyzed using Masson''s Trichrome staining; total collagen was quantified by hydroxyproline assays, collagen III/I+III ratios by delayed reduction SDS PAGE, glycosaminoglycans by dimethylmethylene blue assay, and elastic tissue associated proteins (ETAP) by amino acid analysis. Young''s modulus, maximum stress/strain, and permanent strain following cyclic loading were determined in ovine vagina.

Results

Both sheep and human vaginal tissue showed comparable tissue composition. Ovine vaginal tissue showed significantly higher total collagen and glycosaminoglycan values (p<0.05) nearest the cervix. No significant differences were found along the length of the human vagina for collagen, GAG or ETAP content. The proximal region was the stiffest (Young''s modulus, p<0.05), strongest (maximum stress, p<0.05) compared to distal region, and most elastic (permanent strain).

Conclusion

Sheep tissue composition and mechanical properties showed regional differences along the postmenopausal vaginal wall not apparent in human vagina, although the absolute content of proteins were similar. Knowledge of this baseline variation in the composition and mechanical properties of the vaginal wall will assist future studies using sheep as a model for vaginal surgery.     

How Accurate Is Preoperative Evaluation of Pelvic Organ Prolapse in Women Undergoing Vaginal Reconstruction Surgery?

Objective

To evaluate the differences between the in-office and intraoperative techniques used to evaluate pelvic organ prolapse.

Materials and Methods

A prospective study included 25 women undergoing vaginal reconstruction surgery including vaginal hysterectomy for pelvic organ prolapse. The outpatient pelvic and site-specific vaginal examination was performed in the lithotomy position with the Valsalva maneuver. Repeated intraoperative examination was performed under general anesthesia with standard mild cervical traction. The Pelvic Organ Prolapse Quantification system (POPQ) was used for both measurements and staging. The values found under the two conditions were compared.

Results

The intraoperative POPQ-measurements values were significantly higher than the outpatient values for apical wall prolapse in 17/25 (68%) women and for anterior wall prolapse in 8/25 (32%) women. There was not a significant difference in the posterior wall where increase in staging was shown in 3/25 (12%) patients.

Conclusions

Clinicians and patients should be alert to the possibility that pelvic organ measurements performed under general anesthesia with mild traction may be different from preoperative evaluation.     

Linking hyperelastic theoretical models and experimental data of vaginal tissue through histological data

Mechanical characterization of living tissues and computer-based simulations related to medical issues, has become increasingly important to improve diagnostic processes and treatments evaluation. This work proposes a link between the mechanical testing and the material model predictions through histological data of vaginal tissue. Histological data was used to link tensile testing experiments with material-dependent parameters; the approach was adequate to capture the nonlinear response of ovine vaginal tissue over a large strain range.The experimental data obtained on a previous study, has two main components: tensile testing and histological analysis of the ovine vaginal tissue. Uniaxial tensile test data and histological data were collected from three sheep groups: virgins, pregnant and parous. The distal part of vaginal wall was selected since it is prone to tears induced by vaginal delivery.The HGO (Holzapfel-Gasser-Ogden) model parameters were fitted using a stochastic approach, namely the Simple Genetic Algorithm (SGA). The SGA was able to fit the experimental data successfully (R² > 0.986). The dimensionless coefficient ξ, was highly correlated with histological data. The ratio was seen to increase linearly with increasing collagen content.Coefficient ξ brings a new way of interpreting and understanding experimental data; it connects the nonlinear mechanical behaviour (tensile test) with tissue’s morphology (histology). It can be used as an ‘inverse’ (approximate) method to estimate the mechanical properties without direct experimental measurements, through basic histology.In this context, the proposed methodology appears very promising in estimating the response of the tissue via histological information.     

3D finite element modeling of pelvic organ prolapse

Objectives: The purpose of this study is to develop a validated 3D finite element model of the pelvic floor system which can offer insights into the mechanics of anterior vaginal wall prolapse and have the ability to assess biomedical device treatment methods. The finite element results should accurately mimic the clinical findings of prolapse due to intra-abdominal pressure (IAP) and soft tissues impairment conditions. Methods: A 3D model of pelvic system was created in Creo Parametric 2.0 based on MRI Images, which included uterus, cervix, vagina, cardinal ligaments, uterosacral ligaments, and a simplified levator plate and rectum. The geometrical model was imported into ANSYS Workbench 14.5. Mechanical properties of soft tissues were based on experimental data of tensile test results from current literature. Studies were conducted for IAP loadings on the vaginal wall and uterus, increasing from lowest to extreme values. Results: Anterior vaginal wall collapse occurred at an IAP value corresponding to maximal valsalva and showed similar collapsed shape as clinical findings. Prolapse conditions exhibited high sensitivity to vaginal wall stiffness, whereas healthy tissues was found to support the vagina against prolapse. Ligament impairment was found to have only a secondary effect on prolapse.     

Pelvic Organ Support in Animals with Partial Loss of Fibulin-5 in the Vaginal Wall

Compromise of elastic fiber integrity in connective tissues of the pelvic floor is most likely acquired through aging, childbirth-associated injury, and genetic susceptibility. Mouse models of pelvic organ prolapse demonstrate systemic deficiencies in proteins that affect elastogenesis. Prolapse, however, does not occur until several months after birth and is thereby acquired with age or after parturition. To determine the impact of compromised levels of fibulin-5 (Fbln5) during adulthood on pelvic organ support after parturition and elastase-induced injury, tissue-specific conditional knockout (cKO) mice were generated in which doxycycline (dox) treatment results in deletion of Fbln5 in cells that utilize the smooth muscle α actin promoter-driven reverse tetracycline transactivator and tetracycline responsive element-Cre recombinase (i.e., Fbln5^f/f/SMA⁺⁺-rtTA/Cre⁺, cKO). Fbln5 was decreased significantly in the vagina of cKO mice compared with dox-treated wild type or controls (Fbln5^f/f/SMA⁺⁺-rtTA/Cre^-/-). In controls, perineal body length (PBL) and bulge increased significantly after delivery but declined to baseline values within 6–8 weeks. Although overt prolapse did not occur in cKO animals, these transient increases in PBL postpartum were amplified and, unlike controls, parturition-induced increases in PBL (and bulge) did not recover to baseline but remained significantly increased for 12 wks. This lack of recovery from parturition was associated with increased MMP-9 and nondetectable levels of Fbln5 in the postpartum vagina. This predisposition to prolapse was accentuated by injection of elastase into the vaginal wall in which overt prolapse occurred in cKO animals, but rarely in controls. Taken together, our model system in which Fbln5 is conditionally knock-downed in stromal cells of the pelvic floor results in animals that undergo normal elastogenesis during development but lose Fbln5 as adults. The results indicate that vaginal fibulin-5 during development is crucial for baseline pelvic organ support and is also important for protection and recovery from parturition- and elastase-induced prolapse.     

Expression of matrix metalloproteinase-1 in uterosacral ligaments tissue of women with genital prolapse

Collagen metabolism is altered in the pelvic organ tissues of women with genital prolapse. The aim of this study was to compare collagen metabolism by measuring matrix metalloproteinase-1 (MMP-1) expression in uterosacral ligament tissues of postmenopausal women with and without genital prolapse. Uterosacral ligament tissues were obtained at the time of abdominal or vaginal surgery from twenty-four patients with pelvic organ prolapse (POP) and 21 women who underwent gynecologic surgery for benign indications. The tissue samples were analyzed by immunohistochemistry. There were no differences in age, BMI and parity between two groups. The patients with genital prolapse demonstrated significantly higher occurences of MMP-1 expression compared to controls. These findings indicate that increased MMP-1 expression in uterosacral ligaments is associated with genital prolapse. Our data are consistent with the theory that increased collagen breakdown may play an important role in the onset and development of pelvic organ prolapse (POP).     

Changes in Muscularis Propria of Anterior Vaginal Wall in Women with Pelvic Organ Prolapse

The objective of this study was to evaluate the morphological and immunohistochemical alterations of tissue removed from the upper third of anterior vaginal wall in a sample group of the female population presenting homogenous risk factors associated with pelvic organ prolapse (POP). The case study consisted of 14 patients with POP and there were 10 patients in the control group. Patient selection was carried on the basis of specific criteria and all of the patients involved in the study presented one or more of the recognized POP risk factors. Samples were taken from POP patients during vaginal plastic surgery following colpohysterectomy, and from control patients during closure of the posterior fornix following hysterectomy. Samples were processed for histological and immunohistochemical analyses for Collagen I and Collagen III, α-Smooth Muscle Actin (α-SMA), Platelet-Derived-Growth-Factor (PDGF), matrix metalloproteinase 3 (MMP3), tissue inhibitors metalloproteinase 1 (TIMP1), Caspase3. Immunofluorescence analyses for Collagen I and III and PDGF were also carried out. In prolapsed specimens our results show a disorganization of smooth muscle cells that appeared to have been displaced by an increased collagen III deposition resulting in rearrangement of the muscularis propria architecture. These findings suggest that the increase in the expression of collagen fibers in muscularis could probably be due to a phenotypic switch resulting in the dedifferentiation of smooth muscle cells into myofibroblasts. These alterations could be responsible for the compromising of the dynamic functionality of the pelvic floor.Key words: Pelvic organ prolapse, immunohistochemistry, smooth muscle cells, collagen, PDGF     

经利用网片的前盆底重建术治疗重度盆腔器官脱垂的临床疗效

目的:探讨经利用网片的前盆底重建术治疗重度盆腔器官脱垂的临床效果。方法:选取2013年1月-2015年3月我院妇科收治的以前中盆腔联合缺陷为主的盆底障碍性疾病患者39例(POP-Q分度III-IV度)。实施经阴道Avaulta前盆腔重建术,24例切除子宫,9例保留子宫,其中6例因中盆腔重度脱垂联合行阴道残端骶棘韧带固定术,2例合并尿失禁同时行经尿道无张力悬吊术。术后随访12-25月,根据盆腔脏器脱垂评分(POP-Q)测量及PFIQ-7评分进行客观评价及主观感受评价进行中短期的临床效果分析。结果:39例患者手术过程顺利,术后无严重并发症,POP-Q评分均较术前显著降低,客观疗效理想。术后阴道前壁及子宫、阴道穹窿脱垂等症状显著改善;到目前为止无一例复发。结论:经阴道前盆腔重建术治疗前盆腔重度盆腔脱垂为主以及合并尿失禁等复杂情况的并发症少,治愈率高,复发率低,临床效果好。     

Tissue engineering in female pelvic floor reconstruction

Pelvic organ prolapse is a common and frequently occurring disease in middle‐aged and elderly women. Mesh implantation is an ideal surgical treatment. The polypropylene mesh commonly used in clinical practice has good mechanical properties, but there are long‐term complications. The application of tissue engineering technology in the treatment of pelvic organ prolapse disease can not only meet the mechanical requirements of pelvic floor support, but also be more biocompatible than traditional polypropylene mesh, and can promote tissue repair to a certain extent. In this paper, the progress of tissue engineering was summarized to understand the application of tissue engineering in the treatment of pelvic organ prolapse disease and will help in research.     

Local oestrogen therapy modulates extracellular matrix and immune response in the vaginal tissue of post‐menopausal women with severe pelvic organ prolapse

This study investigates the effect of local oestrogen therapy (LET) on the expression of proteins participating in collagen/elastin biogenesis and immune markers in vaginal tissues of post‐menopausal women with severe pelvic organ prolapse (POP). Vaginal biopsies were collected from the anterior vaginal wall of informed and consented 52 post‐menopausal women with severe POP undergoing total hysterectomy. Twenty‐nine of the 52 women were treated with LET (in the form of vaginal oestrogen cream or tablet), while the remaining 23 untreated patients served as the controls. This study was approved by Sinai Health System REB. Vaginal tissue specimens were analysed for gene and protein expression using real‐time RT‐PCR and Luminex assays, protein localization and immune cell infiltration were assessed by immunohistochemistry. Forty‐four cytokines were detected. We found that LET application: (a) significantly increased (P < 0.05) gene and protein expression levels of extracellular matrix (ECM) structural proteins, collagen and elastin, as well as the expression of ECM maturation enzyme BMP1; (b) decreased protein expression level of ECM degradation enzymes MMP1, MMP2 and MMP3 accompanied by an increase in their tissue inhibitors, TIMP1 and TIMP4; (c) significantly increased (P < 0.05) the gene and protein expression levels of 14 vaginal cytokines involved in leucocyte infiltration, which was confirmed by immunohistochemistry. Our results indicate that LET plays an important role in the activation of immune system within the local vaginal environment, limiting the undesirable ECM degradation, which supports the strengthening of vaginal ECM in post‐menopausal women, therefore resisting menopause/age‐related changes and inducing urogenital tract tissue regeneration.     

Quantification of pelvic organ prolapse in mice: vaginal protease activity precedes increased MOPQ scores in fibulin 5 knockout mice

Two mouse models of pelvic organ prolapse have been generated recently, both of which have null mutations in genes involved in elastic fiber synthesis and assembly (fibulin 5 and lysyl oxidase-like 1). Interestingly, although these mice exhibit elastinopathies early in life, pelvic organ prolapse does not develop until later in life. In this investigation we developed and validated a tool to quantify the severity of pelvic organ prolapse in mice, and we used this tool prospectively to study the role of fibulin 5, aging, and vaginal proteases in the development of pelvic organ prolapse. The results indicate that >90% of Fbln5(-/-) mice develop prolapse by 6 mo of age, even in the absence of vaginal delivery, and that increased vaginal protease activity precedes the development of prolapse.     

补中益气汤联合生物反馈电刺激在产后盆底功能障碍患者的应用研究

目的:研究探讨补中益气汤联合生物反馈电刺激对产后盆底功能障碍患者的临床治疗效果及对其盆底肌肉张力的影响。方法:选择我院就诊的90例产后盆底功能障碍气虚证患者(2017年11月～2018年10月),按照随机数字表法划分为30例/组的A组、B组、C组,A组实施常规盆底肌锻炼,B组采用盆底治疗仪实施生物反馈电刺激治疗,C组实施生物反馈电刺激联合中药补中益气汤治疗,比较三组的疗效、盆底肌力评分、阴道最大收缩压、盆腔器官脱垂、尿失禁、性生活质量评分。结果:(1)组间临床疗效、证候疗效比较,C组的临床总有效率、证候改善总有效率均高于A组、B组(P0.05),而A组与B组之间比较均无统计学差异(P0.05)。(2)治疗后,C组的盆底肌力评分、阴道最大收缩压、性生活质量评分均高于A组、B组(P0.05),A组与B组之间比较均无统计学差异(P0.05)。(3)C组的盆腔器官脱垂、尿失禁等发生率均低于A组、B组(P0.05),而A组与B组比较均无统计学差异(P0.05)。结论:生物反馈电刺激联合补中益气汤治疗可有效改善产后盆底功能障碍患者的盆底肌力和阴道压力,达到良好的临床疗效,有利于预防盆腔器官脱垂、尿失禁的发生,改善其性生活质量。     

Quantitative analysis of 17 amino acids in the connective tissue of patients with pelvic organ prolapse using capillary electrophoresis-tandem mass spectrometry

The simultaneous determination of 17 amino acids in connective tissue using capillary electrophoresis is described in this study. Separation was carried out on a fused silica capillary column (80 cm x 50 mm i.d.) with 1M formic acid as the running electrolyte. The detection was conducted on a mass spectrometer by selective reaction monitoring (SRM) mode via an electrospray ionization source. Tissue samples were prepared by reduction and acid hydrolysis to extract amino acids; over 84.3% recovery was seen for all compounds. The method allowed for sensitive, reproducible, and reliable quantification, and all 17 amino acids were separated using this method. Good linearity over the investigated concentration ranges was observed, with values of R higher than 0.993 for all the analytes. Precision and accuracy examined at three concentration levels ranged from 0.2% to 19.5% and 84.1% to 120.0%, respectively. Matrix effects were also tested and ranged from -9.1% to 15.4%. The validated method was applied to the quantitation of 17 amino acids in pelvic connective tissue of pelvic organ prolapsed patients. Methionine, glutamine, and histidine were significantly higher in the experimental patients compared to the controls. This suggests that changes in the amino acid concentrations within the connective tissue could be a factor in the genesis of pelvic organ prolapse. Therefore, this method is potentially applicable for amino acid analysis in tissue, providing a more complete understanding of pelvic organ prolapse.     

Anisotropic effects of the levator ani muscle during childbirth

Pelvic floor dysfunction and pelvic organ prolapse have been associated with damage to the levator ani (LA) muscle, but the exact mechanisms linking them remain unknown. It has been postulated that factors such as vaginal birth and ageing may contribute to long-term, irreversible LA muscle damage. To investigate the biomechanical significance of the LA muscle during childbirth, researchers and clinicians have used finite element models to simulate the second stage of labour. One of the challenges is to represent the anisotropic mechanical response of the LA muscle. In this study, we investigated the effects of anisotropy by varying the relative stiffness between the fibre and the matrix components, whilst maintaining the same overall stress–strain response in the fibre direction. A foetal skull was passed through two pelvic floor models, which incorporated the LA muscle with different anisotropy ratios. Results showed a substantial decrease in the magnitude of the force required for delivery as the fibre anisotropy was increased. The anisotropy ratio markedly affected the mechanical response of the LA muscle during a simulated vaginal delivery. It is apparent that we need to obtain experimental data on muscle mechanics in order to better approximate the LA muscle mechanical properties for quantitative analysis. These models may advance our understanding of the injury mechanisms of pelvic floor during childbirth.     

Dysregulation of Protease and Protease Inhibitors in a Mouse Model of Human Pelvic Organ Prolapse

Mice deficient for the fibulin-5 gene (Fbln5^−/−) develop pelvic organ prolapse (POP) due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP)-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5^−/− mice, herein named V1 (25 kDa). V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS) 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5^−/− mice. PRSS3 was (a) localized in epithelial secretions, (b) detected in media of vaginal organ culture from both Fbln5^−/− and wild type mice, and (c) cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin) and Elafin] was dysregulated in Fbln5^−/− epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice.     

A biomechanical model to assess the contribution of pelvic musculature weakness to the development of stress urinary incontinence

A biomechanical model of the female pelvic support system was developed to explore the contribution of pelvic floor muscle defect to the development of stress urinary incontinence (SUI). From a pool of 135 patients, clinical data of 26 patients with pelvic muscular defect were used in modelling. The model was employed to estimate the parameters that describe the stiffness properties of the vaginal wall and ligament tissues for individual patients. The parameters were then implemented into the model to evaluate for each patient the impact of pelvic muscular defect on the vaginal apex support and the bladder neck support, a factor that relates to the onset of SUI. For the modelling analysis, the compromise of pelvic muscular support was demonstrated to contribute to vaginal apex prolapse and bladder neck prolapse, a condition commonly seen in SUI patients, while simulated conditions of restored muscular support were shown to help re-establish both vaginal apex and bladder neck supports. The findings illustrate the significance of pelvic muscle strength to vaginal support and urinary continence; therefore, the clinical recommendation of pelvic muscle strengthening, such as Kegel exercises, has been shown to be an effective treatment for patients with SUI symptoms.     

First report of vaginal prolapse in a bitch treated with oestrogen

Vaginal prolapse is the protrusion of edematous vaginal tissue into and through the opening of the vulva occurring during the pro-oestrus and oestrus stages of the sexual cycle. True vaginal prolapse may occur near parturition, as the concentration of serum progesterone declines and the concentration of serum oestrogen increases. In a bitch, true vaginal prolapse is a very rare condition. This case report describes an 18-month-old crossbreed bitch, weighing 40 kg presented with type III vaginal prolapse. The patient had developed vaginal prolapse after receiving oestrogen in order to oestrus induction. Subsequent to unsuccessful attempts for repositioning, ovariohysterectomy (OHE), circumferential excision of the prolapsed tissue and finally vulvoplasty were performed. There was no evidence of recurrence of the prolapse during 30 days after surgery. This case report describes type III vaginal prolapse as an unusual side effect of oestrus induction hormonal therapy in the bitch.     

Two parallel,pragmatic, UK multicentre,randomised controlled trials comparing surgical options for upper compartment (vault or uterine) pelvic organ prolapse (the VUE Study): study protocol for a randomised controlled trial

BackgroundOne in three women who have a prolapse operation will go on to have another operation, though not necessarily in the same compartment. Surgery can result in greater impairment of quality of life than the original prolapse itself (such as the development of new-onset urinary incontinence, or prolapse at a different site). Anterior and posterior prolapse surgery is most common (90 % of operations), but around 43 % of women also have a uterine (34 %) or vault (9 %) procedure at the same time. There is not enough evidence from randomised controlled trials (RCTs) to guide management of vault or uterine prolapse. The Vault or Uterine prolapse surgery Evaluation (VUE) study aims to assess the surgical management of upper compartment pelvic organ prolapse (POP) in terms of clinical effectiveness, cost-effectiveness and adverse events.Methods/designVUE is two parallel, pragmatic, UK multicentre, RCTs (Uterine Trial and Vault Trial). Eligible for inclusion are women with vault or uterine prolapse: requiring a surgical procedure, suitable for randomisation and willing to be randomised. Randomisation will be computer-allocated separately for each trial, minimised on: requiring concomitant anterior and/or posterior POP surgery or not, concomitant incontinence surgery or not, age (under 60 years or 60 years and older) and surgeon. Participants will be randomly assigned, with equal probability to intervention or control arms in either the Uterine Trial or the Vault Trial. Uterine Trial participants will receive either a vaginal hysterectomy or a uterine preservation procedure. Vault Trial participants will receive either a vaginal sacrospinous fixation or an abdominal sacrocolpopexy. Participants will be followed up by postal questionnaires (6 months post surgery and 12 months post randomisation) and also reviewed in clinic 12 months post surgery. The primary outcome is the participant-reported Pelvic Organ Prolapse Symptom Score (POP-SS) at 12 months post randomisation.DiscussionDemonstrating the efficacy of vault and uterine prolapse surgeries is relevant not only to patients and clinicians but also to health care providers, both in the UK and globally.

Trial registration

Current controlled trials ISRCTN86784244 (assigned 19 October 2012), and the first subject was randomly assigned on 1 May 2013 

Electronic supplementary material

The online version of this article (doi:10.1186/s13063-016-1576-x) contains supplementary material, which is available to authorized users.