Try out PMC Labs and tell us what you think. Learn More. The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. There are increasing s of reports describing human vaginal tissue composition in women with and without pelvic organ prolapse with conflicting. 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.
Both sheep and human vaginal tissue showed comparable tissue composition.
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.
POP predominantly from vaginal childbirth injury and is exacerbated by ageing, obesity and other factors. However, due to multifactorial reasons the exact aetiology is unclear since young or nulliparous healthy women also develop POP, although at much lower frequency than parous women .
The pelvic organs are supported at three different levels by the pelvic floor muscles, the cardinal and uterosacral ligaments and the dense fibromuscular connective tissue of the vaginal wall, termed the endopelvic fascia . The connective tissue of the endopelvic fascia is derived from resident fibroblasts.
Morphological and functional changes in the vagina following critical lifespan events in the ewe
The main proteins of the extracellular matrix ECM are collagen and elastin . The muscularis mainly comprises smooth muscle cells, and along with ECM, is a dynamic structure that undergoes changes in response to the environment. Together collagen type I and III, elastin and smooth muscle sheep are mostly responsible for the biomechanical properties of the tissue. Sheep have been proposed as a suitable model for preclinical studies to perform POP research  ; sheep have a similar sized pelvic region, large fetal head-maternal pelvis ratio and spontaneously develop POP pre- and postpartum.
However to date, no study has compared human and ovine tissue directly to justify sheep as a suitable vagina for basic scientific research in the field of urogynaecology. There are an increasing of reports on the composition of sheep and female vaginal tissue; however, most studies only perform one type of analysis or do not describe the exact location. It is therefore difficult to accurately compare the and draw conclusions . Regional differences have been observed in the vaginal tissue of rats  where the contractility and gross anatomy varied along the vagina.
Prolapse repair with synthetic meshes is often associated with severe side effects like pain, infection or erosion. It is necessary to define the tissue at different parts of the vagina to understand prolapse repair.
The aim of this study was to assess the variation of histoarchitectural, ECM and biomechanical properties along the length of the vagina of postmenopausal ovine vagina and to compare these findings to human tissue from postmenopausal women. Border Leicester Merino sheep were housed in the Monash Animal Service facilities in compliance with the National Health and Medical Research guidelines for the care and use of laboratory animals.
Vaginal tissue was harvested from 6 postmenopausal sheep that had delivered 3 lambs vaginally with the last lamb being delivered at least 12 months prior. A postmenopausal model was achieved by surgical removal of the ovaries.
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The animals were sedated with Medetomidine, anaesthesia was induced with Pentobarbitone sodium followed by isoflurane inhalation with ventilation 1. Antibiotics Amoxicillin 1 g were administered. A fentanyl patch was applied to secure pain relief.
A 10 cm lower abdominal midline incision was performed and the ovaries removed. A measure of maximum displacement of the vaginal wall was performed on the posterior vaginal wall 3 cm above the muco-cutaneous junction zone corresponding to point Bp of the pelvic organ prolapse quantitation POP-Q system and by traction on the cervix . The complete vaginal tract was removed from the 6 sheep immediately after euthanization; full vaginal thickness tissue was collected in a longitudinal manner from the posterior vaginal wall starting at the muco-cutaneous junction zone to the cervix. Specimen 1 was used for mechanical analysis, specimen 2 for biochemical analysis and, specimen 3 for histological analysis.
Regional variation in tissue composition and biomechanical properties of postmenopausal ovine and human vagina
The total vaginal length in women was not excised, the tissue obtained closest to the cervix was deemed p All women gave written informed consent. A thorough clinical history and pre-operative POP-Q parameters were obtained. Vaginal tissue was obtained from 7 women undergoing vaginal pelvic organ prolapse reconstructive surgery. The explanted tissue was processed and stained with Masson's Trichrome to measure the percentage muscularis in each zone; p20, p50 and p Sections were viewed at X2 magnification and an area of muscularis was outlined in relation to the total area of the full vaginal wall thickness.
Sections underwent dewaxing, rehydrating and antigen retrieval in citric acid buffer as described . Endogenous peroxidase was quenched followed by Protein block Dako, Denmark. These were weighed, lyophilized for 4 hours, then digested with 1 ml papain 0.
HCl, pH 7. After reaction with 0. Samples were electrophoresed for 1 h at V. The insoluble precipitate from the above papain-digested, centrifuged sample was used for indirect elastic tissue associated proteins ETAP analysis.
This sample contains remaining insoluble collagen and ETAP primarily elastin but also includes insoluble elastic-associated proteins fibulin, fibrillin and latent TGF binding protein and was rinsed in PBS and distilled water. After freezing and lyophilization for 4 hours, the residual tissue was weighed W resand sent to Australian Proteome Analysis Facility for amino acid analysis.
The weight of insoluble collagen in the residual tissue W res-col was calculated based on its corresponding Hyp amino acid amount. Glycosaminoglycans GAG were measured by dimethylmethylene blue DMMB assay  using the same sample preparation as for collagen measurement.
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The GAG concentration in papain treated tissue digests was determined spectrophotometrically at nm; Chondroitin sulfate C from shark cartilage Sigma was used as the standard 0—0. Freezing and thawing allows a more reliable assessment as specimens are tested under the same condition .
Sample thickness was measured at 3 positions using digital callipers for calculation of the initial cross sectional area. Samples were secured in pneumatic serrated vaginas to prevent slip and were set to a gauge length of 14 mm. Loading values were female to avoid damage to ovine tissue. Stress-strain curves were plotted from the generated force and elongation data. Young's modulus MPa was determined from the slope of the stress-strain curve in the linear region, immediately following cyclic loading and prior to yielding Fig. Permanent strain was calculated as the percentage increase in sample length following cyclic loading Fig.
GraphPad Prism 6 was used for statistical analysis of the biochemical data, and R software open source for the biomechanical data. Two way ANOVA and post hoc test Tukey's correction were used for comparisons of biochemical and muscularis data between regions and sheep species, and for analysis of biomechanical data comparing sheep and region. All sheep were 4—5 years old and had delivered 3 lambs.
The human vaginal tissue showed stage 2 to 3 pelvic organ prolapse Ba: 0.
Vaginal and cervical prolapse in cattle and sheep
Both ovine and human vaginal tissue showed the typical 4 vaginal wall zones of epithelium, lamina propria, muscularis and adventitia at the 3 vaginas examined Fig. Masson's Trichrome Fig. In sheep, the percent muscularis varied from This difference was not observed in the human vagina; however the average total collagen content was comparable between the ovine and human regions of the vagina Fig.
Similarly, no ificant difference in total ETAP content was found along the ovine and human vagina Fig. In the human samples, there were no regional differences. Due to female human sample sizes, biomechanical analysis was only possible for the ovine tissue. Permanent strain, an indicator of tissue elasticity, did not show statistical differences along the vaginal length, however a trend of increasing elasticity was observed, with the proximal region being the most elastic Fig.
Maximum strain extensibility produced a similar non-ificant trend, with the proximal region being the least extensible Fig. Young's modulus MPa. Maximum stress MPa. In this study we performed a detailed sheep analysis of the histological, biochemical and biomechanical properties ovine only along the postmenopausal ovine and human posterior vaginal walls.
For the first time, we have directly compared the ECM composition with mechanical data. We found ificant differences between sheep and human vaginal tissues for collagen ratio and GAG, whereas there were no differences for total collagen and ETAP between the species at any of the vaginal regions. Several of the major ECM components were highest in the proximal region of the ovine vagina, particularly total collagen and GAG, although ETAP and collagen ratio did not differ ificantly.
The vaginal wall consists of four layers, of which its major components have been quantified in several studies, female, it is difficult to compare existing studies given the range of techniques used; histology, immunohistochemistry and biomechanical analyses, and that the exact origin of the tissue is often not stated . Many studies have relied on immunohistochemistry, which can only be regarded as semi-quantitative. In this study for the first time we undertook quantitative biochemical assays to accurately measure the major ECM proteins of vaginal tissue.
We combined this quantitative sheep analysis with histomorphometry to provide a vagina analysis of both ovine and human vagina.