Objective
The purpose of this study was to determine the impact of parity on mechanical behavior of the vagina and to correlate these findings with alterations in collagen structure.
Study Design
Mechanical properties of 5 nulliparous and 6 parous rhesus macaques were derived from uniaxial tensile tests. Collagen ratios and alignment were quantified by quantitative fluorescent microscopy and picrosirius red staining. Outcomes were compared by the Student t test or Mann Whitney U test ( P < .05) and Spearman’s rho for correlation coefficients.
Results
Mechanical properties were inferior in a parous vs nulliparous vagina with decreased tangent modulus ( P = .03), tensile strength ( P < .001), and strain energy density ( P = .006). Although no difference in collagen ratios ( P = .26) were observed, collagen alignment decreased with parity ( P = .06). Worsening pelvic organ support negatively correlated with decreasing collagen alignment (r 2 = –0.66) and mechanical properties (r 2 = –0.67).
Conclusion
Vaginal parity is associated with inferior tissue mechanics and loss of collagen alignment. Such behavior likely predisposes to the development of pelvic organ prolapse.
A well-supported vagina is essential for normal pelvic organ support. The vagina, in turn, is supported by the physiologically complex interactions between the levator ani muscles and the connective tissue attachments between the vagina and the pelvic sidewall. Thus, with transient increases in intraabdominal pressure, the vagina and its supportive tissues orchestrate an equally distributed counter pressure to maintain pelvic organ support. Failure of any of the components of this intricate system can lead to loss of support by the vagina and the development of pelvic organ prolapse (POP). Up to 11% of all women in the United States will undergo major surgery to repair prolapse or related conditions by the age of 80 years with a direct cost of surgery of >$1 billion per year.
Unfortunately, the cause of POP is unknown. Injury to the vagina and its supportive tissues at the time of vaginal birth is considered a major risk factor because most parous women have mild asymptomatic prolapse, and vaginal delivery has been shown to result in a quantifiable deterioration of vaginal support as measured by the POP quantitative (POP-Q) examination. However, surprisingly little is known of the mechanism by which vaginal birth alters the supportive function of the vagina and predisposes to POP.
Mechanically, it is well-known that all supportive tissues require a minimum stiffness and strength to meet the demands of physiologic loading. The primary load-bearing protein of supportive tissues is collagen. Indeed, collagen composition and alignment have been shown to be related to the mechanical integrity of soft tissues. For example, a higher collagen I/(III+V) ratio and/or increased alignment of collagen in the direction of applied forces is associated with superior mechanical properties. Collagen III is increased in the vagina of women with advanced prolapse, relative to those women without prolapse; however, whether this is a cause or an effect of prolapse is not clear.
One of the primary limitations in the establishment of a link between vaginal parity and the development of prolapse is access to tissue. Indeed, the small quantity of tissues that are obtained from biopsy samples of women precludes conventional mechanical testing. Thus, studies that investigate how changes in the structural proteins (such as collagen and their impact on the mechanical properties of the vagina and supportive tissues subsequent to vaginal delivery) relate to the pathogenesis of POP have been limited. To overcome these issues, we have turned to animal models; however, choosing the appropriate model for the research question at hand is an important factor to consider. Previously, we have used a rodent model to assess changes that occur during pregnancy and the postpartum period. These studies have proved to be invaluable in our understanding of how the vagina and supportive tissue complex adapts during pregnancy, but have been limited in illustrating the long-term effects of vaginal delivery on vaginal mechanical properties. This is because the rat does not sustain a large degree of maternal birth trauma and completely recovers after a vaginal birth. In contrast, the rhesus macaque is an established model to study the impact of vaginal delivery on vaginal tissue behavior because it has similar reproductive physiologic form to humans and spontaneously experiences prolapse. More importantly, the size of the fetal head relative to the vaginal diameter places the mothers at a high risk to sustain an injury at the time of vaginal delivery, which is similar to humans.
In this study, we hypothesized that vaginal delivery results in long-term changes in the vagina that predispose to the development of POP in the context of specific risk factors. Specifically, we believe that parity-induced changes manifest as inferior mechanical properties that are associated with altered collagen structure. To test this hypothesis, we compared collagen alignment, the amount of the fibrillar collagens I, III, and V, and mechanical properties (tangent modulus or stiffness and tensile strength) in nulliparous and vaginally parous rhesus macaques. We focused our studies on the vagina because of the relative ease of access to this tissue and our contention that any loss of pelvic organ support ultimately manifests itself in the vagina.
Materials and Methods
Animals
The animals that were used in this study were maintained and treated according to experimental protocols approved by the Institutional Animal Care Utilization Committee of the University of Pittsburgh (IACUC # 0603434) and in adherence to the National Institutes of Health Guidelines for the use of laboratory animals. Accordingly, a minimum number of rhesus macaques ( macacca mulatta ) were used to answer our research question (see power analysis later) comprised of 14 cycling animals between 9 and 19 years of age. Parous animals (n = 7) were a minimum of 12 months after their last delivery to allow adequate time to recover from short-term birth injuries. Seven nulliparous control nonhuman primates (NHPs) were used for comparison. Routine laboratory tests and regular examinations by veterinarians during a quarantine period were used to certify that these experimental animals were pathogen-free and in good physical condition. Animals were maintained in standard cages with ad libitum water and a scheduled monkey diet supplemented with fresh fruit, vegetables, and multiple vitamins daily. A 12-hour light/dark cycle (7 am to 7 pm ) was used, and menstrual cycle patterns were recorded daily.
Available demographic data were collected that included age, body mass index (BMI), gravidity, and parity of each NHP. A modified POP-Q examination was developed to take into account the smaller vaginal size in sedated NHPs. Our values were based on 10 previously examined nulliparous NHPs. Using these examinations, we calculated the urethrovesical junction to be 2 cm proximal to the vaginal introitus. Thus, in the fully supported vagina this position that corresponds to point Aa of the POP-Q examination is –2 cm. The corresponding position on the posterior vagina wall (Ap) is –2 cm from the introitus. The GH, PB, Ba, Bp, C, D, and total vaginal length are measured similar to humans. In addition, the descent of the organs in all compartments is reported relative to the vaginal introitus, because the hymen is less prominent in these animals. Before the vagina was harvested, a POP-Q examination was performed as described with a pediatric speculum. A Credé’s maneuver was used to mimic valsalva as a proxy for strain.
After a POP-Q examination, the vagina and supportive tissues were excised en bloc by a transabdominal approach. After the vaginal tissue had been isolated, the middle two-thirds was divided for (1) histomorphologic, (2) biochemical, and (3) biomechanical analyses. Tissue samples were divided for each analysis immediately to minimize the number of freeze-thaw cycles and were stored for an average of 1 month before analysis to maximize the number of specimens per assay to ensure that conditions were as consistent as possible. Tissue samples that were obtained for all analyses avoided the urethra to reduce the influence on collagen alignment, composition, and its effect on the mechanical properties. Because of limited vaginal sizes, several animals were unable to be examined by all described experimental protocols. Each section of this study was performed by 1 of the researchers who was blinded to the identification of the samples.
Trichrome staining
For histomorphologic imaging, tissue was embedded and frozen in optimal cutting temperature compound (Sakura, Tokyo, Japan). Sections of the vaginal cross-section were cut onto slides roughly 5- to 8-μm thick. Trichrome slides were used to ensure full-thickness samples of the vaginal cross-section (epithelium, subepithelium, muscularis, and adventitia). These images were used to assess differences qualitatively between the vaginal cross-sections that may be associated with vaginal parity. Blinded researchers examined each slide for any large changes in cross-sectional area composition (eg, increases in smooth muscle or collagen composition).
Immunofluorescence
To assess the relative amounts of collagen I, III, and V and the ratio of collagen I/(III+V), we followed an established protocol. Briefly, sections that were used for immunofluorescence were also oriented along the longitudinal axis of the vagina, which was consistent with trichrome staining. All animals had 3 separate serial sections (5-7 μm) analyzed per assay; 5-7 sites were quantified per section. Blocks were fixed, rehydrated, and placed into normal donkey serum for 45 minutes. Primary antibodies for collagen type I (mouse anti-Human 1:200; Biodesign, Saco, ME), collagen type III (goat anti-human 1:500; Biodesign), and collagen V (rabbit anti-human 1:1000; Biodesign) were first applied, which was followed by incubation with secondary antibodies for 60 minutes. Each sample was imaged with an Olympus Fluoview microscope (DSS Imagetech, New Dehli, India). Fluorescence signals were digitalized to form a pixel based image displayed on a monitor and quantitated using Metamorph (version 5.0; Universal Imaging Corporation, Downington, PA and represented as a relative collagen I/(III+V) ratio for each specimen. Three slides per animal were analyzed in 5-7 randomly selected sites within the subepithelium (dense connective tissue layer of the vagina). Results for each collagen subtype (I, III, and V) were represented as a threshold area for that particular collagen per total area, along with the collagen I/(III+V) ratio.
Collagen alignment
Picrosirius red staining was performed to assess the overall alignment of the vaginal collagen matrix within the cross-section. Circularly polarized light and image-analysis software were used to quantify collagen alignment by assessing fiber hue. To do this, 5- to 10-mm full-thickness mid-vagina sections from 4 nulliparous and 6 parous animals were oriented along the longitudinal axis, embedded in optimal cutting temperature compound, and placed in liquid nitrogen. Blocks of tissue were then sectioned (7 μm), placed on glass slides, and stored at –70°C. Seven to eight frozen sections per animal were hydrated briefly in distilled water and imaged with a microscope (model BX51; Olympus America, Center Valley, PA). Five polarized light images per sample were taken at ×20 in both the vaginal subepithelium and the muscularis. An area analysis was performed with Metamorph software (version 5.0; Molecular Devices, Dowingtown, PA), where the hue component of the resulting image was obtained, and the number of red, orange, yellow, and green (the colors of collagen fibers in order of decreasing thickness and organization) pixels were calculated.
Biomechanics
For biomechanical analysis, vaginal tissue samples from each NHP were isolated carefully from the surrounding connective tissue and wrapped in saline-soaked gauze, placed in a plastic bag, and stored at –20°C. On the day of testing, the tissue was thawed, and a longitudinal section of tissue was isolated (18.2 ± 3.1 mm). Each sample was then gripped with the use of custom-designed soft-tissue clamps and further dissected to ensure the desired aspect ratio (length/width) of 5 within the mid substance of the tissue. Subsequently, tissue cross-sectional area and geometry were measured with a laser micrometer system in 3 areas along the length of the sample’s trimmed midsubstance. Throughout this protocol, the sample was kept moist with 0.9% saline solution. Contrast markers were placed on the tissue near the midline of the sample at a distance of 1 cm apart for strain measurements with a camera system (LS-3060; Keyence, Osaka, Japan) and motion analysis software (Spicatek, Inc, Maui, HI). The specimen-clamp complex was then placed into a 37°C saline solution bath and attached to a uniaxial tensile testing machine (version 5565; Instron Industrial Products, Grove City, PA). The clamp that secured the proximal end of the vagina was contiguous with a 50-lb load cell (model 31; Honeywell Inc, Morristown, NJ) with 0.1 N resolutions; the distal end was attached to the base of the material testing machine. The specimen was allowed to equilibrate unloaded in the bath for 30 minutes before being tested. Next, a small 0.5-N preload was applied to the tissue, and 10 cycles of preconditioning to 7% clamp-to-clamp strain were performed at a rate of 10 mm/min. A load-to-failure test was carried out immediately after the preconditioning along the specimens longitudinal axis. The load (force, Newton) and elongation (millimeters) of the tissue were recorded and used to generate a load-elongation relationship.
The load-elongation relationship was then converted to a stress-strain relationship from which the parameters that described the mechanical properties of the tissue were determined. Stress was defined as the load per cross-sectional area and was normalized to the average of the 3 cross-sectional area measurements that were obtained from the laser micrometer. Strain was calculated from the motion of the contrast markers on the mid substance of the tissue and was defined as the change in marker position relative to the original marker position (Δl/l o ). The slope of the linear region of the resulting stress-strain curve was defined as the tangent modulus that describes the stiffness on a per-unit of tissue basis; the tensile strength (maximum stress) and ultimate strain (strain in the tissue corresponding to the maximum stress) were recorded at failure. The strain energy density was calculated by measurement of the area underneath the stress-strain curve until failure and is a measure of the “toughness” of a tissue in material science jargon. Each of these mechanical properties were calculated from the stress-strain relationship as previously described. Importantly, the loading conditions that were used in these experiments were not meant to recapitulate the physiologic condition of vaginal delivery but rather to provide insight into fundamental differences in tissue behavior between nulliparous and parous animals.
Statistics
All statistical analyses were performed with SPSS software (version 12.0.1; SPSS Inc, Chicago, IL). All data were examined to determine whether it was distributed normally with the use of a 1-sample Kolmogrov-Smirnov test. The Student t test was used to compare all demographic data (age, height, weight, BMI, and total vaginal length), collagen subtypes (collagen I, III, V, and I/[III+V]), collagen orientation (red:green ratio), and biomechanical properties (tangent modulus, tensile strength, maximum strain, and strain-energy density).
Statistics were aimed to answer our primary hypothesis which states that the mechanical properties of the tangent modulus and tensile strength would be inferior in parous NHPs compared with nulliparous animals. Based on a preliminary study done within our laboratory on the biomechanical properties in the rodent model, 7 animals would be required per group to detect at least a 30% difference between nulliparous and parous animals with an 80% power. Significant differences were found between these parameters with 5 and 6 samples in the nulliparous and parous groups, respectively, which indicate that we had reached 100% power for our primary outcomes. The non-parametric Mann-Whitney test was used to compare POP-Q measurements. A Spearman’s rho rank correlation was performed to assess whether the number of vaginal deliveries in the parous group correlated with any changes in collagen ratios, collagen subtypes, alignment, or biomechanical properties. All statistical tests were evaluated at a significance level of .05. For normally distributed parameters, data are represented as mean (SD) and for nonnormally distributed data as median (interquartile range).
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