Characterization of Structure-Function Relationships in the Murine Vagina Throughout Adolescence

Introduction: : The vagina is a crucial part of the female reproductive system, playing an essential role in key physiological functions such as sexual activity, menstruation, and childbirth. The vaginal wall consists of four distinct layers: the epithelium, lamina propria, muscularis, and adventitia. The epithelium is the innermost layer and is composed of ground substance and epithelial cells, while the lamina propria is highly cellular and primarily contains structural proteins such as elastin and collagen fibers. The muscularis mainly consists of vaginal smooth muscle cells and is surrounded by a layer of loosely organized connective tissue known as the adventitia. It is well established that the vaginal wall undergoes extensive growth and dynamic remodeling due to natural events such as pregnancy, childbirth, aging, and menopause. However, the cellular origin of the secretion and deposition of key extracellular matrix (ECM) proteins in the vaginal wall remains unclear. Thus, in the current study we aim to investigate biomechanical and microstructural changes in developing vaginal tissue by examining its biaxial mechanical properties and evaluating extracellular matrix (e.g., elastin) composition, thereby establishing a baseline for structure-function relationships in the murine vagina throughout adolescence. Findings from this work will elucidate when and where structural proteins are deposited within the developing vaginal wall and may provide critical insight into understanding the cellular mechanisms, and potential therapeutic targets, underlying the onset and progression of pelvic floor–related diseases.

Materials and

Methods: : Animals. Intact reproductive systems from wild-type adult female mice (10-20 weeks) as well as wild-type female pups at 3, 7, 14, 21, and 42 days were extracted, and the vagina (from external skin to cervix) was isolated. Biaxial Mechanical Testing. Mechanical properties of vaginal tissues were assessed using biaxial inflation-extension testing. Briefly, following cannulation in a computer-controlled mechanical testing system, the vaginal outer diameter, length and in vivo axial stretch were estimated. Subsequently, tissues were preconditioned prior to biaxial testing with 3 inflation cycles from 5-80 mmHg while held at 95%, 100%, and 105% of the estimated physiological length and 4 extension cycles from -0.05 to 5g axial force while held at fixed pressures of 5, 25, 55, and 80 mmHg. During both tests, the axial force was recorded, and outer diameter was tracked using a camera. Finally, the unloaded wall thickness was measured after testing using Optical Coherence Tomography (OCT) imaging. Combination of the measured loads and tissue geometry was used to calculate the mean circumferential and axial stresses in the vaginal wall. Elastin Immunofluorescence. To evaluate changes in vaginal elastin structure, tissues were cryosectioned at 8 µm, fixed/permeabilized, and stained with a primary elastin antibody (4 hrs.) and Cy5 secondary antibody (1 hr.) at room temperature before mounting with DAPI. Elastin image analysis was conducted with ImageJ. Statistical Analysis. Changes in vaginal elastin, outer diameter and wall thickness were assessed using one-way ANOVA with p< 0.05 considered significant.

Results, Conclusions, and Discussions:: Geometric comparison of murine vaginal tissue indicated a significant increase in vaginal unloaded outer diameter (p=0.0037; Fig.1A) and wall thickness (p=0.006; Fig.1B) over early aging. Interestingly, normalized vaginal elastin content, defined as the ratio of elastin-positive area to total wall area, decreased with age (p=0.0073; Fig.1C). Biaxial testing revealed dynamic changes in loaded diameter (Fig.1E) and axial stretch (Fig.1F) throughout adolescence. The rightward shift in circumferential stress-stretch at 3 days (3D), relative to adult (Ctrl), suggests an early increase in distensibility consistent with the ~50% elastin area fraction of the vaginal wall (Fig.1G). Similarities in circumferential stress at 7D, 14D, and 42D are an indication of adaptive tissue remodeling with early aging and somatic growth. Interestingly, 21D samples have elevated circumferential stress consistent with an increased outer diameter-to-thickness ratio (Fig.1A,B). Lastly, axial stress-stretch curves (Fig.1H) highlight a reduction in maximum axial force and extensibility with age.
Discussion and Conclusion. It is established that the vagina undergoes extensive extracellular matrix (ECM) remodeling in adulthood due to factors such as aging, pregnancy, and delivery. However, the cellular origins of vaginal ECM deposition – particularly of elastin and collagen – is poorly understood. To provide insight into vaginal ECM regulation, we focused on characterizing the age-related changes in vaginal structure and function in female mice throughout adolescence. Thus, we could localize when and where ECM is deposited in the vaginal wall and compare these microstructural observations to tissue-level biomechanical responses. In this study, we found significant increases in vaginal outer diameter and thickness that were accompanied by decreased elastin area fraction throughout adolescence. Additionally, we observed evidence of adaptive biomechanical remodeling from our biaxial stress-stretch analysis consistent with somatic growth in early aging.
Future work will expand upon this investigation to include other structural components – such as collagen, glycosaminoglycans, and smooth muscle – with the goal of further elucidating the mechanobiology of age-related changes in the vagina. Characterizing these age-associated developmental alterations may identify the cellular contributors to vaginal ECM regulation and thereby identify potential therapeutic targets for the treatment of cases with altered vaginal ECM properties, as in pelvic organ–related disorders.

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