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Womens Health

Women's Health - Poster Session D

Poster L17 - Impact of Fibulin-5 Deficiency on Extracellular Matrix Remodeling of the Non-Pregnant Mouse Uterus

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Pelvic organ prolapse (POP) is a benign medical condition that occurs in women wherein one of the pelvic organs descends into an abnormal position. The risk of women experiencing POP in their lifetime is approximately 12-19%, with roughly 300,000 POP surgeries performed yearly in the United States. Even after one POP surgery, women have a 30-50% chance of needing an additional POP surgery in their lifetime. There are many pelvic organs at risk of experiencing POP, such as the bladder, vagina, and uterus. POP is associated with several leading risk factors, such as aging or parity (the number of birth events a woman experiences). The extracellular matrix (ECM) is a major contributor to the biological regulation of organs throughout the body by providing physical support for cells and tissues. One of the main proteins in the ECM is elastin, which promotes tissue stretch and compliance. An elastin-associated glycoprotein that plays a huge role in elastogenesis is fibulin-5 (gene: Fbln5) due to its high binding affinity to tropoelastin, the monomeric subunit of elastin. Past studies have shown that elastin loss is associated with POP. In particular, researchers have studied vaginal remodeling associated with POP development in Fbln5-deficient mice to better understand the effect of altered fibulin-5 expression on vaginal elastic fiber content and biomechanical function. This prior work motivates our current study focused on understanding the effect of fibulin-5 alterations in other pelvic organs, specifically in the mouse uterus.

Materials and

Methods:  This study consisted of a microstructural analysis of 15 different mouse uterine horns from wildtype mice with normal fibulin-5, Fbln5+/+ (n = 5), mice with fibulin-5 partially knocked out, Fbln5+/- (n = 5), and mice with fibulin-5 completely knocked out, Fbln5-/- (n = 5). Each uterine horn was embedded in optimal cutting temperature (OCT) solution and frozen at -80ºC. The frozen tissue blocks were sectioned into 8 μm slices and placed onto histology microscope slides to be stained using immunofluorescence (IF) staining. The IF staining protocol involved tissue fixation and permeabilization prior to applying an unconjugated primary antibody to target tropoelastin (1:300). Since the primary antibody stain is unconjugated, a conjugated secondary antibody stain (Cy5, 1:300) was used to display fluorescence. Additionally, an alpha smooth muscle actin (αSMA) conjugated antibody (Cy3, 1:300) was used to label smooth muscle cells in the uterine horn tissue. Lastly, a DAPI stain was applied to the uterine horn tissue to target the nuclei.

After IF staining, all 15 IF stained cross-sections of uterine horn tissue were imaged at 4 different regions of interest (ROIs): right, up, left, and down. After images were taken, they were processed through Matlab to remove background or negative staining and highlight areas of positive staining. The preprocessed images were then passed into ImageJ for image analysis. Image analysis consisted of calculating the following: tissue area, myometrium area in tissue, percentage of αSMA stain in tissue and myometrium, percentage of elastin stain in tissue and myometrium.

Results, Conclusions, and Discussions: Immunofluorescence staining and imaging revealed a similar uterine wall structure across genotypes (Fig.1). Namely, each genotype had similar cross-sectional area of the uterine wall and outer myometrium layer (Fig.2A,B). Additionally, quantification of the percent of the uterine wall and myometrium that was positive for αSMA staining was similar across genotypes (Fig.2C,D). However, quantification of elastin in the uterine wall revealed that fibulin-5-deficient Fbln5-/- mice had significantly decreased elastin expression. The percentage of elastin in the entire uterine wall was significantly decreased in both Fbln5+/- (partial knockout; p = 0.0259) and Fbln5-/- mice (complete knockout; p = 0.0016), relative to wildtype Fbln5+/+ mice, based on a one-way ANOVA with post-hoc Tukey test for multiple comparisons (Fig.2E). A similar trend was observed when considering myometrial elastin. There was a decreasing trend in Fbln5+/- heterozygous mice that was found to be significantly reduced in Fbln5-/- homozygous mice (p = 0.0056) based on a non-parametric Kruskal-Wallis test with post-hoc Dunn’s test for multiple comparisons (Fig.2F).

Our analysis revealed a significant downward trend in elastin expression with decreasing expression of Fbln5. Namely, the elastin area fraction was found to be significantly reduced throughout the entire uterine wall, and in particular in the myometrium, of Fbln5-/- mice, relative to Fbln5+/+. This loss of elastin is consistent with prior observations of reduced vaginal elastin and correlates with the observed POP phenotype that naturally occurs in the Fbln5-deficient mice. Here we have focused on uterine elastin and smooth muscle, but to enhance our study and understanding of the impact of Fbln5-deficiency on uterine extracellular matrix proteins, we can perform additional microstructural analysis using picrosirius red (PSR) staining to investigate changes in fibrillar collagens. Furthermore, we can perform biaxial mechanical testing on all the uterine horns to identify changes in the biomechanical properties of the tissue as a consequence of reduced elastin expression. Outcomes from this work have the potential to better define the role of elastin in the uterus and identify how alterations in uterine microstructure may be associated with the development of POP.

Acknowledgements (Optional):  This work was made possible by support from the WashU Summer Engineering Fellowship (WUSEF) program.