An Alternative Approach to Vaginal Expansion-Utilizing Estrogen Coated 3-D Printed Vaginal Expansion Sleeves

Background: Vaginal atresia is characterized by an underdeveloped or absent vaginal canal from genetic disorders such as Mayer-Rokitansky-Kuster-Hauser. Current treatment methods utilize mechanical dilation, requiring patient compliance or reconstructive surgery, which is more invasive than mechanical dilation. Previous proof of concept studies in our lab suggested the use of a novel minimally-invasive vaginal expansion sleeve (VES) successfully lengthened the vaginal canal in Sprague Dawley rats. However, histological analysis demonstrated the thinning of the vaginal wall characterized by compression of the soft tissue and stretching of muscle fibers. A follow-up study utilizing treatment with adjunct GLP-2 showed successful lengthening with a decreased but still present mild to moderate inflammation. Estrogen is associated with antiinflammation through several pathways, including the generation of nitric oxide and the inhibition of tumor necrosis factor-α. Purpose: In this project, we aim to investigate the effects of estrogen coating on the VES sleeve functionality. We hypothesize that estrogen contributes to expansion and thickening of the vaginal wall with decreased inflammation. Methods: This study was conducted using an IACUC-approved protocol. The VES sleeve is a woven polyethylene terephthalate cylindrical sleeve with a 3D-printed biocompatible resin cap featuring suture holes for anchorage to the vaginal canal. Six Sprague Dawley rats were anesthetized with isoflurane. To ensure proper sedation, a toe pinch was used. Vaginal lengths were measured with a pediatric anal dilator using lubricating jelly. A VES was then cut to measure 30% larger than the of the measured rat vaginal canal. A dose of 0.005 mg of topical Premarin estrogen (0.625 mg/g) was inserted into the vagina using a Luer Lock syringe before VES implantation. The plunger of the syringe was removed, lubricating jelly was added, and the mixture was reinserted into the vagina to ensure all estrogen was applied. The VES was inserted with the resin cap positioned externally. Two non-absorbable silk sutures anchored to a surgical pledget were used to secure the device in place on each lateral side of the vaginal canal, ensuring a placement inferolateral to the urethra to avoid impaction of urinogenital tract. After surgery, the rats were left to expand the tissue for a week. Rats were observed daily for signs of infection, pain, and/or damage to the VES. If the VES was removed by the rat during the week, surgery was performed again to reinsert the device. No signs of infection were present during the study period; however, the protocol indicates that antibiotic ointment should be placed on external sutures if signs of infection are present. Sleeves were serially replaced weekly and progressively increased in size of 30% of the vaginal canal to facilitate tissue expansion. Following three consecutive sleeve replacements, the VES was removed for two weeks to allow for post-surgical contraction. Vaginal lengths were longitudinally measured through the five-week trial. Following completion of the expansion trial, vaginal tissues were harvested for histological analysis. Vaginal tissue of five control rats was obtained. Each of the vaginal canals were transected down the midline. The tissue was affixed around a wooden stick into a “Swiss-roll” conformation, ensuring the cervical side was internal. A pin was inserted through the tissue to hold its proper shape. Tissue was fixed in 3.7% formaldehyde for at least 24 hours before processing. Samples were embedded in paraffin before sectioning onto glass slides. Slides were stained using hematoxylin and eosin (H&E) and Masson’s trichrome. Results: Weekly estrogen-coated VES yielded an overall mean retained rat vaginal wall length of: 31.5 ± 01.0 mm (week 1), 34.7 ± 0.21 mm (week 2), 36.3 ± 3.8 mm (week 3), 34.5 ± 2.0 mm (week 4), and 34.8 ± 0.8 mm (week 5). The average control vaginal wall length was 29.3 ± 1.2 mm. An overall increase of 19.0% compared to controls was demonstrated (p < 0.001). Previous studies demonstrated an overall increase of 20.4% when used with no adjunct therapy and 35.0% when used with adjunct GLP-2. Despite similar expansion to VES-use only, estrogen-coated VES yielded significantly less expansion than GLP-2-coated VES. However, both previous studies noted moderate inflammation, thinning of the vaginal wall, and eosinophil infiltration. Preliminary histological analysis appears to show that estrogen adjunct therapy increased the overall thickness of the vaginal wall, including the epithelial, elastic, and muscular layers. Particularly, the cellularity of the epithelial layer is increased demonstrating notable multicellular layers. Morphometric analysis is ongoing. Conclusion: VES expansion with adjunct estrogen successfully lengthens vaginal canals, confirming that the estrogen coating did not affect the functionality of the device. Compared to previous VES studies which noted thinning of the vaginal canal, the addition of estrogen increased the thickness of the vaginal wall in the acquired post-mortem specimens. Further histological analysis will focus on the inflammatory response.