Pamela A. Moalli

Vaginal reconstruction is limited by the lack of biomaterials that replicate the structure, biomechanics, and biochemistry of the native tissues. Synthetic meshes, xenografts, and autologous skin or bowel grafts are hindered by their immunogenicity, poor integration, and non-physiological properties. We report a scalable platform for the fabrication of patient-specific living vaginal grafts from autologous fibroblasts. Using scaffold-free micromolding and automated assembly, fibroblasts from small full-thickness vaginal biopsies self-assembled into a collagen-rich, highly aligned extracellular matrix. To improve mechanical integrity, we create twisted subunit assemblies that displayed highly aligned collagen, dense cellularity, and a predominantly quiescent fibroblast phenotype with minimal myofibroblast activation. This autologous tissue-specific construct addresses the shortcomings of current materials and offers a customizable and biocompatible solution for regenerative gynecology. By combining tissue specificity, immunologic safety, and modular scalability, this approach has the potential to transform surgical options for congenital anomalies, post-oncologic reconstruction, fistula repair, and pelvic organ prolapse.

The vagina undergoes physiologic changes across the menstrual cycle, pregnancy, birth and menopause. Most women will experience vaginal dysfunction at some point during their lives and treatment options are limited. The cyclic regeneration of the vaginal epithelium during each menstrual cycle suggests it is a stem-cell based tissue; however, human vaginal epithelial stem cells (veSCs) have not been identified. We utilized in vitro colony forming and organoid assays to confirm that cells in the human vaginal epithelium have self-renewal and differentiation potential. Specifically, we determined that stem cell activity resides in the ITGA6+ and NGFR+ fractions of the basal epithelium. We performed single-cell RNA sequencing to identify the distinct cellular compartments of the full thickness human vagina, including spatially distinct populations comprising layers of the stratified vaginal epithelium. Markers of these cells within the vaginal epithelium were validated by immunohistochemistry. CD9+ITGA6+NGFR+ and CD9+ITGA6+NGFR- cells were capable of efficient colony formation but only the CD9+ITGA6+NGFR+ fraction produced organoids containing basal, intermediate and superficial layers of the vaginal epithelium. We characterized the premenopausal human vagina at single cell resolution, validated markers and assays to test veSC developmental potential, and identified putative stem cells that may open new avenues for treating vaginal dysfunction.

Background: Midurethral sling placement is often performed during prolapse repair to treat or prevent stress urinary incontinence. However, some women experience persistent or new-onset stress or urgency urinary incontinence after surgery. It is unclear how prolapse repair, with or without a concomitant midurethral sling, alters urethral morphology and support, and how these changes relate to urinary continence outcomes. Objectives: To compare postoperative urethral morphology (dimensions, angles, shape) and support (position, mobility) after transvaginal prolapse repair with vs without a concurrent midurethral sling, and to explore associations between postoperative urethral characteristics and urinary outcomes (stress, urgency symptoms). Study Design: This ancillary analysis used magnetic resonance imaging and urinary outcome data from the Defining Mechanisms of Anterior Vaginal Wall Descent Study conducted across 8 clinical sites within the United States Pelvic Floor Disorders Network. Eighty-two women (median age, 65 years) underwent transvaginal prolapse repair (vaginal mesh hysteropexy or vaginal hysterectomy with uterosacral ligament suspension) with or without a concurrent midurethral sling between April 2013 and February 2015. Postoperative imaging at rest and during strain was performed 30-42 months after surgery (or earlier if they chose reoperation) between June 2014 and May 2018. Prolapse recurrence, defined as descent beyond the vaginal introitus during strain, was recorded. The urethra was segmented from postoperative scans to create 3-dimensional models for measuring urethral diameters, length, surface area, volume, angles, shape (principal component scores from a statistical shape model), position, and mobility (rest-to-strain displacement). Preoperative and 24-48-month postoperative urinary continence outcomes were assessed using validated questionnaires: the Urogenital Distress Inventory, Urinary Impact Questionnaire, and the Incontinence Severity Index. Comparisons of urethral and urinary outcomes by (1) midurethral sling and (2) stress urinary incontinence were made using Wilcoxon rank-sum tests, principal component analysis, and multivariate models as appropriate. Associations between urethral and urinary outcomes were evaluated with Spearman’s rank correlation. Results: Forty-six women (22 hysteropexy, 24 hysterectomy) were in the sling group, and 36 (19 hysteropexy, 17 hysterectomy) were in the no-sling group. Among the 48 women without prolapse recurrence (28 sling, 20 no-sling), those with a sling (vs without) had larger urethral dimensions (all P<.03), a more anterior-superior position of the proximal urethra (indicating better bladder neck support) (P=.04), a straighter urethral shape (P=.006), and reported less bothersome postoperative stress incontinence (P=.02). Overall, 14 women (17%) experienced postoperative stress incontinence. Stress urinary incontinence was linked to a more acute proximal urethral sagittal angle (more aligned with axial plane) (P=.01), and a lower proximal urethra position (P=.04) and mid-urethra position (P=.03). Poorer stress and urgency urinary outcomes were associated with a shorter urethral length (P=.01), a more posterior-inferior urethral position (all P<.05), increased “C” or “S”-shaped urethral concavity (P=.008; P=.006), and smaller rest-to-strain displacement of the proximal (P=.03) and distal (P=.009) urethra. Conclusions: Urethral morphology and support differed with concomitant midurethral sling (vs no sling) and stress urinary incontinence after vaginal surgery. Urethral characteristics were also associated with postoperative urinary symptoms. Urethral configuration may influence urinary outcomes and could be considered during prolapse and stress urinary incontinence repairs. CONDENSATION PAGE: TWEETABLE STATEMENT: Vaginal prolapse repair with midurethral sling (vs without) was linked to a larger, straighter urethra, and better urinary outcomes. Poorer urinary outcomes were linked to a shorter, “C”- or “S”-shaped urethra. SHORT TITLE: Urethral Morphology Associated with Midurethral Sling and Postoperative Urinary Outcomes AJOG AT A GLANCE: A. Why was this study conducted?: Some women experience urinary incontinence after prolapse repair with or without a concomitant midurethral sling. It remains unclear how midurethral sling surgery alters urethral morphology and support, and how these changes relate to postoperative urinary symptoms. B. What are the key findings?: This magnetic resonance imaging study compared postoperative urethral characteristics (morphology, support) and urinary outcomes among women after vaginal prolapse repair with and without concomitant midurethral sling. Among women without prolapse recurrence, midurethral sling (vs no sling) was associated with larger urethral dimensions, a straighter, better-supported urethra, and superior urinary outcomes. Poorer urinary outcomes were associated with a shorter, curvier urethra and worse urethral support. C. What does this study add to what is already known?: Postoperative urethral morphology and support differ by midurethral sling and were associated with urinary outcomes after vaginal prolapse repair. Urethral configuration warrants surgical consideration in prolapse and urinary incontinence repairs.

Pelvic organ prolapse (POP) repair using synthetic mesh introduces mechanical mismatches that significantly influence fibroblast behavior. As the primary mechanosensitive cells in vaginal connective tissue, fibroblasts respond to extracellular stiffness via mechanotransduction pathways, such as focal adhesions (FAs), transforming growth factor β (TGF-β) signaling, and others. These pathways regulate key cellular responses, including proliferation, apoptosis, and differentiation into myofibroblasts. While transient myofibroblast activation is essential for normal wound healing, sustained activation driven by mesh can result in complications—primarily pain and fibrosis. This review highlights the molecular and mechanical mechanisms underlying fibroblast-to-myofibroblast transition (FMT), with a focus on integrin-mediated adhesion complexes and cytoskeletal signaling. It also explores how mesh architecture and local mechanical cues contribute to pathological tissue remodeling in POP. Understanding these processes can inform the design of biomechanically compatible meshes and targeted therapeutics such as ROCK or YAP inhibitors to mitigate adverse fibroblast responses and improve surgical outcomes.

STRUCTURED Importance: Sexual dysfunction can occur after midurethral sling (MUS) and transvaginal prolapse surgery. It remains unclear whether these procedures impact the clitoris, despite its role in sexual function and proximity to the MUS and vagina. Objectives: To compare postoperative sexual function and clitoral features by MUS and vaginal surgery approach after transvaginal prolapse repair with/without concomitant MUS. Design: Cross-sectional ancillary study of magnetic resonance imaging (MRI) and sexual function data from the Defining Mechanisms of Anterior Vaginal Wall Descent study. Setting: Eight clinical sites in the US Pelvic Floor Disorders Network. Participants: 88 women with uterovaginal prolapse who underwent vaginal mesh hysteropexy or vaginal hysterectomy with uterosacral ligament suspension with/without MUS between 2013-2015. Data were analyzed between September 2021-June 2023. Exposures: Between June 2014-May 2018, participants underwent pelvic MRI 30-42 months after surgery, or earlier if reoperation was desired. Sexual activity and function at baseline and 24-48-month follow-up were evaluated using the Pelvic Organ Prolapse/Incontinence Sexual Questionnaire, IUGA-Revised (PISQ-IR). Clitoral features were obtained from postoperative MRI-based 3-dimensional models. Main Outcomes and Measures: PISQ-IR scores and clitoral features (size, position). Results: Eighty-two women (median [range] age, 65 [47-79] years) were analyzed: 45 MUS (22 hysteropexy, 23 hysterectomy) and 37 No-MUS (19 hysteropexy, 18 hysterectomy). Postoperatively, 25 MUS, 12 No-MUS, 20 hysteropexy, and 17 hysterectomy patients were sexually active (SA). Overall, within the MUS and vaginal surgery groups, sexual function remained unchanged or improved (most PISQ-IR change from baseline scores were ≥0) among SA and NSA women. Among SA women after surgery, the MUS group (vs No-MUS) had a poorer PISQ-IR arousal/orgasm (SA-AO) score (median, 3.5 vs 4.3; P=.02). The hysteropexy group (vs hysterectomy) had less improvement in PISQ-IR SA-AO score (median, 0.0 vs 0.3; P=.01). Women with MUS (vs without) had a smaller clitoral glans thickness (median, 9.0 mm vs 10.0 mm; P=.008) and clitoral body volume (median, 2783.5 mm ³ vs 3587.4 mm ³ ; P=.01). Conclusions and Relevance: SA women with MUS (vs without) or hysteropexy (vs hysterectomy) experienced poorer postoperative sexual function. MUS was linked to a smaller clitoris. Future studies should explore surgery-induced changes in clitoral anatomy and sexual function. KEY POINTS: Question: How do sexual function and clitoral anatomy differ by midurethral sling placement and vaginal surgery approach? Findings: This cross-sectional study compared patient-reported sexual function outcomes and 30-42-month postoperative magnetic resonance imaging-based 3-dimensional clitoral models of 82 women after vaginal prolapse surgery with or without concomitant midurethral sling. Midurethral sling (vs no sling) and vaginal mesh hysteropexy (vs vaginal hysterectomy) were associated with poorer postoperative sexual function outcomes. Additionally, midurethral sling was associated with a smaller clitoral glans and body. Meaning: Midurethral sling and vaginal mesh hysteropexy were associated with, and may adversely alter, postoperative sexual function and/or clitoral anatomy. VISUAL ABSTRACT/PROMOTIONAL IMAGE: