Vaginal Smooth Muscle Research Articles

Functional outcome of the anterior vaginal wall in a pelvic surgery injury rat model after treatment with stem cell-derived progenitors of smooth muscle cells.

Background : Stem-cell-derived therapy is a promising option for tissue regeneration. Human iPSC-derived progenitors of smooth muscle cells (pSMCs) have limited proliferation and differentiation, which may minimize the risk of in vivo tumor formation while restoring smooth muscle cell deficiencies. Up to 30 % of women who suffer from recurrence of vaginal prolapse after prolapse surgery are faced with reoperation. Therefore, there is an unmet need for therapies that can restore vaginal tissue function. We hypothesize that human pSMCs can restore vaginal function in a vaginal-injury rat model. Methods : Female immune-compromised RNU rats were divided into 5 groups: intact controls (n=12), VSHAM (surgery + saline injection, n=33), and cell-injection group (surgery + cell injection using three patient pSMCs lines, n=14/cell line). The surgery, similar to what is done in vaginal prolapse surgery, involved ovariectomy, urethrolysis, and vagina injury. The vagina, urethra, bladder dome and trigone were harvested 10 weeks after surgery (5 weeks after injection). Organ bath myography was performed to evaluate the contractile function of vagina, and smooth muscle thickness was examined by tissue immunohistochemistry. Collagen I, collagen III, and elastin mRNA and protein expressions in tissues were assessed. Results : When compared to the VSHAM group, cell-injection groups showed significantly increased vaginal smooth muscle contractions induced by carbachol (groups A and C) and by KCl (group C), and significantly higher collagen I protein expression in the vagina (groups A and B). Elastin mRNA and protein expressions in the vagina did not correlate with injection group. In the urethra, mRNA expressions of collagen I, collagen III, and elastin were all significantly higher in the cell-injection groups compared to the VSHAM group. Collagen I protein expression of the urethra was also higher in the cell-injection group compared to the VSHAM group. Elastin protein expression in the urethra did not correlate with injection group. Conclusions : Human iPSC-derived pSMCs improved contractile function of the post-surgery vagina. Additionally, pSMC injection modulated collagen I, collagen III and elastin mRNA and protein expressions in the vagina and urethra. These findings suggest that pSMCs may be a possible therapy for vaginal prolapse recurrence after surgical intervention.

Biophysical Mechanisms of Vaginal Smooth Muscle Contraction: The Role of the Membrane Potential and Ion Channels.

The vagina is an essential component of the female reproductive system and is responsible for providing female sexual satisfaction. Vaginal smooth muscle contraction plays a crucial role in various physiological processes, including sexual arousal, childbirth, and urinary continence. In pathophysiological conditions, such as pelvic floor disorders, aberrations in vaginal smooth muscle function can lead to urinary incontinence and pelvic organ prolapse. A set of cellular and sub-cellular physiological mechanisms regulates the contractile properties of the vaginal smooth muscle cells. Calcium influx is a crucial determinant of smooth muscle contraction, facilitated through voltage-dependent calcium channels and calcium release from intracellular stores. Comprehensive reviews on smooth muscle biophysics are relatively scarce within the scientific literature, likely due to the complexity and specialized nature of the topic. The objective of this review is to provide a comprehensive description of alterations in the cellular physiology of vaginal smooth muscle contraction. The benefit associated with this particular approach is that conducting a comprehensive examination of the cellular mechanisms underlying contractile activation will enable the creation of more targeted therapeutic agents to control vaginal contraction disorders.

Remodeling of murine vaginal smooth muscle function with reproductive age and elastic fiber disruption

Advanced maternal age during pregnancy is associated with increased risk of vaginal tearing during delivery and maladaptive postpartum healing. Although the underlying mechanisms of age-related vaginal injuries are not fully elucidated, changes in vaginal microstructure may contribute. Smooth muscle cells promote the contractile nature of the vagina and contribute to pelvic floor stability. While menopause is associated with decreased vaginal smooth muscle content, whether contractile changes occur before the onset of menopause remains unknown. Therefore, the first objective of this study was to quantify the active mechanical behavior of the murine vagina with age. Further, aging is associated with decreased vaginal elastin content. As such, the second objective was to determine if elastic fiber disruption alters vaginal contractility. Vaginal samples from mice aged 2–14 months were used in maximum contractility experiments and biaxial extension-inflation protocols. To evaluate the role of elastic fibers with age, half of the vaginal samples were randomly allocated to enzymatic elastic fiber disruption. Contractile potential decreased and vaginal material stiffness increased with age. These age-related changes in smooth muscle function may be due, in part, to changes in microstructural composition or contractile gene expression. Furthermore, elastic fiber disruption had a diminished effect on smooth muscle contractility in older mice. This suggests a decreased functional role of elastic fibers with age. Quantifying the age-dependent mechanical contribution of smooth muscle cells and elastic fibers to vaginal properties provides a first step towards better understanding how age-related changes in vaginal structure may contribute to tissue integrity and healing. Statement of significanceAdvanced maternal age at the time of pregnancy is linked to increased risks of vaginal tearing during delivery, postpartum hemorrhaging, and the development of pelvic floor disorders. While the underlying causes of increased vaginal injuries with age and associated pathologies remain unclear, changes in vaginal microstructure, such as elastic fibers and smooth muscle cells, may contribute. Menopause is associated with fragmented elastic fibers and decreased smooth muscle content; however, how reproductive aging affects changes in the vaginal composition and the mechanical properties remains unknown. Quantifying the mechanical contribution of smooth muscle cells and elastic fibers to vaginal properties with age will advance understanding of the potential structural causes of age-related changes to tissue integrity and healing.

Abstract P164: Montelukast, A Leucine Rich Repeat Containing 8A Inhibitor, Decreases Contractility Of Vaginal Smooth Muscle In Diabetic Mice: A Possible Treatment For Female Sexual Dysfunction In Diabetes

Vaginal smooth muscle (VaSM) is critical to sexual function, desire, and arousal. Individuals with hypertension or diabetes have smooth muscle dysfunction which is associated with elevated superoxide anion production and increased inflammation in smooth muscle. Further, endothelial dysfunction is common in the small arteries, like the pudendal arteries, that supply the sex organs in females. This loss of function could contribute to female sexual dysfunction (FSD). This study aims to bridge the gap to treat hypertensive or diabetic individuals’ sexual dysfunction and discover non-hormonal treatments to increase blood flow to the VaSM and clitoral tissue. In this study, we hypothesize that the inhibition of leucine rich repeat containing 8A (LRRC8A) volume regulated anion channels (VRACs) will decrease the contraction to endothelin-1 (ET-1) in vaginal smooth muscle of lean and diabetic (db/db) mice. LRRC8A channels have been proposed to modulate entry of extracellular superoxide (O •- ) associated with NADPH oxidase activity. The activity of NADPH oxidase is increased in diabetes and hypertension. Female young lean and db/db (12 wks) mice underwent distal vaginal dissection. The VaSM was then mounted on a strip myograph for measurement of isometric force development. Contraction to ET-1 was performed in the absence and presence of the LRRC8A inhibitor, montelukast (1uM) (MONT) after a 30-minute incubation. The weight/length of VaSM (mg/mm) was used to normalize the maximum contraction (mN). Measures of weight/length in db/db mice VaSM tissue were less than in lean mice (p=0.0112). In lean mice, force development to ET-1 was significantly less after incubation with MONT (logEC50 control 8.428 +/- 0.06886 vs. drug 7.859 +/- 0.06083, p= 0.0007 ). In db/db mice, force development to ET-1 was significantly less after incubation with MONT (logEC50 control 8.779 +/- 0.1007 vs. drug 7.278 +/- 0.05490 , p= 0.0004). Treatment with an inhibitor of LRRC8A offers a novel approach to treat FSD and should be further researched to observe its overall impact. LRRC8A may play an important physiological role in the maintenance of the contractile response by managing the flux of O •- in the VaSM, making it an important potential target in conditions like hypertension and diabetes.

(008) Chronic High Fat Diet Decreases Vaginal Smooth Muscle Tone but does not Affect Pelvic Floor Skeletal Muscle in Mice

Abstract Introduction Pelvic organ prolapse (POP) is a disease that affects the physical and mental health of women, as well as their quality of life. POP, regardless of severity, can be a direct cause of female sexual dysfunction (FSD) by producing pain during intercourse or a general reluctance to engage in sexual activity. The largest risk factors for POP include parity, obesity, and advanced age. Obesity, a modifiable risk factor, is believed to increase intra-abdominal pressure and weaken the pelvic floor muscles and fascia. Studies have shown that obesity and a high fat diet (HFD) can weaken skeletal muscle by increasing collagen deposition and inducing muscle apoptosis. Objectives There is paucity in the literature of animal studies that have specifically investigated the effect of obesity on the support structures of the pelvic floor and how it contributes to POP. The objective of this study is to examine the structure and function of the vagina and pelvic floor in female mice fed a chronic HFD. Methods Adult female C57/Blk6N mice (10 weeks, n=32) were fed a control (10% kcal fat) or HFD (45% kcal fat) for 24 weeks. Ex vivo external vaginal sphincter contraction to increasing concentrations of adrenergic agonist norepinephrine and relaxation to nitric oxide donor DEA NONOate and electric field stimulation (EFS) was measured. Iliococcygeus (IC) muscles of the pelvic floor were isolated and set to optimal length and tetanic contractions were performed at increasing frequencies to asses absolute and specific force. Fatigue was measured following repetitive stimulations over 5 minutes. IC muscle fiber types were characterized with anti-myosin antibodies and cross-sectional area (CSA) of each fiber type was measured. Results Vaginal sphincter adrenergic-mediated contraction was a significantly decreased in HFD-fed mice (Fig 1A; p<0.05). The vaginal sphincter smooth muscle relaxation in response to a nitric oxide donor was greater in the HFD mice (Fig 1B; p<0.05). Neurogenic-mediated EFS vaginal relaxations were also enhanced in the HFD mice (p<0.05). Surprisingly, chronic HFD did not impact iliococcygeus muscle specific force, absolute force, or muscle fatigue (Fig 1C). Furthermore, HFD did not affect the CSA or population of type I, IIa, IIX, or IIb iliococcygeus muscle fibers. Conclusions HFD-induced obesity in mice leads to pelvic floor weakness by enhancing relaxation and decreasing tone of the vaginal smooth muscle. Interestingly, the function and composition of pelvic skeletal muscle was unaffected by HFD. As the vagina serves as a primary support structure to the pelvic organs, lack in muscle contractility and increase in relaxation following HFD suggests that obesity-induced POP may play a direct role in FSD. Disclosure

(082) The Effect of Sex Steroids on RhoA/ROCK Pathway in Rat Distal Vagina

Abstract Introduction The RhoA/ROCK calcium-sensitizing pathway plays a role in clitoral contractility, and therefore we deemed interesting to investigate its involvement in the vagina. Objective The aim of this study was to investigate the sex steroid regulation of the vagina contractility through the RhoA/ROCK pathway, using a validated animal model. Methods Ovariectomized Sprague-Dawley rats (OVX) were treated with 17β-estradiol (E2), testosterone (T), with or without letrozole (T+L), and compared with intact animals. Contractility studies were performed on vaginal strips to test the effect of ROCK inhibitor Y-27632 and NO synthase inhibitor L-NAME. In vaginal tissues, ROCK1 immunolocalization was investigated, mRNA expression was analyzed by semi-quantitative RT-PCR, and RhoA membrane translocation was evaluated by Western blot (Wb) analysis. Finally, rat smooth muscle cells (rvSMCs) were isolated from distal vagina of intact animals and the quantification of RhoA inhibitory protein RhoGDI was performed by Wb after stimulation with NO-donor SNP, with or without the soluble guanylate cyclase inhibitor ODQ or the PKGR1 inhibitor KT5823 administration. Results The results show that ROCK1 was immunolocalized in the smooth muscle bundles and in the blood vessel wall of vagina, while a weak positivity was detected in the epithelium. Y-27632 induced a dose-dependent relaxation of noradrenaline pre-contracted vaginal strips, decreased by OVX and completely restored by E2, while T and T+L further decreased it even below OVX level increased it. Accordingly, in Wb analysis, compared to controls, OVX significantly induced RhoA activation, as reveled by its membrane translocation, with T reverting it at a level significantly lower than in controls. This effect was not exerted by E2. Abolishing the NO formation via L-NAME increased Y-27632 responsiveness in the OVX+T group; L-NAME had only a partial effect in controls whilst it did not modulate Y-27632 responsiveness in OVX. Finally, stimulation of rvSMCs with SNP significantly increased the RhoGDI protein expression, an effect counteracted by ODQ and, partially, by the protein kinase inhibitor KT5823 incubation. Conclusions In conclusion, in vagina androgen administration in OVX functionally decreases RhoA/ROCK activity by hampering RhoA membrane translocation and plays a critical role in the inhibitory mechanism of the smooth muscle distal vagina contractility. Accordingly, androgens, by inhibiting the RhoA/ROCK pathway, could positively contribute to vaginal smooth muscle relaxation, favoring the sexual intercourse. Disclosure No

Montelukast, a LRRC8A inhibitor, decreases the contractile response to Endothelin-1 in the vaginal smooth muscle of aged mice

Introduction: Vaginal smooth muscle (VaSM) is an important contributor to female sexual function, desire, and arousal. Increased vaginal blood flow via vasodilation of the vaginal blood vessels is key to the of lubrication and creating an enjoyable sexual experience. Female sexual dysfunction (FSD) is common in hypertensive and diabetic women, as well as many who suffer from hormonal imbalances, including menopause. Despite the substantial population that suffers from FSD, research into the mechanisms and non-hormonal treatments are limited. Leucine rich repeat containing channel 8A (LRRC8A) is a volume regulated anion channel (VRAC) that regulates Nox1 and can modulate the extracellular production of superoxide (O2∙-). In aged tissues, there are increased in extracellular ROS production as well as vascular and smooth muscle dysfunction. The objective of these experiments is to elucidate the mechanisms behind female sexual dysfunction that present with vascular and smooth muscle dysfunction and determine if the LRRC8A inhibitor, Montelukast, can alleviate some of the symptoms of FSD and improve quality of life. In this study, we hypothesize that the inhibition of LRRC8A in will decrease the contraction to Endothelin-1 in the vaginal smooth muscle of the aged mice. Methods: Aged (16 mo and 20 mo old) female lean mice underwent distal vaginal dissection. Vaginal tissue was cleaned and mounted on a strip myograph in Krebs solution and the contraction elicited by Endothelin-1 was performed in the absence and repeated in the presence of the LRRC8A inhibitor, Montelukast (1uM) after a 30-minute incubation. Data was analyzed using Prism. The maximal contraction in the absence of the inhibitor was taken as 100%, and the contraction in the presence of the inhibitor was calculated as a percentage of the maximal contraction attained in the absence of the inhibitor. Results: Contraction induced by Endothelin-1 (10-12 M to 3✕10 -8 M) alone compared to in the presence of Montelukast (1 uM) showed that Montelukast decreased both the potency and the maximum force of contraction in the aged VaSM. After incubation with 1 mM Montelukast, the EC50 and Emax were significantly different (0.0430 and <0.0001, respectively). Conclusion: This is the first study investigating the vaginal smooth muscle from the perspective of ROS production and the influence of LRRC8A in aged animals. The change in the contractile response in the treated animals may be due to the modulation of the production of O2∙-. Patients that suffer from FSD are a large and overlooked community in need of new therapeutic approaches. Treatment with LRRC8A offers a potential novel approach to treat FSD and should be further researched to observe its overall impact in FSD. 1R01DK132948-01, SMSNA Student Grant This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Smooth muscle contribution to vaginal viscoelastic response

Smooth muscle cells contribute to the mechanical function of various soft tissues, however, their contribution to the viscoelastic response when subjected to multiaxial loading remains unknown. The vagina is a fibromuscular viscoelastic organ that is exposed to prolonged and increased pressures with daily activities and physiologic processes such as vaginal birth. The vagina changes in geometry over time under prolonged pressure, known as creep. Vaginal smooth muscle cells may contribute to creep. This may be critical for the function of vaginal and other soft tissues that experience fluctuations in their biomechanical environment. Therefore, the objective of this study was to develop methods to evaluate the contribution of smooth muscle to vaginal creep under multiaxial loading using extension – inflation tests. The vaginas from wildtype mice (C57BL/6 × 129SvEv; 3–6 months; n = 10) were stimulated with various concentrations of potassium chloride then subjected to the measured in vivo pressure (7 mmHg) for 100 s. In a different cohort of mice (n = 5), the vagina was stimulated with a single concentration of potassium chloride then subjected to 5 and 15 mmHg. A laser micrometer measured vaginal outer diameter in real-time. Immunofluorescence evaluated the expression of alpha-smooth muscle actin and myosin heavy chain in the vaginal muscularis (n = 6). When smooth muscle contraction was activated, vaginal creep behavior increased compared to the relaxed state. However, increased pressure decreased the active creep response. This study demonstrated that extension – inflation protocols can be used to evaluate smooth muscle contribution to the viscoelastic response of tubular soft tissues.

The regulatory effect of sex steroids on the RhoA/ROCK pathway in the rat distal vagina.

Sex steroids have been demonstrated as important modulators of vaginal function. The RhoA/ROCK calcium-sensitizing pathway plays a role in genital smooth muscle contractile mechanism, but its regulation has never been elucidated. This study investigated the sex steroid regulation of the vaginal smooth muscle RhoA/ROCK pathway using a validated animal model. Ovariectomized (OVX) Sprague-Dawley rats were treated with 17β-estradiol (E2), testosterone (T), and T with letrozole (T + L) and compared with intact animals. Contractility studies were performed to test the effect of the ROCK inhibitor Y-27632 and the nitric oxide (NO) synthase inhibitor L-NAME. In vaginal tissues, ROCK1 immunolocalization was investigated; mRNA expression was analyzed by semiquantitative reverse transcriptase-polymerase chain reaction; and RhoA membrane translocation was evaluated by Western blot. Finally, rat vaginal smooth muscle cells (rvSMCs) were isolated from the distal vagina of intact and OVX animals, and quantification of the RhoA inhibitory protein RhoGDI was performed after stimulation with NO donor sodium nitroprusside, with or without administration of the soluble guanylate cyclase inhibitor ODQ or PRKG1 inhibitor KT5823. Androgens are critical in inhibiting the RhoA/ROCK pathway of the smooth muscle compartment in the distal vagina. ROCK1 was immunolocalized in the smooth muscle bundles and blood vessel wall of the vagina, with weak positivity detected in the epithelium. Y-27632 induced a dose-dependent relaxation of noradrenaline precontracted vaginal strips, decreased by OVX and restored by E2, while T and T + L decreased it below the OVX level. In Western blot analysis, when compared with control, OVX significantly induced RhoA activation, as revealed by its membrane translocation, with T reverting it at a level significantly lower than in controls. This effect was not exerted by E2. Abolishing NO formation via L-NAME increased Y-27632 responsiveness in the OVX + T group; L-NAME had partial effects in controls while not modulating Y-27632 responsiveness in the OVX and OVX + E2 groups. Finally, stimulation of rvSMCs from control animals with sodium nitroprusside significantly increased RhoGDI protein expression, counteracted by ODQ and partially by KT5823 incubation; no effect was observed in rvSMCs from OVX rats. Androgens, by inhibiting the RhoA/ROCK pathway, could positively contribute to vaginal smooth muscle relaxation, favoring sexual intercourse. This study describes the role of androgens in maintaining vaginal well-being. The absence of a sham-operated animal group and the use of the only intact animal as control represented a limitation to the study.

Distal Vagina Smooth Muscle Contractility Mechanisms: Effects of in Vivo Sex Steroids Treatment in an Animal Experimental Model of Ovariectomy

ABSTRACT Introduction Female sexual response is a complex phenomenon that involves multiple psychophysiological factors. Sex steroids play a fundamental role in the mechanisms of regulation of sexual arousal, modulating the sexual response and preserving the integrity and functionality of the vaginal tissue. For this reason, imbalances in the sexual environment of steroids in women favor the onset of important pathophysiological conditions such as hypoactive sexual desire disorder (HSDD) and genitourinary syndrome of menopause (GSM). Previous research has focused primarily on the effects of estrogen supplementation on vaginal physiology, but only few studies evaluated the effects of androgen administration, particularly on the vaginal smooth muscle compartment. The calcium sensitizing pathway of RhoA/ROCK may play a role in regulating vaginal contractility but, at present, the regulatory mechanisms of RhoA/ROCK signaling in the female genitourinary tract have yet to be fully elucidated. In particular, no specific research has been conducted on the effects of testosterone (T) on the contractility mechanism of smooth muscle in the vagina. Objective The aim of this study was to evaluate the sex steroid regulation on the distal vagina contractility mechanism, mediated by the RhoA/ROCK pathway, using a validated animal model of female ovariectomized rats. Methods Ovariectomized Sprague-Dawley rats (OVX) were treated with 17β-estradiol (OVX+E), testosterone (OVX+T), or testosterone and letrozole (OVX+T+L) and compared with an intact animals’ group (CTRL). In vitro contractility studies were performed on noradrenaline (NA) pre-contracted vaginal strips to investigate the effect of OVX and in vivo treatments on vaginal smooth muscle activity. The mRNA from vagina tissues isolated from each experimental group was analyzed by semi-quantitative RT-PCR, and membrane translocation of RhoA was evaluated by Western blot analysis. Results Y-27632 induced a dose-dependent relaxation on NA pre-contracted vaginal strips that was significantly decreased by OVX; T, with or without L, further decreased OVX-induced hyporesponsiveness to Y-27632, whilst it was fully restored up to control level by 17β-estradiol (E) supplementation. Accordingly, gene expression analysis indicated that OVX decreased the expression of RhoA/ROCK pathway markers, which was normalized by E. However, at variance with contractility results, androgen receptor (AR) activation upregulated, rather than downregulating, RhoA/ROCK expression. In Western blot analysis, OVX induced activation and membrane translocation of the active form of RhoA as compared to controls. Interestingly, T reverted this effect by reducing RhoA translocation to membrane to a level even significantly below the control one. This effect was not exerted by E administration. Considering that AR activation increases NO signaling (as measured by Prkg1 mRNA expression), we tested the effect of abolishing NO formation through the NO synthase inhibitor L-NAME. Pre-incubation with L-NAME substantially increased Y-27632 responsiveness in the OVX +T group and partially in the CTRL one and, but not in the OVX group. Conclusions This study confirms that in vagina estrogens increase the expression of the calcium-sensitizing pathway RhoA/ROCK, while androgen administration functionally decreases RhoA/ROCK activity, by hampering RhoA translocation to the membrane, a mechanism which is most probably driven by NO. Disclosure No

Testosterone positively regulates vagina NO-induced relaxation: An experimental study in rats

ABSTRACT Introduction Female sexual response is a result of a complex interplay between central and peripheral mechanisms and the nitric oxide (NO)-mediated relaxation of clitoris and distal vagina vascular bed leads to clitoris tumescence and vagina lubrification. Objective The aim of this study was to evaluate sex steroids regulation of the relaxant/contractile pathway in vagina, using a validated animal model of ovariectomized (OVX) Sprague-Dawley rats. Methods Subgroups of OVX rats were treated with 17β-estradiol (10 mg/kg/die), testosterone (T, 30 mg/kg/week), or testosterone and letrozole (2.5 mg/kg/die) for 6 weeks. The experimental groups were compared with a group of intact rats. In vitro contractility studies were carried out in order to investigate the effect of OVX and in vivo treatments on vaginal smooth muscle activity. RNA from vaginal tissue was analyzed by semi-quantitative RT-PCR. Results In vitro contractility studies were performed on noradrenaline pre-contracted distal vaginal strips, isolated from each experimental group. Acetylcholine (Ach; 0.001-10 µM) stimulation induced a concentration-dependent relaxation, which was significantly reduced by NO-synthase inhibitor L-NAME (100 µM) and by guanylate cyclase inhibitor ODQ (1 µM). OVX resulted in a decreased responsiveness to Ach, restored by T supplementation, with or without letrozole, but not by 17β-estradiol. Vaginal strips sensitivity to the NO-donor SNP (1 nM - 100 µM) was higher in OVX than in the control. Vardenafil (100 nM), a PDE5 inhibitor, enhanced SNP effect in OVX+T as well as in control, suggesting that T positively modulates PDE5, as supported by RNA expression analysis. Conclusions In vitro contractility studies and mRNA expression analysis highlight the positive regulation of T on Ach- and NO-dependent muscle relaxation by modulating the NO/cGMP/PKG pathways.Our data demonstrate that testosterone improves the NO-mediated vascular smooth muscle vaginal cells relaxation confirming its critical role in maintaining the integrity of muscular relaxant machinery. Our study confirms the decisive role played by androgens, rather than estrogens, in maintaining the functionality and integrity of the vagina. This study might provide support for the use of androgens in the treatment of genitourinary syndrome of menopause (GSM) and/or sexual arousal disorders related to ageing related-hormonal deficiency. Disclosure Work supported by industry: no.

Microenergy acoustic pulse therapy restores urethral wall integrity and continence in a rat model of female stress incontinence.

To determine the outcomes and mechanisms of microenergy acoustic pulse (MAP) therapy in an irreversible rat model of female stress urinary incontinence. Twenty-four female Sprague-Dawley rats were randomly assigned into four groups: sham control (sham), vaginal balloon dilation and ovariectomy (VBDO), VBDO + β-aminopropionitrile (BAPN), and VBDO + β-aminopropionitrile treated with MAP (MAP). MAP therapy was administered twice per week for 4 weeks. After a 1-week washout period, all 24 rats were evaluated with functional and histological studies. The urethral vascular plexus was examined by immunofluorescence staining with antibodies against collagen IV and von Willebrand factor (vWF). The urethral smooth muscle stem/progenitor cells (uSMPCs) were isolated and functionally studied in vivo and in vitro. Functional study with leak point pressure (LPP) measurement showed that the MAP group had significantly higher LPPs compared to VBDO and BAPN groups. MAP ameliorated the decline in urethral wall thickness and increased the amount of extracellular matrix within the urethral wall, especially in the urethral and vaginal elastic fibers. MAP also improved the disruption of the urethral vascular plexus in the treated animals. In addition, MAP enhanced the regeneration of urethral and vaginal smooth muscle, and uSMPCs could be induced by MAP to differentiate into smooth muscle and neuron-like cells in vitro. MAP appears to restore urethral wall integrity by increasing muscle content in the urethra and the vagina and by improving the urethral vascular plexus and the extracellular matrix.

Smooth Muscle Organization and Nerves in the Rat Vagina: A First Look Using Tissue Clearing and Immunolabeling.

Smooth muscle fiberswithin the vagina, as well as the nervefibers that contribute to their control mechanisms, are important for the maintenance and alteration of vaginal length and tone. Vaginal smooth muscle (VaSM) is typically described as being arranged into two distinct concentric layers: an inner circular muscular layer and an outer longitudinal muscular layer. However, the distribution of VaSM oriented in the longitudinal direction (LD) and circumferential direction (CD) has never been quantified. In this study, tissue clearing and immunohistochemistry were performed so that the VaSM, and surrounding nerves, within whole rat vaginas ([Formula: see text]) could be imaged without tissue sectioning, preserving the three-dimensional architecture of the organs. Using these methods, the vagina was viewed through the full thickness of the muscularis layer, from the distal to the proximal regions. The VaSM orientation in the proximal and distal regions and the VaSM content along the LD and CD were quantified. Additionally, a qualitative assessment of vaginal nerves was performed. When compared using a permuted version of the Watson [Formula: see text] test, the orientation of VaSM in the proximal and distal regions were found to be significantly different in 4 of the 6 imaged rat vaginas ([Formula: see text]). While the distal vagina contained a similar amount of VaSM oriented within [Formula: see text] of the LD and within [Formula: see text] of the CD, the proximal vagina contained significantly more VaSM oriented towards the LD than towards the CD. Nerve fibers were found to be wavy, running both parallel and perpendicular to vascular and non-vascular smooth muscle within the vagina. Micro-structural analyses, like the one conducted here, are necessary to understand the physiological function and pathological changes of the vagina.

Testosterone positively regulates vagina NO-induced relaxation: an experimental study in rats

PurposeFemale sexual response involves a complex interplay between neurophysiological mechanisms and the nitric oxide (NO)-mediated relaxation of clitoris and vagina. The aim of this study was to evaluate sex steroids regulation of the relaxant pathway in vagina, using a validated animal model.MethodsSubgroups of OVX Sprague–Dawley rats were treated with 17β-estradiol, testosterone, or testosterone and letrozole, and compared with a group of intact animals. Masson’s trichrome staining was performed for morphological evaluation of the distal vaginal wall, in vitro contractility studies investigated the effect of OVX and in vivo treatments on vaginal smooth muscle activity. RNA from vaginal tissue was analyzed by semi-quantitative RT-PCR.ResultsImmunohistochemical analysis showed that OVX induced epithelial and smooth muscle structural atrophy, testosterone and testo + letrozole increased the muscle bundles content and organization without affecting the epithelium while 17β-estradiol mediated the opposite effects. In vitro contractility studies were performed on noradrenaline pre-contracted vaginal strips from each experimental group. Acetylcholine (0.001–10 µM) stimulation induced a concentration-dependent relaxation, significantly reduced by NO-synthase inhibitor L-NAME and by guanylate cyclase inhibitor ODQ. OVX resulted in a decreased responsiveness to acetylcholine, restored by testosterone, with or without letrozole, but not by 17β-estradiol. OVX sensitivity to the NO-donor SNP was higher than in the control. Vardenafil, a PDE5 inhibitor, enhanced SNP effect in OVX + testosterone as well as in control, as supported by RNA expression analysis.ConclusionsOur study demonstrates that testosterone improves the NO-mediated smooth muscle vaginal cells relaxation confirming its role in maintaining the integrity of muscular relaxant machinery.

Role of fibulin-5 insufficiency and prolapse progression on murine vaginal biomechanical function

The vagina plays a critical role in supporting the pelvic organs and loss of support leads to pelvic organ prolapse. It is unknown what microstructural changes influence prolapse progression nor how decreased elastic fibers contributes to vaginal remodeling and smooth muscle contractility. The objective for this study was to evaluate the effect of fibulin-5 haploinsufficiency, and deficiency with progressive prolapse on the biaxial contractile and biomechanical function of the murine vagina. Vaginas from wildtype (n = 13), haploinsufficient (n = 13), and deficient mice with grade 1 (n = 9) and grade 2 or 3 (n = 9) prolapse were explanted for biaxial contractile and biomechanical testing. Multiaxial histology (n = 3/group) evaluated elastic and collagen fiber microstructure. Western blotting quantified protein expression (n = 6/group). A one-way ANOVA or Kruskal–Wallis test evaluated statistical significance. Pearson’s or Spearman’s test determined correlations with prolapse grade. Axial contractility decreased with fibulin-5 deficiency and POP (p < 0.001), negatively correlated with prolapse grade (ρ = − 0.80; p < 0.001), and positively correlated with muscularis elastin area fraction (ρ = − 0.78; p = 0.004). Circumferential (ρ = 0.71; p < 0.001) and axial (ρ = 0.69; p < 0.001) vaginal wall stresses positively correlated with prolapse grade. These findings demonstrated that fibulin-5 deficiency and prolapse progression decreased vaginal contractility and increased vaginal wall stress. Future work is needed to better understand the processes that contribute to prolapse progression in order to guide diagnostic, preventative, and treatment strategies.

Overexpressing miR-122-5p Inhibits the Relaxation of Vaginal Smooth Muscle in Female Sexual Arousal Disorder by Targeting Vasoactive Intestinal Peptide Receptor 1.

IntroductionFemale sexual arousal disorder (FSAD) is a common issue causing physical and psychological pain, but it has no standard diagnostic criteria or treatment. So its pathogenesis desiderates to be explored.AimTo investigate the specific function of miR-122-5p in FSAD.Methods18 subjects were grouped into FSAD and normal control groups according to the Chinese version of the Female Sexual Function Index, and the expression levels of miR-122-5p and vasoactive intestinal peptide receptor 1 (VIPR1) protein in their tissue were verified through real-time quantitative polymerase chain reaction (qRT-PCR) and western blot (WB) analysis. Then in vitro experiment, miR-122-5p was overexpressed or inhibited in rat vaginal smooth muscle cells (SMCs). The relaxation of rat vaginal SMCs was reflected by the cell morphology, intracellular free cytosolic calcium ion (Ca2+) levels, cell proliferation and apoptosis, together with the cyclic adenosine monophosphate (cAMP) concentration and protein kinase A (PKA) activities. Additionally, the expression levels of relaxation-related proteins, including VIPR1, stimulatory G protein (Gs), adenylate cyclase (AC), and PKA, were detected based on WB analysis. Furthermore, a rescue experiment that simultaneously overexpressed or silenced miR-122-5p and VIPR1 was conducted, and all the indicators were evaluated.Main Outcomes MeasureThe expression level of VIPR1 and downstream proteins, cell morphology, cell proliferation and apoptosis, and intracellular free Ca2+ levels were examined.ResultsWe verified that women with FSAD had higher miR-122-5p and lower VIPR1 protein. Then overexpressing miR-122-5p decreased relaxation of rat vaginal SMCs, which was manifested as a contractile morphology of cells, an increased intracellular free Ca2+ concentration, and lower cAMP concentration and PKA activity. Moreover, by rescue experiments, we inferred that VIPR1 was the target of miR-122-5p and affected the relaxation function of vaginal SMCs.ConclusionmiR-122-5p regulates the relaxation of vaginal SMCs in FSAD by targeting VIPR1, ulteriorly providing an underlying diagnostic and therapeutic target for FSAD.Cong S, Gui T, Shi Q, et al. Overexpressing miR-122-5p Inhibits the Relaxation of Vaginal Smooth Muscle in Female Sexual Arousal Disorder by Targeting Vasoactive Intestinal Peptide Receptor 1. Sex Med 2021;9:100390.

Criteria for risk stratification of vulvar and vaginal smooth muscle tumors: a follow-up study with application to leiomyoma variants, smooth muscle tumors of uncertain malignant potential, and leiomyosarcomas

Data have shown that uterine diagnostic criteria are universal for smooth muscle tumors (SMTs) originating in the ovary, vulva, vagina, broad ligament, and other supportive connective tissueand that uterine criteria outperform site-specific criteria for vulvar and vaginal SMTs. Classic benign and malignant spindled SMTs were well represented in our recent study comparing uterine and site-specific criteria in vulvovaginal SMTs, but leiomyoma variants and smooth muscle tumors of uncertain malignant potential (STUMPs) were relatively few. Therefore, we evaluated additional leiomyoma variants, STUMPs, and leiomyosarcomas from 17 patients (10 vaginal and 7 vulvar). The 10 vaginal tumors (59%) comprised cellular leiomyoma (n=2), leiomyoma with bizarre nuclei (n=3), STUMP (n=1), and leiomyosarcoma (n=4). The 7 vulvar tumors (41%) comprised leiomyoma with bizarre nuclei (n=3), STUMP (n=1), and leiomyosarcoma (n=3). Follow-up was available for 13 patients (76.5%) ranging from 1 to 97 months (mean: 17.3; median: 7). Follow-up for some patients with leiomyosarcoma was limited (≤4 months for 4 patients). One vaginal STUMP locally recurred after 19 months, and 2 patients diagnosed with leiomyosarcoma developed distant metastases. All remaining patients had either no evidence of disease at last follow-up (10 patients, 58.8%) or their status was unknown (4 patients, 23.5%). Uterine criteria are valid for vulvovaginal leiomyoma variants and STUMPsand more appropriately classified these tumors than site-specific criteria. Our combined findings from the current and previous studies support use of uterine diagnostic thresholds for the entire spectrum of vulvovaginal SMTs.

Altered mechanics of vaginal smooth muscle cells due to the lysyl oxidase-like1 knockout

The remodeling mechanisms that cause connective tissue of the vaginal wall, consisting mostly of smooth muscle, to weaken after vaginal delivery are not fully understood. Abnormal remodeling after delivery can contribute to development of pelvic organ prolapse and other pelvic floor disorders.The present study used vaginal smooth muscle cells (vSMCs) isolated from knockout mice lacking the expression of the lysyl oxidase-like1 (LOXL1) enzyme, a well-characterized animal model for pelvic organ prolapse. We tested if vaginal smooth muscle cells from LOXL1 knockout mice have altered mechanics including stiffness and surface adhesion.Using atomic force microscopy, we performed nanoindentations on both isolated and confluent cells to evaluate the effect of LOXL1 knockout on in vitro cultures of vSMCs cells from nulliparous mice. The results show that LOXL1 knockout vSMCs have increased stiffness in pre-confluent but decreased stiffness in confluent cultures (p* < 0.05) and significant decreased surface adhesion in pre-confluent cultures (p* < 0.05). This study provides evidence that the weakening of vaginal connective tissue in the absense of LOXL1 changes the mechanical properties of the vSMCs. Statement of significancePelvic organ prolapse is a common condition affecting millions of women worldwide, which significantly impacts their quality of life. Alterations in vaginal and pelvic floor mechanical properties can change their ability to support the pelvic organs. This study provides evidence of altered stiffness of vaginal smooth muscle cells from mice resembling pelvic organ prolapse. The results from this study set a foundation to develop pathophysiology–driven therapies focused on the interplay between smooth muscle mechanics and extracellular matrix remodeling.

PD36-03 CHRONIC HIGH FAT DIET INCREASES VAGINAL SPHINCTER LAXITY BUT NOT PELVIC FLOOR MUSCLE FUNCTION IN MICE

You have accessJournal of UrologySexual Function/Dysfunction: Female (PD36)1 Apr 2020PD36-03 CHRONIC HIGH FAT DIET INCREASES VAGINAL SPHINCTER LAXITY BUT NOT PELVIC FLOOR MUSCLE FUNCTION IN MICE Lindsey Burleson*, Adam Amorese, Michelle Oyen, Espen Spengenburg, and Johanna Hannan Lindsey Burleson*Lindsey Burleson* More articles by this author , Adam AmoreseAdam Amorese More articles by this author , Michelle OyenMichelle Oyen More articles by this author , Espen SpengenburgEspen Spengenburg More articles by this author , and Johanna HannanJohanna Hannan More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000907.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Pelvic organ prolapse (POP) affects 41% women and is a direct cause of female sexual dysfunction (FSD) by producing pain during intercourse or a reluctance to engage in sexual activity. The largest risk factors for POP include parity, obesity, and advanced age. Obesity, a modifiable risk factor, is believed to increase intra-abdominal pressure and weaken the pelvic floor muscles and fascia. The impact of obesity on vaginal smooth muscle versus pelvic floor skeletal muscle has not been studied in animal models of POP. We will examine the structure and function of the vagina and pelvic floor in female mice fed a chronic high fat diet (HFD). METHODS: Adult female C57/Blk6N mice (10 weeks, n=32) were fed a control (10% kcal fat) or HFD (45% kcal fat) for 24 weeks. Ex vivo external vaginal sphincter contraction to increasing concentrations of adrenergic agonist norepinephrine and relaxation to nitric oxide donor DEA NONOate and electric field stimulation (EFS) was measured. Iliococcygeus muscles of the pelvic floor were isolated and set to optimal length and tetanic contractions were performed at increasing frequencies to asses absolute and specific force. Fatigue was measured following repetitive stimulations over 5 minutes. Iliococcygeus muscle fiber types were characterized with anti-myosin antibodies and cross-sectional area (CSA) of each fiber type was measured. RESULTS: Vaginal sphincter adrenergic-mediated contraction was a significantly decreased in HFD-fed mice (Fig 1A; p<0.05). The vaginal sphincter smooth muscle relaxation in response to a nitric oxide donor was greater in the HFD mice (Fig 1B; p<0.05). Neurogenic-mediated EFS vaginal relaxations were also enhanced in the HFD mice (p<0.05). Surprisingly, chronic HFD did not impact iliococcygeus muscle specific force, absolute force, or muscle fatigue (Fig 1C). Furthermore, HFD did not affect the CSA or population of type I, IIa, IIX, or IIb iliococcygeus muscle fibers. CONCLUSIONS: HFD-induced obesity in mice leads to pelvic floor weakness by enhancing relaxation and decreasing tone of the external vaginal sphincter. Interestingly, the function or composition of pelvic skeletal muscle was unaffected. As the vagina provides significant support to pelvic organs, obesity-induced decreased vaginal sphincter tone and structure may lead to POP and FSD. Source of Funding: International Society for the Study of Women’s Sexual Health, Alpha Omega Alpha Medical Society © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e723-e724 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Lindsey Burleson* More articles by this author Adam Amorese More articles by this author Michelle Oyen More articles by this author Espen Spengenburg More articles by this author Johanna Hannan More articles by this author Expand All Advertisement PDF downloadLoading ...

Simulated vaginal delivery causes transients vaginal smooth muscle hypersensitivity and urethral sphincter dysfunction.

Although pelvic floor dysfunction (PFD) has a multifactorial etiology, pregnancy and childbirth are considered crucial events predisposing to urinary incontinence as well as pelvic organ prolapse, which are highly prevalent. Rats are the most frequently used animal model and pudendal nerve crush (PNC) and vaginal distension (VD) are often used to mimic vaginal delivery. To document the time course of events after simulated vaginal delivery (SVD) on the urethral sphincter and the vaginal smooth muscle layer. Virgin female Sprague-Dawley rats were subjected to SVD (PNC + VD) or sham surgery and evaluated at 7, 14, 21, and 42 days after the injury. Urethral function was determined in vivo by microultrasound during cystometry and vaginal smooth muscle layer was harvested for in vitro pharmacologic investigation by isometric tension recording. Furthermore, vaginal and urethral samples were investigated by immunohistochemistry and real-timequantitative polymerase chain reaction. Microultrasound showed no bursting of the urethral sphincter in the SVD group at 7 days with a functional recovery starting at 14 days, and normal bursting at 21 and 42 days. Vaginal smooth muscle showed higher sensitivity to carbachol at 14 and 21 days after injury; however, at 42 days, its sensitivity decreased when compared with sham. SVD induces urethral dysfunction and a shift in vaginal smooth muscle contractile responses to carbachol.