Recurrent infections drive persistent bladder dysfunction and pain via sensory nerve sprouting and mast cell activity.

Urinary tract infections (UTIs) account for almost 25% of infections in women. Many are recurrent (rUTI), with patients frequently experiencing chronic pelvic pain and urinary frequency despite clearance of bacteriuria after antibiotics. To elucidate the basis for these bacteria-independent bladder symptoms, we examined the bladders of patients with rUTI. We noticed a notable increase in neuropeptide content in the lamina propria and indications of enhanced nociceptive activity. In mice subjected to rUTI, we observed sensory nerve sprouting that was associated with nerve growth factor (NGF) produced by recruited monocytes and tissue-resident mast cells. Treatment of rUTI mice with an NGF-neutralizing antibody prevented sprouting and alleviated pelvic sensitivity, whereas instillation of native NGF into naïve mice bladders mimicked nerve sprouting and pain behavior. Nerve activation, pain, and urinary frequency were each linked to the presence of proximal mast cells, because mast cell deficiency or treatment with antagonists against receptors of several direct or indirect mast cell products was each effective therapeutically. Thus, our findings suggest that NGF-driven sensory sprouting in the bladder coupled with chronic mast cell activation represents an underlying mechanism driving bacteria-independent pain and voiding defects experienced by patients with rUTI.

Characterizing the innervation of the vulvar vestibule and the immunohistochemical features of neuroproliferative vestibulodynia.

BACKGROUND: Provoked vestibulodynia (PVD) is a chronic pain condition characterized by allodynia localized to the vulvar vestibule. The finding of increased densities of nerve fibers in the vestibular mucosa of patients with PVD has led to the identification of a neuroproliferative subtype. The etiology of PVD, including neuroproliferative vestibulodynia (NPV), is not fully understood. The gross and microscopic innervation of the vulvar vestibule remains incompletely described, despite the preliminary data supporting the role of peripheral innervation in PVD. AIM: To characterize the gross anatomic and microscopic innervation of the vulvar vestibule through cadaveric dissection and immunohistochemistry. METHODS: The pudendal nerve and inferior hypogastric plexus (IHP) were dissected using 6 cadaveric donors. Histology and immunohistochemistry were used to confirm patterns of innervation identified gross anatomically. Immunohistochemistry was performed on vestibulectomy specimens obtained from 6 patients diagnosed with NPV and compared with cadaveric vestibular tissues. OUTCOMES: Outcomes included (1) dissection of pelvic innervation and (2) immunohistochemical localization of markers for the following: general innervation protein gene product 9.5 (PGP9.5), sensory innervation (calcitonin gene-related peptide), autonomic innervation (vasoactive intestinal polypeptide, tyrosine hydroxylase), neuroproliferation (nerve growth factor [NGF]), and immune activation (C-kit). RESULTS: Perineal (pudendal) nerve branches were traced to the external wall of the vulvar vestibule. Some anatomic heterogeneity was observed in perineal nerve-branching patterns. Fibers from the IHP were identified in close proximity to the vulvar vestibule. Autonomic and sensory nerve fibers were identified in both patient and cadaveric vulvar vestibule samples. Patient samples were characterized by the proliferation of PGP9.5-positive nerve fibers and C-kit-positive mast cells, which were in proximity to neve bundles and showed coexpression with putative NGF-positive cells. NGF expression was localized to a subset of nerves, including those that demonstrated co-expression of sensory and autonomic nerve markers. Increased densities of autonomic fibers positive for vasoactive intestinal polypeptide and tyrosine hydroxylase were observed in 1 patient sample. CLINICAL TRANSLATION: Heterogeneity in gross and microscopic patterns of innervation could explain variability in clinical response to treatment and should be used to inform future therapeutic interventions. STRENGTHS AND LIMITATIONS: This study used a combination of approaches to elucidate the innervation of the vulvar vestibule, including in NPV. The small sample size is a limitation. CONCLUSION: The vulvar vestibule contains both sensory and autonomic innervation, which may originate from the pudendal nerve and IHP. Our results support the existence of a neuroproliferative subtype that is characterized by the proliferation of sensory and autonomic nerve fibers and neuroimmune interactions.

In vitro high glucose increases apoptosis, decreases nerve outgrowth, and promotes survival of sympathetic pelvic neurons.

Background: Diabetes mellitus (DM) is a common cause of erectile dysfunction (ED), yet the molecular basis of DM neurogenic ED remains unknown. Aim: In this study we examined the impact of high glucose on survival and growth of primary cultured pelvic neurons in a rat model and assessed whether coculturing with healthy Schwann cells (SCs) can rescue pelvic neuron growth in patients with DM. Methods: Major pelvic ganglia (MPGs) from adult male Sprague Dawley rats (n = 8) were dissociated and plated on coverslips. Neurons were exposed to high glucose (45 mM) for 24 or 48 hours and compared to time-matched controls (25 mM). Neurons were stained for neuron-specific beta-tubulin, neuronal nitric oxide synthase, vesicular acetylcholine transferase, tyrosine hydroxylase, and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling) assay. Schwann cells were dissociated from MPGs of healthy male Sprague Dawley rats (n = 4) and grown to confluence. Additional Sprague Dawley rats were made diabetic with streptozotocin (50 mg/kg, n = 4), and 5 weeks later MPGs were collected from these rats, dissociated, and cocultured on healthy SCs. Neurons and SCs were stained with beta-tubulin and S100. Outcomes: Length, branching, and survival of nitrergic, parasympathetic, and sympathetic neurons was assessed in neurons exposed to normal or high glucose concentrations, and neuron length was measured in neuron-SC coculture. Results: The total number of neurons and the length and number of branches were significantly decreased after 24 and 48 hours of high glucose (P < .05). The percentage of nitrergic neurons decreased 10% after 24 hours and 50% after 48 hours of high glucose (P < .05). After 24 hours of high glucose, cholinergic-positive neurons were unchanged; however, these neurons decreased 30% after 48 hours (P < .05). The proportion of sympathetic neurons increased 25% after 48 hours of high glucose (P < .05). At both timepoints, there was a 2-fold increase in the total apoptotic neurons with high glucose (P < .05). Neurite outgrowth recovered to control lengths after coculture of diabetic neurons with healthy SCs (P < .05). Clinical Translation: Glucose can be used as a tool to investigate the direct effects of DM on neuritogenesis. Our data suggest that an effective treatment for DM ED protects and repairs the penile neuronal supply. Strengths and Limitations: Exposing MPG neurons to high glucose offers a quick and, inexpensive proxy for DM-related conditions. A limitation of our study is that our model reflects type 1 DM, whereas clinically, most diabetic ED patients have type 2 DM. Conclusion: Culturing pelvic neurons in high glucose can be used as a tool to elucidate how to protect proerectile neurons from cell death and may lead to new therapeutic strategies for diabetic men suffering from ED.

Patient and provider perspectives on LEEP/LLETZ treatment and outcomes: an interview study.

BACKGROUND: The loop electrosurgical excision procedure (LEEP) and large loop excision of the transformation zone (LLETZ) effectively treat cervical dysplasia, though some women have reported negative outcomes postoperatively (e.g., sexual dysfunction, psychosexual sequalae). There is insufficient understanding of patient experiences with these symptoms and perspectives from the providers who perform LEEP/LLETZ. AIM: To characterize the perceptions and experiences of LEEP/LLETZ treatment from providers and patients, including whether there is a characteristic symptom profile of women who report negative outcomes. METHODS: Patients who had LEEP/LLETZ treatment and reported negative outcomes and providers who perform LEEP/LLETZ completed semistructured interviews about their perceptions and experiences, which were coded through thematic analysis (NVivo 12; QSR International). Patients also completed an online survey assessing demographics, medical history, and sexual function. OUTCOMES: Outcomes included perspectives generated from patient and provider interviews regarding LEEP/LLETZ procedural outcomes, including symptoms and experiences related to sexual functioning. RESULTS: Perspectives and experiences gathered from patient and provider interviews revealed misaligned narratives surrounding LEEP/LLETZ outcomes and treatment. We identified 4 overarching themes encapsulating provider and patient responses: Expectations for Preoperative Consultation; Procedure Experiences; Attitudes; and Resources. Patients reported a unique symptom profile and negative outcome experiences, namely surrounding domains of sexual functioning: decreased physical sensations, orgasm response, and vaginal discharge, as well as loss of arousal, interest, and desire. Patients described changes to overall quality of life, with impacts to interpersonal relationships. Patients discussed preferring open-ended and directed questions to comprehensively elucidate negative outcomes. Provider narratives outlined the current process of care, emphasizing limited experiences with adverse outcomes (e.g., sexual issues) and the use of open-ended questions during counseling. Providers described an evolving intention to create comfortable clinical spaces. Regarding pre- and postoperative resources, patients described seeking support from online patient groups, and providers disclosed limitations to providing resources. CLINICAL IMPLICATIONS: Evidence of discordance between patient and provider perspectives of LEEP/LLETZ reveals a need to reassess clinical practices surrounding this procedure at the level of discussions regarding informed consent, sexual function, and available resources. STRENGTHS AND LIMITATIONS: This study is the first to examine patient and provider perspectives on LEEP/LLETZ treatment. Only patients who self-report negative outcomes were recruited, to elicit narratives from this specific subpopulation. CONCLUSION: Results indicate a characteristic symptom profile of women who undergo LEEP/LLETZ and report negative outcomes and that the perceptions of patients and providers differ regarding several aspects of the treatment experience, supporting the need for directed open conversation and comprehensive pre- and postoperative sexual counseling.

The contribution of the cervix to sexual response: an online survey study.

BACKGROUND: The role of the cervix in sexual response has been poorly studied, despite previous research indicating that some women experience pleasurable sexual sensations from cervical stimulation; given previous reports of sexual issues after cervix electrocautery, it is possible that cervical injury may compromise the role of the cervix in sexual functioning. AIM: The aims of this study were to examine locations of pleasurable sexual sensations, to identify sexual communication barriers, and to investigate if cervical procedures are associated with negative impacts on sexual function. METHODS: Women with (n = 72) and without (n = 235) a history of a gynecological procedure completed an online survey assessing demographics, medical history, sexual function (including locations of sexual pleasure and pain on diagrams), and barriers. The procedure group was divided into subgroups of those who had experienced a cervical (n = 47) or noncervical (n = 25) procedure. Chi-square analyses and t tests were conducted. OUTCOMES: Outcomes included locations and ratings of pleasurable and painful sexual stimulation, as well as sexual function. RESULTS: Over 16% of participants reported experiencing some pleasurable sexual sensations from the cervix. The gynecological procedure group (n = 72) reported significantly higher pain in the vagina and lower rates of pleasure in their external genitals, vagina, deep vagina, anterior and posterior vaginal walls, and clitoris vs the non-gynecological procedure (n = 235) group. The gynecological procedure group and the cervical procedure subgroup (n = 47) reported significant decreases in desire, arousal, and lubrication and increased avoidance of sexual activity due to vaginal dryness. The gynecological procedure group reported significant pain with vaginal stimulation, whereas the cervical subgroup identified significant pain with cervical and clitoral stimulation. CLINICAL IMPLICATIONS: Cervical stimulation elicits some pleasurable sexual sensations for many women, and gynecological procedures that affect the cervix are associated with pain and sexual issues; thus, health care providers should counsel patients about the possibility of related sexual concerns. STRENGTHS AND LIMITATIONS: This study is the first to examine locations of pleasure and pain and experiences of sexual pleasure and function in participants who underwent a gynecological procedure. A hybrid measure was used to assess sexual issues, including symptoms of dysfunction. CONCLUSION: Results indicate an association between cervical procedures and sexual issues, supporting the need to inform patients of this possibility following cervical procedures.

The prostate in women: an updated histological and immunohistochemical profile of the female periurethral glands and their relationship to an implanted midurethral sling.

BACKGROUND: There is evidence of glandular tissue in the region of the anterior vaginal wall-female periurethral tissue (AVW-FPT) that has similar morphology and immunohistochemistry to the prostate in men. Surgical injury to this tissue has been suggested as a potential cause of sexual dysfunction following midurethral sling (MUS) procedures. However, the anatomy and embryology of these glands have not been fully resolved. This has led to difficulties in classifying this tissue as a prostate and defining its clinical significance related to MUS procedures. AIM: To describe the histological and immunohistochemical characteristics of the female periurethral glands using markers of prostate tissue and innervation and to examine their anatomical relationships to an implanted MUS. METHODS: Using gross and fine dissection, the AVW-FPT was dissected from 9 cadavers. Prior to dissection, 2 cadavers underwent simulation of the MUS procedure by a urogynecologist. Samples were paraffin embedded and serially sectioned. Immunohistochemistry was performed using markers of prostate tissue and innervation. OUTCOMES: Immunohistochemical localization of markers for prostatic tissue and innervation of the glandular tissue of the AVW-FPT, including the region of MUS implantation. RESULTS: Female periurethral glands were immunoreactive for markers of male prostatic tissue, including prostate-specific antigen, androgen receptor, HOXB13, and NKX3.1. Markers of innervation (protein gene product 9.5, choline acetyl transferase, and vasoactive intestinal polypeptide) also localized to certain regions of the glandular tissue and associated blood supply. Surgical simulation of the MUS procedure demonstrated that some periurethral glands are located in close proximity to an implanted sling. CLINICAL TRANSLATION: The AVW-FPT contains glandular tissue in the surgical field of MUS implantation. Iatrogenic damage to the female periurethral glands and the associated innervation during surgery could explain the negative impacts on sexual dysfunction reported following MUS procedures. STRENGTHS AND LIMITATIONS: This is the first study to characterize the female periurethral glands using markers of prostatic tissue in concert with markers of general and autonomic innervation and characterize their anatomical relationships within the surgical field of MUS implantation. The small sample size is a limitation of this study. CONCLUSION: We provide further evidence that the AVW-FPT contains innervated glands that are phenotypically similar to the male prostate and may share a common embryonic origin. The microscopic and immunohistochemical features of the periurethral glands may be indicative of their functional capacity in sexual responses. The location of these glands in the surgical field of MUS procedures underscores the clinical significance of this tissue.

Cocultured Schwann Cells Rescue Irradiated Pelvic Neuron Outgrowth and Increase Survival.

BACKGROUND: Prostatic radiation therapy (RT) leads to erectile dysfunction by damaging peri-prostatic pro-erectile nerves of the pelvic ganglion. Schwann cells (SC) facilitate neuronal repair after mechanical injury, however, their role in repair of pelvic neurons post-radiation hasn't been explored. AIM: To determine if SCs cocultured with primary pelvic neurons can rescue neuronal survival and growth after ex vivo RT. METHODS: Major pelvic ganglia (MPG) were collected from adult male Sprague-Dawley rats (n = 12) to isolate SCs. SCs received RT (0 or 8 Gy), were plated on coated coverslips and grown to confluence before the addition of neurons. Additional MPGs were irradiated (0 or 8 Gy) and digested to isolate pelvic neurons. Dissociated neurons were plated alone or atop SC-coated coverslips to create 6 experimental groups (n = 3/grp): (i) Control (CON) MPG, (ii) RT MPG, (iii) CON SC + CON MPG, (iv) CONSC + RT MPG, (v) RT SC + CON MPG, and (iv) RT SC + RT MPG. After 72 hours, coverslips were fixed and stained for beta-tubulin (neuron marker), S100 (SC marker), neuronal nitric oxide synthase (nitrergic marker), tyrosine hydroxylase (sympathetic marker), and terminal deoxynucleotidyl transferase dUTP nick-end labeling. OUTCOMES: We measured neurite length, branching, specific neuron populations and apoptosis. RESULTS: Ex vivo RT decreased MPG neuron length, increased apoptosis and decreased nitrergic neurons in monoculture. Compared to all other groups, CON SC + RT MPG cocultures demonstrated increased neurite outgrowth (P < .001). Neurite branching was decreased in the RT MPG + RT SC coculture, but unchanged in other cocultures. Groups containing RT MPG neurons exhibited increased apoptosis, but coculture with CON SC reduced the degree of RT-induced apoptosis (P < .01). The number of tyrosine hydroxylase positive neurons was unchanged while nitrergic neurons were significantly lower in RT neurons and coculture with CON SCs was unable to prevent nitrergic loss. CLINICAL TRANSLATION: These findings suggest that SCs may be an important target in prostate cancer patients with radiation-induced pelvic neuropathy to promote MPG neuron survival and neuronal repair after RT. STRENGTHS AND LIMITATIONS: This is the first study to characterize the ex vivo ability of SCs to rescue pelvic nerve growth and survival. The study is limited by little supporting mechanistic molecular data and the need to confirm the ability of healthy SCs to promote pelvic neuron survival and repair following prostatic RT in vivo. CONCLUSION: Unirradiated SCs partially mitigated RT-induced MPG apoptosis but did not affect the loss of nitrergic neuron populations suggesting that SCs promote irradiated MPG neuron survival and facilitate intrinsic repair functions. Randolph JT, Pak ES, McMains JC, et al. Cocultured Schwann Cells Rescue Irradiated Pelvic Neuron Outgrowth and Increase Survival. J Sex Med 2022;19:1333-1342.

An Innovative Use of Twitter to Disseminate and Promote Medical Student Scholarship During the COVID-19 Pandemic: Usability Study.

BACKGROUND: Due to the emergence of the COVID-19 pandemic in March 2020, the cancellation of in-person learning activities forced every aspect of medical education and student engagement to pivot to a web-based format, including activities supporting the performance and dissemination of scholarly work. At that time, social media had been used to augment in-person conference learning, but it had not been used as the sole platform for scholarly abstract presentations. OBJECTIVE: Our aim was to assess the feasibility of using Twitter to provide a completely web-based forum for real-time dissemination of and engagement with student scholarly work as an alternative to a traditional in-person poster presentation session. METHODS: The Brody School of Medicine at East Carolina University launched an online Medical Student Scholarship Forum, using Twitter as a platform for students to present scholarly work and prepare for future web-based presentations. A single student forum participant created posts using a standardized template that incorporated student research descriptions, uniform promotional hashtags, and individual poster presentations. Tweets were released over 5 days and analytic data were collected from the Twitter platform. Outcome measures included impressions, engagements, retweets, likes, media engagements, and average daily engagement rate. RESULTS: During the conference, the student leader published 63 tweets promoting the work of 58 students (55 medical and 3 dental students) over 5 days. During the forum and the following week, tweets from the @BrodyDistinctly Twitter account received 63,142 impressions and 7487 engagements, including 187 retweets, 1427 likes, and 2082 media engagements. During the 5 days of the forum, the average daily engagement rate was 12.72%. CONCLUSIONS: Using Twitter as a means of scholarly dissemination resulted in a larger viewing community compared to a traditional in-person event. Early evidence suggests that social media platforms may be an alternative to traditional scholarly presentations. Presenting via Twitter allowed students to receive instantaneous feedback and effectively network with wider academic communities. Additional research is needed to evaluate the effectiveness of knowledge uptake, feedback, and networking.

Cardiometabolic Diseases and Female Sexual Dysfunction: Animal Studies.

BACKGROUND: The association between erectile dysfunction and cardiometabolic disease is well characterized; men are often diagnosed with cardiovascular disease 2-5 years following the incidence of erectile dysfunction. There is evidence that this relationship may also exist for cardiometabolic diseases and female sexual dysfunction (FSD) - particularly sexual arousal disorders. AIM: To provide a summary of the preclinical literature related to the evidence of FSD in animal models of cardiometabolic diseases and indicate where further research is needed. METHODS: A detailed Medline search of peer-reviewed publications was performed on the associations between animal models of cardiometabolic diseases, FSD and underlying mechanisms. OUTCOMES: A summary of the preclinical evidence of FSD in animal models of cardiometabolic diseases. RESULTS: Common methods for assessing female sexual arousal and physiology in animal models include: 1) behavioral assessments (apomorphine-induced genital vasocongestive arousal; hormone-dependent lordosis), 2) nerve-mediated vaginal and clitoral blood flow, 3) pudendal artery, vaginal and clitoral smooth muscle physiology (vasoreactivity and molecular biology), 4) morphology of genital tissues. Twenty-eight studies examined female animal models of atherosclerosis, hypertension, diabetes (type 1 and 2) and obesity. They showed functional alterations, including decreased lordosis, lubrication, or vaginal and clitoral blood flow, and structural impairments, such as increased clitoral and vaginal fibrosis. Several possible mechanisms have been described including increased TGF-ß, renin angiotensin system and endothelin/rho-kinase signaling, increased reactive oxygen species, and decreased nitric oxide/cGMP signaling. CLINICAL TRANSLATION: In line with existing clinical studies, preclinical evidence supports that cardiometabolic diseases alter female genital tissue's function and structure leading to impaired sexual arousal. STRENGTHS AND LIMITATIONS: This masterclass paper gives an overview of the preclinical research assessing FSD in cardiometabolic disease. Limitations include the small number of studies that have assessed sexual function and arousal in female cardiometabolic animal models. CONCLUSION: Preclinical evidence exists showing cardiometabolic diseases alter the structure and function of female genital tissues. However, similar to clinical studies, there are few studies to draw from, particularly in models of type 2 diabetes, obesity and metabolic syndrome. More studies are required using optimized animal models and methodology to confirm the mechanisms underlying cardiometabolic disease-induced FSD. Angulo J, Hannan JL. Cardiometabolic Diseases and Female Sexual Dysfunction: Animal Studies. J Sex Med 2022;19:408-420.

Should We Call It a Prostate? A Review of the Female Periurethral Glandular Tissue Morphology, Histochemistry, Nomenclature, and Role in Iatrogenic Sexual Dysfunction.

INTRODUCTION: There is evidence of glandular tissue within the region of the anterior vaginal wall-female periurethral tissue (AVW-FPT) having similar morphology and immunohistochemistry to the prostate in men and having physiological roles in the female sexual response (FSR). Whether this tissue should be called a prostate in women has been debated. Iatrogenic injury to structures of the AVW-FPT, including these glands and the associated neurovasculature, could be a cause of female sexual dysfunction (FSD). OBJECTIVES: To consolidate the current knowledge concerning the glandular tissue surrounding the urethra in women, evidence was reviewed to address whether: (i) these glands comprise the prostate in women, (ii) they have specific functions in the FSR, and (iii) injury to the AVW-FPT and prostate has sexual dysfunction as a likely outcome. METHODS: A literature review was conducted using keywords including female prostate, Skene's/paraurethral glands, periurethral tissue, Gräfenberg (G)-spot, female ejaculation, mid-urethral sling (MUS), and sexual dysfunction. RESULTS: Histological and immunohistochemical studies of the glandular tissue surrounding the urethra support the existence of prostate in women. Evidence suggests this tissue may have physiologically and clinically relevant autonomic and sensory innervation, and during sexual arousal may contribute to secretions involved in ejaculation and orgasm. Gaps in knowledge relating to the functional anatomy, physiological roles, and embryological origins of this tissue have impeded the acceptance of a prostate in women. Injury to the innervation, vasculature, and/or glandular tissue within the surgical field of MUS implantation suggests iatrogenic sexual dysfunction is plausible. CONCLUSIONS: Continuing to advance our understanding of the morphology, histochemistry, and physiologic capacity of this glandular tissue will clarify the characterization of this tissue as the "prostate" involved in the FSR, and its role in FSD following surgical injury. Tomalty D, Giovannetti O, Hannan J, et al. Should We Call It a Prostate? A Review of the Female Periurethral Glandular Tissue Morphology, Histochemistry, Nomenclature, and Role in Iatrogenic Sexual Dysfunction. Sex Med Rev 2022;10:183-194.

High-fat diet induces obesity in adult mice but fails to develop pre-penile and penile vascular dysfunction.

Obesity can lead to cardiovascular disease, diabetes, and erectile dysfunction (ED), which decreases overall quality of life. Mechanisms responsible for obesity-induced ED are unknown. Current mouse models of high-fat diet (HFD)-induced obesity yield conflicting results. Genetic variants among common "wild type" strains may explain contradictory data. Adult male C57BL/6N and 6J mice were fed a 45% HFD for 12 weeks. Weekly food intake, weight gain, and body-fat percentage were measured. After 12 weeks, ex vivo vascular reactivity was measured in aortas, internal pudendal arteries, and penises. We assessed smooth muscle contractility, endothelial-dependent and -independent relaxation, and penile neurotransmitter-mediated relaxation. C57BL/6N mice developed greater obesity and glucose sensitivity compared to C57BL/6J mice. Aortas from both strains that fed a HFD had decreased contraction, yet contraction was unchanged in HFD pudendal arteries and penises. Interestingly, endothelial-dependent and -independent relaxation was unchanged in both systemic and penile vasculature. Likewise, HFD did not impair penile neurotransmitter-mediated relaxation. Both strains fed 12 weeks of HFD-developed obese phenotypes. However, HFD did not impair pre-penile or penile smooth muscle vasoreactivity as demonstrated in previous studies, suggesting that this preclinical model does not accurately represent the clinical phenotype of obesity-induced ED.

Ex vivo Akt inhibition reverses castration induced internal pudendal artery and penile endothelial dysfunction.

AIMS: Androgen deprivation therapy is a common prostate cancer treatment which causes men to have castrate levels of testosterone. Unfortunately, most testosterone deficient patients will suffer severe erectile dysfunction (ED) and have no effective ED treatment options. Testosterone deficiency causes endothelial dysfunction and impairs penile vasodilation necessary to maintain an erection. Recent evidence demonstrates testosterone activates androgen receptors (AR) and generates nitric oxide (NO) through the Akt-endothelial NO synthase (eNOS) pathway; however, it remains unknown how castration impacts this signaling pathway. MATERIALS AND METHODS: In this study, we used a surgically castrated rat model to determine how castration impacts ex vivo internal pudendal artery (IPA) and penile relaxation through the Akt-eNOS pathway. KEY FINDINGS: Unlike systemic vasculature, castration causes significant IPA and penis endothelial dysfunction associated with a 50% AR reduction. Though testosterone and acetylcholine (ACh) both phosphorylate Akt and eNOS, castration did not affect testosterone-mediated IPA and penile Akt or eNOS phosphorylation. Surprisingly, castration increases ACh-mediated Akt and eNOS phosphorylation but reduces the eNOS dimer to monomer ratio. Akt inhibition using 10DEBC preserves IPA eNOS dimers. Functionally, 10DEBC reverses castration induced ex vivo IPA and penile endothelial dysfunction. SIGNIFICANCE: These data demonstrate how castration uncouples eNOS and provide a novel strategy for improving endothelial-dependent relaxation necessary for an erection. Further studies are needed to determine if Akt inhibition may treat or even prevent ED in testosterone deficient prostate cancer survivors.

Dysfunctional voiding behavior and impaired muscle contractility in a rat model of detrusor underactivity.

AIMS: Detrusor underactivity (DU) is an understudied health concern with inadequate clinical management. The pathophysiology of DU is unclear, and current therapies fail to improve symptoms. The current studies characterized voiding function and contractility of bladder and urethral tissues in a novel rat model of DU. METHODS: Female obese prone (OP) and obese resistant (OR) rats were fed a 60 kcal% fat diet at 8 weeks old. A subset of rats (n = 4/strain) underwent uroflowmetry biweekly for 18 weeks in metabolic cages. At 40-56 weeks old, rats (n = 9-10/strain) underwent instrumented cystometry under urethane anesthesia. Following cystometry, bladder and urethral tissues (n = 8-9/strain) were harvested for in vitro assessments of contractility in response to carbachol, electric field stimulation, atropine, alpha, beta-methylene ATP, and caffeine. RESULTS: OP rats exhibited increased urinary frequency (p = 0.0031), decreased voided volume (p = 0.0093), and urine flow rate (p = 0.0064) compared to OR rats during uroflowmetry. Bethanechol (10 mg/kg) did not alter uroflowmetry parameters. During cystometry, OP rats exhibited decreased bladder emptying efficiency (p < 0.0001), decreased pressure to generate a void (p < 0.0001), and increased EUS activity during filling (p = 0.0011). Bladder contractility was decreased in OP rats when exposed to carbachol (p < 0.0003) and ATP (p = 0.0004), whereas middle urethral contractility was increased when exposed to carbachol (p = 0.0014), EFS (p = 0.0289), and caffeine (p = 0.0031). CONCLUSION: Impaired cholinergic and purinergic signaling in the bladder may contribute to poor voiding function in OP rats. In addition, increased urethral activity may engage a guarding reflex to augment continence and exacerbate incomplete emptying.

Increased Level of Tumor Necrosis Factor-Alpha (TNF-α) Leads to Downregulation of Nitrergic Neurons Following Bilateral Cavernous Nerve Injury and Modulates Penile Smooth Tone.

BACKGROUND: Erectile dysfunction (ED) after injury to peripheral cavernous nerve (CN) is partly a result of inflammation in pelvic ganglia, suggesting that ED may be prevented by inhibiting neuroinflammation. AIM: The aim of this study is to examine temporal changes of TNF-α, after bilateral CN injury (BCNI), to evaluate effect of exogenous TNF-α on neurite outgrowth from major pelvic ganglion (MPG), and to investigate effect of TNF-α signal inhibition to evaluate effects of TNF-α on penile tone with TNF-α receptor knockout mice (TNFRKO). METHODS: Seventy Sprague-Dawley rats were randomized to undergo BCNI or sham surgery. Sham rats' MPGs were harvested after 48 hours, whereas BCNI groups' MPGs were at 6, 12, 24, 48 hours, 7, or 14 days after surgery. qPCR was used to evaluate gene expression of markers for neuroinflammation in MPGs. Western blot was performed to evaluate TNF-α protein amount in MPGs. MPGs were harvested from healthy rats and cultured in Matrigel with TNF-α. Neurite outgrowth from MPGs was measured after 3 days, and TH and nNOS immunofluorescence was assessed. Wild type (WT) and TNFRKO mice were used to examine effect of TNF-α inhibition on smooth muscle function after BCNI. MPGs were harvested 48 hours after sham or BCNI surgery to evaluate gene expression of nNOS and TH. OUTCOMES: Gene expression of TNF-α signaling pathway, Schwann cell and macrophage markers, protein expression of TNF-α in MPGs, and penile smooth muscle function to electrical field stimulation (EFS) were evaluated. RESULTS: BCNI increased gene and protein expression of TNF-α in MPGs. Exogenous TNF-α inhibited MPG neurite outgrowth. MPGs cultured with TNF-α had decreased gene expression of nNOS (P < .05). MPGs cultured with TNF-α had shorter nNOS+ neurites than TH+ neurites (P < .01). Gene expression of nNOS was enhanced in TNFRKO mice compared to WT mice (P < .01). WT mice showed enhanced smooth muscle contraction of penises of WT mice was enhanced to EFS, compared to TNFKO (P < .01). Penile smooth-muscle relaxation to EFS was greater in TNFKO mice compared to WT (P < .01). CLINICAL TRANSLATION: TNF-α inhibition may prevent ED after prostatectomy. STRENGTH/LIMITATIONS: TNF-α inhibition might prevent loss of nitrergic nerve apoptosis after BCNI and preserve corporal smooth muscle function but further investigation is required to evaluate protein expression of nNOS in MPGs of TNFKO mice. CONCLUSIONS: TNF-α inhibited neurite outgrowth from MPGs by downregulating gene expression of nNOS and TNFRKO mice showed enhanced gene expression of nNOS and enhanced penile smooth-muscle relaxation. Matsui H, Sopko NA, Campbell JD, et al. Increased Level of Tumor Necrosis Factor-Alpha (TNF-α) Leads to Downregulation of Nitrergic Neurons Following Bilateral Cavernous Nerve Injury and Modulates Penile Smooth Tone. J Sex Med 2021;18:1181-1190.

Immunohistochemical Investigation of Autonomic and Sensory Innervation of Anterior Vaginal Wall Female Periurethral Tissue: A Study of the Surgical Field of Mid-Urethral Sling Surgery Using Cadaveric Simulation.

BACKGROUND: Female sexual dysfunction, including female orgasm disorder, has been reported following mid-urethral sling (MUS) surgery to treat bothersome stress urinary incontinence. Anterior vaginal wall-female periurethral tissue (AVW-FPT) likely contains autonomic and sensory innervation involved in the female sexual response, and injury to these nerves may result from MUS implantation. AIM: To characterize, using fresh cadaveric tissue, autonomic and sensory nerves in AVW- FPT using immunohistochemistry (IHC), and to assess their proximity to an implanted MUS. METHODS: AVW-FPT was excised following careful dissection from four fresh cadavers. Prior to dissection, one cadaver underwent simulation of the MUS procedure by a urogynegologist, using a fascial sling. All samples were paraffin embedded, sectioned, and stained with hematoxylin. Serial sectioning and IHC were performed to identify nerves. IHC markers were used to characterize the sensory and autonomic innervation. OUTCOMES: IHC localization of autonomic and sensory nerve markers consistent with neural tissue within the region of MUS implantation. RESULTS: IHC of AVW-FPT using protein gene product 9.5 (PGP9.5), a general nerve stain, revealed innervation throughout the region targeted by the MUS implantation. More specifically, immunoreactivity for both autonomic (tyrosine hydroxylase, TH) and sensory (Nav1.8 and S100ß) nerves were found in close proximity (<1 mm) to the implanted MUS. In addition, a subset of S100ß positive nerves also showed immunoreactivity for calcitonin gene-related peptide (CGRP). Combining the IHC findings with the surgical simulation of the MUS implantation revealed the potential for damage to both autonomic and sensory nerves as a direct result of the MUS procedure. CLINICAL TRANSLATION: The identified autonomic and sensory nerves of the AVW-FPT may contribute to the female sexual response, and yet are potentially negatively impacted by MUS procedures. Given that surgeries performed on male genital tissue, including the prostate, may cause sexual dysfunction secondary to nerve damage, and that urologists routinely provide informed consent regarding this possibility, urogynaecologists are encouraged to obtain appropriate informed consent from prospective patients undergoing the MUS procedure. STRENGTHS & LIMITATIONS: This is the first study to characterize the sensory and autonomic innervation within the surgical field of MUS implantation and demonstrate its relationship to an implanted MUS. The small sample size is a limitation of this study. CONCLUSION: The present study provides evidence of potential injury to autonomic and sensory innervation of AVW-FPT as a consequence of MUS implantation, which may help explain the underlying mechanisms involved in the reported post-operative female sexual dysfunction in some women. Giovannetti O, Tomalty D, Gaudet D, et al. Immunohistochemical Investigation of Autonomic and Sensory Innervation of Anterior Vaginal Wall Female Periurethral Tissue: A Study of the Surgical Field of Mid-Urethral Sling Surgery Using Cadaveric Simulation. J Sex Med 2021;18:1168-1180.

Sex Differences in Pulmonary Eicosanoids and Specialized Pro-Resolving Mediators in Response to Ozone Exposure.

Ozone (O3) is a criteria air pollutant known to increase the morbidity and mortality of cardiopulmonary diseases. This occurs through a pulmonary inflammatory response characterized by increased recruitment of immune cells into the airspace, pro-inflammatory cytokines, and pro-inflammatory lipid mediators. Recent evidence has demonstrated sex-dependent differences in the O3-induced pulmonary inflammatory response. However, it is unknown if this dimorphic response is evident in pulmonary lipid mediator metabolism. We hypothesized that there are sex-dependent differences in lipid mediator production following acute O3 exposure. Male and female C57BL/6J mice were exposed to 1 part per million O3 for 3 h and were necropsied at 6 or 24 h following exposure. Lung lavage was collected for cell differential and total protein analysis, and lung tissue was collected for mRNA analysis, metabololipidomics, and immunohistochemistry. Compared with males, O3-exposed female mice had increases in airspace neutrophilia, neutrophil chemokine mRNA, pro-inflammatory eicosanoids such as prostaglandin E2, and specialized pro-resolving mediators (SPMs), such as resolvin D5 in lung tissue. Likewise, precursor fatty acids (arachidonic and docosahexaenoic acid; DHA) were increased in female lung tissue following O3 exposure compared with males. Experiments with ovariectomized females revealed that loss of ovarian hormones exacerbates pulmonary inflammation and injury. However, eicosanoid and SPM production were not altered by ovariectomy despite depleted pulmonary DHA concentrations. Taken together, these data indicate that O3 drives an increased pulmonary inflammatory and bioactive lipid mediator response in females. Furthermore, ovariectomy increases susceptibility to O3-induced pulmonary inflammation and injury, as well as decreases pulmonary DHA concentrations.

Impact of prostatic radiation therapy on bladder contractility and innervation.

AIMS: To determine the effect of prostatic radiation therapy (RT) on bladder contractility and morphology, and axon, or neuron profiles within the detrusor and major pelvic ganglia (MPG) in male rats. METHODS: Male Sprague-Dawley rats (8 weeks) received a single dose of prostatic RT (0 or 22 Gy). Bladders and MPG were collected 2- and 10-weeks post-RT. Detrusor contractile responses to carbachol and electrical field stimulation (EFS) were measured. Bladders were stained with Masson's trichrome, and antibodies for nonspecific neuronal marker, cholinergic nerve marker choline acetyltransferase (ChAT), and alpha-smooth muscle actin. MPG gene expression was assessed by quantitative polymerase chain reaction for ubiquitin carboxy-terminal hydrolase L1 (Uchl1) and Chat. RESULTS: At 2 weeks post-RT, bladder smooth muscle, detrusor cholinergic axon profiles, and MPG Chat gene expression were increased (p < .05), while carbachol and EFS-mediated contractions were decreased (p < .05). In contrast, at 10 weeks post-RT, nerve-mediated contractions were increased compared with control (p < .05), while bladder smooth muscle, detrusor cholinergic axon profiles, MPG Chat expression, and carbachol contractions had normalized. At both 2- and 10-weeks post-RT, there was no change in detrusor nonspecific axon profiles and MPG Uchl1 expression. CONCLUSION: In a rat model, RT of the prostate and MPG was associated with early changes in MPG Chat gene expression, and bladder cholinergic axon profiles and smooth muscle content which resolved over time. After RT recovery, bladder contractility decreased early and increased by 10 weeks. Long-term changes to the MPG and increased bladder cholinergic axons may contribute to RT-induced bladder dysfunction in prostate cancer survivors.

Ex Vivo Radiation Leads to Opposing Neurite Growth in Whole Ganglia vs Dissociated Cultured Pelvic Neurons.

BACKGROUND: Prostatic radiation therapy (RT) often causes erectile dysfunction (ED) and the mechanisms governing RT-induced ED are unclear with a lack of therapeutic strategies. AIM: To determine the effects of ex vivo RT on major pelvic ganglion (MPG) neuron survival, and neurite growth in whole vs dissociated culture. METHODS: MPGs were removed and irradiated (0 or 8 Gy) from male Sprague Dawley rats. For dissociated culture, MPG neurons were digested in collagenase/dispase and cultured on coverslips. Immunofluorescent staining for beta-tubulin III (TUBB3; neuron marker), neuronal nitric oxide synthase (nNOS; nitrergic marker), tyrosine hydroxylase (TH; sympathetic marker), and terminal deoxynucleotidyl transferase dUTP nick end labeling assessed neurite length, branching, autonomic neuron density, and apoptosis. For whole organ culture, MPGs were grown in Matrigel. Gene expression of apoptotic markers (caspase 1, 3), TUBB3, nNOS, TH, and Schwann cells (Sox10, Krox20, glial fibrillary acid protein) was measured in whole organ cultured MPGs by quantitative polymerase chain reaction. OUTCOMES: After 72 hours, neurite length, branching, autonomic neuron density, and apoptosis were assessed, and gene expression was measured. RESULTS: RT increased apoptosis in dissociated neurons measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (P < .001) and whole MPG culture via upregulation of caspase 3 gene expression (P < .05). Nitrergic neurons were markedly decreased in irradiated dissociated culture (P < .05), while nNOS gene expression was upregulated in irradiated whole organ culture (P < .05). The proportion of dissociated sympathetic neurons and whole organ TH gene expression remained unchanged after RT. Interestingly, RT dissociated neurites were 22% shorter than controls, while RT whole organ neurites were 15% longer than controls (P < .01). MPG Schwann cells markers (Sox10, Krox20) were elevated after RT in whole organ culture. CLINICAL TRANSLATION: Prostatic RT leads to increased neuronal cell death and less erectogenic nitrergic neurons contributing to ED. STRENGTHS & LIMITATIONS: The advantages of dissociated neuron culture include distinct neurites which are easily measured for apoptosis, length/branching, and specific neuron types. In contrast, whole MPG culture is advantageous as it contains all the supporting cells present in vivo. CONCLUSION: The 2 different culture methods demonstrated opposing neurite growth after RT indicating the importance of supporting cell network to promote pelvic neuron neuritogenesis and survival following RT. Randolph JT, Pak ES, Koontz BF, et al. Ex Vivo Radiation Leads to Opposing Neurite Growth in Whole Ganglia vs Dissociated Cultured Pelvic Neurons. J Sex Med 2020;17:1423-1433.

Managing female pelvic floor disorders: a medical device review and appraisal.

Pelvic floor disorders (PFDs) will affect most women during their lifetime. Sequelae such as pelvic organ prolapse, stress urinary incontinence, chronic pain and dyspareunia significantly impact overall quality of life. Interventions to manage or eliminate symptoms from PFDs aim to restore support of the pelvic floor. Pessaries have been used to mechanically counteract PFDs for thousands of years, but do not offer a cure. By contrast, surgically implanted grafts or mesh offer patients a more permanent resolution but have been in wide use within the pelvis for less than 30 years. In this perspective review, we provide an overview of the main theories underpinning PFD pathogenesis and the animal models used to investigate it. We highlight the clinical outcomes of mesh and grafts before exploring studies performed to elucidate tissue level effects and bioengineering considerations. Considering recent turmoil surrounding transvaginal mesh, the role of pessaries, an impermanent method, is examined as a means to address patients with PFDs.

Chronic high-fat diet decreased detrusor mitochondrial respiration and increased nerve-mediated contractions.

AIMS: To assess the impact of chronic high-fat diet (HFD) on behavioral voiding patterns, detrusor contractility, and smooth muscle mitochondrial function in male mice. MATERIALS AND METHODS: Male C57BL/6J mice (6 weeks) were fed a control or HFD for 20 weeks. Bladder function was assessed by void spot assays. Bladders were collected and detrusor contractility to carbachol (10-9 -10-5 M), and electrical field stimulation (EFS, 0.5-32 Hz) in the presence and absence of atropine was measured. Homogenized detrusor samples were placed in oxygraphs to assess the rate of oxygen consumption of the mitochondria within the detrusor in the presence of different substrates. Mitochondrial hydrogen peroxide (H2 O2 ) emission was measured fluorometrically. Detrusor citrate synthase activity was measured via enzyme activity kit and Western blots assessed the electron transport chain (ETC) protein content. RESULTS: HFD significantly increased body weight, adiposity, and blood glucose levels. HFD mice demonstrated increased voiding frequency and increased EFS-induced detrusor contractility. There were no changes in detrusor relaxation or cholinergic-medicated contraction. Mitochondrial respiration was decreased with HFD and H2 O 2 emission was increased. The relative amount of mitochondria in the detrusor was similar between groups. However, ETC complexes V and III were increased following HFD. CONCLUSIONS: Chronic HFD increased adiposity, lead to more frequent voiding, and enhanced EFS-mediated detrusor contractions. Mitochondrial respiration was decreased and H2 O 2 emission increased following HFD. Further research is required to determine if alterations in mitochondrial function could play a role in the development of HFD-induced bladder dysfunction.