Immunohistochemical staining with CD117 and PGP9.5 of excised vestibular tissue from patients with neuroproliferative vestibulodynia.

BACKGROUND: Neuroproliferative vestibulodynia (NPV), a provoked genital pain characterized by severe allodynia and hyperalgesia, is confirmed in excised vestibular tissue by immunohistochemical staining (>8 CD117-positive immunostained cells/100× microscopic field) rather than by hematoxylin and eosin staining. AIM: In this study we sought to assess immunostaining of tissue samples obtained during vestibulectomy surgery and to correlate results with patient outcomes. METHODS: Patients (n = 65) meeting criteria for NPV who underwent vestibulectomy during the period from June 2019 through December 2022 formed the study cohort. We performed assessment of pathology of vestibular tissues by use of immunohistochemical staining, including quantitation of mast cells by CD117 (mast cell marker) and nerve fibers by protein gene product (PGP) 9.5 (neuronal marker). We analyzed 725 photomicrographs of immunostained tissue sections (100× and 200×) by manual counting and computer-assisted histometry and correlated these data to clinical assessments. OUTCOMES: Outcomes included density of CD117 and PGP9.5 immunostaining in the 1:00-11:00 o'clock and 12:00 o'clock vestibular regions, and patient-reported outcomes assessing sexual function, pain, distress, and symptom improvement. RESULTS: All 65 NPV patients (median age 26 years), 45 with lifelong and 20 with acquired NPV, had severe pain documented by PROs and vulvoscopy and had >8 CD117-immunopositive cells/100× microscopic field. Median cell count values were similar in the 1:00-11:00 o'clock and 12:00 vestibular regions (28.5 and 29.5/100× field, respectively). Likewise, the marker) and nerve fibers by protein gene product (PGP) 9.5 (neuronal marker). We analyzed 725 photomicrographs of immunostained tissue sections (100× and 200×) by manual counting and computer-assisted histometry and correlated these data to clinical assessments. OUTCOMES: Outcomes included density of CD117 and PGP9.5 immunostaining in the 1:00-11:00 o'clock and 12:00 o'clock vestibular regions, and patient-reported outcomes assessing sexual function, pain, distress, and symptom improvement. RESULTS: All 65 NPV patients (median age 26 years), 45 with lifelong and 20 with acquired NPV, had severe pain documented by PROs and vulvoscopy and had >8 CD117-immunopositive cells/100× microscopic field. Median cell count values were similar in the 1:00-11:00 o'clock and 12:00 vestibular regions (28.5 and 29.5/100× field, respectively). Likewise, the median area of CD117 immunostaining was similar in both regions (0.69% and 0.73%). The median area of PGP9.5 immunostaining was 0.47% and 0.31% in these same regions. Pain scores determined with cotton-tipped swab testing were nominally higher in lifelong vs acquired NPV patients, reaching statistical significance in the 1:00-11:00 o'clock region (P < .001). The median score for the McGill Pain Questionnaire affective subscale dimension was also significantly higher in lifelong vs acquired NPV patients (P = .011). No correlations were observed between hematoxylin and eosin results and density of mast cells or neuronal markers. Of note, 63% of the patient cohort reported having additional conditions associated with aberrant mast cell activity. CLINICAL IMPLICATIONS: The pathology of NPV is primarily localized to the vestibular epithelial basement membrane and subepithelial stroma with no visible vulvoscopic findings, making clinical diagnosis challenging. STRENGTHS AND LIMITATIONS: Strengths of this study include the large number of tissues examined with what is to our knowledge the first-ever assessment of the 12:00 vestibule. Major limitations are specimens from a single timepoint within the disease state and lack of control tissues. CONCLUSIONS: Performing immunohistochemical staining of excised vestibular tissue with CD117 and PGP9.5 led to histometric confirmation of NPV, indications that NPV is a field disease involving all vestibular regions, validation for patients whose pain had been ignored and who had experienced negative psychosocial impact, and appreciation that such staining can advance knowledge.

Unusual Presentation of Lymphangioma Circumscriptum of the Vulva: In Association With Crohn Disease.

ABSTRACT: Lymphangioma circumscriptum (LC) is a rare benign condition, with marked dilation of surface lymphatic vessels in the deep and subcutaneous layers. Vulvar LC can become a highly disabling condition with vulvar discomfort, itching, burning and lymph seeping being the dominant symptoms. Biopsy is mandatory for the diagnosis. There is no consensus on the standard treatment for vulvar LC and recurrence is frequent. In complex cases with wide disease location, combination of different treatment options, such as abrasive methods and surgery, may lead to the best clinical and aesthetical result, with extended disease-free periods. We present a patient with a long history of Crohn disease with multiple pelvic surgeries who developed an extensive vulvar LC.

Exploring Localized Provoked Vulvodynia: Insights from Animal Model Research.

Provoked vulvodynia represents a challenging chronic pain condition, characterized by its multifactorial origins. The inherent complexities of human-based studies have necessitated the use of animal models to enrich our understanding of vulvodynia's pathophysiology. This review aims to provide an exhaustive examination of the various animal models employed in this research domain. A comprehensive search was conducted on PubMed, utilizing keywords such as "vulvodynia", "chronic vulvar pain", "vulvodynia induction", and "animal models of vulvodynia" to identify pertinent studies. The search yielded three primary animal models for vulvodynia: inflammation-induced, allergy-induced, and hormone-induced. Additionally, six agents capable of triggering the condition through diverse pathways were identified, including factors contributing to hyperinnervation, mast cell proliferation, involvement of other immune cells, inflammatory cytokines, and neurotransmitters. This review systematically outlines the various animal models developed to study the pathogenesis of provoked vulvodynia. Understanding these models is crucial for the exploration of preventative measures, the development of novel treatments, and the overall advancement of research within the field.

Innervation of the human vulvar vestibule: A comprehensive review.

Pain of the vulvar vestibule, including provoked vestibulodynia, is prevalent among women, yet challenging to treat due to its multifactorial etiology. Recent evidence indicates a neuroproliferative subtype in which hypersensitivity of the vulvar vestibule is due, in part, to hyperinnervation. Detailed knowledge regarding the innervation of the vulvar vestibule is crucial to understanding and treating pain conditions impacting this region. The purpose of this review is to consolidate the current evidence regarding the innervation of the human vulvar vestibule and discuss the implications of this innervation for pathological conditions affecting this tissue. A comprehensive review of the literature was conducted using keywords including vulvar vestibule, innervation, and vestibulodynia to identify articles concerning the innervation of the vulvar vestibule. Fifteen studies published between 1998 and 2017 were reviewed. Evidence from immunohistochemical investigations support that the vulvar vestibule has nociceptive, mechanosensory, sympathetic, and parasympathetic innervation. In pathological samples, hyperinnervation supports the neuroproliferative etiology of provoked vestibulodynia. Additionally, there is some evidence supporting the role of the pudendal nerve in vulvar vestibule innervation, although no cadaveric studies have been reported to date. Progress has been made in our understanding of the innervation of the vulvar vestibule, though further research into the origin of sensory and autonomic innervation of this region is needed. Advancing the knowledge of vulvar vestibule innervation is crucial towards improving our understanding of the function of this tissue, in addition to informing the etiology and management of pain syndromes impacting this region.

An update on the management of chronic pelvic pain in women.

Chronic pelvic pain represents a major public health problem for women and impacts significantly on their quality of life. Yet it is under-researched and a challenge to manage. Women who suffer from chronic pelvic pain frequently describe their healthcare journey as long, via a variety of specialists and frustrating, with their pain often dismissed. Aetiological factors and associations are best conceptualised using the 'three P's' model of predisposing, precipitating and perpetuating factors. This integrates the numerous biological, psychological and social contributors to the complex, multifactorial nature of chronic pelvic pain. Overall management involves analgesia, hormonal therapies, physiotherapy, psychological approaches and lifestyle advice, which like other chronic pain conditions relies on a multidisciplinary team approach delivered by professionals experienced and trained in managing chronic pelvic pain.

Subcutaneous botulinum toxin type A injections for provoked vestibulodynia: a randomized placebo-controlled trial and exploratory subanalysis.

BACKGROUND: Previous studies using botulinum toxin type A (BT) to treat provoked vestibulodynia (PVD) reported conflicting findings, possibly attributable to singular injections or low doses. We assessed PVD treatment effectiveness with high-dose single injections of BT (50 or 100 units) versus placebo, and then repeat BT 100 U injections over 6 months. METHODS: This was a randomized, double-blind, three-arm, placebo-controlled study with 33 PVD patients. BT 50 U (arm A), 100 U (arm B) or saline (arm C) were injected subcutaneously into the dorsal vulvar vestibulum and pain was assessed after 3 months. The investigation proceeded as an unblinded exploratory analysis, in which symptomatic patients received a BT 100 U injection. Symptomatic patients in arm C received a second BT 100 U injection at the 6-month visit. Symptoms were measured at 3-month cycles using: (1) cotton swab-provoked visual analogue scale (VAS), (2) von Frey filaments, and (3) Marinoff dyspareunia scale. RESULTS: The three groups were comparable in terms of demographics and baseline clinical characteristics. Three months after the initial injection, no significant differences in pain were observed among the study arms, yet significant improvements occurred within all groups using the von Frey filaments test. Results from the exploratory analyses showed repeat injections of 100 U BT over 6 months led to significant pain reduction (VAS and von Frey filaments). Fifty-eight percent (7/12) of patients assessable after repeat injections were symptom-free or had ≥ 2 VAS reduction. Adverse events were minor and no serious adverse events occurred during the RCT or exploratory analysis. CONCLUSIONS: PVD symptoms after one subcutaneous injection of BT (50 or 100 units) did not significantly differ compared to placebo, yet all three study arms experienced a reduction in pain 3 months after a single injection. Exploratory analyses indicated that repeat high-dose BT injections may significantly reduce pain over 6 months. TRIAL REGISTRATION: This trial was registered with the Swiss Medical Agency (reference number: 2007DR2102) in 2007.

Descriptors of Vulvodynia: A Multisocietal Definition Consensus (International Society for the Study of Vulvovaginal Disease, the International Society for the Study of Women Sexual Health, and the International Pelvic Pain Society).

OBJECTIVES: Three scientific societies, the International Society for the Study of Vulvovaginal Disease (ISSVD), the International Society for the Study of Women Sexual Health (ISSWSH), and the International Pelvic Pain Society (IPPS) developed the "2015 ISSVD, ISSWSH, and IPPS Consensus Terminology and Classification of Persistent Vulvar Pain and Vulvodynia" (referred to as the "2015 consensus terminology").The terminology included 11 descriptors of vulvodynia. However, the definitions of the descriptors were not included in the 2015 consensus terminology publications. The objective of this article was to provide these definitions. MATERIALS AND METHODS: The ISSVD led a discussion on the definitions for the 11 vulvodynia descriptors, with participation from the ISSWSH and IPPS. The definitions were created through a consensus process. RESULTS: The definitions are described and the rationale for their choice is elucidated. CONCLUSIONS: The definitions of vulvodynia descriptors were determined by a multistaged process of discussion among health care providers with expertise in the pathophysiology, evaluation, and treatment of vulvodynia. The definitions were approved by the ISSVD, ISSWSH, and IPPS. It is recommended that these definitions of vulvodynia descriptors as well as the 2015 consensus terminology be used for the classification of vulvodynia.

Mindfulness-Based Group Cognitive Behavior Therapy for Provoked Localized Vulvodynia: A Randomized Controlled Trial.

OBJECTIVE: The aim of the study was to compare the effectiveness of mindfulness-based group cognitive behavior therapy (M-gCBT) versus education support group therapy for the pain and distress associated with provoked localized vulvodynia. MATERIALS AND METHODS: Participants were randomized to M-gCBT or education support group therapy. Mindfulness-based group cognitive behavior participants attended 8 weekly sessions. Education support group participants received 8 weeks of online education with 3 in-person group visits. Vaginal insertion pain (tampon test) was the primary outcome. Secondary outcomes (Generalized Anxiety Disorder 7, Beck's Depression Index, Female Sexual Distress Scale, Female Sexual Function Index, and Pain Catastrophizing) were administered before intervention and at the completion of the study period, 3 months, and 6 months. Sample size was based on the ideal number for group dynamics of 6 to 12 participants per group. RESULTS: Participants were enrolled from August 1, 2016, to January 30, 2017. Thirty-two participants were enrolled and 31 were randomized: 14 to M-gCBT and 17 to education support. Baseline characteristics did not differ significantly. Vaginal insertion pain decreased in both groups but was not statistically different between groups (difference of 1.23; 95% CI = -0.52 to 2.98). At 6 months, participants in the M-gCBT group showed statistically significant improvement in the Female Sexual Function Index, Generalized Anxiety Disorder 7, and Beck's Depression Index compared with the education support group. CONCLUSIONS: Mindfulness-based group cognitive behavior and education support group therapy are effective in reducing pain and distress. However, women in the M-gCBT program showed greater improvement in certain secondary outcomes, indicating that M-gCBT may offer some advantages in reducing distress associated with provoked localized vulvodynia.

Abnormal vaginal microbioma is associated with severity of localized provoked vulvodynia. Role of aerobic vaginitis and Candida in the pathogenesis of vulvodynia.

Localized provoked vulvodynia (LPV) causes introital dyspareunia in up to 14% of premenopausal women. Vaginal infections like candidosis may play a initiating role. The aim of this study was to test a possible association of vaginal microbiota alternations such as Candida vaginitis (CV), aerobic vaginitis (AV) and bacterial vaginosis (BV) with severity of vulvodynia and painful intercourse. In an observational study, Q-tip touch test (score 1 (no pain) to 10 (worst possible pain)) was performed on seven vestibular locations in 231 LPV patients presenting in the Vulvovaginal Disease Clinics in Tienen, Leuven and Antwerp, Belgium. Severity of pain upon attempting sexual intercourse was recorded in a similar scale. Both scales were compared to results from fresh wet mount phase contrast microscopy on vaginal fluid smears tested for abnormal vaginal flora (AVF), BV, AV and CV according the standardized microscopy method (Femicare). Fisher's exact test was used. Average age was 31.3 ± 11.6 years, and 58.8% (n = 132) had secondary vestibulodynia. There was an inverse relation between the presence of Candida in the vaginal smears and pain score (p = 0.03). There was no relation of pain score, nor Q-tip score with BV. LPV patients with Q-tip score above 7 at 5 and/or 7 o'clock or at 1 and/or 11 o'clock had more often AV than women with lower pain scores (30 vs 14.5%, p = 0.01, and 39 vs 14.7%, p < 0.005, respectively). Detailed study of the vaginal microflora in patients demonstrates that the most severe patients suffer more from AV and less from Candida. These abnormalities need to be actively looked for and corrected before considering surgery or other therapies.

Systematic Review of Treatment Outcome Measures for Vulvodynia.

OBJECTIVES OF THE STUDY: To systematically evaluate the literature regarding vulvodynia treatment outcome measures. METHODS: A systematic literature search on OVID, PubMed, and PsycINFO databases was conducted from inception until May 2016. Studies were included/excluded based on prespecified criteria. Reported outcome measures were organized into 6 core outcome domains recommended by the Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials (IMMPACT): pain; physical functioning, emotional functioning, participant ratings of global improvement and satisfaction with treatment, symptoms and adverse events, and participant disposition. RESULTS: Of the 206 articles identified for full-text screening, 33 met our criteria. One study adhered to all IMMPACT recommendations. The number of outcomes measured per study ranged from 1 to greater than 20. Patient-reported pain outcomes were found in the majority (27/33; 82%) of studies. Pain severity with intercourse was reported by 24 (73%) of 33 studies-9 different scales were used to measure this outcome. Clinician-reported outcomes were present in 14 (42%) of 33 studies. Methods of measuring vestibular sensitivity by "cotton swab" test were different in 8 of 10 studies. Other domains reported included; physical function (8/33 studies; 24%), sexual function (23/33 studies; 70%), and emotional function (13/33 studies; 39%). Symptoms and adverse events were reported by 15 (45%) of 33 studies. One study formally reported participant disposition using all the information recommended by CONSORT. CONCLUSIONS: Comparison of clinical trial results in vulvodynia is not possible because of a lack of standard treatment outcome measures. Vulvodynia researchers should apply the IMMPACT criteria to guide the development of a minimum core set of standard outcome measures that measure holistic health.

Toll-Like Receptor Signaling Contributes to Proinflammatory Mediator Production in Localized Provoked Vulvodynia.

OBJECTIVES: Localized provoked vulvodynia (LPV) afflicts approximately 8% of women in the United States and represents a huge financial, physical, and psychological burden. Women with LPV experience intense pain localized to the vulvar vestibule (area immediately surrounding vaginal opening). We have identified mechanisms involved in the development of LPV whereby vulvar fibroblasts respond to proinflammatory stimuli to perpetuate an inflammatory response that causes pain. However, these mechanisms are not fully elucidated. Therefore, we explored the role of toll-like receptors (TLRs), a class of innate immune receptors that rapidly respond to microbial assaults. MATERIALS AND METHODS: To determine whether TLRs are expressed by vulvar fibroblasts and whether these contribute to proinflammatory mediator production and pain in LPV, we examined TLR expression and innate immune responses in fibroblasts derived from painful vestibular regions compared with nonpainful external vulvar regions. RESULTS: Human vulvar fibroblasts express functional TLRs that trigger production of inflammatory mediators associated with chronic pain. We focused on the TLR-7-imiquimod proinflammatory interaction, because imiquimod, a ligand of TLR-7, may exacerbate pain in women during treatment of human papillomavirus-associated disease. CONCLUSIONS: Human vulvar fibroblasts express a broad spectrum of TLRs (a new finding). A significantly higher TLR-mediated proinflammatory response was observed in LPV case vestibular fibroblasts, and with respect to the imiquimod-TLR 7 interaction, development of chronic vestibular pain and inflammation may be a possible sequelae of treatment of vulvar human papillomavirus-associated disease. Suppressing enhanced TLR-associated innate immune responses to a spectrum of pathogen-associated molecular patterns may represent a new/effective therapeutic approach for vulvodynia.

Immune activation enhances epithelial nerve growth in provoked vestibulodynia.

BACKGROUND: Provoked vestibulodynia manifests as allodynia of the vulvar vestibular mucosa. The exact mechanisms that result in altered pain sensation are unknown. Recently, we demonstrated the presence of secondary lymphoid tissue, which is the vestibule-associated lymphoid tissue in the vestibular mucosa, and showed that this tissue becomes activated in provoked vestibulodynia. OBJECTIVE: The purpose of this study was to examine whether expression of intraepithelial nerve fibers and nerve growth factor are related to immune activation in provoked vestibulodynia. STUDY DESIGN: Vestibular mucosal specimens were obtained from 27 patients with severe provoked vestibulodynia that was treated by vestibulectomy and from 15 control subjects. We used antibodies against the protein gene product 9.5, the neuron specific neurofilament, and nerve growth factor for immunohistochemistry to detect intraepithelial nerve fibers and nerve growth factor expressing immune cells in the vestibular mucosa. For intraepithelial nerve fibers, we determined their linear density (fiber counts per millimeter of the outer epithelial surface, protein gene product 9.5) or presence (neuron specific neurofilament). Nerve growth factor was analyzed by counting the staining-positive immune cells. Antibodies against CD20 (B lymphocytes) and CD3 (T lymphocytes) were used to identify and locate mucosal areas with increased density of lymphocytes and the presence of germinal centers (ie, signs of immune activation). B-cell activation index was used to describe the overall intensity of B-cell infiltration. RESULTS: We found more protein gene product 9.5-positive intraepithelial fibers in vestibulodynia than in the control samples (6.3/mm [range, 0.0-15.8] vs 2.0/mm [range, 0.0-12.0]; P=.006). Neuron specific neurofilament -positive intraepithelial fibers were found in 17 of 27 vestibulodynia cases (63.0%) and in none of the control cases. Protein gene product 9.5-positive intraepithelial fibers were more common in samples with more pronounced immune activation. The density of these fibers was higher in samples with than without germinal centers (6.1/mm [range, 4.3-15.8] vs 3.0/mm [range, 0.0-13.4]; P=.020). A positive correlation between the fiber density and B-cell activation index score of the sample was found (Spearman's Rho, 0.400; P=.004; R2=0.128). No significant difference, however, was found in the density or presence of nerve fibers between samples with high and low T-cell densities. We identified areas of minor and major vestibular glands in 16 of the patient samples and in 1 control sample. Protein gene product 9.5-positive nerve fibers were found more often in glandular epithelium surrounded by B-cell infiltration than in glands without B cells (P=.013). Also, the presence of neuron specific neurofilament-positive fibers in glandular epithelium was associated with B-cell infiltrates (P=.053). Nerve growth factor-positive immune cells were more common in mucosal areas with than without B-cell infiltration and intraepithelial nerve fibers. CONCLUSION: Excessive epithelial nerve growth in provoked vestibulodynia is associated with increased B-cell infiltration and the presence of germinal centers. This supports the fundamental role of immune activation in provoked vestibulodynia.

Differences in the Biometry of the Levator Hiatus at Rest, During Contraction, and During Valsalva Maneuver Between Women With and Without Provoked Vestibulodynia Assessed by Transperineal Ultrasound Imaging.

INTRODUCTION: Pelvic floor muscle (PFM) involvement is suspected in the pathophysiology of provoked vestibulodynia (PVD); however, the underlying mechanisms are unclear. PFM morphology can be inferred from the biometry of the levator hiatus determined through dynamic ultrasound imaging. AIMS: The aim of this study was to determine the nature of PFM involvement in women with PVD via an evaluation of the biometry of the levator hiatus at rest, upon maximal voluntary contraction (MVC) of the PFMs, and upon maximal Valsalva maneuver (MVM). METHODS: Thirty-eight women with PVD and 39 asymptomatic controls were imaged using 3D transperineal ultrasound. Levator hiatal dimensions (area; left-right [LR] and anteroposterior [AP] diameters) were measured at rest, on MVC, and on MVM. Differences in hiatal dimensions and in relative changes in dimensions from rest to MVC and from rest to MVM were compared between groups using separate 1-way analyses of variance for each measure and task. Analysis of covariance models were used to investigate the impact of levator hiatal dimensions at rest on the relative changes in the levator hiatal dimensions during MVC and MVM. MAIN OUTCOME MEASURES: Levator hiatal area, LR, and AP diameters, at rest, on MVC, and on MVM were the main outcome measures. Relative changes in hiatal dimensions were assessed as the percent change in hiatal area, LR diameter, and AP diameter. RESULTS: In comparison with controls, women with PVD had smaller hiatal areas at rest, on MVC, and on MVM, concurrent with smaller LR diameters on MVM. Women with PVD had a significantly smaller change in hiatal area on MVM than controls, but no differences were evident on MVC. In both groups, smaller levator hiatal dimensions at rest were associated with smaller relative decreases in dimensions on MVC and larger relative increases in dimensions on MVM. CONCLUSION: In comparison to controls, women with PVD appear to have narrower levator hiatus' and less capacity to distend their hiatus on Valsalva. The state of the PFMs at rest appears to significantly influence biometric changes in the PFMs during contraction and Valsalva.

Presence of Spontaneous Pain and Comorbid Pain Conditions Identifies Vulvodynia Subgroups.

OBJECTIVE: The aims of the study were to define the heterogeneity of vulvodynia by determining data-driven subgroups within the vulvodynia diagnosis using exploratory cluster analysis and to characterize the subgroups identified. MATERIALS AND METHODS: Included were participants in the longitudinal population-based study of vulvodynia in southeast Michigan who screened positive for vulvodynia at least once during the study. A cluster analysis using variables reflecting vulvar pain characteristics and comorbid pain conditions was conducted. Variables reflecting best separation of clusters were used to assign participants to subgroup categories. Demographic, psychiatric, general health, and other vulvar pain characteristics were summarized for each subgroup, followed by multinomial regression and pairwise comparisons of subgroups on these factors. RESULTS: Of 441 women screening positive for vulvodynia during the course of the study, 393 were eligible on the basis of data requirements. Cluster analysis suggested that best subgroup separation was based on the following 2 variables: (1) presence or absence of spontaneous vulvar pain and (2) presence or absence of other comorbid pain conditions. Subgroups did not differ by age or ethnicity. The subgroup having spontaneous pain and other comorbid conditions demonstrated greatest morbidity in general health measures, psychiatric disorders, and other vulvar pain measures. Primary versus secondary vulvodynia did not vary between subgroups and did not assist in subgroup separation in the cluster analysis. DISCUSSION: Subgroups based on exploratory cluster analysis demonstrated that presence of spontaneous pain and the presence of comorbid pain conditions resulted in best separation of groups. Presence of both spontaneous pain and other comorbid pain conditions identified the group with greatest morbidity.

Vestibular Mast Cell Density in Vulvodynia: A Case-Controlled Study.

OBJECTIVES: To identify whether mast cell densities in vulvar biopsies from the vestibule are associated with vulvodynia. METHODS: We enrolled 100 women aged 19 to 59 years with confirmed vulvodynia cases, 100 racially matched controls, and 100 black control women. All had vulvar biopsies performed at the 7 o'clock position of the vestibule, which were then immunostained to detect c-KIT protein. The numbers of c-KIT positive mast cells per ×400 magnification field were manually counted, and t tests and logistic regression were used to assess the association with case-control status. RESULTS: Of the biopsies, 235 were adequate samples for c-KIT testing for mast cells. The mast cell density was substantially lower in black control women (13.9 ± 10.9) in comparison to white control women (22.5 ± 13.2 p < 0.001): hence the analysis was confined to white cases and racially matched control women. Compared with racially matched controls, cases were younger, more likely to be married, and reported a higher household income. The average number of mast cells per ×400 magnification field overall was 19.1 ± 13.2 (range, 0-62). There was no difference in the mast cell count between racially matched cases (22.4 ± 13.9 per ×400 field) and controls (22.5 ± 13.2) in either the univariate or multivariable analyses. Within the group of cases, there was no difference in mast cell density based on the presence or absence of a variety of urogenital symptoms. CONCLUSIONS: No difference in mast cell density in biopsies of the vestibule was found between white cases and racially matched controls. Black control women have a lower mast cell density compared with white control women.

Activation of vestibule-associated lymphoid tissue in localized provoked vulvodynia.

OBJECTIVE: Localized provoked vulvodynia (LPV) may have inflammatory etiology. We wanted to find out whether the cell-mediated immune system becomes activated in the vestibular mucosa in LPV. STUDY DESIGN: This was a controlled cross-sectional study. Vestibular mucosal specimens were obtained from 27 patients with severe LPV and 15 controls. Detailed clinical history of the patients was obtained. For immunohistochemistry, antibodies against CD3 (T cells), CD20 (B cells), IgA (mucosal plasma cells), CD163 (dendritic cells [DCs]), CD68 (macrophages), and CD117 (mast cells) were employed. Mann-Whitney U test and χ(2) test were used for statistical analyses. RESULTS: More B lymphocytes and mature mucosal IgA-plasma cells were found in patients than in controls (P < .001 and P < .001, respectively). In LPV samples, B and T cells were arranged into germinal centers representing local immune activation. Germinal centers were not seen in controls. Antigen-presenting DCs and macrophages were found both in patients and controls with similar densities. DCs were found to extend their dendrites into the luminal space through an intact epithelium. Similar amounts of mast cells were found evenly scattered throughout the stroma of vestibular mucosa of both patients and controls. CONCLUSION: We demonstrate here local organized vestibule-associated lymphoid tissue analogous to mucosa-associated lymphoid tissue. Vestibule-associated lymphoid tissue may emerge as a response to local infection or inflammation in LPV.

Active and Passive Components of Pelvic Floor Muscle Tone in Women with Provoked Vestibulodynia: A Perspective Based on a Review of the Literature.

AIM: Pelvic floor muscle (PFM) dysfunctions, especially elevated tone or tension, are suggested to play an important role in the pathophysiology of provoked vestibulodynia (PVD). However, the involvement of the PFMs remains misunderstood as the assessment of muscle tone is complex and requires a thorough understanding of muscle physiology in relation to the characteristics and limitations of current PFM assessment tools. The aim of this review was to describe the structures and mechanisms involved in muscle tone in normally innervated muscle, and to discuss and relate these concepts to the PFM findings in women with PVD. METHODS: A narrative overview of the literature retrieved from searches of electronic databases and hand searches. RESULTS: Muscle tone in a normally innervated muscle comprises both active (contractile) and passive (viscoelastic) components. Current methods for evaluating PFM tone such as digital palpation, ultrasound imaging, pressure perineometry, dynamometry, and electromyography may evaluate different components. Research findings suggestive of PFM hypertonicity in women with PVD include elevated general PFM tone, changes in viscoelastic properties, and at least in some women, abnormal increases in electrogenic activity. CONCLUSION: There is a growing body of evidence to support the involvement of PFM hypertonicity in the pathophysiology of PVD. Limitations of the instruments as well as their properties should be considered when evaluating PFM tone in order to obtain better insight into which component of PFM tone is assessed. Future research is required for further investigating the underlying mechanisms of PFM hypertonicity, and studying the specific effects of physiotherapeutic interventions on PFM tone in women with PVD.

Persistent genital hyperinnervation following progesterone administration to adolescent female rats.

Provoked vestibulodynia, a female pelvic pain syndrome affecting substantial numbers of women, is characterized by genital hypersensitivity and sensory hyperinnervation. Previous studies have shown that the risk of developing provoked vestibulodynia is markedly elevated following adolescent use of oral contraceptives with high progesterone content. We hypothesized that progesterone, a steroid hormone with known neurotropic properties, may alter genital innervation through direct or indirect actions. Female Sprague Dawley rats received progesterone (20 mg/kg subcutaneously) from Days 20-27; tissue was removed for analysis in some rats on Day 28, while others were ovariectomized on Day 43 and infused for 7 days with vehicle or 17beta estradiol. Progesterone resulted in overall increases in vaginal innervation at both Day 28 and 50 due to proliferation of peptidergic sensory and sympathetic (but not parasympathetic) axons. Estradiol reduced innervation in progesterone-treated and untreated groups. To assess the mechanisms of sensory hyperinnervation, we cultured dissociated dorsal root ganglion neurons and found that progesterone increases neurite outgrowth by small unmyelinated (but not myelinated) sensory neurons, it was receptor mediated, and it was nonadditive with NGF. Pretreatment of ganglion with progesterone also increased neurite outgrowth in response to vaginal target explants. However, pretreatment of vaginal target with progesterone did not improve outgrowth. We conclude that adolescent progesterone exposure may contribute to provoked vestibulodynia by eliciting persistent genital hyperinnervation via a direct effect on unmyelinated sensory nociceptor neurons and that estradiol, a well-documented therapeutic, may alleviate symptoms in part by reducing progesterone-induced sensory hyperinnervation.

Morphometry of the pelvic floor muscles in women with and without provoked vestibulodynia using 4D ultrasound.

INTRODUCTION: It has been suggested that pelvic floor muscles (PFMs) play an important role in provoked vestibulodynia (PVD) pathophysiology. Controversy in determining their exact contribution may be explained by methodological limitations related to the PFM assessment tools, specifically the pain elicited by the measurement itself, which may trigger a PFM reaction and introduce a strong bias. AIM: The aim of this study was to compare PFM morphometry in women suffering from PVD to asymptomatic healthy control women using a pain-free methodology, transperineal four-dimensional (4D) ultrasound. METHODS: Fifty-one asymptomatic women and 49 women suffering from PVD were recruited. Diagnosis of PVD was confirmed by a gynecologist following a standardized examination. All the participants were nulliparous and had no other urogynecological conditions. The women were evaluated in a supine position at rest and during PFM maximal contraction. MAIN OUTCOME MEASURES: Transperineal 4D ultrasound, which consists of a probe applied on the surface of the perineum without any vaginal insertion, was used to assess PFM morphometry. Different parameters were assessed in sagittal and axial planes: anorectal angle, levator plate angle, displacement of the bladder neck, and levator hiatus area. The investigator analyzing the data was blinded to the clinical data. RESULTS: Women with PVD showed a significantly smaller levator hiatus area, a smaller anorectal angle, and a larger levator plate angle at rest compared with asymptomatic women, suggesting an increase in PFM tone. During PFM maximal contraction, smaller changes in levator hiatus area narrowing, displacement of the bladder neck, and changes of the anorectal and of the levator plate angles were found in women with PVD compared with controls, which may indicate poorer PFM strength and control. CONCLUSION: Using a reliable and pain-free methodology, this research provides sound evidence that women with PVD display differences in PFM morphometry suggesting increased tone and reduced strength.

The value of histology in predicting the effectiveness of vulvar vestibulectomy in provoked vestibulodynia.

OBJECTIVE: This study aimed to determine whether histology can predict response to vestibulectomy in the management of provoked vestibulodynia. MATERIALS AND METHODS: Inflammatory cell, mast cell, and nerve fiber counts were determined in prospectively collected vulvar vestibulectomy specimens from 30 women treated surgically for provoked vestibulodynia. RESULTS: Twenty-three subjects (77%) had a complete early response to surgery. At 3 years of follow-up, this had increased to 28 (93%), with a 29th showing some improvement. No subject had gotten worse after surgery or in the 3 years of follow-up. When comparing patients with an early complete response with those patients who still had symptoms, no difference in lymphocyte counts (27.6 vs. 37.8 per mm), mast cell counts (110.4 vs. 97.8 per mm), or stromal nerve fiber counts (16.4 vs. 16.4 per mm) was found. CONCLUSIONS: Vestibulectomy is a very effective treatment option in women with provoked vestibulodynia who have had failed conservative treatment. Histology is unable to predict which patients will respond to surgery.