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.

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.

Immune mechanisms in vulvodynia: key roles for mast cells and fibroblasts.

Vulvodynia is a debilitating condition characterized by painful sensitivity to touch and pressure in the vestibular tissue surrounding the vaginal opening. It is often a "diagnosis of exclusion" of idiopathic pain made in the absence of visible inflammation or injury. However, the association between increased vulvodynia risk and a history of yeast infections and skin allergies has led researchers to explore whether immune mechanisms of dysregulated inflammation might underlie the pathophysiology of this chronic pain condition. Here we synthesize epidemiological investigations, clinical biopsies and primary cell culture studies, and mechanistic insights from several pre-clinical models of vulvar pain. Taken together, these findings suggest that altered inflammatory responses of tissue fibroblasts, and other immune changes in the genital tissues, potentially driven by the accumulation of mast cells may be key to the development of chronic vulvar pain. The association of increased numbers and function of mast cells with a wide variety of chronic pain conditions lends credence to their involvement in vulvodynia pathology and underscores their potential as an immune biomarker for chronic pain. Alongside mast cells, neutrophils, macrophages, and numerous inflammatory cytokines and mediators are associated with chronic pain suggesting immune-targeted approaches including the therapeutic administration of endogenous anti-inflammatory compounds could provide much needed new ways to treat, manage, and control the growing global pandemic of chronic pain.

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.

The histopathological results of vestibulectomy specimens in localized provoked vulvodynia in Turkey.

INTRODUCTION: Localized Provoked Vulvodynia (LPV) is a gynecological disease that is difficult to manage. Despite the wide spectrum of pathophysiological mechanisms and treatment modalities, there is limited success in the management of this disease. Surgical treatment is usually performed as the last resort. We aimed to investigate the histopathological results of 38 women with LPV who underwent surgical vestibulectomy. METHODS: of the 55 women that were diagnosed with LPV and underwent vulvar vestibulectomy, 38 patients with complete histopathological results were included in this retrospective study. RESULTS: in 14 patients, the pathological reports revealed Low-Grade Squamous Intraepithelial Lesions (LGSIL) (36.8%) whereas for 21 cases (55.2%), the findings were concordant with vestibulitis. The remaining three patients (7.8%) were diagnosed with lichen simplex chronicus. CONCLUSION: the presence of LGSIL in the surgical specimens of LPV cases is noteworthy. In this group of patients, surgical excision may contribute to the prevention of progression into high-grade lesions. The relationship between Human Papilloma Virus (HPV) infections and LPV should be further investigated.

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.

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.

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.

Disease-Related Microstructural Differences in the Brain in Women With Provoked Vestibulodynia.

Provoked vestibulodynia (PVD) is a chronic pelvic pain disorder affecting 16% of the female population. Neuroimaging studies have highlighted central abnormalities in PVD, similar to other chronic pelvic pain disorders, including brain regions involved in sensory processing and modulation of pain. The aim of the study was to determine alterations in the subvoxel, microstructural organization within tissues in PVD compared with healthy control participants (HCs) and a disease control group (irritable bowel syndrome [IBS]). Diffusion tensor imaging magnetic resonance imaging was conducted in 87 age-matched premenopausal women (29 PVD, 29 HCs, 29 IBS). Statistical parameter mapping of fractional anisotropy (FA) and mean diffusivity (MD) maps were used to identify microstructural difference in the brain specific to PVD or shared with IBS. PVD alterations in microstructural organization of the brain were predominantly observed in fibers associated with sensorimotor integration and pain processing that relay information between the thalamus, basal ganglia, sensorimotor, and insular cortex. PVD, compared with HCs, showed extensive increases in the FA of somatosensory and basal ganglia regions. In contrast, PVD and IBS subjects did not show any FA-related group differences. PVD subjects showed greater MD in the basal ganglia compared with HCs (higher MD in the internal capsule and pallidum) and IBS (higher MD in the putamen and pallidum). Increases in MD were associated with increased vaginal muscle tenderness and vulvar pain. The current findings highlight possible shared mechanisms between 2 different pelvic pain disorders, but also highlight the widespread alterations observed specifically in PVD compared with HCs. PERSPECTIVE: Alterations in microstructure in PVD were observed in fibers associated with sensorimotor integration and pain processing, which were also associated with increased vaginal muscle tenderness and vulvar pain. These alterations may be contributing to increased pain sensitivity and tenderness, highlighting the need for new therapies targeting the central nervous system.

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.

Inflammatory Renin-Angiotensin System Disruption Attenuates Sensory Hyperinnervation and Mechanical Hypersensitivity in a Rat Model of Provoked Vestibulodynia.

Vestibulodynia is characterized by perivaginal mechanical hypersensitivity, hyperinnervation, and abundant inflammatory cells expressing renin-angiotensin system proteins. We developed a tractable rat model of vestibulodynia to further assess the contributions of the renin-angiotensin system. Complete Freund's adjuvant injected into the posterior vestibule induced marked vestibular hypersensitivity throughout a 7-day test period. Numbers of axons immunoreactive for PGP9.5, calcitonin gene-related peptide, and GFRα2 were increased. Numbers of macrophages and T cells were also increased whereas B cells were not. Renin-angiotensin-associated proteins were abundant, with T cells as well as macrophages contributing to increased renin and angiotensinogen. Media conditioned with inflamed vestibular tissue promoted neurite sprouting by rat dorsal root ganglion neurons in vitro, and this was blocked by the angiotensin II receptor type 2 receptor antagonist PD123319 or by an angiotensin II function blocking antibody. Sensory axon sprouting induced by inflamed tissue was dependent on activity of angiotensin-converting enzyme or chymase, but not cathepsin G. Thus, vestibular Complete Freund's adjuvant injection substantially recapitulates changes seen in patients with provoked vestibulodynia, and shows that manipulation of the local inflammatory renin-angiotensin system may be a useful therapeutic strategy. PERSPECTIVE: This study provides evidence that inflammation of the rat vestibule induces a phenotype recapitulating behavioral and cytological features of human vestibulodynia. The model confirms a crucial role of the local inflammatory renin-angiotensin system in hypersensitivity and hyperinnervation. Targeting this system holds promise for developing new nonopioid analgesic treatment strategies.

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.

Local and Systemic Inflammation in Localized, Provoked Vestibulodynia: A Systematic Review.

OBJECTIVE: To synthesize and critically evaluate all available evidence investigating whether localized, provoked vestibulodynia is associated with a specific inflammatory profile at both a local and a systemic level. DATA SOURCES: Comprehensive electronic searches were performed in MEDLINE, EMBASE, Scopus, PubMed, Web of Science, Cumulative Index to Nursing and Allied Health Literature, the Cochrane Collaboration databases, and ClinicalTrials.gov. The search strategy was developed using MeSH terms related to localized, provoked vestibulodynia, and inflammatory markers. METHODS OF STUDY SELECTION: Two independent investigators screened titles and abstracts and performed data extraction and risk of bias assessments. Studies were included if they reported at least one baseline inflammatory marker in women with localized, provoked vestibulodynia and compared them with healthy women. Reference lists from published reviews on localized, provoked vestibulodynia were screened for additional studies. TABULATION, INTEGRATION, AND RESULTS: There were 1,619 studies identified. Eighteen studies met the inclusion criteria, including 400 women with localized, provoked vestibulodynia and 212 healthy women in a control group. Risk of bias assessment revealed that the methodologic quality was generally low. Fifteen studies investigated local inflammation and three studies investigated systemic inflammation. On a local level, the number of mast cells expressed in vestibular tissues was greater in women with localized, provoked vestibulodynia expressed than in women in the control group. Several studies reported undefined inflammatory infiltrate in vestibular tissues to a greater level in women with localized, provoked vestibulodynia than in women in the control group. Systemically, levels of natural killer cells were lower in women with localized, provoked vestibulodynia than in women in the control group. There were no systemic differences in systemic interferon-α and interferon-υ levels between groups. CONCLUSION: There is limited and contradictory evidence regarding the characteristics of local and systemic inflammation in women with localized, provoked vestibulodynia.

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.

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.