Vestibulodynia presentation is differentiated by the presence of additional chronic primary pain conditions.

This paper presents data from one of five subprojects within the larger collaborative initiative, Complex Persistent Pain Conditions: Unique and Shared Pathways of Vulnerability , approved by the Biomedical Institutional Review Board of the University of North Carolina at Chapel Hill (#10–0499). The overarching study aimed to identify both unique and shared pathways associated with five overlapping chronic primary pain conditions: episodic migraine, fibromyalgia, irritable bowel syndrome, temporomandibular disorder, and VBD. All participants enrolled in the study completed a visit with the Phenotyping Core in addition to participating in condition-specific procedures at the subproject specialty clinics led by clinicians specialized in that area. Participants were recruited through community-based advertising and an existing UNC registry, comprised of patients referred from pain-specialty clinics, between December 2010 and November 2014, with final study size determined by maximum participant recruitment during this time frame. Pain-free controls were individuals previously recruited from a similar catchment area as these clinics, who were confirmed to not have any of the five CPPCs. Interested individuals underwent an initial telephone screening interview, which informed selection of a preliminary primary diagnostic category, this being one of the five pain conditions or a non-case control. This initial primary categorization directed the assignment of the subproject/clinic with which eligible participants would have their first condition-specific visit. Initial comorbid conditions were also recorded if applicable. The initial condition classification criteria are found in Supplemental Table 1 . On the day of this visit, the research coordinator verified the participant’s responses from the phone interview and inspected the questionnaires for missing data. Once the informed consent process was completed, the rest of the parent study protocol, as described in detail below, was performed. This included expanded diagnostic criteria, and participants classifications were confirmed or adjusted as needed. Ultimately, participants were categorized as 1) controls, eligible to participate in any subproject as such, or 2) individuals diagnosed with one or more of the target conditions, eligible to participate in the additional subproject(s) representing their condition(s). Thus, there was a subsequent bidirectional flow of participants to and from respective clinical sites, coordinated by the Clinical Core, which was housed at the UNC Center for Pain Research and Innovation (since relocated to Duke University as the Center for Translational Pain Medicine). This study model is depicted in Supplemental Figure 1 . A positive response to any of the following additional criteria led to initial exclusion from the parent study: history of significant medical conditions (e.g. seizure disorder, diabetes, thyroid disorder, heart disease, kidney disease, rheumatoid arthritis, ulcerative colitis); currently pregnant or nursing; currently undergoing chemotherapy or orthodontic treatment; having experienced a facial injury or surgery within the preceding six months. A positive response to any of the following additional criteria led to exclusion from the VBD subproject: Age 64; menopausal; known diagnosis of comorbid urogenital pain conditions (e.g. non-menstrual daily pain localized to the pelvic region, interstitial cystitis); history of pelvic re-constructive surgery (except obstetrical repair); unprovoked pain/discomfort (such as burning, itching, knife-like shooting pain) with or without concomitant provoked pain. All participants self-identified as female. Before their first appointment, participants completed a series of online questionnaires administered by the Phenotyping Core. These questionnaires assessed various physical and psychological symptoms, as detailed in Supplemental Table 2 . 51 – 57 Participants also reported their current medication use. CPPC case status for all participants was confirmed using a combination of in-person questionnaires and physical exams, as required by the following standard diagnostic criteria. 58 Examiners were naïve to the participant’s original classification. Episodic migraine with and without aura were diagnosed using the International Classification of Headache Disorders, 2 nd edition, including a neurologic exam. 59 Fibromyalgia was diagnosed using both the 1992 and 2010 versions of the American College of Rheumatology Diagnostic Criteria for Fibromyalgia. 60 , 61 Irritable bowel syndrome was diagnosed using the Rome III Diagnostic Criteria. 62 Temporomandibular disorder was diagnosed using The Research Diagnostic Criteria for Temporomandibular Disorder (RDC/TMD). 63 Included in this assessment was a standardized palpation assessment of pain and the collection of pain pressure thresholds (PPTs), measured using a 1 cm 2 flat-tipped algometer (Pain Test ™ Force Dial FPK/N, Wagner Instruments, Greenwich, CT), as described in previous work. 64 , 65 Neck sites (upper, middle, and lower sternocleidomastoid muscles, upper splenius capitus, upper semispinalis capitus, mid splenius capitus C3, and lower splenius capitus C5) were palpated using 2 lbs. of pressure, and general body sites (trapezius, supraspinatus, second rib, lateral epicondyle, gluteal, greater trochanter, knee) were palpated using 3 lbs. of pressure, bilaterally, for which positive or negative pain responses were recorded. PPTs were recorded for the temporalis (anterior), masseter (body), TMJ (lateral pole), trapezius (mid upper border), and lateral epicondyle. Beyond diagnostic categorization, these measurements were also analyzed to assess mechanical sensitivity remote from the site of chief complaint. Vestibulodynia was diagnosed using a gynecologic health questionnaire. Participants had to endorse isolated complaints of provoked pain with intercourse and/or tampon use, not caused by an underlying skin condition or infection. Participants reported their pain using the Gracely Box Pain Scale. 43 , 66 , 67 They were asked to rate the lowest, average and maximal pain in association with tampon use or intercourse using a Likert Scale of 0–20, and select verbal descriptors most representative of their pain experience, which have a predetermined numeric value. 67 – 69 These measures were completed by a trained examiner naïve to CPPC case status. Heat threshold and tolerance. A computer-controlled thermode (2.56 cm 2 ATS Pathway thermode, Medoc Inc) was applied to participants’ left ventral forearm. The device would gradually heat up from room temperature at a rate of 0.5°C/s, and participants were instructed to click a button on a mouse when they first felt a painful sensation. Once the button was clicked, the thermal device quickly decreased to room temperature. After a practice trial to ensure the subject understood the task, this procedure was repeated 4 times. The participants’ final recorded measurements were the average temperature of the 4 testing trials. The heat tolerance procedure and measurement recording were identical to the threshold procedure, except participants were asked to click a button when they could no longer tolerate the heat rather than at first sensation of pain. For both threshold and tolerance, testing concluded when the thermode reached 52°C. This temperature was recorded if the participant had not yet indicated pain. Cold threshold. The cold pain perception threshold protocol was identical to that of the heat threshold, but the temperature of the thermode dropped from room temperature at a rate of 0.5°C/s until the participants clicked the button. This protocol was run on the participants’ right ventral forearm. Testing concluded when the thermode reached 0°C. This temperature was recorded if the participant had not yet indicated pain. Participants’ blood was drawn and placed into BD Vacutainer ® EDTA Tubes, inverted 8 times, put into ice water, and stored at −80°C within 1 hour of collection. Genomic DNA purification was performed using the Qiagen Extraction Kit following the manufacturer’s recommended protocol. Genome-wide SNP polymorphisms were genotyped using the Thermo Fisher Scientific Applied Biosystems ™ Axiom ™ Precision Medicine Research Array microarray kit (Santa Clara, CA) by the Génome Québec Innovation Centre. The quality of samples was assessed using checks for missing call rate, chromosomal anomalies, cryptic relatedness, and concordance between self-reported and genetic sex and ancestry. Genotype quality for each SNP was assessed by missingness rate, duplicate sample concordance, and Mendelian errors; Hardy-Weinberg equilibrium was tested separately in homogenous European and African ancestry groups. Genotype calls from the cleaned dataset were used for imputation to the 1000 Genomes Project phase 3 reference panel, using SHAPEIT 70 for pre-phasing and IMPUTE2 71 for imputation. We used our previously described standardized approach to assess pain of the vulvar vestibular mucosa. 66 The mucosal surface is conventionally described using an imaginary clock face from the perspective of the examiner. Thus, sites are described with numbers based o’clock positions. For example, sites 12 and 6 correspond to the anterior and posterior positions on the vestibule midline in the dorsal lithotomy position, respectively. The sites can be grouped into two clinically relevant regions - the superior vestibule, corresponding to sites 10, 12 and 2, and the inferior vestibule, corresponding to sites 7, 6, and 5. Anatomical landmarks are utilized to standardize the location of vestibular sites between patients. The superior vestibule was examined first, followed by the inferior vestibule. Within the respective region, the examination was conducted in a specified order: right, left, and midline, as viewed by the examiner. With an inter-stimulus interval of 2 seconds, we delivered an approximate force rate of 1 N per second to each of the six tested sites using a Wagner Instruments FDIX Force One ™ digital algometer affixed to a disposable cotton swab. In effect, each site was slightly depressed for approximately 1 second, after which the participants were prompted to 1) indicate whether the stimulus was painful and 2) rate the pain intensity from 1 (least pain) to 10 (most painful), with rating 0 reserved for those who reported no pain. This is depicted in Figure 1A . Similarly, we utilized the description and assessment of pelvic muscle anatomy established in our previous work. 50 , 66 Using the same clock face orientation and o’clock naming convention, the right puborectalis levator ani muscle is described as muscle site 9, the perineal muscle complex is described as site 6, and the left puborectalis levator ani muscle is described as site 3. Thus, the sites are labeled analogously to the mucosa, with site 6 position on the midline, site 9 lateral and to the left as viewed by the examiner, and 3 lateral and to the right as viewed by the examiner. The examiner used their gloved finger affixed with a standard 1.26 cm 2 force-sensing resistor to palpate each pelvic muscle site in isolation at an approximate force rate of 1 N per second. For each muscle site, the participants were asked if they found the palpation painful, and if so, to rate the severity. This corresponds to a 0–10 scale, with 0 reserved for those who reported no pain. This is depicted in Figure 1B . Summary scores were analyzed with 2-tailed t-tests, Benjamini-Hochberg procedure for multiple testing correction. Self-reported medication data was coded by the Slone Epidemiology Center (Boston University) using the Slone Drug Dictionary. 72 Medication classes commonly used for pain relief where tabulated, the differences in medication use between controls, VBD, and VBD+ were analyzed using the Fisher exact test, followed by Bonferroni correction for multiple testing. As not all participants completed all surveys and QST measures, the number of participants varies across assays. In examining pain rating data, we determined the use of non-parametric analyses by Shapiro-wilk test for normality. Lateral site pairs (mucosal 10 and 2, mucosal 7 and 5, muscular 9 and 3) were averaged for each woman. Cross-group pain intensity scores were analyzed using one-way ANOVA on ranks (Kruskal–Wallis test, Dunn’s adjusted p-values). Within-group (cross-site) pain intensity scores were analyzed using two-tailed Wilcoxon matched-pairs signed rank test to account for intra-participant site pairings. Pain intensity statistics and graphs were generated using GraphPad Prism 10 (GraphPad Software, La Jolla, CA, USA). To visualize and compare the magnitude of site pain differences compared to controls for each group, we utilized the output of the Kruskal Wallis/Dunn’s tests. Pain intensity ANOVA Z-scores for VBD vs. control and VBD+ vs. control for each individual site were normalized internally with standard linear scaling: x’ = x – x min / x max – x min . A color gradient was applied to the resulting values, with 0 (white) representing the site with the smallest pain difference compared to controls, and 1 (magenta) representing the site greatest pain difference compared to controls within each group respectively. The resulting colors are overlayed on a diagram of the corresponding anatomical location, using the average of the values for lateralized site pairs. Genome-wide association analyses of DNA from the whole blood collected during the QST visit were conducted using logistic regression as implemented by the “dosage” method in PLINK v1.9 on the imputed genotypes. Age and three principal components representing racial eigenvectors were included in regression models as covariates. Regression analyses tested differences in allele dosage between the respective case group (VBD or VBD+) and controls. The resulting association p-values were filtered using thresholds for genome-wide association of p<5×10 −8 , and for suggestive association with a threshold of p<5×10 −5 . 73 , 74 SNPnexus was used to annotate the SNPs meeting this threshold from via the GRCh Build 38 human genome (GRCh38). 75 – 79 To explore the potential biological relevance or shared molecular mechanisms contributing to phenotypic variation in VBD and VBD+, the annotated genes were uploaded to the Ingenuity Pathway Analysis Software version 134816949 (QIAGEN Inc., https://digitalinsights.qiagen.com/IPA ). 80 All annotated genes were included as input for core analysis. The “Grow to Diseases and Functions” tool was applied to identify significantly enriched biological pathways and functions based on the input gene set. Enrichment significance was calculated using Fisher’s Exact Test (right-tailed), and results were ranked by p-value. To focus on the most relevant findings, we selected the top five enriched disease/function categories represented by more than one gene and lacking redundancy in canonical pathways. Non-connected molecule nodes were removed from the visual outputs to improve interpretability.

On average, women in the VBD group were slightly younger than those in the control and VBD+ groups. Recruited participants of all three groups were majority White and African American, with similar self-reported racial and ethnic distributions ( Table 1 ). Gravidity was recorded for women in the VBD subproject with no differences found between control, VBD, and VBD+ groups (median [IQR]: 0 [0–2]). Women with VBD+ reported the greatest active use of medications for pain relief, including skeletal muscle relaxants, analgesics and antipyretics, anticonvulsants, and antidepressants ( Supplemental Table 3 ). Data on the presence of comorbid CPPCs was collected in 151 women with VBD ( Figure 2 ). 70% presented with at least one additional chronic pain condition. Episodic migraine, temporomandibular disorder, and irritable bowel syndrome each presented in about two-thirds of these women, whereas fibromyalgia only presented in 27% ( Supplemental Table 4 ). Women with VBD reported higher pain intensity than controls at all superior mucosal sites, while those with VBD+ showed significant differences only at medial site 12 ( Figure 3A ). In the inferior vestibular mucosa, both groups reported significantly higher pain ratings than controls at all sites ( Figure 3B ). Medial mucosal pain ratings were higher than lateral pain ratings for both VBD and VBD+ groups ( Supplemental Figure 2A ). Mucosal pain ratings were higher for the inferior vestibule than the superior vestibule for women with VBD+ ( Supplemental Figure 2B ). Both groups reported significantly higher pain intensities at the lateral puborectalis levator ani muscles compared to controls ( Figure 3C ). There were no significant differences in the pain intensities between the VBD and VBD+ groups at any one given site, or for averaged ratings. Within-group normalized Z-Scores for each site compared to controls represents the variation in the magnitude of increased sensitivity ( Figure 4A ). Among women with VBD, this magnitude as was the largest at the midline mucosal sites 12 and 6 and at muscle site 6. Among women with VBD+, this magnitude was largest at lateral muscular sites 9 and 3 ( Figure 4B ). Overall, the VBD group did not report significantly higher mechanical pain sensitivity at remote body sites compared to controls. In contrast, the VBD+ group reported more painful neck and body palpation sites and significantly lower pressure pain thresholds across all examined muscles compared to both the control and VBD groups. Thermal stimulus results were analogous, as though the VBD group reported slightly lower thermal threshold and tolerance than controls, only the VBD+ group’s differences indicated statistically significantly heightened sensitivity ( Table 2 ). In assessment of mood state, scores scaled in severity from control to VBD to VBD+, with the VBD+ group exhibiting the most pronounced negative affect. This trend, representing increasingly worse outcomes across the groups, was reported repeatedly for a wide array of somatic and psychological symptoms. The VBD+ group experienced the highest sleep dysfunction, reactivity to everyday stimuli, and awareness of common somatic symptoms. They further reported perceived stress equating to a ‘moderate to high’ level, whereas the VBD and control groups reported ‘moderate’ levels. Finally, this also held true for symptoms of depression, anxiety, and pain catastrophizing ( Table 3 , Supplemental Tables 5 – 6 ). Case-control GWAS results showed 71 SNPs meeting the threshold for suggestive association for VBD and 97 SNPs meeting this threshold for VBD+. None were common between the groups. For VBD, these were highly condensed to chromosome 1, which included 52 associated SNPs ( Figure 5A ). The VBD+ associated SNPs were more distributed, with 17 in chromosome 3, 28 in chromosome 5, and 13 in chromosome 20 ( Figure 5B ). Of the VBD markers, 27 SNPs mapped directly to 11 genes, 3 of which were represented by more than one SNP (LMX1A, CIDEA, and NKAIN2). LMX1A was the most represented, with 13 SNPs. For VBD+, 60 SNPs mapped to 18 genes/gene regions, of which 7 genes (CDH18, ARMC8, PAPPA, ADGRB3, CCDC3, KCNMB2, and LARGE1) and 4 gene regions ( AC013762.1 , C20orf181, AL049536.1 , and LINC02588) were represented by more than one SNP. CDH18 was the most represented, with 24 SNPs ( Figure 5C ). The SNPs of the highest significance for VBD (p≈2×10 −7 ) did not map directly to a gene, rather, they were upstream of the processed pseudogene FTLP18, and downstream of lincRNA AL596257.1 . Those with the highest significance for VBD+ mapped to ARMC8 (p≈1×10 −6 ). The full annotated list of SNPs meeting the suggestive association threshold is found in Supplemental SNP Data File . Ingenuity Pathway Analysis identified categories of diseases and disorders linked to the gene sets represented by differentially expressed SNPs for VBD and VBD+. For VBD, these included abnormal cellular growth of the endometrium (7 genes/13 sources) and non-central nervous system (8 genes/72 sources), as well as familial neurological disorders (3 genes/84 sources) ( Figure 6A ). For VBD+, these included migraines (3 genes/3 sources) and extraintestinal functional disorders (5 genes/7 sources) as well as similarly identified abnormalities of the pelvis/endometrium ( Figure 6B ).

Vestibulodynia is a prevalent and multifaceted pain condition which profoundly impacts millions of women each year. While numerous studies highlight the heterogeneity of VBD, women are commonly grouped together for the purpose of evaluating causes and treatments. Our prior work found utility in stratifying VBD case status based on absence or presence of co-occurring irritable bowel syndrome, 44 but was limited in its sample size, demographic representation, and characterization of just one CPPC. Here, we enrolled over 300 women, representing a wider range of ages, racial backgrounds, and CPPC combinations than has previously been reported. Of note, we provide new data on medication use, as women with VBD+ reported taking significantly more medications to treat their pain, including skeletal muscle relaxants, analgesics and antipyretics, anticonvulsants, antidepressants. Heightened inferior vestibular mucosa pain sensitivity aligns with results from the original pilot study validating our pelvic exam protocol 50 , however, our finding that midline mucosal sites are more sensitive than lateral sites for women with both VBD subtypes has not previously been documented. These findings highlight the importance of anatomic specificity in the assessment of pelvic pain and may provide context for future work evaluating pathological vulvar hyperinnervation and nociceptor sensitization as an explanation of provoked vestibular pain. 30 , 32 , 81 – 83 The discussion of whether mucosa or muscle dysfunction underlies provoked VBD is ongoing. 49 , 50 Evidence suggests that pain attributed to the muscles of the pelvic floor is associated with the presence of overlapping CPPCs, whereas isolated VBD may be more attributable to the mucosa. 49 Comparisons of individual site pain ratings to controls within subtype groups showed that women with VBD reported the highest relative pain at the medial mucosal sites, while women with VBD+ reported the highest relative pain at the inferior medial mucosa and lateral pelvic floor muscles. This work suggests CPPC case status could be a useful tool in the early steps of investigating a woman’s particular anatomic drivers of provoked vulvo-vestibular pain. Consideration of this information may then also help inform first-line treatment decisions. For example, a topical therapy, such as lidocaine cream or gel, may work better for someone with pain localized to the vestibular mucosa, whereas pelvic floor physical therapy could prove more fruitful for patients with specified muscular pain. 84 , 85 Assessment of pain at remote body sites, revealed pronounced mechanical pain sensitivity for women with VBD+ compared to those with VBD and controls. Further, women with VBD+, but not VBD, had increased sensitivity to both heat and cold stimuli. These results are in line with prior work by our group and others demonstrating that individuals with co-occurring CPPCs have enhanced widespread bodily pain related to dysregulations in central processing of pain, irrespective of the index condition. 42 – 44 , 58 , 86 – 90 . Further, compared to women with VBD alone, those with VBD+ consistently reported poorer sleep quality, heightened somatic awareness, worse negative affect, and more stress, depression, and anxiety. This finding is in line with our prior work, 44 underscoring the impact of multiple co-occurring pain conditions on quality of life. Relationships between psychosocial variables, sleep quality, stress and pain experience have been shown to act reciprocally in the context of CPPCs. 91 – 93 Management of mental health, sleep hygiene, and stress levels have been shown to meaningfully improve chronic pain outcomes, and vice versa. 94 – 96 Therefore, women with VBD+ may benefit from additional support addressing these symptoms as part of their personalized pain management. Finally, the findings of our GWAS by case status group offer compelling avenues for further investigation into the genetic risk factors for VBD. Despite the shared chief complaint of pelvic pain, identified case-associated variants meeting the suggested associative threshold (p<5×10 −5 ) were entirely exclusive. We identified 3 genes with multiple SNPs associated with VBD. LMX1A, represented by the most SNPs in this group, is a member of the LIM homeodomain (LIM-HD) family of transcription factors that play crucial roles in a variety of biological functions, including the development of the reproductive system and midbrain dopamine neuron differentiation. 97 – 99 Mesolimbic dopamine signaling has been implicated in the salience of pain stimuli and chronic pain leads to a hypodopaminergic state. 100 CIDEA is a transcriptional coactivator regulating lipid production, with genetic variants linked to obesity, 101 which highly increases risk of developing chronic pain, potentially through inflammatory signaling pathways mediated by adipocytes. 102 – 105 The function of NKAIN2 not well known, though it has been linked to tumor suppression and nervous system development. 106 The IPA results further supported these individual gene-level findings by highlighting biological pathways related to neurodevelopment and reproductive system diseases, 107 – 112 but this is the first study, to date, to link LMX1A, CIDEA, and NKAIN2 to VBD as well as to pain in general. We identified 7 genes with multiple SNPs associated with VBD+, with CDH18, ARMC8, and PAPPA represented by the most. CDH18 has been implicated in the development of post-surgical neuropathic pain, migraine, and endometrial carcinoma. 113 – 116 ARMC8 has not been directly implicated in pain, but was found to co-express with the AMPA glutamate receptor (GRIA) that mediates excitatory synaptic transmission, 117 potentially playing a role in heightened neurotransmission. 118 , 119 PAPPA has been implicated in the pathogenesis of other CPPCs like endometriosis 120 and immune activation processes, like those resulting from COVID-19 infection, which may serve as a trigger for the development of chronic pain, 121 , 122 including infiltration of memory T-cells and macrophages. 123 Further, the overall associated SNP gene set was found by IPA to be linked to analogous diseases and disorders, including CPPCs like migraine, and their common comorbid psychiatric conditions, such as insomnia. 90 , 112 , 124 – 129 Together, these exploratory analyses identified genes associated with VBD subtype, with those involved in reproductive and neurological development unique to VBD and those involved in central sensitization and immune cell activation unique to VBD+. Yet, future studies are needed to confirm any individual genetic associations. Despite this study’s many strengths and important new findings, there were several limitations. First, while this study was open to participants of all ethnic and racial backgrounds, only non-Hispanic/Latino White and African American women were represented in significant numbers. Increased representation of other racial groups should be prioritized in the recruitment of future cohorts. Second, it’s possible that mucosal and muscular pain sensations may be conflated, such as via stretching of the skin during muscle manipulation. Localized anesthetic delivery may prove useful in avoiding this phenomenon, providing increased confidence in direct comparability of the assessments. Third, given our sample size, we were not powered to detect SNP associations at the standard of p<5×10 −8 . Despite this limitation, we believe this information holds value, as there is a substantial gap in the existing literature regarding the genetic basis of VBD, while recognizing that our findings should be viewed as exploratory rather than definitive. Similarly, pathway analyses depend on existing literature and thus are by no means exhaustive but rather serve as a tool for exploring a breadth of established findings with more efficiency and objectivity. In summary, this study confirms the importance of distinguishing VBD subtypes based on the presence of additional CPPCs for testing underlying biological mechanisms of chronic provoked vulvar pain. The observed differences in pain profiles coupled with preliminary genetic findings underscore the multifactorial nature of VBD and the need for tailored diagnostic and therapeutic approaches. Future research prioritizing recruitment of diverse cohorts and integrating genetic, psychosocial, and anatomical perspectives will be essential for advancing our understanding and improving care for those affected by this complex condition.

Vestibulodynia (VBD) is a chronic primary pain condition (CPPC) characterized by the presence of vulvovaginal pain lasting at least 3 months with no obvious root cause. 1 – 3 VBD is common, with prevalence estimates ranging from 8 to 16% globally, and is not limited by age, race, ethnicity or socioeconomic status. 4 – 7 VBD negatively impacts physical and emotional health, interpersonal relations, and daily activity. 8 – 12 Further, the personal nature of the topic of sexual health lends itself to feelings of shame and guilt. 8 , 13 As further evidence of its public health significance, VBD treatment can cost patients thousands of dollars annually, and poses a national burden upwards of $20 billion a year in the United States alone. 14 , 15 Despite its high prevalence and significant economic burden, VBD remains ineffectively treated due to its unclear etiology and heterogeneous clinical presentation. The pathophysiology of VBD is complex and assumed to be multifactorial, 16 – 18 including psychosocial risk factors, central nervous system sensitization, hyper-innervation, neuroinflammation, and heightened immune response. 19 – 35 Further, the clinical presentation of VBD varies widely with respect to the quality, intensity, and temporal pattern of pain, 1 , 2 , 36 making it challenging to identify underlying mechanisms and evidence-based treatments. To effectively evaluate potential mechanisms and treatments, it is essential to establish greater specificity in the definition and description of the VBD pain experience. Case stratification has proven fruitful in directing investigation of the underlying biological mechanisms of complex disorders, such as CPPCs. 37 – 41 In prior work, we stratified VBD case status based on absence or presence of co-occurring CPPCs. While some women present with localized vulvovaginal pain, upwards of 75% have additional CPPCs. 42 , 43 We found that compared to women with VBD alone, those with VBD + irritable bowel syndrome experienced greater pain at local and remote body sites and poorer overall self-reported health and mood. Women with VBD + irritable bowel syndrome also had a shift towards pro-inflammatory immune responses and dysregulation of miRNAs predicted to be involved in pain processing and central sensory pathways. However, this study was limited in its sample size, enrollment of predominantly White women between the ages of 21 and 45, and adequate characterization of just one CPPC. 44 Further, vaginal mucosa and pelvic muscle exam data were not differentiated by anatomical site and genetic risk factors were not examined. Though preliminary genetic investigations of VBD have been run, they are limited in scope. 45 – 48 Finally, differences between VBD predominated by mucosal versus muscular pain have been identified that could inform first-line treatment decisions, motivating further investigation specifying this distinction. 49 , 50 Thus, to more comprehensively characterize VBD phenotypic differences by CPPC case status, we conducted an extensive multi-modal clinical assessment in over 300 women, with greater demographic diversity. Our cohort was comprised of pain-free controls, women with localized VBD, and those with VBD + one or more co-occurring CPPCs (VBD+), namely, episodic migraine, fibromyalgia, irritable bowel syndrome, and temporomandibular disorder. We hypothesized that, compared to women with VBD alone, women with VBD+ experience 1) greater pain at local vulvovaginal sites, 2) greater pain at remote body sites, and 3) greater self-reported pain and poorer self-reported health and mood. In addition to these primary clinical aims, we conducted an exploratory genetic analysis to examine potential associations between VBD subgroups and underlying genetic variants, and to identify enriched biological pathways that may contribute to distinct pain phenotypes. These findings may inform future investigations into underlying biological mechanisms.