Dysregulation in Sphingolipid Signaling Pathways is Associated With Symptoms and Functional Connectivity of Pain Processing Brain Regions in Provoked Vestibulodynia.

Table 1 presents the demographic characteristics of the PVD and HC subjects. The mean age for PVD (~28 years) was about 3 years older than the mean age for HC (~25 years). HC were more likely to endorse consuming diets other than American or Mediterranean. Body mass index did not differ between the groups. Less than half of participants were taking systemic hormonal contraceptives [PVD (~40%, N plasma = 23; N vaginal = 22), HC (29%, N = 14)]. Few women were using a local hormone [13 % PVD (N plasma = 8, N vaginal = N =7), 13% (HC N = 6)]. About half the sample report no hormone contraceptive use [~47%, PVD (N plasma = 29, N vaginal = 25), 60% HC (N = 29). Table 2 contains the clinical characteristics of the woman with PVD by sample type. There were no differences between the hormone subgroups in pain duration, total vulvar vestibule pain score on exam, total vaginal muscle tenderness, or symptom intensity in the past 24 hours. The estimated mean and 95% confidence intervals for the functional connectivity strength of each cortical and subcortical region of interest for women with PVD are depicted in the bar plots in Fig. 1 and 2 . All regions of interest showed significant connectivity strength (95% confidence intervals do not include zero). Pathway enrichment analysis of metabolomic data was utilized to identify the biochemical processes specifically altered in the PVD group. The results of this approach are summarized in Table 3 and show the top 15 sub-pathways with the highest enrichment score for the PVD to HC no hormone subgroup comparison. Importantly, sphingosines, sphingolipid synthesis, hexosylceramides (HCER) and sphingomyelins showed enrichment scores greater than 1. These sub-pathways are integral to sphingolipid biosynthesis and degradation, suggesting that they may be preferentially altered under conditions of PVD. The enrichment analysis performed for the entire dataset identified the same sub-pathways among the top 15 enriched processes (data not shown). Based on this analysis and the role of sphingolipid metabolism pathways in developing peripheral hyperalgesia and central sensitization, a detailed analysis of this pathway is presented below. In the vaginal fluid samples, PVD compared to HC had significantly (q < .05) higher levels of metabolites involved in sphingolipid synthesis (e.g., sphinganine, sphingadienine) and sphingosines (e.g., sphingosine, eicosanoylsphingosine (d20:1)*). As shown in Table 4 (All Subjects), in general, there was a decrease in vaginal levels of several sphingomyelins [e.g., sphingomyelin (d18:1/20:0, d16:1/22:0)*, behenoyl sphingomyelin (d18:1/22:0)*, tricosanoyl sphingomyelin (d18:1/23:0)*], ceramides [i.e., ceramide (d18:1/17:0, d17:1/18:0)*, ceramide (d18:1/20:0, d16:1/22:0, d20:1/18:0)*, ceramide (d18:2/24:1, d18:1/24:2)*] and hexosylceramides (HCER) [ e.g., glycosyl ceramide (d18:1/20:0,d16:1/22:0)*]. Fig 3 displays the box plots and raw data for all subjects for each of the altered vaginal sphingolipid metabolites (organized by subpathways shown in Table 4 ). In contrast to significant differences in vaginal metabolites, we did not see strong evidence for any significant group differences in plasma samples. Rather, we only observed higher levels (q < .02) of a single biochemical in PVD compared to HC, i.e., lactosyl-N-behenoyl-sphingosine (d18:1/22:0). As can be seen in Table 4 (Subgroup analysis), many of the differences observed in the vaginal fluid samples of the larger sample were maintained in the subgroup comparison of PVD to HC in women not using hormonal contraceptives. However, no significant differences were observed between PVD and HC using systemic hormones, or between PVD women taking systemic hormones versus no hormones. In plasma samples, for women not reporting hormone usage, PVD compared to HC only showed trends (q’s = .08) for higher levels of two biochemicals in the lactosyl ceramide pathways (i.e., lactosyl-N-behenoyl-sphingosine (d18:1/22:0), lactosyl-N-nervonoyl-sphingosine). As shown in Fig 4 , moderate effect size associations were observed between many of the PVD-specific alterations in vaginal concentrations in the sphingolipid pathway metabolites and increased severity of PVD symptoms ( Table 5 ). For example, PVD-specific increases in sphingolipid pathway metabolites correlated with increased vaginal muscle tenderness total scores. Further, increases in sphingosines showed moderate effect size correlations with increased vulvar vestibular pain. Finally, many of the alterations in sphingolipid metabolite concentrations observed in PVD compared to HC correlated with greater sexual dysfunction. Several of the altered metabolites in PVD also showed moderate associations with the connectivity of brain regions involved in pain processing/modulatory brain regions ( Table 6 ). In particular, PVD-specific increases in sphingosines and sphingolipid synthesis pathway metabolites were associated with decreased connectivity strength in the right amygdala and right hippocampus. Increases in sphingosine metabolite concentration observed in PVD versus HC also correlated with decreased connectivity in the bilateral thalamus, and the posterior insula, bilaterally. On the other hand, PVD-specific decreases observed in dihydroceramides and ceramides were associated with increased functional connectivity strength of the superior part of the right precentral sulci and the right caudate nucleus. We also observed decreases in sphingomyelin (d18:0/18:0, d19:0/17:0)* which were associated with decreased connectivity strength in the brainstem. Several symptom measures showed direct correlations with the connectivity of brain regions ( Table 7 ). For example, decreased connectivity strength in the right hippocampus and left posterior insula were associated with increased vulvar vestibular pain and muscle tenderness scores. Also, decreased connectivity in the right amygdala correlated with increased vulvar vestibular pain and greater sexual dysfunction. Furthermore, increased connectivity in the precentral cortex and paracentral lobule was associated with increased 24-hour symptom intensity.

Premenopausal women with PVD and healthy controls (HC), ages 18 to 50, were identified. A diagnosis of PVD was confirmed by clinical examination by a gynecologist or specialized nurse practitioner, both with recognized expertise and examination of women with vulvar pain. Participants were recruited via advertisement on social media, Craigslist, campus-wide emails via the Registrar’s office at UCLA, onsite recruiting by the study coordinator at the UCLA Division of Digestive Diseases general GI clinics and OB/GYN clinics, and the UCLA and other local college newspapers. All subjects provided written informed consent to participate and were compensated for participating in the study. The study was approved by the University of California, Los Angeles (UCLA) Institutional Review Board (IRB#13–001113) and was conducted in accordance with the institutional guidelines regulating human subject research. A total of 60 women with PVD and 49 HC were enrolled in the study based on the desire to detect moderate effect size differences (Cohen’s d~=.50), if they existed, in metabolite concentrations between PVD and HC. Inclusion criteria for women with PVD included: at least 6 months of vulvar vestibular pain, at least 4 out of 10 rating in pain severity during intercourse or other activities involving vestibular pressure (e.g., tampon use), and findings on cotton swab exam consistent with provoked vestibulodynia. Infections (such as candida, bacterial vaginosis or herpes simplex), estrogen deficiency or dermatological disorders were ruled out by history, visual inspection, vaginal pH and saline and potassium hydroxide slide prep. Speculum examination of the vagina and bimanual pelvic examination was performed to exclude other potential etiologies or pathology such as vaginal or pelvic masses or inflammation. We excluded patients who met criteria for generalized vulvodynia and those with only spontaneous but not provoked vestibulodynia. 10 All subjects were asked to abstain from intercourse for 7 days prior to the visit, and to avoid the use of intravaginal products, including douches, sprays, tampons, spermicides, gels, foams, and diaphragms. Women were excluded from the study if they were pregnant, breastfeeding, or postpartum less than 4 months. In addition, subjects were excluded if they have received systemic or vaginal antimicrobial or probiotic therapy within 1 month before the study visit. Inclusion/exclusionary criteria for healthy control subjects were the same as for those with PVD, with the exception of inclusion for pain with vaginal penetration and vestibular allodynia. The subjects’ menstrual history was obtained during phone screening so that the first visit could be scheduled around the follicular phase (or day 5–14 after starting first day of the last menstrual period). During Visit 1, all subjects completed study questionnaires and provided biological samples. Only women with PVD participated in a second visit, during which brain imaging was obtained. Time between Visit 1 and Visit 2 for these patients averaged 9 (SD = 9.1) days (median = 7 days). During the clinical assessment, PVD subjects were mapped for pain in the vulvar vestibule by touching the vestibule perpendicularly with the cotton end of the swab (enough to indent the mucosa to a depth of less than 1/3 of the cotton end) for 1 second at 5, 6, 7 (posterior vestibule), 10, 12 and 2 o’clock (peri-urethral). Participants were asked to rate the pain at each site from 0 to 10/10. Pain scores across all sites on the cotton swab test were summed for a total vulvar vestibular pain score [0–60, numeric rating scale (NRS)]. Internal muscle pain was assessed in PVD subjects with a single lubricated digit, applying approximately 2 kg of pressure for 2 seconds (The examiner’s finger pressure was calibrated before the exam with an algometer). The right and left levator ani muscles (in the vagina) and the perineal complex (at the vaginal entrance) were assessed. The participant was asked to rate the pain severity at each site from 0 to 10/10 (0; none −10; most severe pain imaginable. Scores were summed across all locations to compute a total vaginal muscle tenderness score (0–30, NRS). 2 The study physician or nurse practitioner recorded whether, in addition to provoked pain, pain might also arise spontaneously (unprovoked) and whether the onset of symptoms was primary or secondary (at or after first introital penetration, respectively). 10 Patients also reported the duration of their PVD symptoms. Female sexual functioning was assessed in women with PVD using the Female Sexual Functioning Index (FSFI). The FSFI is a multi-dimensional self-report instrument consisting of six subscales: desire, arousal, lubrication, orgasm, global satisfaction and pain. Items are summed to indicate a total “sexual function” score, where higher scores indicate greater sexual function, and where women who score below the clinical cut-off of 26 are considered at-risk for sexual dysfunction. 67 For all subjects, we obtained information on ethnicity, race, and subjective social status. 1 Subjective social status is 1-item assessment that captures a subject’s sense of social status and has been linked to psychological factors that may predispose individuals to better health trajectories. Participants are asked to place themselves on a drawing of a latter that is anchored at the top by those in society who are best off in terms of income, education, and occupation and at the bottom by those who are worst off (highest score = 9, lowest score = 1). Detailed data on current use of contraceptives or hormones were used to categorize hormone use as “no hormone” (i.e., condoms, abstinence), “local hormones” (e.g., levonorgestrel containing intrauterine device, estrogen or testosterone vaginal cream, or “systemic hormones” (e.g., oral contraceptive pills, vaginal contraceptive ring, transdermal contraceptive patch, etonogestrel sub-dermal contraceptive implant, depo medroxyprogesterone acetate injection). Diet type was assessed by asking subjects to endorse their usual diet. Choice included Standard American (high consumption of processed, frozen, and packaged foods, pasta and breads, and red meat; infrequent vegetable and fruit consumption), Modified American (high consumption of whole grains including some processed, frozen, and packaged foods; limited intake of red meat; moderate vegetable and fruit consumption), Mediterranean (high consumption of fruits, vegetables, beans, nuts, and seeds; olive oil as the key monounsaturated fat source; low to moderate intake of dairy products, fish, and poultry, infrequent red meat consumption), and other (all other diets that do not fit into the above categories, e.g. vegan, vegetarian, pescatarian, dairy-free). Plasma samples were obtained in 60 women with PVD and 49 HC women. From these women, vaginal swab samples were obtained from 51 of the 60 PVD women providing plasma samples and all 49 HC women. Additionally, 3 women with PVD only provided vaginal swab samples. Vaginal swab and plasma samples were collected during the mid to late follicular phase based on last menstrual period in women not on hormonal contraceptives or in days 5 to 14 of the active pill phase, considering day 1 as the first day of bleeding. When swabs arrive from the manufacturer, the study coordinator labeled each sterile tube containing the swabs with two cryo-freezer safe labels. One label was for patient study ID and date of sample, and the second label was for PRE and POST collection weight, in grams. The coordinator weighed each dry swab and container and provided PRE gram weight on the swab label. At Visit 1, the physician/nurse practitioner collected the vaginal sample after gently retracting the outer labia with gloved fingers and inserting a sterile swab approximately 1 inch into the vaginal vault. Swab was rotated 360 degrees, removed, and inserted into the sterile tube (without liquid medium). This procedure was repeated for the remaining 2 swabs. The swabs were immediately weighed post collection (using the same gram scale), and the POST weight was documented on the cryo-freezer label. The samples were immediately placed into a small lab cooler filled with ice and brought to the lab where they were stored in a −80 degree Celsius freezer within 20 minutes of collection. Samples were shipped on dry ice with a collection log (vaginal sample log) to Metabolon for further processing and analysis on their global metabolomics and bioinformatics platform (Metabolon, 617 Davis Drive, Durham, NC). Using standard venipuncture procedure, blood was collected in EDTA tubes from individuals after an overnight fast. Plasma was centrifuged, aliquoted to 6 (1 mL) sterile polypropylene cryotubes, and immediately stored at −80°C until shipped to Metabolon. Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS) was used to perform discovery-based, untargeted global metabolomic profiling to characterize biochemical pathways in vaginal fluid and plasma samples that were altered in PVD compared to HC (Metabolon, 617 Davis Drive, Durham, NC). Specific details on the Metabolomics platform can be found in Supplemental methods I . Datasets provided by Metabolon identified a total of 824 compounds of known identity (named biochemicals) detected in plasma, and 952 in vaginal swabs. Before the brain imaging scan during Visit 2, PVD patients rated the intensity of their vulvodynia symptoms in the past 24 hours on a verbal descriptor visual analog scale ranging from 0 to 20 (No Sensation to Extremely Intense). Using a 3.0T MRI scanner (Siemens Trio; Siemens, Erlangen, Germany), high resolution structural brain images and a ten-minute 6 seconds eyes-closed resting-state scan obtained in women with PVD. Using established computational pipelines, 30 , 31 , 57 we applied graph theory to this data to compute the connectivity strength or global influence of each of the pain processing/modulatory brain regions of interest (ROIs) on brain functioning. As defined by the Destrieux (cortical) 19 and Harvard-Oxford Subcortical Atlases, 18 a priori specified individual ROIs included the sensorimotor cortex [precentral gyrus, inferior part of the precentral sulcus, superior part of the precentral sulcus, paracentral lobule and sulcus, subcentral sulci and gyrus (central operculum), central sulcus, postcentral sulcus, postcentral gyrus] including the primary the posterior insula (long insular gyrus and central sulcus of the insula, and the inferior segment of the circular sulcus of the insula) and subcortical regions [amygdala, hippocampus, basal ganglia (nucleus accumbens, caudate nucleus, globus pallidus, putamen), thalamus and brainstem]. Strength represents the weighted sum of connections (i.e., Fisher Z transformed correlations >.30) for a given ROI. High compared to low values for strength indicate greater centrality or influence on the global state of brain functioning. For details on image acquisition, quality control and preprocessing, and computation of connectivity strength, see Supplemental Methods II . Within the framework of our preprocessing and computational workflows, this subject-specific network metric has been shown to have good and reproducible reliability. 65 , 85 Pathway enrichment analysis was utilized to identify pathways that differentiated study groups. For each individual pair-wise comparison, this analysis displays the number of statistically significantly different compounds relative to all detected compounds in a subpathway, compared to the total number of statistically significantly different compounds relative to all detected compounds in the study, according to the formula: Enrichment Value = (k/m)/((n-k)/(N-m)), where m = number of metabolites in the pathway, k = number of significant metabolites in the pathway, n = total number of significant metabolites, and N = total number of metabolites. A pathway enrichment value greater than one indicates that a given pathway contains more significantly changed compounds relative to the number of changes observed in the entire set of metabolites measured in samples. Pathways represented by ≥4 metabolites were analyzed to focus on biochemical processes that showed adequate coverage in the global metabolomic profiling. Based on the enrichment analysis, we reported the group differences observed in sphingolipid metabolism pathways, specifically 39 validated compounds identified in vaginal fluid and 63 identified in plasma. Following log transformation and imputation of missing values, if any, with the minimum observed value for each compound, Welch’s two-sample t-test was used to identify biochemicals that differed significantly between healthy women and women with PVD. Conservative error control for false positives (Type I errors) for discovery-based analysis across all metabolites was realized by calculating the false discovery rate-adjusted p-value (q), based on the total number of compounds identified during global metabolomic profiling in each sample type (952 in vaginal swabs, and 824 in plasma samples). Due to differences in use of hormone contraceptives in the sample, we performed an additional sensitivity analysis to explore the influence of taking hormonal contraceptives on observed differences in the sphingolipid metabolism pathways. Specifically, we categorized women by hormone usage (no hormones, systemic hormones, local hormones) and performed a contrast analysis within the framework of the general linear model 36 , 68 , 74 testing the following contrasts of interest: 1) PVD no hormones – HC no hormones, 2) PVD systemic hormones – HC systemic hormones and 3) PVD systemic hormones – PVD no hormones. We present the significant results from the PVD-HC analysis including fold change (i.e., ratio of the means), uncorrected p values, and FDR corrected p values (q) in Table 4 (see All Subjects ). FDR corrected significance levels for the sensitivity analyses are also shown in Table 4 (see Subgroup Analysis). A full list of the biochemical compounds detected in the sphingolipid metabolism pathways in vaginal swabs and plasma is provided in Supplemental Tables 1 and 2 , respectively. These tables also contain the fold change estimates and uncorrected p values for the subgroup analyses, as well as a comparison between HC systemic hormones – HC no hormones. Finally, where available, the supplemental tables contain links to the Kyoto Encyclopedia of Genes and Genomes (KEGG), Human Metabolome Database (HMDB), and PubChem databases containing detailed information about the identified metabolites. Correlational analysis was performed to provide insights into the functional mechanisms linked to the identified alterations in sphingolipid metabolism. In women with PVD, Spearman correlations were used to estimate the association between untransformed concentrations of altered metabolites with symptom measures (i.e., pain intensity in the last 24 hours, total vulvar vestibular pain and vaginal muscle tenderness scores, sexual functioning) as well as the global functional connectivity of pain processing/modulatory brain regions. We used Cytoscape v. 3.8.0 24 and implemented multidimensional scaling using a compound spring embedded algorithm 20 to visualize the metabolite-brain, metabolite-symptom, metabolite-brain and brain-symptom correlations as a tripartite association network at P < .05. 43 , 56 , 58 We considered the exploratory cross-sectional correlation analyses important for generating future hypotheses and reported uncorrected p values. Although we reported correlations as significant at P < .05, emphasis should be placed on interpretation of the effect sizes. As a rule of thumb, Cohen (1988) suggested the following guidelines for interpreting effect size, r: small, r = .10 (explain 1% of the variance), medium, r = .30 (explain 9% of the variance), and large, r = .50 (explain 25% of the variance). We used independent t-tests and 2-sided Fisher’s exact tests to compare groups on continuous and categorical descriptive variables.

In conclusion, this is the first study to use unbiased, untargeted global metabolomics profiling to identify novel biomarkers in PVD. Although the results from this study are novel and compelling, the cross-sectional and exploratory nature of the study design necessitates validation in independent data sets with thorough patient phenotyping. Women with PVD had significant modulations in sphingolipid signaling pathway metabolites previously linked to peripheral and central sensitization, neuropathic pain and inflammatory pain conditions. These modified metabolites were associated with increased vulvar vestibule pain on exam, vaginal muscle tenderness and sexual dysfunction. Additionally, these modified metabolites were found to be in parallel with modified global connectivity of brain regions critical for pain processing. Ultimately, modified levels of metabolites comprising sphingolipid signaling pathways may be an important molecular marker for understanding the pathophysiology of symptoms in PVD and could lead to new targets for therapeutic intervention.

Untargeted and unbiased metabolomics profiling revealed that women with PVD, compared to HC, demonstrated differences primarily in vaginal (but not plasma) concentrations of metabolites from sphingolipid signaling pathways, suggesting localized rather than systemic effects in vagina and vulvar vestibule. Consistent with the clinical importance of these findings, the observed alterations in sphingolipid metabolism were associated with increased vulvar pain, muscle tenderness, and sexual dysfunction as well as primarily decreased functional connectivity strength in pain processing/modulatory brain regions. We observed that while the use of systemic hormonal contraceptives resulted in altered plasma steroid metabolites in PVD and HC, these associations were unlikely to explain the subsequent directionality nor vaginal specificity and link to pain observed in PVD subjects. Sphingolipid metabolites, including ceramides and sphingosines, have been associated with neuropathic pain and are thought to play a critical role in several inflammatory diseases, attributed to their anti– or pro–inflammatory and pain modulatory effects. 12 , 26 , 27 , 54 , 59 , 69 , 74 Ceramide is generated from three pathways including de novo biosynthesis (involving sphinganine), hydrolysis of sphingomyelins, and a salvage pathway involving degradation of complex sphingolipids (including sphingomyelins) from ceramides. 7 Once produced, ceramide is converted to sphingosine which is subsequently converted to sphingosine 1-phosphate (S1P). This ceramide to S1P pathway has been implicated in the development of peripheral sensitization and hyperalgesia. 38 , 54 , 69 , 74 , 82 , 31 In this study, there were significantly higher levels of sphinganine but lower levels of sphingomyelins in vaginal swabs in PVD, suggesting alterations in ceramide synthesis. Downstream we observed decreased vaginal fluid levels of ceramide species and an increase of sphingosine. Although S1P, a major bioactive lipid species, was not detected in this study, we observed increases in sphingosine, the precursor to S1P. The alterations in the ceramide to S1P pathway may reflect alterations in the balance of pro and anti-inflammatory cytokines levels in the vaginal vestibule and/or in circulating blood of women with PVD. 4 , 10 , 13 , 22 , 23 , 25 , 71 , 77 , 86 Consistent with an involvement in nociception, animal models of allodynia have demonstrated an association between sphingolipid signal metabolites, increased sensitivity of dorsal root ganglia and overexpression of inflammatory cytokines with peripheral and central pain sensitization 21 , 54 , 69 Together, these alterations suggest inflammatory processes which may contribute to both peripheral and mechanical hypersensitivity 59 as well as indicate altered sphingosine kinase activity and hence production of the signaling molecule S1P in PVD. Metabolomic profiling of biological tissue and fluid has emerged as an important for examining biological processes in chronic diseases including chronic pain [for a review see 52 , 75 , 76 , 84 ]. In particular, evidence of altered sphingolipid metabolism has been reported in several chronic pain conditions often found to be comorbid with PVD. 17 , 50 In fibromyalgia, one study reported an upregulation of plasma levels SP1, 51 and another demonstrated increased plasma sphingosines which correlated with pain and fatigue. 47 Compared to HC, endometriosis patients showed increased concentrations of sphingolipids in both follicular fluid and peritoneal and endometrial tissue samples. 1 , 46 , 79 , 80 Similar to PVD, alterations in pro-inflammatory mediators 8 , 15 , 45 , 53 , 61 , 72 have been reported in these chronic pain conditions supporting the hypothesis that observed alterations in sphingolipid metabolism may reflect ongoing inflammatory processes and a shared mechanism. The use of systemic hormone contraceptives appeared to reduce PVD - HC differences observed in vaginal fluid levels of sphingolipids. Use of hormonal contraceptives did not change the lack of group differences observed between PVD and HCs in circulating levels of sphingolipid metabolites. However, within PVD, use of hormonal contraceptives appeared to lower plasma concentrations of lactosyl ceramides and increase levels of dihydrosphingomyelins and sphingomyelins. This finding is consistent with research demonstrating that steroid hormones are involved in the regulation of sphingolipid metabolism. 49 Symptom-associated alterations in gray matter density, regional activity during evoked pain studies, and intrinsic resting-state connectivity of primary and secondary sensory and motor cortices, posterior insula, hippocampus, basal ganglia and thalamus have been reported in women with PVD compared to HC. 5 , 32 , 35 , 70 We have previously shown that women with PVD showed microstructural alterations in cortico-thalamicbasal ganglia white matter tracts associated with ascending nociceptive processing (i.e., primary sensory cortex, thalamus, basal ganglia). 33 The connectivity strength of a brain region is a topological measure reflecting its global influence on brain functioning. In the present study, PVD-specific alterations in metabolites comprising sphingosines, sphingolipid synthesis, HCER and dihydrosphingomyelins sub pathways showed moderate linear associations with decreased connectivity strength for pain processing (thalamus, posterior insula, nucleus accumbens of the basal ganglia, brainstem) and modulatory (amygdala, hippocampus) regions. In turn, the reduced connectivity of many of the regions (posterior insula, amygdala, hippocampus) also showed direct correlation with increased symptomatology. PVD-specific decreases observed in one sphingomyelin and a few ceramide species were associated with increased connectivity strength of the caudate nucleus, hippocampus, and the precentral sulci. The amygdala and hippocampus connections are key features the emotional arousal network. The observed associations with the connectivity strength of these regions may reflect the neurobiological substrates of pain-related anxiety and fear responses secondary to central sensitization that increase the facilitatory descending modulation. 3 , 29 , 42 The observed correlations between alterations in vaginal concentration of sphingolipid metabolites and the functional connectivity strength of pain processing/modulatory regions support the hypotheses that sphingolipid species enhance ascending transmission and modulation of pain signals. The current study has identified sphingolipid metabolites associated with vestibular pain but was not designed to determine the causal nature of these associations nor their directionality. These biochemical alterations could reflect biological markers of persistent inflammatory processes and modulation of peripheral nerve fibers 34 of the vagina and vulvar vestibule of PVD patients leading to long-lasting allodynia and hyperalgesia. The hypothesized nociceptive signaling to the brain may result in structural remodeling of pain processing regions, and alter the balance of facilitatory and inhibitory descending pain modulation contributing to the production and maintenance of vulvar pain. 39 This interpretation is consistent with the moderate to large effect size association observed between vaginal concentrations of sphingolipid metabolites with pain and muscle tenderness, symptom intensity and sexual dysfunction as well as functional connectivity strength of brain regions associated with pain processing and pain modulation. Together, our findings suggest that sphingolipids may serve as biomarkers for patient stratification in PVD. Emerging evidence identified the S1P receptor 1 subtype (S1PR1), one of the five known G protein-coupled S1P receptor subtypes, as being critical in transducing the effects of S1P in pain. 12 , 21 Noteworthy, over the last decade, several orally bioavailable and CNS penetrant, S1PR1 functional and competitive antagonists have been developed. 6 , 14 , 28 , 78 Two S1PR1 functional antagonists are now FDA-approved for the treatment of multiple sclerosis: the pro-drug FTY720 (fingolimod; Gilenya, Novartis) approved in 2010 11 and ozanimod (RPC1063, Zeposia, Celgene) approved in 2020. 44 , 73 We enrolled women with PVD, the most common form of vulvodynia. Therefore, inferences should not be made to women with generalized vulvodynia. The presence of comorbid chronic overlapping pain conditions should be addressed in future studies. Due to the heterogeneity of patient characteristics, it will be important to have larger sample sizes for well-powered subgroup analyses. Larger samples are also important to ensure unbiased parameter estimates and avoiding type II error. Future studies using targeted sphingolipid metabolomics profiling (i.e., absolute quantification of a pre-defined set of metabolites) and S1P receptor expression studies in vaginal tissue in PVD will be critical to further validate these findings. Research may also benefit from examining the impact of hormone-based therapy which specifically and significantly affects ceramide pathways. Ultimately, longitudinal multi-omics studies comprising concurrent measurement of pro-and anti-inflammatory markers as well as micro-RNA expression associated with inflammation (including gonadotropin-releasing hormone signaling pathways, steroid hormone levels, and sphingolipid metabolite concentrations) are needed to delineate the relationship between these distinct but interacting mechanisms as they relate to peripheral and central sensitization, brain alterations and symptoms in PVD. Finally, metabolomics for chronic overlapping pain syndromes is still in its infancy, 15 , 16 as a considerable amount of work is required to validate specific metabolites or pathways as candidate biomarkers.