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Familiality analysis of provoked vestibulodynia treated by vestibulectomy supports genetic predisposition

Published:November 25, 2015DOI:https://doi.org/10.1016/j.ajog.2015.11.019

      Background

      Provoked vestibulodynia is a poorly understood disease that affects 8–15% of women in their lifetime. There is significant inflammation and nerve growth in vestibular biopsies from affected women treated by vestibulectomy compared with matched female population controls without vestibulodynia. The triggers leading to this neurogenic inflammation are unknown, but they are likely multifactorial.

      Objective

      Our objective was to determine whether vestibulodynia is more common in close and distantly related female relatives of women diagnosed with the disease and those specifically treated by vestibulectomy. Excess familial clustering would support a potential genetic predisposition for vestibulodynia and warrant further studies to isolate risk alleles.

      Study Design

      Using population-based genealogy linked to University of Utah Hospital CPT coded data, we estimated the relative risk of vestibulectomy in female relatives of affected women. We also compared the average pairwise relatedness of cases to the expected relatedness of the population and identified high-disease-burden pedigrees.

      Results

      A total of 183 potential vestibulectomy probands were identified using CPT codes. The relative risk of vestibulectomy was elevated in first-degree (20 [6.6–47], P < .00001), second-degree (4.5 [0.5–16], P = .07), and third-degree female relatives (3.4 [1.2–8.8], P = .03). Seventy of these 183 CPT-based probands had available clinical history to confirm a diagnosis of moderate to severe vestibulodynia. Notably, this smaller group of confirmed probands (n = 70) revealed a similar familiality in first-degree (54 [17.5–126], P < .00001), second-degree (19.7 [2.4–71], P = .005), and third-degree relatives (12 [3.3–31], P = .0004), despite less statistical power for analysis. Overall, the average pairwise relatedness of affected women was significantly higher than expected (P < .001) and a number of high-disease-burden Utah families were identified.

      Conclusion

      Our data suggest that vestibulodynia treated by vestibulectomy has a genetic predisposition. Future studies will identify candidate genes by linkage analysis in affected families and sequencing of distantly related probands.

      Key words

      Provoked vestibulodynia (PVD) occurs in approximately 8–15% of women during their lifetime and is the leading cause of dyspareunia (painful intercourse).
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      have shown that vestibulectomy tissue specimens have increased chronic inflammation, mast cell recruitment, and conspicuous submucosal nerve growth compared with vestibular biopsies from unaffected women. There are also more CD4-positive T cells, which may provide insights into the triggers of this neurogenic inflammation;
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      in some cases, a plasma cell–mediated process may be involved.
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      Activation of vestibule-associated lymphoid tissue in localized provoked vulvodynia.
      Unfortunately, therapies aimed at treating the chronic inflammation, or providing neuromodulation, are often ineffective.
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      Vulvar vestibulitis: prevalence and historic features in a general gynecologic practice population.
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      Critique of new gynecologic surgical procedures: surgery for the vulvar vestibulitis.
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      Our prior studies have largely been based on women with chronic disease recalcitrant to alternative therapies, prompting vestibulectomy for relief.
      • Goetsch M.F.
      Vulvar vestibulitis: prevalence and historic features in a general gynecologic practice population.
      • Goetsch M.F.
      • Morgan T.K.
      • Korcheva V.B.
      • Peters D.
      • Li H.
      • Leclair C.M.
      Histologic and receptor analysis of primary and secondary vestibulodynia and controls: a prospective study.
      • Leclair C.
      • Goetsch M.
      • Korcheva V.
      • Anderson R.
      • Peters D.
      • Morgan T.K.
      Differences in primary compared with secondary vestibulodynia by immunohistochemistry.
      • Leclair C.
      • Goetsch M.
      • Li H.
      • Morgan T.K.
      Histopathologic characteristics of menopausal vestibulodynia.
      • Leclair C.
      • Leeborg N.
      • Jacobson-Dunlop E.
      • Goetsch M.
      • Morgan T.K.
      CD4-positive T-cell recruitment in primary provoked localized vulvodynia: potential insights into disease triggers.
      The problem is that PVD is usually diagnosed after years of symptoms and the tissue is already remodeled.
      • Leclair C.
      • Goetsch M.
      • Korcheva V.
      • Anderson R.
      • Peters D.
      • Morgan T.K.
      Differences in primary compared with secondary vestibulodynia by immunohistochemistry.
      • Leclair C.
      • Goetsch M.
      • Li H.
      • Morgan T.K.
      Histopathologic characteristics of menopausal vestibulodynia.
      New predictive markers are needed to identify women at risk for PVD and provide early interventions to prevent the chronic neurogenic inflammation and tissue remodeling in these select patients.
      We hypothesize that PVD may have a genetic predisposition. Many chronic diseases have a genetic predisposition with risk alleles that may serve as both diagnostic aides and insights into pathogenic mechanisms. PVD is not currently considered a genetic disease, but there are reports of a potential association with seasonal allergies,
      • Harlow B.
      • He W.
      • Nguyen R.
      Allergic reactions and risk of vestibulodynia.
      proinflammatory cytokines (eg, mannose-binding lectin, IL-6, IL-1β
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      Interleukin-1 receptor antagonist gene polymorphism in women with vulvar vestibulitis.
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      Association between vulvar vestibulitis syndrome, defective induction of tumor necrosis factor-a, and carriage of the mannose-binding lectin codon 54 gene polymorphism.
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      Impact of genetic variation in interleukin-1 receptor antagonist and melanocortin-1 receptor genes on vulvar vestibulitis syndrome.
      ), and aberrant hormone regulation.
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      • et al.
      Polymorphisms of the androgen receptor gene and hormonal contraceptive induced provoked vestibulodynia.
      Genetic testing in women with PVD is only in the early stages of discovery, but other potential candidate genes may include common mediators of neurogenic inflammation.
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      Autoimmune diseases: insights from genome-wide association studies.
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      A comprehensive evaluation of IL-4 variants in ethnically diverse populations: association of total serum IgE levels and asthma in white subjects.
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      • Ogawa Y.
      Molecular analysis of adrenergic receptor genes and interleukin-4/interleukin-4 receptor genes in patients with interstitial cystitis.
      Population-based analysis of the familial clustering of a disorder is an informative approach to test for evidence of a genetic contribution to common chronic diseases like PVD.
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      The familiality of pelvic organ prolapse in the Utah Population Database.
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      • et al.
      A family-based paradigm to identify candidate chromosomal regions for isolated congenital diaphragmatic hernia.
      Once evidence for a genetic contribution to disease risk is determined, studies of affected individuals in high-risk pedigrees can identify risk alleles.
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      • et al.
      A family-based paradigm to identify candidate chromosomal regions for isolated congenital diaphragmatic hernia.
      For example, a number of familial studies have determined that interstitial cystitis,
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      which is commonly associated with PVD,
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      • Chiesa A.
      • Spinillo A.
      Interstitial cystitis is associated with vulvodynia and sexual dysfunction-a case control study.
      has a genetic predisposition. This is important, because both diseases present with pain and neurogenic inflammation.
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      Mast cells and nerve fibers in interstitial cystitis (IC): an algorithm for histologic diagnosis via quantitative image analysis and morphometry (QIAM).
      Perhaps these 2 common diseases share genetic risk factors and pathogenic triggers? Indeed, risk allele studies of interstitial cystitis have revealed promising candidate genes like IL-4 and IL-10 variants
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      involved in T-cell regulation that we suspect may also play a role in PVD.
      • Leclair C.
      • Leeborg N.
      • Jacobson-Dunlop E.
      • Goetsch M.
      • Morgan T.K.
      CD4-positive T-cell recruitment in primary provoked localized vulvodynia: potential insights into disease triggers.
      To test whether there is a significant heritable contribution to PVD, we employed the Utah Population Database (UPDB), which links genealogy information for millions of individuals to medical diagnostic data (ICD-9 and CPT codes). This approach has been validated and revealed genetic contributions to a number of common diseases.
      • Norton P.
      • Allen-Brady K.
      • Cannon-Albright L.
      The familiality of pelvic organ prolapse in the Utah Population Database.
      • Arrington C.
      • Bleyl S.
      • Matsunami N.
      • et al.
      A family-based paradigm to identify candidate chromosomal regions for isolated congenital diaphragmatic hernia.
      • Gibson S.B.
      • Figueroa K.P.
      • Bromberg M.B.
      • Pulst S.M.
      • Cannon-Albright L.
      Familial clustering of ALS in a population-based resource.

      Materials and Methods

      The Utah Population Database

      The UPDB (www.healthcare.utah.edu/huntsmancancerinstitute/research/updb/) computerized genealogic data for the Utah pioneers and their descendants (from the mid-1800s) has been linked to diagnoses and procedural outcomes from the University of Utah Health Sciences Center (UUHSC) for patients treated from 1994 to present. The genealogy includes over 8 million unique individual records; 1.3 million of these individuals have at least 3 generations of genealogy data available meeting inclusion criteria. Inclusion criteria for familial clustering analyses required all subjects (cases and matched population controls) to have genealogy information available for at least 12 of 14 of their immediate ancestors (ie, parents, all grandparents, and at least 6 of 8 great-grandparents). These inclusion criteria have been determined to provide sufficient sensitivity to reasonably detect affected first-, second-, and third-degree relatives.
      • Norton P.
      • Allen-Brady K.
      • Cannon-Albright L.
      The familiality of pelvic organ prolapse in the Utah Population Database.
      • Arrington C.
      • Bleyl S.
      • Matsunami N.
      • et al.
      A family-based paradigm to identify candidate chromosomal regions for isolated congenital diaphragmatic hernia.
      • Gibson S.B.
      • Figueroa K.P.
      • Bromberg M.B.
      • Pulst S.M.
      • Cannon-Albright L.
      Familial clustering of ALS in a population-based resource.
      All analyses used coded data (nonidentifiable).

      Identification of PVD subjects

      The study was approved by the Oregon Health & Science University (IRB #011064) and the University of Utah (IRB #068774 and #53474) Institutional Review Boards. It was also approved by the Utah Resource for Genetic and Epidemiological Research, which oversees the UPDB. We used 2 approaches to identify cohorts of women with dyspareunia related to vestibulodynia. First, we employed a broad search using all potential CPT codes employed for vestibulectomy: 56620 (n = 150), 56625 (n = 0), 56630 (n = 14), 56631 (n = 3), 56632 (n = 8), 56633 (n = 1), 56634 (n = 3), 56635 (n = 0), 56637 (n = 3), and 56640 (n = 1), for a total of 183 potentially affected vestibulodynia cases diagnosed at the University of Utah and treated by vestibulectomy. Second, we had a cohort of 216 cases of vestibulodynia treated by vestibulectomy by 1 surgeon (H.S.) for cross-referencing with the UPDB. Seventy (70/216) of these confirmed probands linked to the UPBD had adequate ancestry to be included in a separate analysis. These clinically confirmed vestibulectomy cases were all diagnosed using Friedrich criteria
      • Friedrich E.
      Vulvar vestibulitis syndrome.
      and they had no other identifiable reason for dyspareunia (eg, vulvar dermatosis, infectious vaginitis).
      • Stockdale C.
      • Lawson H.W.
      2013 vulvodynia guideline update.
      All of these confirmed cases had reported severe or moderate dyspareunia for at least 6 months; they were all refractory to conservative management (eg, topical anesthetics, corticosteroids, physical therapy, and neuromodulators); and they all had vestibulectomies.
      • Goetsch M.F.
      Simplified surgical revision of the vulvar vestibule for vulvar vestibulitis.

      Population controls and CPT code–based vestibulectomy rates

      Female population controls were randomly selected from UUHSC patients that were matched to cases based on sex, birthplace (urban/rural based on Utah birth county residence), and birth year (5-year birth cohorts), similar to our other previous publications.
      • Norton P.
      • Allen-Brady K.
      • Cannon-Albright L.
      The familiality of pelvic organ prolapse in the Utah Population Database.
      • Allen-Brady K.
      • Norton P.A.
      • Cannon-Albright L.
      Risk of associated conditions in relatives of subjects with interstitial cystitis.
      Population-based rates of vestibulectomy were estimated by using all UUHSC data linked to the UPDB. All 1.3 million UPDB individuals with at least 12 of their 14 immediate ancestors were assigned to 1 of 205 cohort “bins” based on cohort characteristics. UUHSC population cohort-specific rates of vestibulectomy were estimated by dividing the number of vestibulectomy cases identified by CPT code at UUHSC in each cohort by the total number of UUHSC female subjects in the cohort.

      Relative risk

      We estimated the relative risk of vestibulectomy among relatives of cases as the ratio of the number of observed cases to the number of expected cases in the population. The observed number of affected relatives of probands treated at UUHSC with CPT code-based vestibulectomy was counted among the specific degree of relatives of interest (eg, first-degree). The expected number of cases was calculated by multiplying the number of relatives of interest in each cohort (eg, first-degree) who were patients at UUHSC times the estimated rate of vestibulectomy for each cohort, and then summing across all of the cohorts as described.
      • Norton P.
      • Allen-Brady K.
      • Cannon-Albright L.
      The familiality of pelvic organ prolapse in the Utah Population Database.
      • Gibson S.B.
      • Figueroa K.P.
      • Bromberg M.B.
      • Pulst S.M.
      • Cannon-Albright L.
      Familial clustering of ALS in a population-based resource.
      The relative risk is assumed to follow a Poisson distribution, with the mean value equal to the expected number of cases (ie, null hypothesis has a relative risk = 1.0). Significance was determined by Fisher exact test.

      Genealogical Index of Familiality statistic

      The Genealogical Index of Familiality (GIF) statistic uses the Malécot coefficient of kinship to measure the probability that a pair of individuals share an identical copy of a chromosome region by descent from a common ancestor in the UPDB.
      • Norton P.
      • Allen-Brady K.
      • Cannon-Albright L.
      The familiality of pelvic organ prolapse in the Utah Population Database.
      GIF analysis tests the hypothesis that affected cases may be more related to each other than expected compared with the relatedness of matched sets of similar women in the population. In turn, we compared the average pairwise relatedness of CPT code-based vestibulectomy cases with the mean average pairwise relatedness of 1000 sets of randomly selected but age- and region-matched female controls. The distribution of the GIF statistic for the 1000 sets of population controls represented the expected pairwise relatedness of these women in the Utah population. The GIF statistic for the 183 vestibulectomy cases represented their relatedness to each other. Since the GIF statistic may also reflect nongenetic shared influences such as environment, which would be most evident in close relationships, we also computed the distant GIF statistic (dGIF), which excludes first- and second-degree relatives and tests for a significant excess of distant relationships to reduce the effects of potential ascertainment bias.

      Comorbidity analysis

      We also tested for a number of potentially comorbid conditions in cases compared with matched controls using the same relative risk method. The conditions tested included interstitial cystitis,
      • Cardella B.
      • Porru D.
      • Nappi R.
      • Dacco M.
      • Chiesa A.
      • Spinillo A.
      Interstitial cystitis is associated with vulvodynia and sexual dysfunction-a case control study.
      seasonal allergies,
      • Harlow B.
      • He W.
      • Nguyen R.
      Allergic reactions and risk of vestibulodynia.
      and T cell-mediated diseases.
      • Leclair C.
      • Leeborg N.
      • Jacobson-Dunlop E.
      • Goetsch M.
      • Morgan T.K.
      CD4-positive T-cell recruitment in primary provoked localized vulvodynia: potential insights into disease triggers.
      • Lettre G.
      • Rioux J.D.
      Autoimmune diseases: insights from genome-wide association studies.
      • Sugaya K.
      • Nishijima S.
      • Yamada T.
      • Miyazato M.
      • Hatano T.
      • Ogawa Y.
      Molecular analysis of adrenergic receptor genes and interleukin-4/interleukin-4 receptor genes in patients with interstitial cystitis.
      In addition, since our recent work suggested a relationship between interstitial cystitis, fibromyalgia, and constipation,
      • Allen-Brady K.
      • Norton P.A.
      • Cannon-Albright L.
      Risk of associated conditions in relatives of subjects with interstitial cystitis.
      we added these conditions to the risk analysis. We used ICD-9 codes to identify these comorbidities in the UPDB: interstitial cystitis (ICD-9 595.1), asthma/airway obstruction (ICD-9 493.*, 496.*), lupus erythematosus (ICD-9 695.4), myalgia and myositis unspecified (ICD-9 729.1), and constipation (ICD-9 564.0). Because there is no ICD-9 code for fibromyalgia, patients with this diagnosis were identified using codes for myalgia and myositis.

      High-risk pedigrees

      High-risk vestibulectomy pedigrees were determined by identifying founders with an excess observed number of affected descendants compared with the expected number of affected descendants in the UUHSC population. The expected number of affected descendants was calculated by counting all descendants who have UUHSC data by cohort, multiplying the number of cases in each cohort times the cohort-specific rate for vestibulectomy, and summing over all cohorts. Pedigrees with significantly more affected women than expected (P < .05) are considered high risk.

      Results

      CPT-based vestibulectomy cohort

      We identified 183 probands in the UPDB using strict CPT codes most commonly used for vestibulectomies at UUHSC between 1994 and 2014 and who met our strict geneaology requirements. Relative risk estimates for vestibulectomy in the first-, second-, and third-degree relatives of the 183 probands are reported in Table 1. It appears that women recalcitrant to conservative therapy may be a special population with a strong genetic contribution to disease risk. We observed significant excess risk for vestibulectomy among first-, second-, and third-degree relatives. Increased relative risk was supported by GIF analysis (Figure 1), which showed a significant excess of relationships among affected cases (case GIF =11.1, control GIF = 4.0, P < .001) compared to expected, even after removing first- and second-degree relatives (case dGIF = 5.8, control dGIF = 3.6, P = .001).
      Table 1Relative risk estimates for vestibulectomy in first-, second-, and third-degree relatives of 183 probands based on CPT codes
      Relationship to probandNumber of relativesObserved no. vestibulectomiesExpected no. vestibulectomiesRelative risk [95% CI]; P value
      First-degree49850.25
      Calculated expected frequencies are much less than 10%, because not all women diagnosed with vestibulodynia are treated by vestibulectomy, and fewer women are linked to the UPDB and meet strict ancestry requirements within the 1994–2015 search window. These caveats are assumed to affect both the case and control populations equally.
      20 [6.6–47]; <.0001
      Second-degree955<5-4.5 [0.5–16]; .07
      Third-degree2194<5-3.4 [1.2–8.8]; .03
      CPT codes for vestibulectomy were used to identify probands diagnosed at the University of Utah Health Sciences Center and linked to the Utah Population Database (UPDB). Significance was determined by Fisher exact test to measure whether there were more observed cases in relatives of the 183 probands compared with the expected number (no.) in matched population controls.
      Note: UPDB regulations require “<5” to be used rather than precise numbers to protect patient privacy. Similarly, the paired expected frequencies are not shown.
      Morgan et al. Provoked vestibulodynia is familial. Am J Obstet Gynecol 2016.
      a Calculated expected frequencies are much less than 10%, because not all women diagnosed with vestibulodynia are treated by vestibulectomy, and fewer women are linked to the UPDB and meet strict ancestry requirements within the 1994–2015 search window. These caveats are assumed to affect both the case and control populations equally.
      Figure thumbnail gr1
      Figure 1Average pairwise relatedness of vestibulectomy cases compared with 1000 sets of matched Utah hospital population female controls
      Vestibulectomy was defined using CPT specific codes linked to the Utah Population Database. The distribution of the contribution to the GIF statistic (y-axis) is shown by the genetic distance between the pair of relatives (x-axis). Genetic distance of “1” represents parent/offspring pairs, “2” represents primarily siblings, “4” represents primarily first cousins, and so forth. The large dips that occur at the odd values of genetic distance (eg, “1,” “3,” and “5”) are the result of the narrow window of view we have for data from 1994 to present and the disparity of data available for individuals in different generations, rather than the same generation. Since nongenetic environmental effects are more likely in first- or second-degree relatives, P values are also calculated for dGIF (>2 genetic distance).
      dGIF, distant Geneaological Index of Familiality; GIF, Geneaological Index of Familiality.
      Morgan et al. Provoked vestibulodynia is familial. Am J Obstet Gynecol 2016.
      Using these CPT codes, we also identified a number of high-disease-burden Utah families with significant risk of vestibulectomy compared with the general Utah population. These families all had at least 3 generations of documented ancestry and all of the affected women in these kindreds were treated at the University of Utah hospital. An example of 1 of these high-risk pedigrees is displayed in Figure 2.
      Figure thumbnail gr2
      Figure 2Example of a high-risk vestibulectomy pedigree
      Male subjects are represented as squares, female subjects as circles. Female subjects shaded in black have been treated with vestibulectomy. There are 5 affected female subjects in this extended pedigree, compared with the less than 1 case (0.53) that would be expected according to Utah Population Database (UPDB)-linked University of Utah Hospital population rates in a pedigree with a similar number of descendants (P value <.001). Diamonds are used for study subject parents to protect patient privacy per UPDB requirements.
      Morgan et al. Provoked vestibulodynia is familial. Am J Obstet Gynecol 2016.

      Clinically confirmed vestibulodynia treated by vestibulectomy cohort

      In a separate analysis, we had records of 216 clinically confirmed cases of moderate to severe vestibulodynia treated by vestibulectomy. Seventy of these cases met our strict geneaology requirements to test for familiality. Relative risk estimates in their relatives used CPT codes similar to our CPT-based analysis. Although the clinically confirmed proband cohort (n = 70) was one-third the size of the CPT-based cohort (n = 183), there was sufficient power to detect significantly increased risk in first-, second-, and third-degree relatives (Table 2). GIF statistic analysis, comorbidity analysis, and familial pedigree analysis were not performed on this smaller cohort.
      Table 2Relative risk for vestibulectomy in first-, second-, and third-degree relatives of 70 clinically confirmed cases of vestibulodynia treated by vestibulectomy
      Relationship to probandNumber of relativesObserved no. vestibulectomiesExpected no. vestibulectomiesRelative risk [95% CI]; P value
      First-degree20350.09
      Calculated expected frequencies are much less than 10%, because not all women diagnosed with vestibulodynia are treated by vestibulectomy, and fewer women are linked to the Utah Population Database (UPDB) and meet strict ancestry requirements within the 1994–2015 search window. These caveats are assumed to affect both the case and control populations equally.
      54 [17.5–126]; <.00001
      Second-degree353<5-20 [2.4–71]; .005
      Third-degree693<5-12 [3.3–31]; .0004
      From a separate database of 216 clinically confirmed cases of vestibulodynia treated by vestibulectomy, we identified 70 that met strict genealogy requirements to test for familiality. Relative risk estimates with 95% confidence intervals (CI) in their relatives used CPT codes similar to the CPT-based analysis (Table 1). Significance was determined by Fisher exact test to measure whether there were more observed cases in relatives of the probands compared with the expected number (no.) in matched population controls.
      Note: UPDB regulations require “<5” to be used rather than precise numbers to protect patient privacy. Similarly, their expected frequencies are not shown.
      Morgan et al. Provoked vestibulodynia is familial. Am J Obstet Gynecol 2016.
      a Calculated expected frequencies are much less than 10%, because not all women diagnosed with vestibulodynia are treated by vestibulectomy, and fewer women are linked to the Utah Population Database (UPDB) and meet strict ancestry requirements within the 1994–2015 search window. These caveats are assumed to affect both the case and control populations equally.

      Potential comorbidities

      In Table 3 we report the risk of various comorbid conditions among CPT-based vestibulectomy probands (n = 183). Subjects were at increased risk for interstitial cystitis, myalgia/myositis, and constipation. There may be a trend for an association with asthma.
      Table 3Risk of comorbid conditions in vestibulectomy probands
      Comorbid conditionObserved numberExpected numberRelative risk [95% CI]P value
      Interstitial cystitis<5-
      The expected number was calculated from the frequency observed in matched female controls. Significance was determined by Fisher exact test to measure whether there were more observed cases in probands compared with the expected number in matched population controls.
      10.6 [2.2–31].003
      Asthma148.61.6 [0.9–2.7].08
      Myalgia/myositis186.82.6 [1.6–4.2].0003
      Constipation176.72.6 [1.5–4.1].0006
      We estimated relative risk with 95% confidence intervals (CI) of comorbid conditions among vestibulectomy probands using ICD-9 and CPT codes, respectively.
      Note: Utah Population Database regulations require “<5” to be used rather than precise numbers to protect patient privacy. Similarly, their paired expected frequencies are not shown. No lupus/autoimmune cases were identified (n = 0).
      Morgan et al. Provoked vestibulodynia is familial. Am J Obstet Gynecol 2016.
      a The expected number was calculated from the frequency observed in matched female controls. Significance was determined by Fisher exact test to measure whether there were more observed cases in probands compared with the expected number in matched population controls.

      Comment

      This is the first familial-based population study of PVD. Our data suggest that vestibulodynia may have a heritable contribution to risk. Moreover, we have identified a number of high-risk Utah families with an excess number of affected women, including distant relatives, that may be employed for future linkage analysis and gene discovery studies.
      Genetic predisposition is only one part of the puzzle, and these results also support a role for environmental factors. Environmental triggers like infection,
      • Tommola P.
      • Butzow R.
      • Unkila-Kallio L.
      • Paavonen J.
      • Meri S.
      Activation of vestibule-associated lymphoid tissue in localized provoked vulvodynia.
      allergies,
      • Harlow B.
      • He W.
      • Nguyen R.
      Allergic reactions and risk of vestibulodynia.
      and aberrant hormone receptor signaling
      • Eva L.
      • Maclean A.
      • Reid W.
      • Rolfe K.
      • Perrett C.
      Estrogen receptor expression in vulvar vestibulitis syndrome.
      • Leclair C.
      • Goetsch M.
      • Li H.
      • Morgan T.K.
      Histopathologic characteristics of menopausal vestibulodynia.
      • Goldstein A.T.
      • Belkin Z.R.
      • Krapf J.M.
      • et al.
      Polymorphisms of the androgen receptor gene and hormonal contraceptive induced provoked vestibulodynia.
      • Ting A.Y.
      • Blacklock A.D.
      • Smith P.G.
      Estrogen regulates vaginal sensory and autonomic nerve density in the rat.
      may all play a role in vestibulodynia. A hormonal mechanism is intriguing, although not entirely clear. Some speculate it may involve a combination of lowered free testosterone, less efficient androgen receptor, and oral contraceptives.
      • Goldstein A.T.
      • Belkin Z.R.
      • Krapf J.M.
      • et al.
      Polymorphisms of the androgen receptor gene and hormonal contraceptive induced provoked vestibulodynia.
      We have previously shown that secondary PVD appears to have fewer numbers of progesterone receptors in the vestibular tissue
      • Leclair C.
      • Goetsch M.
      • Korcheva V.
      • Anderson R.
      • Peters D.
      • Morgan T.K.
      Differences in primary compared with secondary vestibulodynia by immunohistochemistry.
      • Leclair C.
      • Goetsch M.
      • Li H.
      • Morgan T.K.
      Histopathologic characteristics of menopausal vestibulodynia.
      and have speculated that since progestins inhibit estrogen receptor signaling, an aberrant relative increase in estrogen activity may lead to neurogenic inflammation in this tissue.
      • Leclair C.
      • Goetsch M.
      • Korcheva V.
      • Anderson R.
      • Peters D.
      • Morgan T.K.
      Differences in primary compared with secondary vestibulodynia by immunohistochemistry.
      T cell regulators like IL-4 variants involved in asthma
      • Basehore M.
      • Howard T.
      • Lange L.
      • et al.
      A comprehensive evaluation of IL-4 variants in ethnically diverse populations: association of total serum IgE levels and asthma in white subjects.
      and interstitial cystitis
      • Sugaya K.
      • Nishijima S.
      • Yamada T.
      • Miyazato M.
      • Hatano T.
      • Ogawa Y.
      Molecular analysis of adrenergic receptor genes and interleukin-4/interleukin-4 receptor genes in patients with interstitial cystitis.
      are also intriguing, since PVD appears to have more CD4 T cells than control biopsies
      • Leclair C.
      • Leeborg N.
      • Jacobson-Dunlop E.
      • Goetsch M.
      • Morgan T.K.
      CD4-positive T-cell recruitment in primary provoked localized vulvodynia: potential insights into disease triggers.
      and aberrant Th1-to-Th2 switching via IL-4 stimulation could provide a mechanism to explain why PVD is associated with allergies.
      • Harlow B.
      • He W.
      • Nguyen R.
      Allergic reactions and risk of vestibulodynia.
      We also suspect the JAK/STAT pathway, which plays a key role in neurogenic inflammation and interstitial cystitis.
      • Kisseleva T.
      • Bhattacharya S.
      • Braunstein J.
      • Schindler C.W.
      Signaling through the JAK/STAT pathway, recent advances and future challenges.
      • Keay S.K.
      • Zhang C.O.
      • Shoenfelt J.
      • et al.
      Sensitivity and specificity of antiproliferative factor, heparin-binding epidermal growth factor-like growth factor, and epidermal growth factor as urine markers for interstitial cystitis.
      In fact, inhibition of IL-4/EGFR-mediated stimulation of the JAK/STAT pathway may explain why heparin-based interventions seem to ameliorate PVD in select patients.
      • Ji S.-L.
      • Cui H.-F.
      • Shi F.
      • Chi Y.-Q.
      • Cao J.-C.
      • Geng M.-Y.
      • Guan H.-S.
      Inhibitory effect of heparin-derived oligosaccharides on secretion of interleukin-4 and interleukin-5 from peripheral blood T lymphocytes.
      • Farajun Y.
      • Zarfati D.
      • Abramov L.
      • Livoff A.
      • Bornstein J.
      Enoxaparin treatment for vulvodynia: a randomized controlled trial.
      This remains to be tested.
      Alternative pathways may also be suggested by comorbidity analysis. We have already discussed the importance of interstitial cystitis in terms of both genetic risk and potentially involved genes. And in fact, we, too, have observed an association between PVD and interstitial cystitis in women with vestibulectomy. The frequency of interstitial cystitis in vestibulectomy cases was less than expected,
      • Cardella B.
      • Porru D.
      • Nappi R.
      • Dacco M.
      • Chiesa A.
      • Spinillo A.
      Interstitial cystitis is associated with vulvodynia and sexual dysfunction-a case control study.
      but this may be related to insufficient coding for this specific disease in the UUHSC database.
      • Allen-Brady K.
      • Norton P.A.
      • Cannon-Albright L.
      Risk of associated conditions in relatives of subjects with interstitial cystitis.
      Our working hypothesis predicts that other common diseases involving neurogenic inflammation should also be associated with PVD. For example, Dr Harlow’s group has reported an association with seasonal allergies.
      • Harlow B.
      • He W.
      • Nguyen R.
      Allergic reactions and risk of vestibulodynia.
      Since IL-4 variants play a role in atopy,
      • Basehore M.
      • Howard T.
      • Lange L.
      • et al.
      A comprehensive evaluation of IL-4 variants in ethnically diverse populations: association of total serum IgE levels and asthma in white subjects.
      and cystitis is associated with asthma,
      • Allen-Brady K.
      • Norton P.A.
      • Cannon-Albright L.
      Risk of associated conditions in relatives of subjects with interstitial cystitis.
      we tested for an association between PVD and asthma. Our comorbidity analysis reveals a potential association with vestibulectomy (P = .08), but a larger sample size will be required to confirm this hypothesis. Similarly, we tested whether lupus may be more common in our cases, but we detected no association.
      Interstitial cystitis is associated with constipation and fibromyalgia;
      • Allen-Brady K.
      • Norton P.A.
      • Cannon-Albright L.
      Risk of associated conditions in relatives of subjects with interstitial cystitis.
      therefore, we also tested whether they are associated with PVD. We observed strong associations between vestibulectomy and fibromyalgia and constipation (Table 3). These associations are fascinating because they raise new potential pathways and candidate genes. Perhaps sensitivity to pain is increased in women with PVD, which may support a recently reported association with the 5HT-2A serotonin receptor variant.
      • Heddini U.
      • Bohm-Starke N.
      • Gronbladh A.
      • Nyberg F.
      • Nilsson K.
      • Johannesson U.
      Serotonin receptor gene (5HT-2A) polymorphism is associated with provoked vestibulodynia and comorbid symptoms of pain.
      A mechanism involving increased risk for constipation is less clear, but could involve serotonin activity or aberrant sex hormone regulation—both involved in bowel activity.
      Employing high-risk Utah pedigrees for linkage analysis and genomic sequencing of distantly related affected women will be the key to testing these many hypotheses. Indeed, an important strength of the UPDB and this study is not only identification of large Utah pedigrees, but the availability of archived tissue and DNA samples at UUHSC. For example, our group has 216 vestibulectomy cases with available tissue and 70 of these were linked to the UPDB with at least 3 generations of genealogy. Many of these 70 clinically confirmed cases are distantly related and their DNA is available for high-density genotyping to isolate shared genetic variants.
      Finally, it must be acknowledged that our conclusions have limitations that are common to most genetic studies. First of all, the precision and accuracy of phenotyping is critical. Our analyses relied on CPT codes recorded in UUHSC electronic records. Whether we employed CPT codes to define vestibulectomy probands or we employed these codes to search for affected relatives of clinically confirmed probands, we relied on the precision and accuracy of CPT codes for vestibulectomy. There is no code specific for vestibulodynia treated by vestibulectomy. In our experience, CPT code 56620 is the most commonly employed (150/183) and was the code used for the 70 clinically confirmed cases. If we run the analysis using only CPT code 56620, the relative risks are greater than those reported in Table 1 but less than those reported in Table 2 (data not shown). In addition, although these 70 cases provide data supporting familiality for clinically confirmed vestibulodynia treated by vestibulectomy, there may be ascertainment bias. Proband treatment success may lead to a similar treatment of their family members for this common condition, while other women outside of their family may not receive treatment. Our CPT code–based analysis of 183 cases suggests significant familiality out past 2 meioses (dGIF), providing some data against ascertainment bias, but we cannot exclude this possibility.
      Another question is why was the number of vestibulectomies in the relatives of our cases and controls so much less than the prevalence of vestibulodynia (∼10%)? First of all, not all women diagnosed with vestibulodynia have vestibulectomy to treat their condition. In fact, the majority of these patients currently do not have vestibulectomies (our experts cite that ∼5–10% of patients may have vestibulectomies, which translates into <1% of women in the population). Second, only cases captured after 1994 and meeting our genealogy inclusion criteria would be recorded; therefore, cases diagnosed before this time, or women who were not diagnosed at UUHSC, or women not linked to the UPDB, or women who failed to meet the strict ancestry requirements would not be recorded in our analysis. This is our interpretation of why the calculated expected frequency of vestibulectomy is less than 10% in our UUHSC female population (Table 2, Table 3). Nonetheless, our inclusion criteria were identical for cases and controls; therefore, our null hypothesis assumed that disease frequency would be similar in both groups. It was not.
      In conclusion, this is the first large familiality analysis to test for a heritable contribution to PVD. Our data support the hypothesis that vestibulodynia has a genetic predisposition, but linkage analysis and genetic sequencing of distantly related women with PVD will be required to confirm this result. The importance of identifying genetic variants associated with PVD will be their value for early diagnostic testing and new, less invasive therapeutic interventions.

      Acknowledgments

      We appreciate the support of the National Vulvodynia Association and the Interstitial Cystitis Association. Partial support for the Utah Population Database was provided by the University of Utah Huntsman Cancer Institute and the Huntsman Cancer Institute Cancer Center Support grant, P30 CA2014 from the National Cancer Institute.

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