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Serotonin Receptor Gene (5HT‐2A) Polymorphism is Associated with Provoked Vestibulodynia and Comorbid Symptoms of Pain
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Serotonin Receptor Gene (5HT-2A) Polymorphism is Associated with Provoked Vestibulodynia and Comorbid Symptoms of Pain Ulrika Heddini, MD, PhD,* Nina Bohm-Starke, MD, PhD,* Alfhild Grönbladh, PhD,† Fred Nyberg, PhD,† Kent W. Nilsson, PhD,‡ and Ulrika Johannesson, MD, PhD* *Karolinska Institutet, Division of Obstetrics and Gynecology, Department of Clinical Sciences, Danderyd Hospital, Stockholm, Sweden; †Department of Pharmaceutical Biosciences, Division of Biological Research on Drug Dependence, Uppsala University, Uppsala, Sweden; ‡Center for Clinical Research, County Council of Västmanland Central Hospital, Uppsala University, Västerås, Sweden DOI: 10.1111/jsm.12685
ABSTRACT
Introduction. Provoked vestibulodynia (PVD) is a common type of dyspareunia among young women. The pathophysiology remains largely unclear. Women with PVD have general pain hypersensitivity and often report additional pain symptoms. Signs point towards PVD being a chronic pain disorder similar to other syndromes of longstanding pain, including a common comorbidity of anxiety and depression. Polymorphism in the serotonin receptor gene, 5HT-2A, has been associated with other chronic pain disorders such as fibromyalgia but has not been investigated in PVD patients. Aim. We aimed to investigate a possible contribution of polymorphism in the 5HT-2A gene to the etiology of PVD as well as a potential influence on pain sensitivity. Methods. In this case-control study 98 women with PVD and 103 healthy controls between 18 and 44 years and in the same menstrual cycle phase completed questionnaires and underwent quantitative sensory testing. Venous blood samples were collected for DNA isolation. Main Outcome Measures. Concomitant pain was reported, a bodily pain score was created and pressure pain thresholds (PPTs) on the arm, leg, and in the vestibule were measured. Intensity of coital pain was rated on a visual analog scale, range 0–100. The T102C (rs6313) and A-1438G (rs6311) single nucleotide polymorphisms (SNPs) in the 5HT-2A gene were analyzed. Results. The probability of PVD was elevated in participants carrying the 1438G- and 102C-alleles of the 5HT-2A gene (OR 2.9). The G-/C- genotypes were also associated with more concomitant bodily pain in addition to the dyspareunia, but not with experimental PPTs or coital pain ratings. PVD patients reported more concomitant bodily pain and had lower PPTs compared with controls. Conclusion. The results indicate a contribution of alterations in the serotonergic system to the patho-genesis of PVD and gives further evidence of PVD being a general pain disorder similar to other chronic pain disorders. Heddini U, Bohm-Starke N, Grönbladh A, Nyberg F, Nilsson KW, and Johannesson U. Serotonin receptor gene (5HT-2A) polymorphism is associated with provoked vestibulodynia and comorbid symptoms of pain. J Sex Med 2014;11:3064–3071. Key Words. Provoked Vestibulodynia; Fibromyalgia; Pain Sensitivity; Serotonin; 5HT-2A; Genetic Polymorphism; Chronic Pain; Dyspareunia
J Sex Med 2014;11:3064–3071
© 2014 International Society for Sexual Medicine
5HT-2A Polymorphism Associated with PVD Introduction
D
yspareunia is a common pain problem among women. The prevalence has been estimated to be 10–15% [1,2]. The most common type of dyspareunia among premenopausal women is provoked vestibulodynia (PVD) [3]. PVD is characterized by pain upon touch, pressure and stretch of the vestibular tissue in spite of the absence of other gynecological or dermatological disease [4]. The pain and its associated sexual consequences have a severe negative impact on the quality of life of affected women [5,6]. Currently the etiology, although still not completely clarified, is considered to be multi-factorial involving biomedical and psychosexual causes [7,8]. Two sub-categories of PVD has been identified: primary PVD, where pain occurs at the first attempt of vaginal entry (intercourse or tampon use); and secondary PVD, where pain occurs after a period of normal functioning [9,10]. There is evidence of patho-physiological changes in three interdependent systems; the vestibular tissue, the pelvic floor muscles and the pain regulatory pathways of the central nervous system [11]. Recurrent vulvo-vaginal Candida infections and hormonal effects on the vestibular mucosa have been suggested as triggers of PVD [12–15]. Signs of a neurogenic inflammation in the vestibular mucosa, with neural hyperplasia and peripheral sensitization have been found along with central pain hypersensitivity with more painful tender points and a higher sensitivity to experimental pain stimuli at non-genital regions in PVD patients [16–21]. Several studies have reported different psychological traits including anxiety, depression, harm avoidance and catastrophizing being more common in PVD patients than in healthy controls [9,18,22]. Additionally, there are signs of PVD being a general pain disorder, associated to other chronic pain syndromes [2,11,23]. Although still not proven in PVD patients there is a familial aggregation in other pain conditions sharing common features with PVD, such as fibromyalgia and irritable bowel syndrome (IBS) [24,25]. Some efforts have been made to explore the genetic background of PVD. Correlations to genetic variants involved in the inflammatory response have been shown, possibly contributing to the neurogenic inflammation seen in the vestibular mucosa of these patients [26]. For instance, a higher presence of an allele of the gene coding for the interleukin 1 (IL-1) receptor antagonist protein, associated with a number of inflammatory
3065 diseases, was found in women with PVD [27,28]. PVD patients have also been reported to be more likely to be homozygous for allele 2 of the IL-1 receptor antagonist gene and to carry at least one of six loss-of-function polymorphisms in the melocortin-1 receptor gene with an additive effect on the risk of developing PVD [29]. A few studies have investigated a contribution to the etiology of PVD by polymorphisms in genes affecting endogenous pain processing. The 118A allele of the A118G SNP in the μ-opioid receptor gene (OPRM1) has been found to be more common in PVD patients than in healthy controls, with an odds ratio of 1.8 to be diagnosed with PVD in carriers [30]. Further, there was a lack of correlation between PVD and a single nucleotide polymorphism (SNP) combination in the GCH1gene, previously suggested as being pain protective [31]. Serotonin (5-hydroxytryptophan) is the key neurotransmitter in the serotonergic system. This system has wide-ranging actions through-out the body, including an anti-nociceptive role [32,33]. Low levels of serum serotonin have been associated with high scores of self reported pain [34]. Selective serotonin reuptake inhibitors (SSRI) have been shown to be effective in the treatment of pain conditions such as fibromyalgia and chronic widespread pain (CWP) [35]. However, it is unclear whether the effect of SSRI-treatment in fibromyalgia is due to effects on pain processing or on the common comorbid symptoms of depression. The serotonin receptor gene, 5HT-2A, has been well researched and two common SNPs in this gene; the A-1438G and T102C have been reported. These SNPs appear always to be co-inherited [36]. A review by Lee in 2012 concludes a significant association between the CC + CT genotype of the T102C SNP and fibromyalgia [37]. There are findings of the T-allele being associated with a decrease in the number of somatic symptoms contradicted by findings of higher pain scores in a group of fibromyalgia patients carrying the TT genotype [34,38]. There are also reports of associations between these SNPs and depression [39,40]. PVD treatment is often longstanding and outcomes vary. To elucidate the background mechanisms of PVD is important in order to develop more efficient treatments. PVD shares many overlapping features with other pain conditions associated to 5HT-2A polymorphism but the possible involvement of this polymorphism in the pathophysiology of PVD has not been explored. J Sex Med 2014;11:3064–3071
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Heddini et al.
Aims
PVD patient/control status and genotype, using a pressure algometer (Somedic Sales AB, Hörby, Sweden) [21]. In brief, the arm on the nondominant side was tested on the deltoid muscle, 3 cm proximal to the tendon insertion of the muscle and the leg on the same side was tested on the anterior tibial muscle, approximately 5 cm below and 3 cm lateral to the tibial tuberosity. Pressure was increased until the participant reported the PPT defined as the first sensation of pain. All participants received an explanation of the procedure and a training session on the opposite arm before testing. PPTs in the vestibule were measured in PVD patients only, using vulvar algesiometers consisting of cylindrical devices containing metal springs of varying compression rates with a cotton swab top, exerting a pressure ranging from 3 to 1,000 g [41,42]. Two areas of the vestibule on the right side of the vaginal opening were tested; area A, close to the urethra and area B, close to the opening of the Bartholin’s glands. Increasing pressure was applied until the PPT was reported. The measurements were repeated twice with a few minutes time interval on each of the four testing areas, and the median values were used for analyses.
We aimed to investigate the possible correlation between the A-1438G and T102C SNPs in the 5HT-2A gene and a PVD diagnose as well as pain sensitivity. Methods
Ninety-eight women with PVD were recruited between May 2008 and May 2010. Inclusion criteria for PVD patients were: age ≥18 years, PVD defined as dyspareunia or, for those not currently engaging in vaginal intercourse, pain at vestibular contact, with duration of symptoms of ≥6 months, based on the initial exam at the time of diagnosis. Exclusion criteria were: local infection or dermatological causes of dyspareunia or vestibular pain, major psychiatric or medical disease (not including mild depression and anxiety), and pregnancy. Controls consisted of 103 healthy women in the same age span and from the same geographical region with regular menstruation. Exclusion criteria were: dyspareunia, regular use of analgesics or anti-depressants, major medical or psychiatric disease (not including mild depression or anxiety) and pregnancy. Participants completed study specific questionnaires and underwent quantitative sensory testing. A venous blood sample was collected for the genetic analysis. Testing was carried out on one occasion during days 3–13 of the menstrual cycle. All participants received oral and written information about the study and provided informed consent and the study was approved by the local ethical committee. Main Outcome Measures
A comprehensive questionnaire surveyed medical (including psychosocial and gynecological) history as well as presence of dyspareunia and other frequent bodily pain symptoms in non-genital areas. A bodily pain score with a range from 0 (no other pain)—5 (five or more other pain symptoms) was created for each participant. Dysmenorrhea was not included in the pain score since the use of hormonal contraceptives varied among participants which can influence menstrual pain. PVD patients engaging in vaginal intercourse scored the intensity of coital pain during the last month on a visual analog scale (VAS) with a range from 0 (no pain)—100 (worst pain imaginable). Pressure pain thresholds (PPTs) on the arm and leg were measured with an examiner blinded to J Sex Med 2014;11:3064–3071
Genetic Analyses Venous blood samples were collected in EDTAtubes. Total genomic DNA was isolated from the whole blood samples using the Magtration 12GC system (Precision System Science, Chiba, Japan) and the Magazorb® DNA Common Kit-200 (PSS, Chiba, Japan). The DNA concentration was determined using a Nanodrop Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). Two SNPs in the 5HT-2A gene, T102C (rs6313) and A-1438G (rs6311), were analyzed using the TaqMan SNP genotyping assay, assay numbers C_3042197_1 and C_8695278_10 respectively (Applied Biosystems, Foster City, USA). Briefly, in each well of a 384-well plate, genomic DNA (5 ng), water, TaqMan Universal PCR master mix, and TaqMan genotyping assay mix was added, in a total volume of 5 μL. Target specific primers and TaqMan MGB probes labeled with two special dyes, FAMTM and VIC®, were included in the assay mix. The reaction was performed using an ABI17900HT genetic detection system (Applied Biosystems, Foster City, USA) with the following amplification protocol: 10 minutes at 95°C followed by 40 cycles of 15 seconds at 92°C and 1 minute at 60°C.
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5HT-2A Polymorphism Associated with PVD Table 1
Clinical background data
Variables
PVD patients (n = 98)
Controls (n = 103)
P value
Current age, years (range) Caucasian ethnicity; n (%) Duration of PVD, years (range) Primary PVD Secondary PVD Menstrual cycle day (range) Current use of HCs Dysmenorrhea Previous depression treatment; n (%) Present SSRI treatment; n (%)
29 (19–44) 95 (97) 8 (0.5–18) 35 (36%) 63 (64%) 7.9 (4–13) 35 (33%) 67 (71%) 64 (65) 6 (6)
24 (18–35) 99 (96) — — — 8.0 (3–13) 55 (54%) 55 (54%) 29 (28) 0 (0)
P < 0.001, t = −7.266 ns — — — ns P = 0.005 P = 0.02 P < 0.001, χ2 = 27.879 P = 0.011, χ2 = 6.500
Statistics The Statistica program (version 10, StatSoft Inc., Tulsa, OK, USA) and the Statistical package for the Social Sciences program (version 20, SPSS Inc., Chicago, IL, USA) were used. The student’s t-test or, for ordinal and non-normally distributed data, the Mann–Whitney U-test, were used for comparisons between groups regarding age and pain measurements. The chi-squared test was used for analyzing frequencies of previous depression treatment, pain disorders, and SNPs. For associations and interactions between the 5HT-2A polymorphism, pain measurements and PVD, univariate and multivariate logistic regression was used. A significance level of P < 0.05 and a confidence interval of 95% were used. Results
Clinical background data are summarized in Table 1. Controls were younger than PVD patients with a mean age difference of 5 years. There were no ethnical differences between the groups. PVD patients had previous or current treatment for depression to a higher degree than controls. PVD patients reported more frequent pain symptoms from non-genital body areas and Table 2
displayed higher pain sensitivity than controls in all pain modalities measured, see Table 2. Genotyping for the studied T102C (rs6313) and A-1438G (rs6311) SNPs in the 5HT-2A gene was completed in 201 subjects. The frequencies were in accordance with the Hardy-Weinberg equilibrium (all: P = 0.377, PVD patients: P = 0.058, controls P = 0.719). See Table 3. In accordance with previous studies the CT/CC- and AG/GG carriers were grouped together for further analyses [37]. The AA and TT genotypes were more common among controls compared with PVD patients. The odds ratio (OR) of having PVD was 2.9 among participants homo- or heterozygous for the G- or C-alleles compared with participants who were homozygous for the A- or T-alleles (OR = 2.9, CI: 1.2–6.9, P = 0.017). The bodily pain score in participants who were homo- or heterozygous for the G allele were higher than in those who were homozygous for the A allele, see Table 4. No other allele-linked differences in the pain measurements were found in the whole population or when PVD patients and controls were analyzed separately. There were no interaction effects of 5HT-2A polymorphism and PVD-patients vs. controls status with respect to the pain measurements.
Pain measurements
Concomitant bodily pain
PVD patients (n = 98)
Controls (n = 103)
P value
Headache; no (%) GI pain and dysfunction; no (%) Back pain; no (%) Muscle pain; no (%) Other pain; no (%) Bodily pain, score 0–5; mean (SD) median (Q1–Q3) Pain measurements PPT leg (kPa); mean (SD) median (Q1–Q3) PPT arm (kPa); mean (SD) median (Q1–Q3) PPT vestibule A (g); mean (SD) median (Q1–Q3) PPT vestibule B (g); mean (SD) median (Q1–Q3) Coital pain, VAS 0–100; mean (SD) median (Q1–Q3)
59 53 48 31 26 2.1
(60%) (54%) (49%) (32%) (27%) (1.2) 2 (1–3)
30 22 20 2 1 0.7
<0.001, <0.001, <0.001, <0.001, <0.001, <0.001,
405 268 48 42 53
(161) 390 (299–499) (124) 238 (189–331) (31) 40 (25–60) (44) 28 (15–50) (32) 54 (23–78)
474 (152) 457 (361–575) 309 (116) 298 (227–355) — — —
(29%) (22%) (20%) (2%) (1%) (0.9) 0 (0–1)
χ2 = 19.659 χ2 = 22.989 χ2 = 28.096 χ2 = 36.731 χ2 = 28.214 z = 7.859
0.001, z = −3.213 0.002, z = −3.142 — — —
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Heddini et al. Genotype frequencies
T102C (rs6313)
TT
TC
CC
P value
PVD patients (n = 97)*; n (%) Controls (n = 103); n (%)
8 (8) 21 (20)
53 (55) 49 (48)
36 (37) 33 (32)
0.051 χ2 = 5.940
A-1438G (rs6311)
AA
AG
GG
PVD patients (n = 98); n (%) Controls (n = 103); n (%)
8 (8) 21 (20)
53 (54) 49 (48)
37 (38) 33 (32)
0.047 χ2 = 6.092
*One missing due to error in analysis.
Conclusions
We aimed to investigate a possible correlation between the A-1438G and T102C SNPs in the serotonin receptor gene, 5HT-2A, and a diagnosis of PVD as well as pain sensitivity. In participants homo- or heterozygous for the G- and C-alleles we found a higher probability of PVD with an OR of 2.9. The results of the C-allele of the T102C SNP being more common among PVD patients compared with controls is concordant with previous result of a similar genotype distribution among female fibromyalgia patients [34]. The T102C polymorphism does not result in any alteration in the amino acid composition and has no influence on receptor protein; therefore so called linkage disequilibrium (i.e. co-inheritance) with the causative mutation has been proposed as a mechanism for the reported associations [34]. A meta-analysis of candidate gene studies of fibromyalgia found an overall OR of the association between the CT/CC genotype and fibromyalgia of 1.3–1.8 [37]. Our finding of an association between the same genotypes and PVD gives further support to the similarity of these conditions. There was also a correlation between concomitant bodily pain and the 5HT-2A SNPs with more pain symptoms from other body areas than the vestibule among G-/C- carriers. However, the Table 4
5HT-2A polymorphism was only correlated to the reported concomitant bodily pain symptoms and not to the experimental PPTs or the reported intensity of coital pain. This raises the question whether the observed association is correlated to the PVD diagnosis per se or to the other pain symptoms present in this group. On the other hand, the correlation to the concomitant bodily pain strengthens the similarity between PVD and syndromes such as fibromyalgia and CWP. Another shared feature of these conditions, apart from the unexplained pain, is the common comorbidity of anxiety and depression [9,22,43]. The serotonergic systems’ contribution to mood disorders and sexual desire is well-known [44] and it is therefore interesting to speculate whether differences in serotonin receptor gene polymorphism might, at least partly, explain both differences in pain sensitivity and psychological traits between women with PVD and healthy women. However, it is also possible that the higher reports of other pain symptoms among PVD patients are a result of the comorbid mood disorders [33]. The causative relationship between longstanding pain and sexual disability and heightened symptoms of anxiety and depression still remains unclear and needs to be further explored. Among the strengths of this study is a very reliable PVD diagnosis among cases, since they were
Pain measurements in relation to 5HT-2A polymorphism 5HT-2A genotype
P value
Pain measurements
AA/TT
n
AG + GG/TC + CC
n
PPT leg (kPa); mean(SD)/median(Q1–Q3) PPT arm (kPa); mean(SD)/median(Q1–Q3) Bodily pain, score 0–5; mean(SD)/ median(Q1–Q3) PPT vestibule A (g); mean(SD)/ median(Q1–Q3) PPT vestibule B (g); mean(SD)/ median(Q1–Q3) Coital pain VAS (0–100); mean(SD)/ median(Q1–Q3)
445 (151) 423 (326–560) 284 (100) 264 (225–334) 1.0 (1.0) 1 (0–3)
29 29 29
440 (161) 411 (329–529) 290 (125) 275 (196–344) 1.5 (1.3) 1 (0–4)
171 171 172
J Sex Med 2014;11:3064–3071
55 (36) 45 (29–75)
8
47 (31) 39 (25–60)
90
ns ns 0.049 z = −1.962 ns
36 (44) 20 (6–50)
8
43 (45) 30 (15–50)
90
ns
56 (29) 61 (32–76)
8
53 (32) 54 (23–78)
90
ns
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5HT-2A Polymorphism Associated with PVD all examined by only a few physicians with very good knowledge of the condition. In addition, the menstrual cycle coordinated examinations reduces the risk of differences in pain sensitivity due to cycle variations. Nevertheless, there are several limitations. The controls were slightly younger than the PVD patients (see Table 1). Although genetic expression has been shown to change with ageing, comparing newborns with middle aged and elderly people [45], it is unlikely that a mean age difference of only 5 years would affect our results. Same age-span, ethnicity, geographical origin (in this case a larger city setting) and menstrual cycle phase was considered the most important matching variables and no other matching was made due to feasibility reasons. To avoid possible effects on pain sensitivity regular use of analgesic and anti-depressive drugs was an exclusion criterion for controls, which might have resulted in a control group with lower pain sensitivity and less depression than the normal population. Moreover, the confidence interval of the reported association between the 5HT-2A polymorphism and PVD was quite wide and approaching 1. The frequencies of the studied 5HT-2A SNPs have not previously been investigated in PVD patients and therefore the sample size was based on a previously outlined method to optimize sample size in candidate gene studies. Based on the frequency of the studied SNP and phenotype in the normal population 100 participants in each group was estimated to achieve a power of 80% to detect differences in SNP frequencies between PVD patients and controls [46]. Although the study group is quite large in the field of PVD research, in a candidate gene study an even larger material would be preferable to give more reliable results and allow for further sub analyses. Different etiologies of the two sub categories of PVD have been suggested, where women with primary PVD have displayed general pain hypersensitivity and mood disorders to a higher extent than women with secondary PVD [9,10]. In this study sub analyses were not performed due to the limitations in sample size (see Table 1) but there is a possibility that mixing the sub categories reduces the strength to find associations that might be stronger with one of the sub groups. Furthermore, susceptibility factors that might be inherited is not the sole requirement for a chronic pain condition to develop, most likely a triggering insult such as an infection or trauma is also necessary. This geneenvironment interaction could lower the sensitivity of genetic studies.
Another aspect is that coital pain and vestibular PPTs were only surveyed among PVD patients, resulting in a reduced power to detect possible differences in these measurements. This was decided in the design of the study since vestibular pain tolerance has previously been found to be consistently much higher in healthy women than in women with PVD and dyspareunia was an exclusion criterion for controls [17,22,42]. For example, a mean vestibular PPT of approximately 300 g in the posterior part of the vestibule of healthy women as compared with 20 g in women with PVD has been reported [47]. Our results indicate an involvement of the serotonergic system in the patho-physiology of PVD pointing towards an effect on pain sensitivity by polymorphism in the serotonin receptor 2A gene. These findings offer a possible genetic explanation to the similarity between PVD and fibromyalgia and give further evidence of PVD being part of a general pain disorder. The knowledge contributes with important information to help caregivers accurately select treatment options for PVD, including possible preventive measures to avoid aggravated pain problems. To continue to explore serotonergic signaling might possibly lead to new pharmacological treatment alternatives for this patient group. Corresponding Author: Ulrika Heddini, MD, Karolinska Institutet, Division of Obstetrics and Gynecology, Department of Clinical Sciences, Danderyd Hospital, Stockholm 182 88, Sweden. Tel: (+46) 8-12355000; Fax: (+46) 8-7531832; E-mail: [email protected] Conflict of Interest: The authors declare that they have no competing interests. Statement of Authorship
Category 1 (a) Conception and Design Nina Bohm-Starke; Ulrika Johannesson; Ulrika Heddini (b) Acquisition of Data Ulrika Heddini; Fred Nyberg; Alfhild Gronbladh (c) Analysis and Interpretation of Data Ulrika Heddini; Kent W. Nilsson
Category 2 (a) Drafting the Article Ulrika Heddini (b) Revising It for Intellectual Content Nina Bohm-Starke; Ulrika Johannesson; Fred Nyberg; Alfhild Gronbladh; Kent W. Nilsson J Sex Med 2014;11:3064–3071
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Category 3 (a) Final Approval of the Completed Article Ulrika Heddini; Nina Bohm-Starke; Ulrika Johannesson; Fred Nyberg; Alfhid Gronbladh; Kent W. Nilsson
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Heddini et al. 18 Granot M, Friedman M, Yarnitsky D, Zimmer EZ. Enhancement of the perception of systemic pain in women with vulvar vestibulitis. BJOG 2002;109:863–6. 19 Pukall CF, Baron M, Amsel R, Khalife S, Binik YM. Tender point examination in women with vulvar vestibulitis syndrome. Clin J Pain 2006;22:601–9. 20 Johannesson U, de Boussard CN, Brodda Jansen G, Bohm-Starke N. Evidence of diffuse noxious inhibitory controls (DNIC) elicited by cold noxious stimulation in patients with provoked vestibulodynia. Pain 2007;130:31–9. 21 Heddini U, Bohm-Starke N, Gronbladh A, Nyberg F, Nilsson KW, Johannesson U. GCH1-polymorphism and pain sensitivity among women with provoked vestibulodynia. Mol Pain 2012;8:68. doi: 10.1186/1744-8069-8-68. 22 Sutton KS, Pukall CF, Chamberlain S. Pain ratings, sensory thresholds, and psychosocial functioning in women with provoked vestibulodynia. J Sex Marital Ther 2009;35:262–81. 23 Arnold LD, Bachmann GA, Rosen R, Kelly S, Rhoads GG. Vulvodynia: Characteristics and associations with comorbidities and quality of life. Obstet Gynecol 2006;107: 617–24. 24 Buskila D, Sarzi-Puttini P, Ablin JN. The genetics of fibromyalgia syndrome. Pharmacogenomics 2007;8:67–74. 25 Saito YA, Petersen GM, Larson JJ, Atkinson EJ, Fridley BL, de Andrade M, Locke GR 3rd, Zimmerman JM, Almazar-Elder AE, Talley NJ. Familial aggregation of irritable bowel syndrome: A family case-control study. Am J Gastroenterol 2010;105:833–41. 26 Babula O, Linhares IM, Bongiovanni AM, Ledger WJ, Witkin SS. Association between primary vulvar vestibulitis syndrome, defective induction of tumor necrosis factor-alpha, and carriage of the mannose-binding lectin codon 54 gene polymorphism. Am J Obstet Gynecol 2008;198:e1–4. 27 Jeremias J, Ledger WJ, Witkin SS. Interleukin 1 receptor antagonist gene polymorphism in women with vulvar vestibulitis. Am J Obstet Gynecol 2000;182:283–5. 28 Arend WP, Malyak M, Guthridge CJ, Gabay C. Interleukin-1 receptor antagonist: Role in biology. Annu Rev Immunol 1998;16:27–55. 29 Foster DC, Sazenski TM, Stodgell CJ. Impact of genetic variation in interleukin-1 receptor antagonist and melanocortin-1 receptor genes on vulvar vestibulitis syndrome. J Reprod Med 2004;49:503–9. 30 Heddini U, Johannesson U, Gronbladh A, Nyberg F, Nilsson WN, Bohm-Starke N. A118G polymorphism in the μ-opioid receptor gene and levels of β-endorphin are associated with provoked vestibulodynia and pressure pain sensitivity. Scand J Pain 2013;5:10–6. 31 Tegeder I, Costigan M, Griffin RS, Abele A, Belfer I, Schmidt H, Ehnert C, Nejim J, Marian C, Scholz J, Wu T, Allchorne A, Diatchenko L, Binshtok AM, Goldman D, Adolph J, Sama S, Atlas SJ, Carlezon WA, Parsegian A, Lotsch J, Fillingim RB, Maixner W, Max MB, Woolf CJ. GTP cyclohydrolase and tetrahydrobiopterin regulate pain sensitivity and persistence. Nat Med 2006;12:1269–77. 32 Mense S. Neurobiological concepts of fibromyalgia—The possible role of descending spinal tracts. Scand J Rheumatol Suppl 2000;113:24–9. 33 Graeff FG. Serotonergic systems. Psychiatr Clin North Am 1997;20:723–39. 34 Bondy B, Spaeth M, Offenbaecher M, Glatzeder K, Stratz T, Schwarz M, de Jonge S, Kruger M, Engel RR, Farber L, Pongratz DE, Ackenheil M. The T102C polymorphism of the 5-HT2A-receptor gene in fibromyalgia. Neurobiol Dis 1999;6:433–9. 35 Nicholl BI, Holliday KL, Macfarlane GJ, Thomson W, Davies KA, O’Neill TW, Bartfai G, Boonen S, Casanueva FF, Finn JD, Forti G, Giwercman A, Huhtaniemi IT, Kula K, Punab M,
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Silman AJ, Vanderschueren, D. Association of HTR2A polymorphisms with chronic widespread pain and the extent of musculoskeletal pain: Results from two population-based cohorts. Arthritis Rheum 2011;63:810–8. Saiz PA, Garcia-Portilla MP, Arango C, Morales B, Alvarez V, Coto E, Fernandez JM, Bascaran MT, Bousono M, Bobes J. Association study of serotonin 2A receptor (5-HT2A) and serotonin transporter (5-HTT) gene polymorphisms with schizophrenia. Prog Neuropsychopharmacol Biol Psychiatry 2007;31:741–5. Lee YH, Choi SJ, Ji JD, Song GG. Candidate gene studies of fibromyalgia: A systematic review and meta-analysis. Rheumatol Int 2012;32:417–26. Holliday KL, Macfarlane GJ, Nicholl BI, Creed F, Thomson W, McBeth J. Genetic variation in neuroendocrine genes associates with somatic symptoms in the general population: Results from the EPIFUND study. J Psychosom Res 2010;68:469–74. Eley TC, Sugden K, Corsico A, Gregory AM, Sham P, McGuffin P, Plomin R, Craig IW. Gene-environment interaction analysis of serotonin system markers with adolescent depression. Mol Psychiatry 2004;9:908–15. Choi MJ, Lee HJ, Ham BJ, Cha JH, Ryu SH, Lee MS. Association between major depressive disorder and the -1438A/G polymorphism of the serotonin 2A receptor gene. Neuropsychobiology 2004;49:38–41.
3071 41 Bohm-Starke N, Brodda-Jansen G, Linder J, Danielsson I. The result of treatment on vestibular and general pain thresholds in women with provoked vestibulodynia. Clin J Pain 2007;23:598–604. 42 Pukall CF, Young RA, Roberts MJ, Sutton KS, Smith KB. The vulvalgesiometer as a device to measure genital pressure-pain threshold. Physiol Meas 2007;28:1543–50. 43 Benjamin S, Morris S, McBeth J, Macfarlane GJ, Silman AJ. The association between chronic widespread pain and mental disorder: A population-based study. Arthritis Rheum 2000;43:561–7. 44 Pfaus JG. Pathways of sexual desire. J Sex Med 2009;6:1506– 33. 45 Christensen BC, Houseman EA, Marsit CJ, Zheng S, Wrensch MR, Wiemels JL, Nelson HH, Karagas MR, Padbury JF, Bueno R, Sugarbaker DJ, Yeh RF, Wiencke JK, Kelsey KT. Aging and environmental exposures alter tissue-specific DNA methylation dependent upon CpG island context. PLoS Genet 2009;5:e1000602. 46 Belfer I, Wu T, Kingman A, Krishnaraju RK, Goldman D, Max MB. Candidate gene studies of human pain mechanisms: Methods for optimizing choice of polymorphisms and sample size. Anesthesiology 2004;100:1562–72. 47 Pukall CF, Binik YM, Khalife S. A new instrument for pain assessment in vulvar vestibulitis syndrome. J Sex Marital Ther 2004;30:69–78.
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Serotonin Receptor Gene (5HT-2A) Polymorphism is Associated with Provoked Vestibulodynia and Comorbid Symptoms of Pain Ulrika Heddini, MD, PhD,* Nina Bohm-Starke, MD, PhD,* Alfhild Grönbladh, PhD,† Fred Nyberg, PhD,† Kent W. Nilsson, PhD,‡ and Ulrika Johannesson, MD, PhD* *Karolinska Institutet, Division of Obstetrics and Gynecology, Department of Clinical Sciences, Danderyd Hospital, Stockholm, Sweden; †Department of Pharmaceutical Biosciences, Division of Biological Research on Drug Dependence, Uppsala University, Uppsala, Sweden; ‡Center for Clinical Research, County Council of Västmanland Central Hospital, Uppsala University, Västerås, Sweden DOI: 10.1111/jsm.12685
ABSTRACT
Introduction. Provoked vestibulodynia (PVD) is a common type of dyspareunia among young women. The pathophysiology remains largely unclear. Women with PVD have general pain hypersensitivity and often report additional pain symptoms. Signs point towards PVD being a chronic pain disorder similar to other syndromes of longstanding pain, including a common comorbidity of anxiety and depression. Polymorphism in the serotonin receptor gene, 5HT-2A, has been associated with other chronic pain disorders such as fibromyalgia but has not been investigated in PVD patients. Aim. We aimed to investigate a possible contribution of polymorphism in the 5HT-2A gene to the etiology of PVD as well as a potential influence on pain sensitivity. Methods. In this case-control study 98 women with PVD and 103 healthy controls between 18 and 44 years and in the same menstrual cycle phase completed questionnaires and underwent quantitative sensory testing. Venous blood samples were collected for DNA isolation. Main Outcome Measures. Concomitant pain was reported, a bodily pain score was created and pressure pain thresholds (PPTs) on the arm, leg, and in the vestibule were measured. Intensity of coital pain was rated on a visual analog scale, range 0–100. The T102C (rs6313) and A-1438G (rs6311) single nucleotide polymorphisms (SNPs) in the 5HT-2A gene were analyzed. Results. The probability of PVD was elevated in participants carrying the 1438G- and 102C-alleles of the 5HT-2A gene (OR 2.9). The G-/C- genotypes were also associated with more concomitant bodily pain in addition to the dyspareunia, but not with experimental PPTs or coital pain ratings. PVD patients reported more concomitant bodily pain and had lower PPTs compared with controls. Conclusion. The results indicate a contribution of alterations in the serotonergic system to the patho-genesis of PVD and gives further evidence of PVD being a general pain disorder similar to other chronic pain disorders. Heddini U, Bohm-Starke N, Grönbladh A, Nyberg F, Nilsson KW, and Johannesson U. Serotonin receptor gene (5HT-2A) polymorphism is associated with provoked vestibulodynia and comorbid symptoms of pain. J Sex Med 2014;11:3064–3071. Key Words. Provoked Vestibulodynia; Fibromyalgia; Pain Sensitivity; Serotonin; 5HT-2A; Genetic Polymorphism; Chronic Pain; Dyspareunia
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© 2014 International Society for Sexual Medicine
5HT-2A Polymorphism Associated with PVD Introduction
D
yspareunia is a common pain problem among women. The prevalence has been estimated to be 10–15% [1,2]. The most common type of dyspareunia among premenopausal women is provoked vestibulodynia (PVD) [3]. PVD is characterized by pain upon touch, pressure and stretch of the vestibular tissue in spite of the absence of other gynecological or dermatological disease [4]. The pain and its associated sexual consequences have a severe negative impact on the quality of life of affected women [5,6]. Currently the etiology, although still not completely clarified, is considered to be multi-factorial involving biomedical and psychosexual causes [7,8]. Two sub-categories of PVD has been identified: primary PVD, where pain occurs at the first attempt of vaginal entry (intercourse or tampon use); and secondary PVD, where pain occurs after a period of normal functioning [9,10]. There is evidence of patho-physiological changes in three interdependent systems; the vestibular tissue, the pelvic floor muscles and the pain regulatory pathways of the central nervous system [11]. Recurrent vulvo-vaginal Candida infections and hormonal effects on the vestibular mucosa have been suggested as triggers of PVD [12–15]. Signs of a neurogenic inflammation in the vestibular mucosa, with neural hyperplasia and peripheral sensitization have been found along with central pain hypersensitivity with more painful tender points and a higher sensitivity to experimental pain stimuli at non-genital regions in PVD patients [16–21]. Several studies have reported different psychological traits including anxiety, depression, harm avoidance and catastrophizing being more common in PVD patients than in healthy controls [9,18,22]. Additionally, there are signs of PVD being a general pain disorder, associated to other chronic pain syndromes [2,11,23]. Although still not proven in PVD patients there is a familial aggregation in other pain conditions sharing common features with PVD, such as fibromyalgia and irritable bowel syndrome (IBS) [24,25]. Some efforts have been made to explore the genetic background of PVD. Correlations to genetic variants involved in the inflammatory response have been shown, possibly contributing to the neurogenic inflammation seen in the vestibular mucosa of these patients [26]. For instance, a higher presence of an allele of the gene coding for the interleukin 1 (IL-1) receptor antagonist protein, associated with a number of inflammatory
3065 diseases, was found in women with PVD [27,28]. PVD patients have also been reported to be more likely to be homozygous for allele 2 of the IL-1 receptor antagonist gene and to carry at least one of six loss-of-function polymorphisms in the melocortin-1 receptor gene with an additive effect on the risk of developing PVD [29]. A few studies have investigated a contribution to the etiology of PVD by polymorphisms in genes affecting endogenous pain processing. The 118A allele of the A118G SNP in the μ-opioid receptor gene (OPRM1) has been found to be more common in PVD patients than in healthy controls, with an odds ratio of 1.8 to be diagnosed with PVD in carriers [30]. Further, there was a lack of correlation between PVD and a single nucleotide polymorphism (SNP) combination in the GCH1gene, previously suggested as being pain protective [31]. Serotonin (5-hydroxytryptophan) is the key neurotransmitter in the serotonergic system. This system has wide-ranging actions through-out the body, including an anti-nociceptive role [32,33]. Low levels of serum serotonin have been associated with high scores of self reported pain [34]. Selective serotonin reuptake inhibitors (SSRI) have been shown to be effective in the treatment of pain conditions such as fibromyalgia and chronic widespread pain (CWP) [35]. However, it is unclear whether the effect of SSRI-treatment in fibromyalgia is due to effects on pain processing or on the common comorbid symptoms of depression. The serotonin receptor gene, 5HT-2A, has been well researched and two common SNPs in this gene; the A-1438G and T102C have been reported. These SNPs appear always to be co-inherited [36]. A review by Lee in 2012 concludes a significant association between the CC + CT genotype of the T102C SNP and fibromyalgia [37]. There are findings of the T-allele being associated with a decrease in the number of somatic symptoms contradicted by findings of higher pain scores in a group of fibromyalgia patients carrying the TT genotype [34,38]. There are also reports of associations between these SNPs and depression [39,40]. PVD treatment is often longstanding and outcomes vary. To elucidate the background mechanisms of PVD is important in order to develop more efficient treatments. PVD shares many overlapping features with other pain conditions associated to 5HT-2A polymorphism but the possible involvement of this polymorphism in the pathophysiology of PVD has not been explored. J Sex Med 2014;11:3064–3071
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Heddini et al.
Aims
PVD patient/control status and genotype, using a pressure algometer (Somedic Sales AB, Hörby, Sweden) [21]. In brief, the arm on the nondominant side was tested on the deltoid muscle, 3 cm proximal to the tendon insertion of the muscle and the leg on the same side was tested on the anterior tibial muscle, approximately 5 cm below and 3 cm lateral to the tibial tuberosity. Pressure was increased until the participant reported the PPT defined as the first sensation of pain. All participants received an explanation of the procedure and a training session on the opposite arm before testing. PPTs in the vestibule were measured in PVD patients only, using vulvar algesiometers consisting of cylindrical devices containing metal springs of varying compression rates with a cotton swab top, exerting a pressure ranging from 3 to 1,000 g [41,42]. Two areas of the vestibule on the right side of the vaginal opening were tested; area A, close to the urethra and area B, close to the opening of the Bartholin’s glands. Increasing pressure was applied until the PPT was reported. The measurements were repeated twice with a few minutes time interval on each of the four testing areas, and the median values were used for analyses.
We aimed to investigate the possible correlation between the A-1438G and T102C SNPs in the 5HT-2A gene and a PVD diagnose as well as pain sensitivity. Methods
Ninety-eight women with PVD were recruited between May 2008 and May 2010. Inclusion criteria for PVD patients were: age ≥18 years, PVD defined as dyspareunia or, for those not currently engaging in vaginal intercourse, pain at vestibular contact, with duration of symptoms of ≥6 months, based on the initial exam at the time of diagnosis. Exclusion criteria were: local infection or dermatological causes of dyspareunia or vestibular pain, major psychiatric or medical disease (not including mild depression and anxiety), and pregnancy. Controls consisted of 103 healthy women in the same age span and from the same geographical region with regular menstruation. Exclusion criteria were: dyspareunia, regular use of analgesics or anti-depressants, major medical or psychiatric disease (not including mild depression or anxiety) and pregnancy. Participants completed study specific questionnaires and underwent quantitative sensory testing. A venous blood sample was collected for the genetic analysis. Testing was carried out on one occasion during days 3–13 of the menstrual cycle. All participants received oral and written information about the study and provided informed consent and the study was approved by the local ethical committee. Main Outcome Measures
A comprehensive questionnaire surveyed medical (including psychosocial and gynecological) history as well as presence of dyspareunia and other frequent bodily pain symptoms in non-genital areas. A bodily pain score with a range from 0 (no other pain)—5 (five or more other pain symptoms) was created for each participant. Dysmenorrhea was not included in the pain score since the use of hormonal contraceptives varied among participants which can influence menstrual pain. PVD patients engaging in vaginal intercourse scored the intensity of coital pain during the last month on a visual analog scale (VAS) with a range from 0 (no pain)—100 (worst pain imaginable). Pressure pain thresholds (PPTs) on the arm and leg were measured with an examiner blinded to J Sex Med 2014;11:3064–3071
Genetic Analyses Venous blood samples were collected in EDTAtubes. Total genomic DNA was isolated from the whole blood samples using the Magtration 12GC system (Precision System Science, Chiba, Japan) and the Magazorb® DNA Common Kit-200 (PSS, Chiba, Japan). The DNA concentration was determined using a Nanodrop Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). Two SNPs in the 5HT-2A gene, T102C (rs6313) and A-1438G (rs6311), were analyzed using the TaqMan SNP genotyping assay, assay numbers C_3042197_1 and C_8695278_10 respectively (Applied Biosystems, Foster City, USA). Briefly, in each well of a 384-well plate, genomic DNA (5 ng), water, TaqMan Universal PCR master mix, and TaqMan genotyping assay mix was added, in a total volume of 5 μL. Target specific primers and TaqMan MGB probes labeled with two special dyes, FAMTM and VIC®, were included in the assay mix. The reaction was performed using an ABI17900HT genetic detection system (Applied Biosystems, Foster City, USA) with the following amplification protocol: 10 minutes at 95°C followed by 40 cycles of 15 seconds at 92°C and 1 minute at 60°C.
3067
5HT-2A Polymorphism Associated with PVD Table 1
Clinical background data
Variables
PVD patients (n = 98)
Controls (n = 103)
P value
Current age, years (range) Caucasian ethnicity; n (%) Duration of PVD, years (range) Primary PVD Secondary PVD Menstrual cycle day (range) Current use of HCs Dysmenorrhea Previous depression treatment; n (%) Present SSRI treatment; n (%)
29 (19–44) 95 (97) 8 (0.5–18) 35 (36%) 63 (64%) 7.9 (4–13) 35 (33%) 67 (71%) 64 (65) 6 (6)
24 (18–35) 99 (96) — — — 8.0 (3–13) 55 (54%) 55 (54%) 29 (28) 0 (0)
P < 0.001, t = −7.266 ns — — — ns P = 0.005 P = 0.02 P < 0.001, χ2 = 27.879 P = 0.011, χ2 = 6.500
Statistics The Statistica program (version 10, StatSoft Inc., Tulsa, OK, USA) and the Statistical package for the Social Sciences program (version 20, SPSS Inc., Chicago, IL, USA) were used. The student’s t-test or, for ordinal and non-normally distributed data, the Mann–Whitney U-test, were used for comparisons between groups regarding age and pain measurements. The chi-squared test was used for analyzing frequencies of previous depression treatment, pain disorders, and SNPs. For associations and interactions between the 5HT-2A polymorphism, pain measurements and PVD, univariate and multivariate logistic regression was used. A significance level of P < 0.05 and a confidence interval of 95% were used. Results
Clinical background data are summarized in Table 1. Controls were younger than PVD patients with a mean age difference of 5 years. There were no ethnical differences between the groups. PVD patients had previous or current treatment for depression to a higher degree than controls. PVD patients reported more frequent pain symptoms from non-genital body areas and Table 2
displayed higher pain sensitivity than controls in all pain modalities measured, see Table 2. Genotyping for the studied T102C (rs6313) and A-1438G (rs6311) SNPs in the 5HT-2A gene was completed in 201 subjects. The frequencies were in accordance with the Hardy-Weinberg equilibrium (all: P = 0.377, PVD patients: P = 0.058, controls P = 0.719). See Table 3. In accordance with previous studies the CT/CC- and AG/GG carriers were grouped together for further analyses [37]. The AA and TT genotypes were more common among controls compared with PVD patients. The odds ratio (OR) of having PVD was 2.9 among participants homo- or heterozygous for the G- or C-alleles compared with participants who were homozygous for the A- or T-alleles (OR = 2.9, CI: 1.2–6.9, P = 0.017). The bodily pain score in participants who were homo- or heterozygous for the G allele were higher than in those who were homozygous for the A allele, see Table 4. No other allele-linked differences in the pain measurements were found in the whole population or when PVD patients and controls were analyzed separately. There were no interaction effects of 5HT-2A polymorphism and PVD-patients vs. controls status with respect to the pain measurements.
Pain measurements
Concomitant bodily pain
PVD patients (n = 98)
Controls (n = 103)
P value
Headache; no (%) GI pain and dysfunction; no (%) Back pain; no (%) Muscle pain; no (%) Other pain; no (%) Bodily pain, score 0–5; mean (SD) median (Q1–Q3) Pain measurements PPT leg (kPa); mean (SD) median (Q1–Q3) PPT arm (kPa); mean (SD) median (Q1–Q3) PPT vestibule A (g); mean (SD) median (Q1–Q3) PPT vestibule B (g); mean (SD) median (Q1–Q3) Coital pain, VAS 0–100; mean (SD) median (Q1–Q3)
59 53 48 31 26 2.1
(60%) (54%) (49%) (32%) (27%) (1.2) 2 (1–3)
30 22 20 2 1 0.7
<0.001, <0.001, <0.001, <0.001, <0.001, <0.001,
405 268 48 42 53
(161) 390 (299–499) (124) 238 (189–331) (31) 40 (25–60) (44) 28 (15–50) (32) 54 (23–78)
474 (152) 457 (361–575) 309 (116) 298 (227–355) — — —
(29%) (22%) (20%) (2%) (1%) (0.9) 0 (0–1)
χ2 = 19.659 χ2 = 22.989 χ2 = 28.096 χ2 = 36.731 χ2 = 28.214 z = 7.859
0.001, z = −3.213 0.002, z = −3.142 — — —
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3068 Table 3
Heddini et al. Genotype frequencies
T102C (rs6313)
TT
TC
CC
P value
PVD patients (n = 97)*; n (%) Controls (n = 103); n (%)
8 (8) 21 (20)
53 (55) 49 (48)
36 (37) 33 (32)
0.051 χ2 = 5.940
A-1438G (rs6311)
AA
AG
GG
PVD patients (n = 98); n (%) Controls (n = 103); n (%)
8 (8) 21 (20)
53 (54) 49 (48)
37 (38) 33 (32)
0.047 χ2 = 6.092
*One missing due to error in analysis.
Conclusions
We aimed to investigate a possible correlation between the A-1438G and T102C SNPs in the serotonin receptor gene, 5HT-2A, and a diagnosis of PVD as well as pain sensitivity. In participants homo- or heterozygous for the G- and C-alleles we found a higher probability of PVD with an OR of 2.9. The results of the C-allele of the T102C SNP being more common among PVD patients compared with controls is concordant with previous result of a similar genotype distribution among female fibromyalgia patients [34]. The T102C polymorphism does not result in any alteration in the amino acid composition and has no influence on receptor protein; therefore so called linkage disequilibrium (i.e. co-inheritance) with the causative mutation has been proposed as a mechanism for the reported associations [34]. A meta-analysis of candidate gene studies of fibromyalgia found an overall OR of the association between the CT/CC genotype and fibromyalgia of 1.3–1.8 [37]. Our finding of an association between the same genotypes and PVD gives further support to the similarity of these conditions. There was also a correlation between concomitant bodily pain and the 5HT-2A SNPs with more pain symptoms from other body areas than the vestibule among G-/C- carriers. However, the Table 4
5HT-2A polymorphism was only correlated to the reported concomitant bodily pain symptoms and not to the experimental PPTs or the reported intensity of coital pain. This raises the question whether the observed association is correlated to the PVD diagnosis per se or to the other pain symptoms present in this group. On the other hand, the correlation to the concomitant bodily pain strengthens the similarity between PVD and syndromes such as fibromyalgia and CWP. Another shared feature of these conditions, apart from the unexplained pain, is the common comorbidity of anxiety and depression [9,22,43]. The serotonergic systems’ contribution to mood disorders and sexual desire is well-known [44] and it is therefore interesting to speculate whether differences in serotonin receptor gene polymorphism might, at least partly, explain both differences in pain sensitivity and psychological traits between women with PVD and healthy women. However, it is also possible that the higher reports of other pain symptoms among PVD patients are a result of the comorbid mood disorders [33]. The causative relationship between longstanding pain and sexual disability and heightened symptoms of anxiety and depression still remains unclear and needs to be further explored. Among the strengths of this study is a very reliable PVD diagnosis among cases, since they were
Pain measurements in relation to 5HT-2A polymorphism 5HT-2A genotype
P value
Pain measurements
AA/TT
n
AG + GG/TC + CC
n
PPT leg (kPa); mean(SD)/median(Q1–Q3) PPT arm (kPa); mean(SD)/median(Q1–Q3) Bodily pain, score 0–5; mean(SD)/ median(Q1–Q3) PPT vestibule A (g); mean(SD)/ median(Q1–Q3) PPT vestibule B (g); mean(SD)/ median(Q1–Q3) Coital pain VAS (0–100); mean(SD)/ median(Q1–Q3)
445 (151) 423 (326–560) 284 (100) 264 (225–334) 1.0 (1.0) 1 (0–3)
29 29 29
440 (161) 411 (329–529) 290 (125) 275 (196–344) 1.5 (1.3) 1 (0–4)
171 171 172
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55 (36) 45 (29–75)
8
47 (31) 39 (25–60)
90
ns ns 0.049 z = −1.962 ns
36 (44) 20 (6–50)
8
43 (45) 30 (15–50)
90
ns
56 (29) 61 (32–76)
8
53 (32) 54 (23–78)
90
ns
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5HT-2A Polymorphism Associated with PVD all examined by only a few physicians with very good knowledge of the condition. In addition, the menstrual cycle coordinated examinations reduces the risk of differences in pain sensitivity due to cycle variations. Nevertheless, there are several limitations. The controls were slightly younger than the PVD patients (see Table 1). Although genetic expression has been shown to change with ageing, comparing newborns with middle aged and elderly people [45], it is unlikely that a mean age difference of only 5 years would affect our results. Same age-span, ethnicity, geographical origin (in this case a larger city setting) and menstrual cycle phase was considered the most important matching variables and no other matching was made due to feasibility reasons. To avoid possible effects on pain sensitivity regular use of analgesic and anti-depressive drugs was an exclusion criterion for controls, which might have resulted in a control group with lower pain sensitivity and less depression than the normal population. Moreover, the confidence interval of the reported association between the 5HT-2A polymorphism and PVD was quite wide and approaching 1. The frequencies of the studied 5HT-2A SNPs have not previously been investigated in PVD patients and therefore the sample size was based on a previously outlined method to optimize sample size in candidate gene studies. Based on the frequency of the studied SNP and phenotype in the normal population 100 participants in each group was estimated to achieve a power of 80% to detect differences in SNP frequencies between PVD patients and controls [46]. Although the study group is quite large in the field of PVD research, in a candidate gene study an even larger material would be preferable to give more reliable results and allow for further sub analyses. Different etiologies of the two sub categories of PVD have been suggested, where women with primary PVD have displayed general pain hypersensitivity and mood disorders to a higher extent than women with secondary PVD [9,10]. In this study sub analyses were not performed due to the limitations in sample size (see Table 1) but there is a possibility that mixing the sub categories reduces the strength to find associations that might be stronger with one of the sub groups. Furthermore, susceptibility factors that might be inherited is not the sole requirement for a chronic pain condition to develop, most likely a triggering insult such as an infection or trauma is also necessary. This geneenvironment interaction could lower the sensitivity of genetic studies.
Another aspect is that coital pain and vestibular PPTs were only surveyed among PVD patients, resulting in a reduced power to detect possible differences in these measurements. This was decided in the design of the study since vestibular pain tolerance has previously been found to be consistently much higher in healthy women than in women with PVD and dyspareunia was an exclusion criterion for controls [17,22,42]. For example, a mean vestibular PPT of approximately 300 g in the posterior part of the vestibule of healthy women as compared with 20 g in women with PVD has been reported [47]. Our results indicate an involvement of the serotonergic system in the patho-physiology of PVD pointing towards an effect on pain sensitivity by polymorphism in the serotonin receptor 2A gene. These findings offer a possible genetic explanation to the similarity between PVD and fibromyalgia and give further evidence of PVD being part of a general pain disorder. The knowledge contributes with important information to help caregivers accurately select treatment options for PVD, including possible preventive measures to avoid aggravated pain problems. To continue to explore serotonergic signaling might possibly lead to new pharmacological treatment alternatives for this patient group. Corresponding Author: Ulrika Heddini, MD, Karolinska Institutet, Division of Obstetrics and Gynecology, Department of Clinical Sciences, Danderyd Hospital, Stockholm 182 88, Sweden. Tel: (+46) 8-12355000; Fax: (+46) 8-7531832; E-mail: [email protected] Conflict of Interest: The authors declare that they have no competing interests. Statement of Authorship
Category 1 (a) Conception and Design Nina Bohm-Starke; Ulrika Johannesson; Ulrika Heddini (b) Acquisition of Data Ulrika Heddini; Fred Nyberg; Alfhild Gronbladh (c) Analysis and Interpretation of Data Ulrika Heddini; Kent W. Nilsson
Category 2 (a) Drafting the Article Ulrika Heddini (b) Revising It for Intellectual Content Nina Bohm-Starke; Ulrika Johannesson; Fred Nyberg; Alfhild Gronbladh; Kent W. Nilsson J Sex Med 2014;11:3064–3071
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Category 3 (a) Final Approval of the Completed Article Ulrika Heddini; Nina Bohm-Starke; Ulrika Johannesson; Fred Nyberg; Alfhid Gronbladh; Kent W. Nilsson
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