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Intervention Study Type Comparison Main outcome F/U Effect Evidence Year of N measures time Quality 5 Publication / Ref Topical lidocaine cream DB, RPCT p.o.desipramine Tampon test 12 w Pain reduction: lidocaine 1 6 2010 (173) 5% for 12 w 133 150mg + (NRS) + 20%, desipramine 24%, (33+33+33+34) combination of 40 w 4 combination 36%, both + matching PBO 33% PBO Topical lidocaine cream RCT EMG for 4 m PPT, VAS 1 12 m Similar positive effect in both 2 6 2006 (172) 2–5% for 4 m 37 (18+19) groups Topical lidocaine cream Case series, - VAS 2 6 m In 57% ≥ 50% reduction of 3 7 2003 (171) 5% overnight for 7 w Prospective dyspareunia 61 Topical capsaicin cream Case series, - Marinoff 6 w Improvement 4 7 2005 (182) 0.025% for 12 w retrospective dyspareunia scale 47 Botulinum toxin A DB,RPCT Saline injections VAS 2, FSFI, 6 m No difference between 1 6 2009 (183) injection (20 U) 64 (32+32) SF-36 groups Botulinum toxin A Case series, - VAS 2, FSFI, 6 m VAS 2 decrease 3 7 2011 (184) injection (100 U) prospective DLQI from 8.3 to 2.7 20 Botulinum toxin A Case series, - VAS 2, FSFI, 24 m VAS2 decrease 47 2016 (185) injection (100 U) retrospective DLQI from 8.7 to 3.1 19 37% CR Topical gabapentin Case series, - VAS 2 8 w VAS2 decrease 4 7 2008 (186) cream 2% or 6% for 8 w retrospective from 7.3 to 2.5 32 Topical amitriptyline Case series, - Symptoms 3 m 10% CR, 46% PR, 3 7 2012 (187) cream 2% for 3 m Prospective and signs 44% NR 150 Sodium chromoglycate DB,RPCT PBO Dyspareunia 3 m No difference 1 6 2001 (188) cream 4% for 12 w 26 (13+13) between groups

 

 
 

 
 
  
 
Intervention Study Type Comparison Main outcome F/U Effect Evidence Year of N measures time Quality 5 Publication / Ref Potent steroid cream DB,RCT Mild steroid Pain, vestibular 6 w No difference 3 6,8 2004 (174) (clobetasol 0.05%) for 4 w 7 + 8 cream, HC 0.5% tenderness between groups Fibroblast cutaneous lysate DB,RPCT, PBO cream Dyspareunia 12 w Greater reduction of dyspareunia in 1 7 2012 (128) skin cream Crossover Vestibular active group, for 12 w 26 sensitivity no difference in sensitivity Topical baclofen 5% + Case report - Dyspareunia N/A Improvement 4 7 2014 (175) palmitoylethanolamid 1 cream for 12 w S.c. enoxaparin 40 mg DB,RPCT Saline Dyspareunia 6 m Reduction of dyspareunia 1 7 2012 (189) injections for 90 days 40 (20+20) injections CST 29% vs. 4%; Decrease in sensitivity 30% vs.11% Intralesional lidocaine + Case series - Dyspareunia 6 – 32% CR, 36% PR, 32% NR 3 7 2001 (190) methylprednisolone Prospective CST 24 m injections weekly for 3 w 19 Topical steroid (1% HC) RCT GCBT NRS 3, 6 m Significant reduction in pain in both 2 7 2016 (166) for 13 w 97 (45+52) McGill, FSFI groups Intralesional interferon Case series, - Dyspareunia 49% partial improvement 3 7 1993 (191) alpha prospective (verbal) Topical nifedipine cream RPCT PBO cream VAS 2 3 m All groups improved, No difference 1 6 2010 (192) 2% or 4% for 6 w 30(10+10+10) Topical estradiol 0.03% + Case series - Vestibular pain - 73% decrease 3 7 2013 (103) testosterone 0.01% cream Prospective score for 20 w 50 w=weeks, m = months, s.c. = sub-cutaneously, HC = Hydrocortisone, DB = double-blinded, RCT = randomized controlled trial, RPCT = randomized placebo controlled trial, PBO = placebo, EMG = electromyographic biofeedback, GCBT = group cognitive behavioral therapy, NRS = numerical rating scale, VAS = visual analogue scale, PPT = pressure pain threshold, SF-36 = Short Form general health survey, QOL=Quality of Life (VAS 0-100), FSFI = Female Sexual Function Index, DLQI = Dermatology Life Quality Index, CST = cotton swab test, F/U=follow-up, N/A=not available, CR=complete response, PR=partial response, NR=no response, Ref = reference. 1 VAS for pain in PPT testing, 2 VAS for dyspareunia (0-10), 3 NRS for dyspareunia, 4 Open label phase, 5 Level of Evidence: 1 = high, 2 = moderate, 3 = low, 4 = very low (Oxford Center of Evidence-Based Medicine – Levels of Evidence), 6 Evidence of no efficacy, 7 Evidence of efficacy, 8 Study interrupted

 
   

  
 




 


  
  
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Antibody/ Clone, Pre-treatment buffer Dilution, Detection system, Catalog Staining Evaluation/ Antigen Catalog number, (pH), Catalog number, Incubatio number, Manufacturer Instrument Analysis Manufacturer Manufacturer n time/°C CD3 2GV6, 790-4341, CC1 (pH 8.0), 950- RTU UltraView Universal DAB Ventana A single number score of T lymphocyte Roche 124, 32 Detection Kit, 760-500, Roche Benchmark the overall cell density for Diagnostics Ltd, Roche Diagnostics min/RT Diagnostics Ltd, Switzerland XT each section1 Switzerland Ltd, Switzerland

CD20 L26, 760-2531, CC1 (pH 8.0), Roche RTU UltraView Universal DAB Ventana Mean number of staining B lymphocyte Roche Diagnostics Ltd, 4 min/RT Detection Kit, 760-500, Roche Benchmark positive cells 2 Diagnostics Ltd, Switzerland Diagnostics Ltd, Switzerland XT Switzerland

IgA Polyclonal, Trypsin 0.5%, 1:2000 EnVision Detection Systems LabVision Mean number of staining 2 Plasma cell A0262, Agilent 37°C/25 min, Dicfo 30min/RT Peroxidase/DAB, K5007, Agilent positive cells Technologies, 250, 215310, Technologies, USA USA BD Bioscience, USA

CD163 NCL- Tris-EDTA (pH 9.0), 1:100 EnVision Detection Systems LabVision Mean number of staining Dendritic cell CD163,10D6, S2367, Agilent 40 Peroxidase/DAB, K5007, Agilent positive cells 2 Leica Biosystems Technologies, USA min/RT Technologies, USA Inc. USA

CD68 M0876, PG-M1, CC1 (pH 8.0), Roche 1:1000 UltraView Universal DAB Ventana Mean number of staining Macrophage Agilent Diagnostics Ltd., 24min/RT Detection Kit, 760-500, Roche Benchmark positive cells 2 Technologies, Switzerland Diagnostics Ltd., Switzerland XT USA

CD117 Polyclonal, CC1 (pH 8,0), Roche 1:400 UltraView Universal DAB Ventana Mean number of staining Mast cell A4502, Agilent Diagnostics Ltd., 32 Detection Kit, 760-500, Roche Benchmark positive cells 2 Technologies, Switzerland min/RT Diagnostics Ltd., Switzerland XT USA



  
  

 


 

  



 

Antibody/ Clone, Pre-treatment buffer Dilution, Detection system, Catalog Staining Evaluation/ Antigen Catalog number, (pH), Catalog Incubation number, Manufacturer Instrument Analysis Manufacturer number, time/°C Manufacturer CD117 Polyclonal, A4502, CC1 (pH 8,0), Roche 1:400 UltraView Universal DAB Ventana Mean number of staining Mast cell Agilent Diagnostics Ltd., 32 min/RT Detection Kit, 760-500, Roche Benchmark positive cells 2 Technologies, USA Switzerland Diagnostics Ltd., Switzerland XT

PGP9.5 Polyclonal, No pre-treatment 1:1000 EnVision Detection Systems LabVision Linear density of Small nerve RA95101, Ultra 30min/RT Peroxidase/DAB, K5007, IENFs 3; overall stromal fiber (C fiber) Clone Ltd., UK Agilent Technologies, USA density of nerve bundles

NF2F11 2F11, M0762 Tris-EDTA 1:200 EnVision Detection Systems LabVision + / - 4; overall stromal Light chain Agilent (pH 9.0),S2367, 30min/RT Peroxidase/DAB, K5007, density of nerve bundles neurofilament Technologies, USA Agilent Technologies, Agilent Technologies, USA USA

NGF Polyclonal, SC-548, EnVision FLEX 0.5 mg/ml MACH 4™ Universal AP Manual Mean number of staining 5 Nerve growth Santa Cruz Target Retrieval ON/4°C Polymer Kit, M4U536, Biocare positive cells factor Biotehnology Inc., Solution (pH 6.1) Medical Inc., USA USA K8005, Agilent Technologies, USA 1 A single number score of the overall cell density both in the epithelium and stroma for each section (from one to three, 1= low density, less than 50 cells/high power field (HPF x40 objective;); 2= moderate density, 50 – 100 cells/HPF; 3 = high density, more than 100 cells/HPF), 2 The mean number of identified positive cells per visual field (HPF x40 objective) calculated from 2 – 4 HPFs, 3 Number of PGP9.5-positive fibers / mm of epithelial outer surface, 4 + = presence of NF2F11-positive fibers, - = absence of NF2F11-positive fibers, 5 Positive immune cells per visual field (HPF x20 objective) were counted from 2-4 HPFs, each representing one of the 4 different types of areas (1. areas with increased B cell infiltration without intraepithelial nerve fibers (IENF), 2. areas without increased B cell infiltration with IENFs present, 3. areas with both increased B cell infiltration and IENFs, and 4. areas lacking both B cell infiltration and IENFs) CD = Cluster of Differentiation, PGP = Protein Gene Product, NF = neurofilament, CC = cell conditioning, RTU = ready to use, RT = room temperature, ON = overnight





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Vestibulectomy Conservative N=39 treatment, N=27 p value
Treatment modality

Discontinuation of COCs 9 / 25 (36.0) 12 / 17 (70.5) 0.094
Physical therapy 10 / 30 (33.3) 15 / 6 (57.7) 0.067

Long-term antifungal treatment 17 / 35 (48.6) 11 / 26 (42.3) 0.627

Submucosal or topical corticosteroids 18 / 31 (58.1) 9 / 26 (34.6) 0.077

Topical podophyllotoxin 1 19 / 31 (61.3) 19 / 26 (73.1) 0.347

Amitriptyline 2 13 / 31 (41.9) 7 / 21(33.3) 0.532

Duration of the conservative treatment period before
opting for surgery 9.0 (2–55) / 27 16.0 (3–129) / 22 0.052

Values are medians (minimum - maximum) / number of data available for continuous
variables, medians (IQR 25%–75%) for VAS values, and number of cases / data available (percentage) for categorical variables 1 Application of podophyllotoxin 5mg/ml (Wartec ®, Glaxo Smith Kline) on tender points of vestibular mucosa following 5% acetic acid application, repeated three times every four
weeks, 2 Amitriptyline 10 mg–50 mg, dose titrated on the basis of pain relief and tolerance COC = combined oral contraceptive


 
  



   
 
 


 

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Duration of operation, minutes 45 (20–90) / 58 Short-term complications 15 1 / 69 (21.4) Postoperative hematoma 6 (8.6) Wound pain or infection 11 (15.7) Bartholin’s duct cyst 2 4/70 (5.7) Absence from work, days 10.5 (3–24) / 60

Duration of wound pain, days 3 14 (0–90) / 49

Time to full recovery, weeks 3 5 (1–25) / 50

Values are medians (minimum - maximum)/ number of data available for continuous variables, medians (IQR 25–75%) for VAS for dyspareunia, and number of cases / data available (percentage) for categorical variables 1 Two patients had both hematoma and infection, 2 At 4, 16, and 24 months post-surgery 3 Data from questionnaire


 
  



  
   


 
 
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AC TA Obstetricia et Gynecologica

MAIN RESEARCH ARTICLE Long-term follow up of posterior vestibulectomy for treating vulvar vestibulitis PAIVI¨ TOMMOLA, LEILA UNKILA-KALLIO & JORMA PAAVONEN Department of Obstetrics and Gynecology, Helsinki University Central Hospital, University of Helsinki, Helsinki, Finland

Key words Abstract Dyspareunia, surgical treatment, sexual problems, vulvar vestibulitis, vestibulectomy, Objective. To evaluate the safety and the effectiveness of posterior vestibulectomy in vulvar pain the treatment of vulvar vestibulitis syndrome. Design. A retrospective cohort study. Setting. University Hospital, tertiary referral center. Population. Seventy women Correspondence treated by posterior vestibulectomy for severe vulvar vestibulitis syndrome during Paivi¨ Tommola, MD, Department of Obstetrics 1995–2007 at the Department of Obstetrics and Gynecology, University Hospital, and Gynecology, University Hospital Helsinki, Helsinki. Methods. All operated women were invited to a long-term follow-up study. PO Box 140, 00290 Helsinki, Finland. E-mail: paivi.tommola@kolumbus.fi¨ Patient characteristics, baseline visual analog scale (VAS) for dyspareunia and data from the postoperative period were collected. Of the 70 women, 57 attended the Conflicts of interest follow-up visit including face-to-face interview, gynecological examination with The authors have stated explicitly that there swab-touch test for vestibular tenderness, current VAS score for dyspareunia and are no conflicts of interest in connection with McCoy questionnaire for sexual problems. Main Outcome Measures. Short-term this article. and long-term complication rates, dyspareunia by VAS score, vestibular tenderness, sexual problem index and overall patient satisfaction. Results. Ninety-one per cent Received: 27 January 2011 were satisfied with the outcome. The VAS for dyspareunia decreased from a median Accepted: 21 July 2011 of 9 to 3 (66.7% decrease; p<0.001). Posterior vestibular tenderness was absent in DOI: 10.1111/j.1600-0412.2011.01248.x 34 patients (64.2%). Six (8.6%) patients developed postoperative bleeding and 11 (15.7%) mild wound infection. Bartholin’s cysts occurred in four (5.7%) patients. Conclusions. Posterior vestibulectomy is effective and safe in the treatment of severe vulvar vestibulitis syndrome and provides long-term patient satisfaction.

Abbreviations: IQR, interquartile range; ISSVD, International Society for the Study of Vulvovaginal Disease; SPSS, Statistical Package for Social Sciences; VAS, visual analog scale; VVS, vulvar vestibulitis syndrome

of this original operation have been developed (8). However, Introduction studies on surgical treatment have been criticized for being Vulvar vestibulitis syndrome (VVS) is a subset of vulvody- non-randomized, having poor outcome definitions and lack nia. It causes vulvar pain and dyspareunia, ruining the sexual of data of complications. In most studies, success is defined life of young premenopausal women (1,2). Vulvar vestibu- only by patients’ self report, with data collected by postal or litis syndrome is characterized by severe pain in the vul- telephone interviews. Here we report systematic short-term var vestibule provoked by touch or attempted vaginal entry. and long-term outcome of surgical treatment of severe VVS. Prevalence rate up to 15% in general gynecologic practice population has been reported (3). The International Society Material and methods for the Study of Vulvovaginal Disease (ISSVD) defines VVS as localized, provoked vulvodynia (4). The study group consisted of 70 women who had undergone Treatment of VVS is challenging because the etiology is vestibulectomy during 1995–2007 in Helsinki University unknown. Treatment guidelines are mostly based on anecdo- Central Hospital, Department of Obstetrics and Gynecol- tal clinical observations or uncontrolled data from case series ogy, Vulva Clinic. Data on preoperative symptoms, clinical (5,6). Surgery as a treatment option for severe VVS was first status, short-term recovery and complications were col- documented by Woodruff et al. in 1981 (7). Modifications lected from the hospital charts. All women were invited to a

C 2011 The Authors Acta Obstetricia et Gynecologica Scandinavica C 2011 Nordic Federation of Societies of Obstetrics and Gynecology 90 (2011) 1225–1231 1225 Vestibulectomy follow-up study P. Tommola et al. follow-up visit in the same clinic. The local Ethical Commit- teehadapprovedthestudy. The Vulva Clinic is a tertiary referral center for patients with vulvar conditions, including vulvar vestibulitis. All study patients were managed according to a predefined algorithm, which is in line with the current vulvodynia treatment guide- lines (5). The diagnosis of VVS was based on Friedrich’s classical criteria: pain on vestibular touch or attempted vagi- nal entry causing dyspareunia and positive swab-touch test of the vestibular gland openings with or without erythema of the involved mucosa (9). Swab-touch test result was recorded by using categories ‘significant’ (verbal expression of intense pain or expression of pain by sudden move), ‘mild’ (verbal expression of mild pain) or ‘none’ (no pain). Dyspareunia was recorded by visual analog scale (VAS) from 0 (no pain) to 10 (worst possible pain; 10). Dermatological conditions were ruled out by histopathological examination of punch biopsies when appropriate, and vaginal infections were ruled out by bedside wet-mount microscopy. Patients were also referred to a physical therapist. These visits included both electrostimulation to activate pelvic floor muscles and to re- lieve pain, and biofeedback therapy for better control and voluntary relaxation of pelvic floor muscles. Education was provided to relieve penetration pain and fear of intercourse. Patients who did not respond to the conservative manage- ment but continued to report VAS score 7 or more were counseled for vestibulectomy. Vestibulectomies were performed under general anesthesia Figure 1. (A) Modified posterior vestibulectomy technique. (B) Postop- by three senior gynecological surgeons. A modified posterior erative appearance, immediately (left) and after two months (right). vestibulectomy technique was used (Figure 1). Briefly, two parallel incisions with cutting electrocautery were made in All women were invited by letter to participate in a long- the posterior vestibule reaching from 2 to 10 o’clock after term follow-up study. The follow-up visits were conducted infiltrating the submucous layer with lidocaine–adrenaline during September 2005 and May 2008 by one senior gyne- 0.05% solution. The inner incision line was made just inside cologist (P.T.), who had not been involved in the preoper- the hymenal ring and the peripheral incision to the Hart’s line. ative management or operations. At the follow-up visit the The posterior vestibular mucosa was then excised by super- women were interviewed face to face using a structured ques- ficial skinning vestibulectomy using cutting electrocautery. tionnaire. They estimated dyspareunia by VAS score. Their The vaginal mucosa was undermined, leaving tissue bridges current general health and the effect of vestibulectomy on for adequate blood supply. The denuded area was covered symptoms, as well as their preference for choosing the same with the undermined vaginal mucosa. The wound was closed operation again were reviewed. Thereafter, a thorough gyne- with interrupted stitches using 2–0 absorbable Dexon R .A cological examination was carried out. Vestibular tenderness single dose of intravenous metronidazol (500 mg) followed by was evaluated with a swab-touch test separately in the ante- 400 mg three times daily for three days was given. Postopera- rior (from 11 and to 1 o’clock) and posterior region (from tive pain was managed with non-steroidal anti-inflammatory 10 to 2 o’clock) with (equal) light touch administered each drugs (ibuprofen 400–600 mg or paracetamol 1g three times time.Thetestresultwasdocumentedasdescribedabove. daily). Showering of the wound frequently was advised. The A follow-up visit to a physical therapist for evaluation of patients were told to avoid physical distress for three weeks. pelvic floor muscle function (Pelvimed 932, Enraf Nonius Sick leave was recommended for 7–21days on an individual B.V., Rotterdam, the Netherlands; Periform vaginal probe, basis. Routine follow-up visits took place at one and two Patterson Medical Ltd., Nottinghamshire, UK) was offered. months. At the first visit the women were encouraged to Resting tone value 10μV or less was defined as good ability perform daily dilatation therapy with a vaginal probe, and in voluntary relaxation. The McCoy questionnaire for sex- resumption of intercourse was permitted. ual well-being (11) was completed. To assess sexual problems

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Table 1. Baseline characteristics and short-term recovery after cephalosporin and metronidazole for one week. Thus, any vestibulectomy (n=70). adverse effect with revisit occurred in 15 (21.4%) patients.

Age at operation (years) 25.5 (18–50)/70 Unilateral Bartholin’s cyst was detected in four (5.7%) Birth control pill use at onset of symptoms 48/67 (68.6) women four, 16 and 24months after surgery. All four were Parous 7/69 (10.1) managed by day surgery. History of psychiatric problems 7/67 (10.4) History of dyspareunia (years) 4.0 (1–18) Baseline VAS score 9.0 (8.0–10.0)/64 Long-term follow up Median operation time (minutes) 45 (20–90)/46 Of the 70 women invited to attend the long-term follow Short-term complications 15∗/70 (21.4) Postoperative hematoma 6 (8.6) up, 57 (81.4%) consented. The only difference between the Wound pain or infection 11 (15..7) 57 attendees and 13 non-attendees was history of psychi- Bartholin’s cyst 4/70 (5.7) atric problems (5.4 vs. 36.4%, p=0.005). Fifty-three (93.0%) Primary sick leave (days) 10.5 (3–24)/62 women were interviewed and examined, and 55 returned Duration of wound pain (days) 14 (0–90)/49 the questionnaire. Two of those examined did not return Time to full recovery (weeks) 5 (1–25)/50 the questionnaire. Four patients returned the questionnaire Note: Values are medians (minimum–maximum)/number of data avail- only, and two of them participated in a structured telephone able for continuous variables, median (interquartile range 25–75%) for interview. The follow-up visit took place after a median of visual analog scale (VAS) values, and numbers of cases/data available 36months (range 5–158months), at the age of 29years (range (percentage) for categorical variables. 20–57years). Of the women, 39 (68.4%) were married or co- ∗ Two patients had both hematoma and infection. habiting and 35 (63.6%) had the same partner as before. An additional five women had a regular sexual relationship and we used a sexual problem index, a calculated sum of reported 11 (20%) were single. Women living in a relationship had a frequency of dryness during sexual acts and frequency of dys- median of two (range 0–20) sexual acts during the preceding pareunia (scale from 1 to 7 representing never to always; 12). month. Eight (14.5%) women had delivered vaginally after Frequency of sexual acts during the preceding one month vestibulectomy. was recorded. The data were analyzed by Statistical Pack- Topical anesthetic for dyspareunia was used by seven (13%) age for Social Sciences (SPSS version 16; SPSS Inc., Chicago, of 51 women. Fourteen (25.9%) women reported some de- IL, USA). We report medians with interquartile range (IQR gree of constant pain without touch. This did not depend on = 25–75%) for VAS values and medians with minimum and the duration of symptoms before treatment (p 0.573). Two maximum values for other continuous data. For comparisons (3.7%) women used oral amitriptyline alone or in combi- we used Mann–Whitney U-test or Kruskall–Wallis test and nation with pregabalin. A second vestibulectomy operation Wilcoxon signed rank test for continuous data and χ 2 anal- had been performed on two women, both after four and a ysis or Fischer’s exact test for categorical data. A two-tailed half years, one in the anterior vestibule with a Bartholin’s cyst p-value of <0.05 was considered significant. For correlations extirpation and the other in the posterior vestibule. Pearson’s correlation coefficient was used. The median VAS score for dyspareunia at follow up was 3.0 (IQR 0.6–4.9), significantly lower than the baseline score Results of 9.0 (IQR 8.0–10.0; p<0.001; Figure 2). The decrease in VAS score was 50% or more in 36 (69.2%) women and less Clinical characteristics of the study population are given in than 15% in three (5.8%) women. The VAS score remained Table 1. Most women were relatively young nulliparas with a unchanged in two (3.8%) women, but no woman reported long history of severe VVS. an increase in VAS score. Follow-up VAS was not associated with the patient’s age, length of history prior to operation Short-term recovery and complication analysis or time after operation, previous history of constant pain, Data on short-term recovery and complications are shown occurrence of a postoperative complication, vaginal delivery in Table 1. Vestibulectomy was performed as day surgery, after the operation or current use of contraceptive pills (data but 14 (20%) women needed an overnight stay because not shown). of pain, nausea, bleeding or difficulty in voiding. Half of Tenderness in the posterior vestibule was absent in 34 the operations were performed in 45minutes or less. Post- (64.2%) women, mild in 11 (20.8%) and significant in eight operative bleeding occurred in six women, of whom four (15.1%) women. The corresponding rates in the anterior (5.7%) required surgery. Eleven (15.7%) patients visited the vestibule, i.e. the area not included in the operation, were 15 outpatient clinic for postoperative pain or symptoms con- (28.3%), 14 (26.4%) and 24 (45.3%). Significant tenderness sistent with non-febrile wound infection or inflammation, in both regions was present in six (11.3%) women. Gyneco- two with hematomas and received a combination of oral logical examination revealed no scarring. Four women had

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complete response, 30 (55.6%) partial response and five (9.3%) no response. The response did not depend on du- ration of the follow up (p=0.401), short-term complications (p=0.320) or the surgeon in charge (p=0.970). Table 2 shows other outcome measures based on subjective experience. Of the 54 women, 49 (90.7%) would have chosen the oper- ation again, four (7.4%) were uncertain and one (1.9%) would have declined. This patient developed a postopera- tive hematoma and wound infection followed by refractory pain syndrome. All four women who were uncertain were in the partial response group, with significant tenderness in the anterior vestibule.

Cases with non-optimal outcome Overall, 18 women had a non-optimal outcome (defined as no response by subjective estimation, follow-up VAS score ≥7, significant tenderness present in the posterior vestibule, or reporting persistent dyspareunia; (Table S1). Such an out- come did not depend on duration of the disease before Figure 2. Visual analog scale (VAS) score for dyspareunia of 52 women treatment (p=0.343) or duration of postoperative follow up at baseline and at follow-up visit. (p=0.213). Of the eight patients with significant remaining posterior tenderness, four reported VAS scores ≥4, three re- persistent vulvar fissure; three of the four had fissure already ported VAS scores <4 and one was not sexually active because at baseline. Women with significant tenderness in the swab- of fear of intercourse. This patient and four other patients re- touch test at follow up reported higher VAS scores for dys- ported no response by surgery. Two of these had significant pareunia (4.0; IQR 2.0–5.0) than those with a non-tender test anterior tenderness with dysesthetic pain, one reported dyses- (0.75; IQR 0.0–3.75; p=0.035). thetic pain and one had a posterior vulvar fissure. Five other Resting tone of the pelvic muscles was measured in 27 patients reported persistent dyspareunia, four of whom had (51%) women. Fourteen (51.8%) showed adequate volun- anterior tenderness and one had a vulvar fissure. One patient tary relaxation (resting tone 10μV or less). They reported reported a VASscore of 7 without tenderness. Thus, only four a median VAS score of 3.0 (IQR 0.8–6.3), while the score patients were considered as pure surgical failures. for those with resting tone more than 10μVwas4.5(IQR = 0.8–5.5; p 0.837). Thirty-four (92%) of 37 women consid- Discussion ered their current voluntary pelvic floor muscle relaxation as satisfactory. Posterior vestibulectomy resulted in 91% long-term satisfac- Forty-four (80%) women reported a regular sexual rela- tion. Significant improvement of posterior vestibular tender- tionship, 50 estimated current dyspareunia and 52 completed ness was achieved in 85% of the patients. Earlier studies on the McCoy questionnaire. Seven (14%) women reported no surgicaltreatmenthavereportedsuccessratesupto89%of dyspareunia and 29 (58%) reported at least half of the inter- cases (13). However, the outcome has been poorly defined in courses as painless. Dyspareunia was persistent in 10 (20%) most previous studies, based on patients’ self reports with no women. Median problem index score was 8.0 (range 2–14). objective measures, and many previous studies lack data on Women with tenderness in the anterior vestibule reported complications, sexual functioning, residual tenderness and higher problem index scores (median 8.0; range 2–14) than short-term recovery (8); however, such information is im- those with a non-tender anterior vestibule (median 6.0; range portant when counseling patients for surgery. 2–13; p=0.046). The current VAS score and the problem in- We aimed to evaluate the short- and long-term well-being dex score were significantly correlated (Pearson’s correlation of 70 women who underwent modified posterior vestibulec- coefficient 0.71; p<0.001). Also, the VAS score correlated tomy. In our study, the inclusion criteria were similar to other with the intensity of dyspareunia in the McCoy question- studies (Friedrich’s criteria) and surgery was performed as the naire (Pearson’s correlation coefficient 0.77; p<0.001). last treatment option after conservative treatment modalities The women were asked whether they had been cured by had failed. This cohort study provides unique new data on the operation (complete response), still had some complaints the safety and effectiveness of vestibulectomy. The thorough (partial response) or were the same or worse than before op- gynecological examination and the interview at the follow-up eration (no response). Of 54 women, 19 (35.2%) reported visit were both conducted by a senior gynecologist who had

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Table 2. Outcome measures and patient satisfaction of 54 patients with vestibulectomy.

Complete response Partial response No response (n=19; 35.2%) (n=30; 55.6%) (n=5; 9.3%) p-Value

VAS score (n)19303 Median VAS score 0.5 (0.0–1.0) 4.0 (2.5–5.3) 10 (7.0–10.0) <0.001 Change of VAS score of >50% 19 (100) 16 (53.3) 1 (33.3) <0.001 Swab touch-test results (n)17 30 5 Positive 10 (58.8) 26 (86.7) 3 (60) 0.001 Negative 7 (41.2) 4 (13.3) 2 (40.0) 0.001 Anterior vestibule tenderness 0.155 Significant 4 (23.5) 17 (56.6) 3 (60) Mild 5 (29.4) 8 (26.7) 0 No 8 (47.1) 5 (16.7) 2 (40.0) Posterior vestibule tenderness 0.086 Significant 0 7 (23.3) 1 (20.0) Mild 1 (5.9) 8 (26.7) 2 (40.0) No 16 (94.1) 15 (50.0) 2 (40.0) Pelvic floor muscles (n)7 17 3 Resting tone (μV) 11 (5.7–28) 10 (2.9–24) 7 (5–15) 0.699 Resting tone ≤10μV 3 (42.8) 9 (52.9) 2 (66.7) 1.000 Problem index score∗ (n)19 28 1 5.0 (2–10) 9.0 (2–14) 10.0 <0.001

Note: Values are medians (minimum–maximum)/number of data available for continuous variables, median (interquartile range 25–75%) for visual analog scale (VAS) values, and numbers of cases/data available (percentage) for categorical variables. ∗ Scale 2–14 (less problems–more problems).

notbeeninvolvedinthetreatmentofthepatients,further well with other measures of satisfactory response, i.e. lack increasing validity of the study. of vestibular tenderness and low problem index score. Thus, Owing to the retrospective design, there are some lim- the VAS score and its change can be recommended as a main itations. Our study lacks detailed baseline data on sexual outcome measures in sexual pain research. problems; however, all women entering operative treatment Surgery did not totally correct posterior tenderness in eight reported high VASscores, indicating problems which severely (15%) of the patients as evaluated by the swab test. Although interfered or totally prohibited sexual intercourse. A long in- posterior vestibulectomy aims to cover the whole posterior terval between the operation and the follow-up visit may have vestibule from 2 to 10 o’clock, it is possible that the surgeon biased some subjective statements. in some cases misjudges the peripheral extension, which may The rate of immediate postoperative adverse effects was result in residual tenderness in the posterior vestibule. The relatively high (21.7%). In this type of operation, postoper- high rate of residual anterior tenderness (45%) is not sur- ative hemorrhage or mild wound infections are not unex- prising, because the anterior vestibule is not included in this pected (14). Also, these patients may be anxious about their operation. It is worth considering whether total vestibulec- symptoms, leading to a low threshold for attending the out- tomy might be a better choice. Some previous studies have patient clinic. It is noteworthy that all wound infections were used techniques other than posterior vestibulectomy without non-febrile and mild. Some cases of wound inflammation demonstrating superior results. Our choice of the posterior may have been overdiagnosed as infections. Our rate of 5.7% vestibulectomy technique was supported by this being the of postoperative Bartholin’s cysts is in line with previous region where most patients locate the symptoms (16) and by reports (15). an assumption that posterior tenderness has more impact on A VAS score was used to evaluate the severity of dyspareu- dyspareunia than anterior tenderness. nia (10), enabling objective comparison of preoperative and Wedisagree with previous studies claiming that pelvic floor postoperative conditions. The VAS score was obtained both muscle dysfunction is the main reason for residual dyspare- at baseline and at follow up, and showed a 66.7% reduc- unia (17). This is because our patients in all response groups tion. The VAS is a simple, reproducible and valid indicator of had similar voluntary pelvic floor muscle relaxation. How- pain severity. In our hands, a VAS score ≥7reflectednore- ever, undoubtedly the whole process of physical therapy, in- sponse to conservative management followed by counseling cluding counseling, caring and psychological support, aug- for vestibulectomy. A low VAS score at follow up correlated ments the healing process.

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Overall patient satisfaction is an important outcome mea- the prevalence of vulvodynia? J Am Med Womens Assoc. sure of surgery. Women’s subjective evaluation of the out- 2003;58:82–8. come most likely reflects the experience of pain and quality 3. Goetsch MF. Vulvar vestibulitis: prevalence and historic of sexual life, expressed by the VAS and problem index score. features in a general gynecologic practice population. Am J In our material, a VAS score of 0.5 and problem index score Obstet Gynecol. 1991;164:1609–14; discussion of 5.0 in the complete response group predicted high pa- 1614–16. tient satisfaction. The problem index score was only slightly 4. Moyal-Barracco M, Lynch PJ. 2003 ISSVD terminology and higher than the score after hysterectomy (4.1) performed for classification of vulvodynia: a historical perspective. J Reprod menorrhagia (12) in a population which presumably has less Med. 2004;49:772–7. sexual problems than our VVS patients. However, the higher 5. Haefner HK, Collins ME, Davis GD, Edwards L, Foster DC, VAS score and problem index in the partial response group Hartmann ED, et al. The vulvodynia guideline. J Low Genit suggest that other treatment modalities beyond surgery are Tract Dis. 2005;9:40–51. needed. Eight patients had residual posterior tenderness, but 6. Mandal D, Nunns D, Byrne M, McLelland J, Rani R, only four of them were considered surgical failures. Although Cullimore J, et al. Guidelines for the management of surgery decreased tenderness, other co-existing problems ex- vulvodynia. Br J Dermatol. 2010;162:1180–5. plained poor outcome in other cases. 7. Woodruff JD, Genadry R, Poliakoff S. Treatment of It is generally known that substantial relief of symptoms dyspareunia and vaginal outlet distortions by perineoplasty. can be obtained by conservative treatment modalities as well. Obstet Gynecol. 1981;57:750–4. In some women, symptoms may even resolve spontaneously 8. Tommola P, Unkila-Kallio L, Paavonen J. Surgical treatment of vulvar vestibulitis: a review. Acta Obstet Gynecol Scand. (6,18,19). This suggests that not all patients need surgery. 2010;89:1385–95. In addition, a relatively large placebo effect is involved in all 9. Friedrich EG Jr. Vulvar vestibulitis syndrome. J Reprod Med. treatments of VVS, as demonstrated in a recent randomized 1987;32:110–14. placebo-controlled trial of oral desipramine and topical lido- 10. Huskisson EC. Measurement of pain. Lancet. caine (20). So far, surgery has been offered as the last resort 1974;2:1127–31. to the most severe cases. In fact, women not responding to 11. McCoy NL, Davidson JM. A longitudinal study of the effects conservative treatment modalities may ultimately represent of menopause on sexuality. Maturitas. 1985;7:203–10. a specific subtype of severe VVS. Randomization of poor re- 12. Hurskainen R, Teperi J, Rissanen P, Aalto AM, Grenman S, sponders to surgery, to continue conservative treatment or to Kivela A, et al. Quality of life and cost-effectiveness of observation only might best reveal whether surgery is supe- levonorgestrel-releasing intrauterine system versus rior. However, these patients may be difficult to recruit into hysterectomy for treatment of menorrhagia: a randomised randomized trials because they are already frustrated with trial. Lancet. 2001;357:273–7. several ineffective treatment attempts. In the open label phase 13. Bornstein J, Zarfati D, Goldik Z, Abramovici H. Vulvar of the recent randomized controlled trial (20), patients opt- vestibulitis: physical or psychosexual problem? Obstet ing for vestibulectomy instead of continuing medical therapy Gynecol. 1999;93:876–80. reported greatest improvement. Likewise, our patients opted 14. Smith EM, Ritchie JM, Galask R, Pugh EE, Jia J, for surgery after a long conservative treatment period with Ricks-McGillan J. Case-control study of vulvar vestibulitis poor response. This may have eliminated the placebo effect, risk associated with genital infections. Infect Dis Obstet at least to some extent. Posterior vestibulectomy seems a safe, Gynecol. 2002;10:193–202. effective and well-tolerated treatment option for severe VVS 15. Goetsch MF. Incidence of Bartholin’s duct occlusion after when conservative treatment modalities have failed. superficial localized vestibulectomy. Am J Obstet Gynecol. 2009;200:688.e1–6. Funding 16. Kehoe S, Luesley D. Vulvar vestibulitis treated by modified vestibulectomy. Int J Gynaecol Obstet. 1999;64:147–52. The study was supported by grants from the Helsinki 17. Goetsch MF. Surgery combined with muscle therapy for University Hospital Research Funds. dyspareunia from vulvar vestibulitis: an observational study. J Reprod Med. 2007;52:597–603. 18. Landry T, Bergeron S, Dupuis MJ, Desrochers G. The References treatment of provoked vestibulodynia: a critical review. Clin J 1. Meana M, Binik YM, Khalife´ S, Cohen D. 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20. Foster DC, Kotok MB, Huang LS, Watts A, Oakes D, Howard Table S1. Details of 18 patients with non-optimal outcome FM, et al. Oral desipramine and topical lidocaine for (defined as ‘no response’, VAS ≥7 at follow up, significant vulvodynia: a randomized controlled trial. Obstet Gynecol. residual tenderness in the posterior vestibule or persistent 2010;116:583–93. dyspareunia).

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AC TA Obstetricia et Gynecologica

AOGS MAIN RESEARCH ARTICLE Long-term well-being after surgical or conservative treatment of severe vulvar vestibulitis PAIVI¨ TOMMOLA, LEILA UNKILA-KALLIO & JORMA PAAVONEN Department of Obstetrics and Gynecology, Helsinki University Central Hospital, University of Helsinki, Helsinki, Finland

Key words Abstract Beck, dyspareunia, McCoy, posterior vestibulectomy, sexual well-being, Objective. To compare long-term well-being of women who needed surgery or vestibulodynia, vulvar pain did not need surgery in the treatment of severe vulvar vestibulitis syndrome. We also attempted to identify factors explaining differences in the treatment response. Correspondence Design. An observational case-control study. Setting. University Hospital Vulva Paivi¨ Tommola, Department of Obstetrics and clinic. Population. Sixty-six women diagnosed with severe vulvar vestibulitis Gynecology, University Hospital Helsinki, P.O. Box 140, 00029 Helsinki, Finland. E-mail: and treated initially by conservative management during 1994–2005. Thirty-nine paivi.tommola@kolumbus.fi women did not respond and underwent posterior vestibulectomy and 27 were managed without surgery. Methods. Baseline patient characteristics, degree of dys- Conflict of interest pareunia, and details of management were collected from hospital charts. At the The authors have stated explicitly that there follow-up visit current dyspareunia, sexual well-being, somatic and mental health, are no conflicts of interest in connection with and social support were analyzed. Vestibular tenderness was measured by swab- this article. touch test. Main outcome measures. Visual analogue scale for dyspareunia, sexual well-being, vestibular tenderness, and overall patient satisfaction at follow-up. Re- Please cite this article as: Tommola P, Unkila-Kallio L, Paavonen J. Long-term sults. Dyspareunia decreased significantly in both groups. The visual analogue scale well-being after surgical or conservative decreased 66.7% in the surgery group and 78.1% in the conservative treatment group treatment of severe vulvar vestibulitis. Acta (p = 0.407). Posterior swab-touch test was negative more frequently after vestibulec- Obstet Gynecol Scand 2012;91:1086–1093. tomy. Long-term sexual well-being did not differ between the two groups. Overall, 89% of the women in both groups were satisfied with the treatment. Women with Received: 18 November 2011 atopic skin problems were less likely to need surgery (odds ratio 0.2; 95% confidence Accepted: 6 May 2012 interval 0.1–0.7). Conclusion. Women with severe vulvar vestibulitis syndrome who do not respond to conservative management achieve good long-term well-being DOI: 10.1111/j.1600-0412.2012.01466.x and decrease of dyspareunia by posterior vestibulectomy. The response is compara- ble to that achieved by conservative management among women who do not need surgery.

Abbreviations: VAS, visual analogue scale; VVS, vulvar vestibulitis syndrome.

follow-up (7), and there are few randomized trials (8,9). Introduction Surgery, i.e. vestibulectomy (10,11), is usually offered to In vulvar vestibulitis syndrome (VVS), also called localized provoked vulvodynia, pain by touch in the vulvar vestibule Key message causes dyspareunia and ruins the sexual life of many young women (1,2). The etiopathogenesis of the disease is unknown Women with severe vulvar vestibulitis syndrome who do and treatment is challenging. The main symptom, dyspare- not respond to conservative management can achieve unia, has been managed by topical anesthetics (3), corticos- good long-term well-being and a decrease of dyspareunia teroids (4), topical or systemic neuropathic pain medications by posterior vestibulectomy. The response is comparable (5,6) based on current guidelines of VVS treatment. How- to conservative management among women who do not ever,the effectiveness and safety of most non-surgical inter- need surgery. ventions to treat VVS are poorly verified, with only short

C 2012 The Authors 1086 Acta Obstetricia et Gynecologica Scandinavica C 2012 Nordic Federation of Societies of Obstetrics and Gynecology 91 (2012) 1086–1093 P. Tommola et al. Vulvar vestibulitis treatment study women refractory to conservative management with good All VVS women at the Vulva clinic are managed accord- success; the proportion of women satisfied after surgery ing to a predefined algorithm (Supporting Information Ap- ranges from 78 to 91% (12,13). Most previous studies of pendix S1) in line with international vulvodynia treatment surgical treatment place an emphasis only on the decrease of guidelines (17). The diagnosis of VVS was based on the dyspareunia and few studies report sexual well-being or qual- Friedrich’s classical criteria (18). Vestibular tenderness was ity of life in the long-term (14). Few studies compare con- evaluated with swab-touch test and intensity was recorded as servative treatment and surgery (9,15,16). Toour knowledge, ‘none’ (no pain), ‘mild’ (verbal expression of pain) or ‘sig- no study has compared the results of conservative manage- nificant’ (expression of pain by sudden move). Dyspareunia ment and surgery in women with severe symptoms. Patient was recorded by VAS from zero (no pain) to 10 (worst possi- characteristics predicting response to conservative manage- ble pain). Vaginal infections were ruled out by bedside wet- ment are unknown. To further evaluate the role of surgical mount microscopy. Dermatological diseases were excluded treatment in severe VVS we report this case-control study by obtaining a punch biopsy when clinically indicated. After on long-term well-being of severe VVS women managed by the diagnosis of VVS was confirmed, women were counseled surgery or without surgery. about the treatment options available. All women were ad- vised about general vulvar care measures. Oral contraceptives Material and methods were withdrawn if possible. Long-lasting antifungal therapy The study population consisted of 66 women diagnosed with with fluconazole was prescribed for women with history of severe vulvar vestibulitis syndrome (VVS) at the Vulva clinic, recurrent yeast infections. When appropriate, local corticos- Department of Obstetrics and Gynecology, University Hos- teroid (injected into the submucosa in a mixture with local pital, Helsinki, Finland during 1994–2005. The Vulva clinic anesthetic or as a topical cream) was used, or amitriptyline orally was prescribed. In some women, mucosal neuromod- is a tertiary referral center for women with vulvar conditions. R Originally, we identified from hospital records 52 women ulation with topical podophyllotoxin 5 mg/mL (Wartec ) treated by posterior vestibulectomy for severe VVS after failed application was used. Women were also referred to a physical conservative management. As controls we searched for 50 therapist for bio-feedback therapy of the pelvic floor muscle women with the same inclusion criteria (severe VVS symp- dysfunction, sexual counseling, and education. Women who toms with visual analogue scale (VAS) score for dyspareunia did not respond to conservative management were offered ≥7, duration of symptoms at least 12 months, absence of surgery, a modified posterior vestibulectomy (13). vulvar dermatosis, and equal timing of treatment period) Details of the conservative treatment modalities used dur- who had not undergone vestibulectomy. Thirty-nine (75%) ing the initial treatment period (before opting for surgery of 52 vestibulectomy women (surgery group) and 27 (54%) or at dismissal after satisfactory conservative treatment) in of 50 conservatively treated women (conservative treatment both groups were collected from the hospital charts. The group) consented and were enrolled in a long-term follow-up long-term follow-up visit of the surgery group took place study (Figure 1). The local Ethical Committee approved the in October 2005 and of the conservative treatment group in study. September 2009. Both visits were conducted by one senior gynecologist (P.T.) who had not been involved previously in the patient management. The difference in follow-up period between the groups was due to practical and funding limita- tions. At follow-up the women were interviewed face-to-face using a structured questionnaire. The effect of individual conservative treatments on symptoms was reviewed and the reason for opting for or refusing surgery was asked. Also, women reported possible additional treatments or medica- tions used to treat symptoms after the Vulva clinic visits. The degree of dyspareunia was estimated by VAS score. Vestibular tenderness was evaluated with swab-touch test separately in the anterior (from 11 and to 1 o’clock) and posterior region (from 10 to 2 o’clock) with a light touch administered each time. Somatic and mental health issues and psychosocial charac- teristics were evaluated with questionnaires. Subjective gen- eral health was assessed with the EuroQol-5-Dimension- Figure 1. Study design. VAS, visual analogue scale. visual analogue scale (EQ5D-VAS) with VAS scores

C 2012 The Authors Acta Obstetricia et Gynecologica Scandinavica C 2012 Nordic Federation of Societies of Obstetrics and Gynecology 91 (2012) 1086–1093 1087 Vulvar vestibulitis treatment study P. Tommola et al. ranging from 0 to 100, a score of 100 representing the best Table 1. Baseline characteristics and conservative treatment modalities possible health state (19). Depression was measured by the used by vulvar vestibulitis syndrome women by study group

13-item version of the Beck depression inventory (20) and Conservative availability of social support by nine items from the vali- Vestibulectomy treatment dated Finnish version (21) of the Medical Outcome Study n = 39 n = 27 p value (MOS) Support Scale (22). Sexual well-being was assessed Age at first visit, years 24.5 (16–48)/301 23.5 (18–32)/24 0.3462 by a modification of McCoy questionnaire (23,24) consist- Nulliparous at baseline 32/39 (82.1) 25/26 (96.1) 0.1313 ing of three indexes: sexual satisfaction index (five items on On OC at onset of 24/37 (61.5) 19/25 (73.1) 0.6283 a scale from 1 to 7 representing the worst to the best sit- symptoms uation), partnership satisfaction index (two items, similar History of 5.0 (1 -18)/35 4.0 (1–16)/23 0.0602 scale), and index for sexual problems (a sum of reported dyspareunia, years frequency of dryness during sexual acts and frequency of Primary vestibulitis4 7/27 (25.9) 8/26 (30.8) 0.6963 2 dyspareunia; scale of 1 to 7 representing never to always). VAS for dyspareunia 9.0 (8.0–10.0)/35 8.0 (8.0–9.0)/25 0.046 at baseline The modification of the McCoy questionnaire for sexual Discontinuation of OC 9/25 (36) 12/17 (70.5) 0.0943 well-being is presented as Supporting Information in Ap- Physical therapy 10/30 (33.3) 15/26 (57.7) 0.0673 pendix S2. The individual overall response at long-term Long-term anti-fungal 17/35 (48.6) 11/26 (42.3) 0.6273 was reviewed by asking whether the patient had been cured treatment (complete response), still had some complaints (partial re- Neuromodulation sponse) or experienced no improvement (no response or Submucous or 18/31 (58.1) 9/26 (34.6) 0.0773 worse). topical corticosteroids Power analysis of VAS difference for dyspareunia at follow- Topical podophyl- 19/31 (61.3) 19/26 (73.1) 0.3473 up indicated that 24 women were needed in both groups to lotoxin5 detect a clinically relevant 20% difference between the groups Amitriptyline 13/31 (41.9) 7/21(33.3) 0.5323 (α = 0.05 and power (1–β) = 0.8). Based on previous re- sults of vulvar vestibulitis treatment studies we estimated 1Values are medians (minimum – maximum)/number of data available that decrease in VAS for dyspareunia could be 66% with for continuous variables, median (interquartile range 25–75%) for VAS values, and numbers of cases/data available (percentage) for categorical surgery and 44% with conservative management (12,25). variables. The data were analyzed by the Statistical Package for So- 2Mann–Whitney U-test. cial Sciences (SPSS version 16; SPSS Inc., Chicago, IL, USA). 3Chi-squared test. We report medians with interquartile range of 25–75% for 4Pain from first attempt of vaginal entry VAS values and medians with minimum and maximum val- 5Application of podophyllotoxin 5 mg/mLl (Wartec R ) on tender points ues for other continuous data. For comparisons we used the of vestibular mucosa following 5% acetic acid application, repeated Mann–Whitney U-test or Kruskal–Wallis test and Wilcoxon three times every four weeks (Appendix S1). Abbreviations; OC, oral contraceptives; VAS, visual analogue scale. signed rank test for continuous data and chi-squared anal- ysis or Fischer’s exact test for categorical data. Odds ratios with 95% confidence interval were calculated when appro- No significant differences were found between the groups. priate. A two-tailed p-value of <0.05 was considered signif- However, the VAS score for dyspareunia at baseline tended icant. For correlations, Pearson’s correlation coefficient was to be higher in the surgery group than in the conservative used. treatment group (9.0 vs. 8.0, p = 0.046). All women had long history of dyspareunia, most of them were nulliparous and Results under 25 years of age. The number of cases with primary vestibulitis (symptoms from first attempt of vaginal entry) All 39 surgical women had opted for surgery because of in- was similar in both groups. sufficient relief of symptoms by conservative management. The duration of the conservative treatment period was Of the 27 conservatively treated women, 23 had satisfac- 18.5 (4–55) months in the surgery group and 16.0 (3–129) tory resolution of symptoms, one patient did not report months in the conservative treatment group (p = 0.596). The her response, and three had had unsatisfactory treatment time from treatment initiation to choosing operation was 9.0 result but refused surgery – one because of fear and two (2–55) months and the median age of the women was 26.0 reported no reason. The drop-out women in either group years (range 17–48, mean 27.9) when making the decision. did not differ from the other women on any aspect at base- The operation was performed at a median age of 27.0 years, line. No data on their long-term outcome are available. Base- (range 18–50, mean 28.6). The different treatment modalities line clinical characteristics of the study groups are shown in used during the conservative treatment period were similar Table 1. in both groups (Table 1).

C 2012 The Authors 1088 Acta Obstetricia et Gynecologica Scandinavica C 2012 Nordic Federation of Societies of Obstetrics and Gynecology 91 (2012) 1086–1093 P. Tommola et al. Vulvar vestibulitis treatment study

The duration of follow-up was 47 (11–114) months in the the surgery group (p = 0.065) but was not associated with surgery group and 77 (34–131) months in the conservative recovery. treatment group (p < 0.001). There was no difference be- The treatment response was reported as complete by 13 tween the two groups in the use of systemic medication or of 36 (36.1%) women in the surgery group and seven of 27 topical anesthetics to control dyspareunia during follow-up (25.9%) women in the conservative treatment group, as par- and none had tried any other treatment options (data not tial by 19 (52.8%) and 17 (63.0%), and as no response by four shown). (11.1%) and two (7.4%), respectively, (p = 0.567). No woman Gynecological examination with swab-touch test was per- in the surgery group was worse than at baseline, whereas one formed in 35 vestibulectomy women and 24 conservatively (3.7%) in the conservative treatment group reported a worse treated women. Seven women refused examination for prac- situation. The response did not depend on the duration of tical reasons but returned questionnaires and attended to the follow-up (p = 0.441). Four of the seven (57.1%) women interview part of the study. No other vulvar diseases were with primary vestibulitis in the surgery group and one of the found, except that three (7.7%) women in the surgery group eight (12.5%) women in the conservative treatment group had a fissure in the posterior fourcette (one recent and two re- reported complete response. current fissures). Anterior tenderness did not differ between Atfollow-up,lessthanoptimaloutcome(definedasnore- the groups but significant posterior tenderness was clearly less sponse by subjective estimation, follow-up VAS score ≥ 7, or common in the surgery than in the conservative treatment presence of constant dyspareunia) was found in nine (23.1%) group (p < 0.001) (Figure 2). womeninthesurgerygroupandfour(14.8%)womeninthe VAS scores for dyspareunia decreased significantly in both conservative treatment group, (p = 0.534). Three of these groups from baseline to follow-up visit; from 9.0 (8.0–10.0) four control women were those who had had unsatisfactory to 3.0 (0.5–4.8), (p < 0.001) in the surgery group and result from the initial conservative treatment. from 8.0 (8.0–9.0) to 2.0 (0.00–3.0), (p < 0.001) in the conservative treatment group. The median changes in both Discussion groups were similar: 6.0 (3.8–8.0) (66.7%) and 6.3 (5.3–8.0) (78.1%), respectively (p = 0.407). Change of VAS did not This study showed that in women with severe VVS who do not depend on the duration of follow-up (R2 = 0.007). In a respond to conservative management, posterior vestibulec- subgroup analysis, women with significant posterior tender- tomy can provide long-term satisfaction and an enjoyable ness reported VAS scores of 5.25 (2.75–7.75) in the surgery sexual life, comparable with the well-being of those initially group and 2.0 (1.0–4.0) in the conservative treatment group responding to conservative management who do not need (p = 0.229). surgery. This supports the prevailing opinion that surgery Somatic or mental health, social, or partnership charac- be offered to the most severe VVS cases when conservative teristics did not differ between the surgery and the conserva- management has failed. tive treatment groups except that atopic skin problems were To our knowledge, this is the first study comparing long- more common among women in the conservative treatment term results of surgery and conservative treatment of women group (Table 2). Need for surgery was less likely in women with uniform definition of equally severe VVS at baseline. with atopic skin problems (odds ratio 0.2; 95% confidence Also this is the first study reporting the very long-term well- interval 0.1–0.7). Pain related to urinary tract, symptoms being of these women and reveals that satisfactory treatment suggestive of irritable bowel syndrome, and frequent muscu- results are preserved for at least up to six years. loskeletal pains were equally common in both groups, as was One advantage of the study was the use of a pragmatic the number of women with symptoms of two or more other treatment algorithm in the management of all study women pain disorders than vestibulodynia. Susceptibility to urogeni- as well as the use of several and versatile outcome mea- tal infections (candidiasis, bacterial vaginosis, other vaginitis, sures. Women’s well-being and health determinants were or urinary tract infection) did not differ between the groups broadly surveyed, including somatic and mental health, so- (data not shown). In both groups around 75% of women were cial characteristics and sexual well-being. The thorough inter- sexually active. Results of the McCoy instrument for sexual view and gynecological examination conducted by an expert well-being were similar in both groups (Table 3). Conserva- was crucial in obtaining comparable clinical data. Further, tively treated women reported use of coital lubricants more as the great majority of the women were sexually active in often than surgically treated women (43.5 vs.14.7%, respec- both groups we also obtained relevant data on sexual well- tively) (p = 0.030) (Table 3). Ten (52.6%) of 19 women in being. the conservative treatment group had changed partner dur- A major limitation of the study is probably the relatively ing the follow-up period. Change of partner tended to be small number of women. However, keeping to strict inclusion more common in the conservative treatment group than in criteria enabled the comparison of women with as similar a

C 2012 The Authors Acta Obstetricia et Gynecologica Scandinavica C 2012 Nordic Federation of Societies of Obstetrics and Gynecology 91 (2012) 1086–1093 1089 Vulvar vestibulitis treatment study P. Tommola et al.

Figure 2. Long-term (4–6 years) results of swab-touch test tenderness of 59 women treated for severe vulvar vestibulitis syndrome by posterior vestibulectomy (surgery group) or by conservative management (conservative treatment group). U, uretra; C, clitoris, V, vagina; – - hymen.

disease history and treatment history as possible and this efficacy (27). Unfortunately, evaluation of personality traits may actually be viewed as an advantage. According to the was not included in our study. power analysis the sample size was large enough to detect a A majority of the women were living in a relationship 20% difference between the two groups’ current VAS scores but reported low monthly frequency of sexual acts. Still, for dyspareunia, the main symptom of VVS. The similar they experienced reasonably good sexual satisfaction. Pos- decrease of dyspareunia with both managements was striking. sibly, women or couples with a long history of dyspareunia Of concern may be the relatively large proportion (46%) of have adapted to be satisfied with having intercourse less fre- womenintheconservativetreatmentgroupwhodidnot quently than healthy couples. Our finding of relatively high agree to participate. This may have introduced a drop-out sexual and partnership satisfaction is consistent with results bias as many unsatisfied women may have been reluctant to of previous studies on vulvodynia women and partners (28). participate. Theproblemindexscoresinbothgroupswerequitehigh, It is also worth considering whether the long duration of revealing residual pain and dryness. However, dryness dur- follow-up of the conservatively treated women had some im- ing the sexual act did not constitute a problem more often pact on the results. It might, for example, have increased after vestibulectomy than after conservative treatment. The the proportion of women whose symptoms resolved sponta- sensation of dryness may be a fundamental symptom of VVS neously. Spontaneous recovery from VVS has been demon- rather than a complication of surgery and may be managed strated recently (26). However, extending the duration of with adequate lubricant use. The less frequent need of lu- follow-up from six months to two and half years did not af- bricants in the surgery group may be explained by less pain fect the results in another study (16). This is also supported consistent with a favorable outcome. by our findings as a change of VAS during follow-up was not Atopic skin problems predicted the response to conserva- time-dependent. tive treatment. We were unable to identify any other explana- Significant posterior vestibular tenderness was more fre- tory factors. Conservative treatments were carried out rather quent in the conservative treatment group. Surprisingly, these similarly, and the women did not show any other differences women still reported quite low VAS scores. The long follow- regarding somatic or mental health, or social characteris- up time of this group may have had a role in making a better tics. Previous reports of primary vestibulitis suggest poor adjustment to pain. Also, the ability to cope depends on spe- response to surgery (14,29). This was not supported by our cial personality traits. Women with better self-efficacy tend results. More than half of the women with primary disease to have higher pain tolerance than women with lower self- reported complete response to surgery.

C 2012 The Authors 1090 Acta Obstetricia et Gynecologica Scandinavica C 2012 Nordic Federation of Societies of Obstetrics and Gynecology 91 (2012) 1086–1093 P. Tommola et al. Vulvar vestibulitis treatment study

Table 2. Long-term somatic and mental health, social, and partnership Table 3. Sexual health of vulvar vestibulitis syndrome women by study characteristics of vulvar vestibulitis syndrome women by study group group

Conservative Conservative Vestibulectomy treatment Vestibulectomy treatment n = 39 n = 27 p value n = 39 n = 27 p value

Age at follow-up, 31 (22–57)/391 32 (24–43)/27 0.5612 Sexually active 30/37 (81.1) 18/25 (72) 0.4023 years Use of coital lubricant 5/34 (14.7) 10/23 (43.5) 0.0303 VAS for dyspareunia 3.0 (0.5–4.8)/35 2.0 (0.0–3.0)/25 0.1742 Use of topical 6/36 (16.7) 1/24 (4.2) 0.2253 at follow-up anesthetic Pain related to 16/34 (47.1) 8/26 (33.3) 0.2023 Use of other ointment 12/36 (33.3) 7/23 (30.4) 1.0003 urinary tract or cream Symptoms suggestive 19/35 (54.3) 10/26 (38.5) 0.2213 Frequency of sexual 2 (0–20)/35 4 (0–15)/24 0.1352 of IBS acts during Frequent joint pains 3/36 (8.3) 3/26 (11.5) 1.0003 preceding month Two or more other 11/36 (30.6) 6/26 (23.1) 0.5153 Sexual satisfaction4 22.5 (11–31)/34 25 (9–29)/25 0.7182 pain disorders Partnership 12 (8–14)/29 12 (7–14)/18 0.2502 Atopic skin problems 5/35 (14.3) 12/26 (46.2) 0.0093 satisfaction4 EQ5D-VAS 80 (48–100)/35 82.5 (40–100)/26 0.2642 Sexual problems4 7 (2–14)/34 7 (2–12)/23 0.4322 Depression (Beck) 3.0 (0–15)/37 3.0 (0–15)/26 0.9942 Pain problem5 15/35 (42.9) 8/24 (33.3) 0.4613 Antidepressant use 1/38 (2.6) 4/26 (15.4) 0.1493 Dryness problem5 19/34 (55.9) 11/23 (47.8) 0.5503 Social support (MOS) 36.5 (21–45)/36 36.5 (28–45)/26 0.9543 Arousal problem5 7/34 (20.6) 6/24 (25.0) 0.6923 Need for more social 9/37 (24.3) 5/26 (19.2) 1.0003 1 support Values are medians (minimum – maximum)/number of data available for Married or cohabiting 27/39 (69.2) 18/27 (66.7) 1.0003 continuous variables and numbers of cases/data available (percentage) Same sexual partner 22/36 (61.1) 9/26 (34.6) 0.1063 for categorical variables 2 than before Mann-Whitney U-test 3 2 Single 7/37 (18.9) 7/25 (28) 0.4023 χ -test 4 Nulliparous 22/37 (59.5) 15/26 (57.7) 0.6133 Index in McCoy instrument 5 Vaginal delivery 7/37 (18.9) 11/27 (40.7) 0.0903 Pain, dryness, or arousal difficulty interfering more than half of the during follow-up intercourses

1Values are medians (minimum – maximum)/number of data available for continuous variables, median (interquartile range 25%-75%) for VAS for dyspareunia, and numbers of cases/data available (percentage) for further study. Change of partner had an impact on recovery categorical variables in one study (14) but in our study, change of partner was 2 Mann-Whitney U-test not associated with outcome. Since it is difficult to predict 3χ 2-test the response to conservative management an initial conser- Abbreviations; VAS, visual analogue scale; IBS, irritable bowel syndrome; EQ5D-VAS EuroQol-5-Dimension-visual analogue scale; MOS, Medical vative treatment period should be planned for all women Outcome Study support scale for availability of social support. at baseline. In this study, women refractory to conserva- tive management were ready to choose surgery after median of nine months of failed conservative treatment attempts. Women with good response needed a treatment period of Symptoms consistent with at least four pain syndromes 16 months to render vestibulectomy unnecessary. Random- other than vulvodynia predicted a poor response to conserva- ization of severe VVS women in the beginning might thus tive treatment in a recent study (30). In our study population not be appropriate. Rather, randomization after a systematic such tendency was not obvious. Also, a history of sexual or conservative treatment period of poor responders either to physical abuse which was not surveyed in our women pre- surgery or to continue conservative treatment might be rea- dicted a poor response to medical treatment in another recent sonable, but even this may be ethically problematic. Women study (31). It is possible that different subgroups of VVS exist withlonghistoryofsevereVVSmaybedifficulttorecruitinto with specific histopathologic features that determine respon- randomized trials. Furthermore, in our study, three of four siveness or influence the natural course of the disease. Our conservatively treated women with non-optimal outcome finding that atopic skin problems were more common among in the long-term had aleady reported an unsatisfactory re- women who responded to conservative treatment suggests a sponse after initial conservative management. This may mean specific immunological background in the pathophysiology that women refractory to conservative management possibly of VVS, or a different immunological response of atopic skin represent a specific subtype of VVS and should be encouraged type, or merely better tolerance to pain. This finding deserves to undergo vestibulectomy. In favor of the safety of surgery

C 2012 The Authors Acta Obstetricia et Gynecologica Scandinavica C 2012 Nordic Federation of Societies of Obstetrics and Gynecology 91 (2012) 1086–1093 1091 Vulvar vestibulitis treatment study P. Tommola et al. stands our recent study on posterior vestibulectomy report- 10. Bergeron S, Bouchard C, Fortier M, Binik YM, Khalife S. The ing the rarity of long-term adverse outcome and the fact that surgical treatment of vulvar vestibulitis syndrome: A over 90% of women would consent to vestibulectomy again follow-up study. J Sex Marital Ther. 1997;23:317–25. (13). 11. Goldstein A. Surgical techniques: Surgery for vulvar In conclusion, posterior vestibulectomy is a good option vestibulitis syndrome. J Sexual Med. 2006;3:559–62. for those severe VVS women who do not respond to conser- 12. Tommola P, Unkila-Kallio L, Paavonen J. Surgical treatment vative treatment. Both successful conservative management of vulvar vestibulitis: A review. Acta Obstet Gynecol Scand. and surgery among refractory women result in an equally 2010;89:1385–95. good long-term outcome. 13. Tommola P, Unkila-Kallio L, Paavonen J. Long-term follow up of posterior vestibulectomy for treating vulvar vestibulitis. Acknowledgments Acta Obstet Gynecol Scand. 2011;90:1225–31. 14. Bohm-Starke N, Rylander E. Surgery for localized, provoked Wethank RN Pirkko Timonen for technical support and Nina vestibulodynia: A long-term follow-up study. J Reprod Med. Hedkrok for secretarial assistance. 2008;53:83–9. 15. Bergeron S, Binik YM, Khalife S, Pagidas K, Glazer HI, Funding Meana M, et al. A randomized comparison of group cognitive–behavioral therapy, surface electromyographic The study was supported by grants from the Helsinki Uni- biofeedback, and vestibulectomy in the treatment of versity Hospital Research Funds. dyspareunia resulting from vulvar vestibulitis. Pain. References 2001;91:297–306. 16. Bergeron S, Khalife S, Glazer HI, Binik YM. Surgical and 1. Meana M, Binik YM, Khalife S, Cohen D. Dyspareunia: behavioral treatments for vestibulodynia: Two-and-one-half Sexual dysfunction or pain syndrome? J Nerv Ment Dis. year follow-up and predictors of outcome. Obstet Gynecol. 1997;185:561–9. 2008;111:159–66. 2. Harlow BL, Stewart EG. A population-based assessment of 17. Haefner HK, Collins ME, Davis GD, Edwards L, Foster DC, chronic unexplained vulvar pain: Have we underestimated Hartmann ED, et al. The vulvodynia guideline. J Low Genit the prevalence of vulvodynia? J Am Med Womens Assoc. Tract Dis. 2005;9:40–51. 2003;58:82–8. 18. Friedrich EG, Jr. Vulvar vestibulitis syndrome. J Reprod Med. 3. Zolnoun DA, Hartmann KE, Steege JF. Overnight 5% 1987;32:110–4. lidocaine ointment for treatment of vulvar vestibulitis. 19. Sculpher MJ, Dwyer N, Byford S, Stirrat GM. Randomised Obstet Gynecol. 2003;102:84–7. trial comparing hysterectomy and transcervical endometrial 4. Murina F, Tassan P, Roberti P, Bianco V. Treatment of vulvar resection: Effect on health related quality of life and costs vestibulitis with submucous infiltrations of two years after surgery. Br J Obstet Gynaecol. 1996;103: methylprednisolone and lidocaine. An alternative approach. J 142–9. Reprod Med. 2001;46:713–6. 20. Beck A, Rial W, Rickels K. Short form of depression 5. Boardman LA, Cooper AS, Blais LR, Raker CA. Topical inventory: Cross validation. Psychol Rep. 1974;34: gabapentin in the treatment of localized and generalized 1184–6. vulvodynia. Obstet Gynecol. 2008;112:579–85. 21. Hurskainen R, Aalto AM, Teperi J, Grenman S, Kivela A, 6. Reed BD, Caron AM, Gorenflo DW, Haefner HK. Kujansuu E, et al. Psychosocial and other characteristics of Treatment of vulvodynia with tricyclic antidepressants: women complaining of menorrhagia, with and without Efficacy and associated factors. J Low Genit Tract Dis. actual increased menstrual blood loss. Br J Obstet Gynaecol. 2006;10:245–51. 2001;108:281–5. 7. Andrews JC. Vulvodynia interventions – systematic review 22. Sherbourne CD, Stewart AL. The MOS social support survey. and evidence grading. Obstet Gynecol Surv. Soc Sci Med. 1991;32:705–14. 2011;66:299–315. 23. McCoy NL, Davidson JM. A longitudinal study of the effects 8. Danielsson I, Torstensson T, Brodda-Jansen G, Bohm-Starke of menopause on sexuality. Maturitas. 1985;7:203–10. N. EMG biofeedback versus topical lidocaine gel: A 24. Hurskainen R, Teperi J, Rissanen P, Aalto AM, Grenman S, randomized study for the treatment of women with Kivela A, et al. Quality of life and cost-effectiveness of vulvar vestibulitis. Acta Obstet Gynecol Scand. 2006;85: levonorgestrel-releasing intrauterine system versus 1360–7. hysterectomy for treatment of menorrhagia: A randomised 9. Foster DC, Kotok MB, Huang LS, Watts A, Oakes D, Howard trial. Lancet. 2001;357:273–7. FM, et al. Oral desipramine and topical lidocaine for 25. Landry T, Bergeron S, Dupuis MJ, Desrochers G. The vulvodynia: A randomized controlled trial. Obstet Gynecol. treatment of provoked vestibulodynia: A critical review. Clin 2010;116:583–93. J Pain. 2008;24:155–71.

C 2012 The Authors 1092 Acta Obstetricia et Gynecologica Scandinavica C 2012 Nordic Federation of Societies of Obstetrics and Gynecology 91 (2012) 1086–1093 P. Tommola et al. Vulvar vestibulitis treatment study

26. Reed BD, Haefner HK, Sen A, Gorenflo DW. Vulvodynia Supporting Information incidence and remission rates among adult women: A 2-year follow-up study. Obstet Gynecol. 2008;112:231–7. Additional supporting information may be found in the on- 27. Rokke PD, Fleming-Ficek S, Siemens NM, Hegstad HJ. line version of this article. Self-efficacy and choice of coping strategies for tolerating Appendix S1. Algorithm for treatment of women with severe acute pain. J Behav Med. 2004;27:343–60. vulvar vestibulitis syndrome at Helsinki University Hospi- 28. Van Lankveld JJ, Weijenborg PT, ter Kuile MM. Psychologic tal Vulva Clinic. Algorithm for treatment of women with profiles of and sexual function in women with vulvar severe vulvar vestibulitis syndrome, Helsinki University Hos- vestibulitis and their partners. Obstet Gynecol. pital Vulva Clinic. ∗Podophyllotoxin 5 mg/mL (Wartec R )is 1996;88:65–70. applied to tender points of vestibular mucosa following 5% 29. Bornstein J, Goldik Z, Stolar Z, Zarfati D, Abramovici H. acetic acid application, treated area is covered with cream and Predicting the outcome of surgical treatment of vulvar women are advised to wash the area after 24 hours. Applica- vestibulitis. Obstet Gynecol. 1997;89:695–8. tion is repeated three times every four weeks. 30. Heddini U, Johannesson U, Nilsson K, Bohm-Starke N. Provoked vestibulodynia – predictors and evaluation of AppendixS2. Modified McCoy questionnaire for sexual well- treatment outcome. J Low Genit Tract Dis. 2011;Suppl Oct being. 2011:1. Please note: Wiley-Blackwell is not responsible for the content 31. Foster DC, Poleshuck EL. Does sexual and physical abuse or functionality of any supporting materials supplied by the predict treatment response in vestibulodynia? J Low Genit authors. Any queries (other than missing material) should be Tract Dis. 2011;Suppl Oct 2011:12. directed to the corresponding author for the article.

C 2012 The Authors Acta Obstetricia et Gynecologica Scandinavica C 2012 Nordic Federation of Societies of Obstetrics and Gynecology 91 (2012) 1086–1093 1093 ,,,

Research ajog.org

GYNECOLOGY Activation of vestibule-associated lymphoid tissue in localized provoked vulvodynia Pa¨ivi Tommola, MD; Ralf Bu¨tzow, MD, PhD; Leila Unkila-Kallio, MD, PhD; Jorma Paavonen, MD, PhD; Seppo Meri, MD, PhD

OBJECTIVE: Localized provoked vulvodynia (LPV) may have inflam- germinal centers representing local immune activation. Germinal matory etiology. We wanted to find out whether the cell-mediated centers were not seen in controls. Antigen-presenting DCs and immune system becomes activated in the vestibular mucosa in LPV. macrophages were found both in patients and controls with similar densities. DCs were found to extend their dendrites into the luminal STUDY DESIGN: This was a controlled cross-sectional study. Vestib- space through an intact epithelium. Similar amounts of mast cells were ular mucosal specimens were obtained from 27 patients with severe found evenly scattered throughout the stroma of vestibular mucosa of LPV and 15 controls. Detailed clinical history of the patients was ob- both patients and controls. tained. For immunohistochemistry, antibodies against CD3 (T cells), CD20 (B cells), IgA (mucosal plasma cells), CD163 (dendritic cells CONCLUSION: We demonstrate here local organized vestibule- [DCs]), CD68 (macrophages), and CD117 (mast cells) were employed. associated lymphoid tissue analogous to mucosa-associated Mann-Whitney U test and c2 test were used for statistical analyses. lymphoid tissue. Vestibule-associated lymphoid tissue may emerge as a response to local infection or inflammation in LPV. RESULTS: More B lymphocytes and mature mucosal IgA-plasma cells were found in patients than in controls (P < .001 and P < Key words: immune activation, inflammation, vestibulodynia, vulvar .001, respectively). In LPV samples, B and T cells were arranged into pain, vulvar vestibulitis

Cite this article as: Tommola P, Bu¨tzow R, Unkila-Kallio L, et al. Activation of vestibule-associated lymphoid tissue in localized provoked vulvodynia. Am J Obstet Gynecol 2015;212:476.e1-8.

ocalized provoked vulvodynia consequences to the quality of life of often reveals increased lymphocytic L (LPV) is a subset of vulvodynia, affected young women. LPV prevalence infiltrates in the vestibular mucosa7,8 associated with induced pain by touch rates can be up to 8-15% in premeno- but the characteristics of the inflamma- on vulvar mucosa in the absence of any pausal population.2,3 tory response have remained unclear. other recognizable disease.1 LPV is The etiopathogenesis of LPV and the However, many studies have found further classified according to the loca- mechanisms resulting in the altered pain no evidence of a classic active inflam- tion of pain. Vestibulodynia (formerly sensation are unknown. Different vul- mation.9,10 Instead, a tendency to an vulvar vestibulitis syndrome) represents vovaginal infections, such as recurrent exaggerated inflammatory response and the pain sensation in the vulvar vestib- candidiasis as well as urinary tract in- dysregulation of inflammation in af- ular mucosa and results in severe dys- fections have been considered as risk fected women have been suggested.11-14 pareunia. This can have devastating factors for the disease.4-6 Histopathology The vestibular mucosa of LPV patients characteristically has an increased num- ber of exceptionally superficial nerve 15 From the Department of Obstetrics and Gynecology, Helsinki University Central Hospital endings. An increased number of mast (Drs Tommola, Unkila-Kallio, and Paavonen); Department of Pathology (Dr Bützow); Research cells producing heparanase enzyme has Programs Unit, Program of Immunobiology (Dr Meri); and Department of Bacteriology and been suggested to boost nerve growth.16 Immunology, Haartman Institute (Dr Meri), University of Helsinki, Helsinki, Finland. However, the role of mast cells has not Received June 5, 2014; revised Aug. 9, 2014; accepted Oct. 27, 2014. been fully proven.17 As the immune and This study was supported by grants from the Finnish Society of Pediatric and Adolescent Gynecology neuronal systems are often closely and Helsinki University Hospital Research Funds (grant nos. TYH2012237, TYH2013340, TYH interrelated, an increased inflammatory 2011128, and TYH 2013308). response may well predispose to the fl The authors report no con ict of interest. development of a pain syndrome. Preliminary results were presented at the VIII European Conference of the European Society for LPV is a pain syndrome with a sus- Infectious Diseases in Obstetrics and Gynecology, London, United Kingdom, Oct. 25-27, 2013, and fl at the XXII World Congress of the International Society for the Study of Vulvovaginal Disease, Rome, pected in ammatory background but Italy, Oct. 6-13, 2013. the detailed characteristics of the in- fl Corresponding author: Päivi Tommola, MD. paivi.tommola@kolumbus.fi ammatory response are unknown. In fi 0002-9378/$36.00 ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajog.2014.10.1098 particular, the role of speci c immune system cells has not been defined. In

476.e1 American Journal of Obstetrics & Gynecology APRIL 2015 ajog.org Gynecology Research this study we performed a thorough was 70. It was not possible to reach health, comorbidity, medications, and immunohistochemical analysis of the 13 women, or they denied participating. detailed disease history including dura- vestibular mucosal tissue focusing on Of the remaining 57 women we chose tion and severity of symptoms, treat- differences in the characteristics of the 27 with sufficient clinical data to form ments before surgery, and response to local immune system between LPV pa- the patient population of this study. All surgery were systematically collected.18,19 tients and controls. the included patients were diagnosed As controls we recruited 15 premeno- as having localized provoked vestibulo- pausal volunteers with no vulvar symp- MATERIALS AND METHODS dynia with long disease history (mean, toms undergoing benign gynecological Study subjects 5.3 years; 95% confidence interval [CI], surgery under general anesthesia. No We recruited 27 women with represen- 3.8e6.8; range, 2e18). The diagnoses clinical data were collected. We obtained tative archival vestibulectomy specimens in 8 patients were classified as primary 4-mm punch biopsies from the posterior after surgically treated LPV during a (symptoms already at the first vaginal vestibule at 5 o’clock position from the period of 13 years from 1995 through entry), in 15 patients as secondary controls. The study was approved by the 2007. The women were identified in the (symptoms appearing later after an in- local ethical committee. All participants Helsinki University Central Hospital terval of painless intercourses), and in 4 provided informed consent. patient registry by matching the diag- patients the classification was unknown. nosis (vulvar vestibulitis, vulvodynia, Clinical data of these 27 LPV patients Tissues and dyspareunia) and the surgical pro- were collected from the patient records All vestibular tissues were routinely cedure (posterior vestibulectomy). The and by a face-to-face interview. Data embedded in paraffin after a maximum total number of the original cohort on age, premenopausal status, general of 24 hours fixation in 10% buffered formalin. We first stained 5-mm sections FIGURE 1 with hematoxylineeosin for evaluation Antigen-presenting cells (DCs and macrophages) in vulvar vestibular to exclude dermatological diseases, to mucosa confirm the quality of samples, and to grade lymphocytic inflammation. Immunohistochemistry on consecutive sections (5 mm in thickness; 10 mm for dendritic cells [DCs] and macrophages) was performed in the Helsinki Univer- sity Central HospitaleHuslab tissue laboratory with routine staining pro- cedures according to the manufacturers’ instructions. We employed antibodies against the following antigens: CD3 (clone 2GV6, RTU; Roche Diagnostics Ltd, Rotkreuz, Switzerland) for T lym- phocytes, CD20 (clone L26, RTU; Roche Diagnostics Ltd) for B lymphocytes, CD68 (clone PG-M1, 1:1000; Dako, Glostrup, Denmark) for macrophages, CD117 (polyclonal, 1:1000; Dako) for mast cells, IgA (polyclonal, 1:2000; Dako) for mucosal plasma cells, CD163 (clone 10D6, 1:100; Leica Biosystems, Nussloch, Germany) for dendritic cells. For the first 4 antibodies above, we used a biotin-free polymer-based detection kit Ultraview (Roche Diagnostics Ltd). CD163 immunostaining for stromal and epithelial DCs in A, localized provoked vulvodynia patient For the last 2 antibodies, we used a and B, control sample. Inserts show DCs extending their projections through intact epithelial surface polymer-based detection kit Envision in greater magnification. CD68 immunostaining for macrophages in C, patient and D, control sample. (Dako). All sections were counterstained Histological sections were counterstained with hematoxylin. Objectives A and B, 40; C and D, 20 with hematoxylin. (Nikon Eclipse E800). DCs, dendritic cells. Tissue analyses Tommola. Activation of lymphoid tissue in localized provoked vulvodynia. Am J Obstet Gynecol 2015. The entire material was analyzed blind- ed to clinical data. Histopathological

APRIL 2015 American Journal of Obstetrics & Gynecology 476.e2 Research Gynecology ajog.org inflammation was analyzed in hematoxylineeosin sections and graded TABLE 1 as none, mild, or moderate (R.B.) Immune cells in vestibular mucosa in localized provoked vulvodynia based on the amount of mononuclear LPV (n [ 27) Controls (n [ 15) a a b infiltrates in stromal tissue beneath the Cell type Mean (95% CI) Mean (95% CI) P value vestibular epithelium at low-power Dendritic cells 98 (86e110)c 129 (101e157)d .053 fi magni cation ( 10). Macrophages 23 (17e29)c 19 (15e24)d .780 Immunohistochemical scoring of the B lymphocytes 121 (97e145) 38 (15e60) < .001 representative sections was performed under light microscopy (Nikon Eclipse Germinal centerse 14 (51.9) 0 (0) .001 E800). The stainings of all antigens were Plasma cells 41 (31e52)c 11 (8e15)f < .001 analyzed for localization (stromal, epi- Mast cells 35 (31e40) 33 (26e39)f .512 thelial, both) and for densities of indi- CI, confidence interval; LPV, localized provoked vulvodynia. vidual cell types in the vestibular a Values are mean cell counts per microscopy field, analyzed from 2e4 fields (40 objective); b Mann-Whitney U test; mucosa. The scoring of each section was c N ¼ 24e26; d N ¼ 8e10; e No. of samples (%) with 1 germinal center; f N ¼ 12e14. based on a consensus of 2 investigators Tommola. Activation of lymphoid tissue in localized provoked vulvodynia. Am J Obstet Gynecol 2015. (P.T. and J.P. or L.U-K. or S.M.) and disagreements were resolved by a joint review. All immune cells, except T lympho- we used Mann-Whitney U test or correlations Spearman rho test for cytes, were quantified from the most Kruskal-Wallis test for continuous data nonparametric data was used. A P value representative areas of inflammation by and c2 analysis or Fisher exact test for of < .05 was considered significant in calculating the mean number of identi- categorical data when appropriate. For a 2-tailed analysis. fied positive cells per field from 2-4 high- power fields (hpf) (40 objective). T lymphocytes were scored by the overall cell density both in the epithe- FIGURE 2 lium and stroma. A single number score T cells and B cells in vulvar vestibular mucosa was given from 1-3 (1 ¼ low density, <50 cells/hpf; 2 ¼ moderate density, 50- 100 cells/hpf; 3 ¼ high density, >100 cells/hpf). Germinal centers were iden- tified and counted from each section. For IgA, the overall staining intensity was additionally scored as 1 (mild in- tensity), 2 (moderate intensity), or 3 (strong intensity). For statistical purposes and to reflect the overall level of B-cell infiltration we created a B-cell activation index (BAI) as follows. The most typical den- sity of B cells in each section scored points from 0 (no B cells) to 4 (high amount of B cells), separately in the epithelium and in the stroma. The exis- tence of at least 1 germinal center scored 4 points and absence scored 0 points. The sum of these 3 values gave a BAI from 0-12. Statistical analyses of the data were performed with software (SPSS, version 20; IBM Corp, Armonk, NY). For com- High density (score 3) of CD3þ T cells in A, localized provoked vulvodynia patient and B, control parisons between patients and controls, sample. CD20 staining for B cells shows greater density in C, patients than in D, controls. Histological between patients with primary and sec- sections were counterstained with hematoxylin and photomicrographed using 20 objective. ondary LPV, and between patients Tommola. Activation of lymphoid tissue in localized provoked vulvodynia. Am J Obstet Gynecol 2015. with different clinical characteristics,

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þ collectively both helper (CD4 ) and FIGURE 3 þ killer (CD8 ) T cells. These were ex- Germinal centers in vulvar vestibular mucosa pressed both in the stromal and epithe- lial layers of the vestibular mucosa with no significant difference in densities be- tween patients and controls (Figure 2,A and B). T-cell density was high in 26.9% (7/26) of patients vs 21.4% (3/14) of controls, moderate in 30.8% (8/26) vs 57.1% (8/14), and low in 42.3% (11/26) vs 21.4% (3/14) of patients and controls, respectively (P ¼ .280). T-cell densities were similar in primary and secondary LPV (P ¼ .512; data not shown). CD20 antigen is expressed on B cells at all stages of development, except the early pro-B cells and mature plasma cells capable of secreting antibodies. Anti- CD20 antibody showed that B lympho- cytes were present mainly in the stromal layer of the mucosa. Small amounts were also found in the epithelium in the most populated areas (Figure 2, C and D). B lymphocytes were more numerous in patients than in controls (Table 1). In areas with highest lymphocyte densities, T cells and B cells clearly formed lymphoid follicle-like structures. These were identified as germinal centers representing secondary lymphoid tissue. These germinal centers were visualized by both the CD3 (Figure 3, A through C) and CD20 (Figure 3, D through F) an- tibodies showing the typical zonewise arrangement of the individual cell types.20 Germinal centers were found only in LPV patients, but not in controls. Germinal centers in 3 patient samples A through C, CD3 immunostaining for T cells and D through F, The number of germinal centers in pa- CD20 immunostaining for B cells. Histological sections were counterstained with hematoxylin and tients varied from 1-4 centers in 14 sec- photomicrographed using 10 objective. tions (52%), but was 0 in 13 sections. Tommola. Activation of lymphoid tissue in localized provoked vulvodynia. Am J Obstet Gynecol 2015. BAI was significantly higher in patients than in controls (median 5.0, range 1.0e9.0 and median 1.0, range 0e4.0, RESULTS Staining for macrophages demon- respectively; P ¼ .001). Antigen-presenting cells strated the chubby shape of the cell body Mature mucosal plasma cells were DCs can capture foreign antigens and and revealed the typical intracellular stained on the basis of their ability to þ process them for presentation to CD4 cytoplasmic granules (Figure 1, C and produce IgA-class antibodies. Compared helper T cells. CD163 immunostain re- D). Macrophages were evenly scattered to B cells, the plasma cells were bigger, vealed the typical morphology of DCs throughout the vestibular stroma with had oval shape, showed high nucleus-to- withtheantigen-capturingdendrites.DCs similar densities in patients and controls cytoplasm ratio, and their nuclei had were occasionally found to extend their (Table 1). typical round cartwheel appearance. dendrites through the epithelial surface to IgA-plasma cells were found in the sub- the luminal space (Figure 1, Aand B). DCs T and B lymphocytes and plasma epithelial layer of the stroma (Figure 4). were located both in the epithelium and cells These were more numerous in patients stroma and tended to be more numer- T-cell staining (using the cytoplasmic than in controls (Table 1). In addition ous in controls than in patients (Table 1). pan T-cell marker CD3) visualized to the plasma cells’ cytoplasm, IgA was

APRIL 2015 American Journal of Obstetrics & Gynecology 476.e4 Research Gynecology ajog.org also seen diffusely in the epithelial sur- FIGURE 4 faces and stromas of mucosal samples. Mucosal plasma cells in vulvar vestibular mucosa In a semiquantitative analysis no differ- ence in the densities of IgA between pa- tients and controls was found (data not shown).

Mast cells Mast cells are tissue granulocytes. Typi- cally they are prominent near the boundaries between the outside and in- side environments, such as underneath mucosal surfaces, and adjacent to blood þ vessels. CD117 mast cells were located in the stroma mostly as evenly distrib- uted single cells, often adjacent to small capillaries (Figure 5) showing similar densities in patients and controls (Table 1).

Clinical correlates of immunological parameters Histopathological inflammation was more pronounced in LPV than in con- trols and tended to be more severe in secondary than in primary LPV (Table 2). The grade did not associate with severity (visual analog scale score for preoperative dyspareunia) (P ¼ .550) or duration of symptoms (P ¼ .650). The presence of germinal centers in patients was not associated with the histological grade of inflammation. Germinal centers were found in 35.7% (5/14) of patients with mild inflamma- tion and in 69.2% (9/13) of patients with moderate inflammation (P ¼ .082). The occurrence of germinal centers was not associated with the severity of dyspar- eunia or duration of symptoms (P ¼.550 and P ¼ .402, respectively). IgA immunostaining to detect mucosal IgA-plasma cells in A through C, patient samples and BAI scores were higher in patients D through F, control samples. Histological sections were counterstained with hematoxylin. Objectives with moderate than mild inflammation A, B, D, and E, 20; C and F, 40. (5.9; 95% CI, 4.5e7.2 vs 3.3; 95% CI, Tommola. Activation of lymphoid tissue in localized provoked vulvodynia. Am J Obstet Gynecol 2015. 1.9e4.6, respectively; P ¼ .011). No correlation was found between BAI and severity of dyspareunia (Spearman rho genitourinary infection, history of LPV patients and healthy controls. We e0.172, P ¼ .392) or duration of symp- autoimmune or inflammatory disease, were able to demonstrate the presence toms (Spearman rho 0.103, P ¼ .610). or clinical response to surgery was not of key immune players and found evi- Associations of selected patient char- associated with the presence of germinal dence of immune activation in LPV. acteristics and immunological parame- centers or with the BAI scores (Table 3). The vestibular area thus appears to have ters are presented in Table 3. Germinal a localized immune system, which we centers were equally common in cases COMMENT call the vestibule-associated lymphoid with primary or secondary LPV. BAI In the present study we conducted a tissue (VALT). scores did not differ between these pa- thorough analysis of different immune All patients had a clinically and his- tient categories. History of recurrent cell types in the vestibular mucosa of topathologically confirmed diagnosis of

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on the effect of surgical treatment of FIGURE 5 LPV.18,23 Mast cells in vulvar vestibular mucosa Mucosal surfaces of the human body are known to contain secondary lym- phoid tissue, the mucosa-associated lymphoid tissue (MALT).20 In MALT, B and T lymphocytes organize to form germinal centers, which play a central role in initiating an immune response against foreign antigens such as those from viruses and bacteria. The crucial point in the initiation of an immune response is the presentation of foreign antigens to T lymphocytes. Macrophages, DCs, and B cells are called professional antigen-presenting cells. By the antigen presentation, the T cells become activated. The activated T cells, in turn, are capable of activating a whole range of immune cells to mount different types of responses depending on the nature of the initial insult. This part of the activation of an adaptive immune response takes place in germinal centers. A proportion of the CD117 immunostaining for mast cells in A, localized provoked vulvodynia patient and B, control activated cells are B cells developing sample. Mast cells typically appear adjacent to C-E, small blood vessels or capillaries. Histological into antibody producing mature plasma sections were counterstained with hematoxylin. Objectives A and B, 20; C, D, and E, 40. cells. In mucosal areas, B cells switch Tommola. Activation of lymphoid tissue in localized provoked vulvodynia. Am J Obstet Gynecol 2015. into plasma cells producing dimeric IgA-class antibodies that will subse- quently be transported across the epi- thelium to the mucosal surfaces. severe LPV with accurate clinical data controls.8,16,21 Control biopsies were DCs capture different antigens with and good-quality archival tissue sam- taken exactly from the 5 o’clock position distinct receptors on their surfaces, ples. The control group consisted of 15 of the vestibule to secure appropriate especially on their dendrites. As shown healthy age-matched women. This is a comparison. This posterior site is the in Figure 1, A and B, DCs can extend major strength of our study since most representative vestibular area in their dendrites into the luminal space many previous studies on the histopa- LPV, as supported by a study on vulvar through the epithelium in an intact thology of LPV reported very few or no pain mapping22 and by the studies mucosa to survey the mucosal surface and to capture potential antigens. Antigen-primed DCs in turn transport the antigen for presentation to T cells in TABLE 2 the secondary lymphoid tissue. Our Lymphocytic inflammation in vestibular mucosa in localized provoked finding of a tendency towards fewer DCs vulvodynia in patients than in controls may be LPV Controls Primary LPV Secondary LPV caused by the migration of these trans- [ a [ b [ [ c Inflammation (n 27) (n 15) (n 8) (n 15) porter cells to germinal centers. Simi- (HE staining) n (%) n (%) n (%) n (%) larly, in another study, loss of DCs was None 0 (0) 7 (46.7) 0 (0) 0 (0) found in the vaginal and cervical Mild 14 (51.9) 8 (53.3) 7 (87.5) 6 (40) epithelia of patients with an ongoing fl Moderate 13 (48.1) 0 (0) 1 (12.5) 9 (60) infection or in ammation compared to those without signs of inflammation.24 HE, hematoxylin-eosin; LPV, localized provoked vulvodynia. a b In a recent study dense lymphocytic Total no. of LPV patients is 27 (8 primary, 15 secondary, and 4 not defined); P < .001, LPV vs controls, exact sign; fi c P ¼ .074, primary vs secondary LPV, Fisher exact sign test. in ltrates were seen in hematoxylin- Tommola. Activation of lymphoid tissue in localized provoked vulvodynia. Am J Obstet Gynecol 2015. eosin-stained sections of vestibulodynia specimens.25 The authors speculated

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TABLE 3 Clinical characteristics and immune activation in vulvodynia patients No. of patients P value for existence Characteristic positive (%) GC D N (%) of GCa BAI scoreb P value for BAIc Primary LPV 8/23 (34.8) 4 (50) 4.0 (1e8) Secondary LPV 15/23 (65.2) 7 (47) 1.000d 5.0 (1e7) .636 History of recurrent 21/27 (77.8) 12 (57) .089 5.0 (1e9) vs 2.5 (1e7) .289 genitourinary infection History of autoimmune or 16/26 (61.5) 7 (44) .686 5.0 (1e9) vs 5.5 (1e8) .391 inflammatory diseasee Complete response to surgery 11/27 (40.7) 7 (64) .252 4.0 (1e8) vs 5.5 (1e9) .111 BAI, B-cell activation index; GC, germinal center; LPV, localized provoked vulvodynia. a c2 analysis; b Comparative BAI scores of patients with and without characteristic, median (range); c Mann-Whitney U test; d Fisher exact test; e Thyroid disease, rheumatic disease, irritative bowel syndrome, bronchial asthma, atopic skin disease. Tommola. Activation of lymphoid tissue in localized provoked vulvodynia. Am J Obstet Gynecol 2015.

the possibility of MALT in the vestibular close association with blood vessels Increasing interest in the role of im- mucosa. Our immunohistochemical (Figure 5). This suggests a link between mune response in pain context is analysis reveals that the lymphocytic in- the mast cell activation and vascular emerging. The immune and neuronal filtrates in LPV indeed represent MALT, hyperemic response and possibly the systems are often interrelated and this containing cells and structures needed to interplay with neurons as well. may predispose to the development of initiate a local immune response. Thus, Although the immune activation in LPV with disturbed pain perception. the vestibular mucosa seems to have its LPV was evident we found no associa- In this study we demonstrated a locally own organized immune system that tion with the patients’ clinical charac- organized VALT, which may emerge as could be called VALT. Furthermore, our teristics, eg, severity of symptoms or a response to local infection or inflam- findings of germinal centers and large history of infectious or autoimmune mation. Although these results do not numbers of B cells and plasma cells diseases. Similarly, there was no associ- definitely prove that inflammatory support immune activation in LPV. In ation between immune activation and response is related to pain, VALT contrast, other types of immune cells, response to surgical treatment. In the may play an essential role in the devel- like macrophages and mast cells, were above-mentioned recent study histology opment of LPV. Potential interplay be- present in equal densities both in the did not predict response to surgery, tween activated immune cells and patient tissues and healthy mucosal either.25 However, 27 study subjects may biomodulators of the pain sensation tissues. not be enough for the subgroup analyses poses a challenge to future research on Acute inflammation is associated with although they serve well for immuno- the mechanisms of neurogenic inflam- the release of cytokines, activation of histochemical evaluation of the cellular mation. Better understanding of the link complement, polymorphonuclear leu- details of inflammation. Also, since the between immune system and pain may kocytes, and mast cells. Acute inflam- clinical data were collected retrospec- also provide new ways to treat LPV, eg, mation is needed to create a milieu for tively, recall bias may affect these results. by immunomodulatory therapy. - activation of the adaptive immune Several studies and clinical observations response. Manifest LPV, however, rep- suggest a susceptibility of LPV patients resents a chronic condition with slow to vulvovaginal infections. Specifically, REFERENCES development and the acute phase of recurrent Candida albicans infection has 1. Moyal-Barracco M, Lynch PJ. 2003 ISSVD inflammation has undoubtedly already been considered as a risk factor for terminology and classification of vulvodynia: a faded away. Our patients all had a long LPV.4-6,29 By nature, the vestibule is historical perspective. J Reprod Med 2004;49: 772-7. history of disease. This may partly extensively exposed to foreign antigens 2. fi Harlow BL, Kunitz CG, Nguyen RH, explain why we did not nd any excess of during sexual and reproductive life. Rydell SA, Turner RM, MacLehose RF. Preva- mast cells in contrast to some previous Thus, both protective and adaptive lence of symptoms consistent with a diagnosis studies.8,26 In other tissues, eg, in the characteristics of the immune response of vulvodynia: population-based estimates from mucosa of gastrointestinal tract and in are required. While protecting against 2 geographic regions. Am J Obstet Gynecol 2014;210:40.e1-8. coronary arteries, a close association of local infection an activated VALT could 3. fl Goetsch MF. Vulvar vestibulitis: prevalence mast cells with small vessels and nerve cause an exaggerated in ammatory re- and historic features in a general gynecologic 27,28 endings has been observed. In our action and contribute to the sensitized practice population. Am J Obstet Gynecol material, mast cells were observed in pain perception in the vestibular mucosa. 1991;164:1609-16.

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4. Harlow BL, Wise LA, Stewart EG. Prevalence 13. Gerber S, Bongiovanni AM, Ledger WJ, 22. Zolnoun D, Bair E, Essick G, Gracely R, and predictors of chronic lower genital tract Witkin SS. Defective regulation of the proin- Goyal V, Maixner W. Reliability and reproduc- discomfort. Am J Obstet Gynecol 2001;185: flammatory immune response in women with ibility of novel methodology for assessment 545-50. vulvar vestibulitis syndrome. Am J Obstet of pressure pain sensitivity in pelvis. J Pain 5. Smith EM, Ritchie JM, Galask R, Pugh EE, Gynecol 2002;186:696-700. 2012;13:910-20. Jia J, Ricks-McGillan J. Case-control study of 14. Gerber S, Bongiovanni AM, Ledger WJ, 23. Lavy Y, Lev-Sagie A, Hamani Y, Zacut D, vulvar vestibulitis risk associated with genital in- Witkin SS. A deficiency in interferon-alpha pro- Ben-Chetrit A. Modified vulvar vestibulectomy: fections. Infect Dis Obstet Gynecol 2002;10: duction in women with vulvar vestibulitis. Am J simple and effective surgery for the treatment of 193-202. Obstet Gynecol 2002;186:361-4. vulvar vestibulitis. Eur J Obstet Gynecol Reprod 6. Farmer MA, Taylor AM, Bailey AL, et al. 15. Tympanidis P, Terenghi G, Dowd P. Biol 2005;120:91-5. Repeated vulvovaginal fungal infections cause Increased innervation of the vulval vestibule in 24. Pudney J, Quayle AJ, Anderson DJ. Immu- persistent pain in a mouse model of vulvodynia. patients with vulvodynia. Br J Dermatol 2003; nological microenvironments in the human va- Sci Transl Med 2011;3:101ra91. 148:1021-7. gina and cervix: mediators of cellular immunity 7. Pyka RE, Wilkinson EJ, Friedrich EG Jr, 16. Bornstein J, Goldschmid N, Sabo E. are concentrated in the cervical transformation Croker BP. The histopathology of vulvar vesti- Hyperinnervation and mast cell activation may zone. Biol Reprod 2005;73:1253-63. bulitis syndrome. Int J Gynecol Pathol 1988;7: be used as histopathologic diagnostic criteria 25. Brokenshire C, Pagano R, Scurry J. The 249-57. for vulvar vestibulitis. Gynecol Obstet Invest value of histology in predicting the effectiveness 8. Goetsch MF, Morgan TK, Korcheva VB, Li H, 2004;58:171-8. of vulvar vestibulectomy in provoked vestibulo- Peters D, Leclair CM. Histologic and receptor 17. Halperin R, Zehavi S, Vaknin Z, Ben-Ami I, dynia. J Low Genit Tract Dis 2014;18:109-1. analysis of primary and secondary vestibulody- Pansky M, Schneider D. The major histopatho- 26. Chaim W, Meriwether C, Gonik B, nia and controls: a prospective study. Am J logic characteristics in the vulvar vestibulitis Qureshi F, Sobel JD. Vulvar vestibulitis subjects Obstet Gynecol 2010;202:614.e1-8. syndrome. Gynecol Obstet Invest 2005;59: undergoing surgical intervention: a descriptive 9. Bohm-Starke N, Falconer C, Rylander E, 75-9. analysis and histopathological correlates. Eur J Hilliges M. The expression of cyclooxygenase 2 18. Tommola P, Unkila-Kallio L, Paavonen J. Obstet Gynecol Reprod Biol 1996;68:165-8. and inducible nitric oxide synthase indicates no Long-term follow up of posterior vestibulectomy 27. Forsythe P, Bienenstock J. The mast celle active inflammation in vulvar vestibulitis. Acta for treating vulvar vestibulitis. Acta Obstet nerve functional unit: a key component of Obstet Gynecol Scand 2001;80:638-44. Gynecol Scand 2011;90:1225-31. physiologic and pathophysiologic responses. In: 10. Eva LJ, Rolfe KJ, MacLean AB, et al. Is 19. Tommola P, Unkila-Kallio L, Paavonen J. Bienestock J, ed. Allergy and the nervous sys- localized, provoked vulvodynia an inflammatory Long-term well-being after surgical or conser- tem. Vol 98. Basel (Switzerland): Karger; 2012: condition? J Reprod Med 2007;52:379-84. vative treatment of severe vulvar vestibulitis. 196-221. 11. Foster DC, Hasday JD. Elevated tissue Acta Obstet Gynecol Scand 2012;91:1086-93. 28. Lappalainen H, Laine P, Pentikainen MO, levels of interleukin-1 beta and tumor necrosis 20. Brandtzaeg P, Kiyono H, Pabst R, Sajantila A, Kovanen PT. Mast cells in neo- factor-alpha in vulvar vestibulitis. Obstet Gyne- Russell MW. Terminology: nomenclature of vascularized human coronary plaques store and col 1997;89:291-6. mucosa-associated lymphoid tissue. Mucosal secrete basic fibroblast growth factor, a potent 12. Foster DC, Piekarz KH, Murant TI, Immunol 2008;1:31-7. angiogenic mediator. Arterioscler Thromb Vasc LaPoint R, Haidaris CG, Phipps RP. Enhanced 21. Leclair CM, Goetsch MF, Korcheva VB, Biol 2004;24:1880-5. synthesis of proinflammatory cytokines by vulvar Anderson R, Peters D, Morgan TK. Differences 29. Sarma AV, Foxman B, Bayirli B, Haefner H, vestibular fibroblasts: implications for vulvar in primary compared with secondary vestibulo- Sobel JD. Epidemiology of vulvar vestibulitis vestibulitis. Am J Obstet Gynecol 2007;196: dynia by immunohistochemistry. Obstet Gyne- syndrome: an exploratory case-control study. 346.e1-8. col 2011;117:1307-13. Sex Transm Infect 1999;75:320-6.

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Original Research ajog.org

GYNECOLOGY Immune activation enhances epithelial nerve growth in provoked vestibulodynia Päivi Tommola, MD; Leila Unkila-Kallio, MD, PhD; Anders Paetau, MD, PhD; Seppo Meri, MD, PhD; Eija Kalso, MD, PhD; Jorma Paavonen, MD, PhD

BACKGROUND: Provoked vestibulodynia manifests as allodynia of the specific neurofilament epositive intraepithelial fibers were found in 17 of vulvar vestibular mucosa. The exact mechanisms that result in altered pain 27 vestibulodynia cases (63.0%) and in none of the control cases. sensation are unknown. Recently, we demonstrated the presence of Protein gene product 9.5epositive intraepithelial fibers were more secondary lymphoid tissue, which is the vestibule-associated lymphoid common in samples with more pronounced immune activation. The tissue in the vestibular mucosa, and showed that this tissue becomes density of these fibers was higher in samples with than without germinal activated in provoked vestibulodynia. centers (6.1/mm [range, 4.3e15.8] vs 3.0/mm [range, 0.0e13.4]; OBJECTIVE: The purpose of this study was to examine whether P¼.020). A positive correlation between the fiber density and B-cell expression of intraepithelial nerve fibers and nerve growth factor are activation index score of the sample was found (Spearman’s Rho, 0.400; related to immune activation in provoked vestibulodynia. P¼.004; R2¼0.128). No significant difference, however, was found in STUDY DESIGN: Vestibular mucosal specimens were obtained from the density or presence of nerve fibers between samples with high and 27 patients with severe provoked vestibulodynia that was treated by low T-cell densities. We identified areas of minor and major vestibular vestibulectomy and from 15 control subjects. We used antibodies against glands in 16 of the patient samples and in 1 control sample. Protein gene the protein gene product 9.5, the neuron specific neurofilament, and nerve product 9.5epositive nerve fibers were found more often in glandular growth factor for immunohistochemistry to detect intraepithelial nerve fi- epithelium surrounded by B-cell infiltration than in glands without B cells bers and nerve growth factor expressing immune cells in the vestibular (P¼.013). Also, the presence of neuron specific neurofilamentepositive mucosa. For intraepithelial nerve fibers, we determined their linear density fibers in glandular epithelium was associated with B-cell infiltrates (fiber counts per millimeter of the outer epithelial surface, protein gene (P¼.053). Nerve growth factorepositive immune cells were more product 9.5) or presence (neuron specific neurofilament). Nerve growth common in mucosal areas with than without B-cell infiltration and factor was analyzed by counting the staining-positive immune cells. An- intraepithelial nerve fibers. tibodies against CD20 (B lymphocytes) and CD3 (T lymphocytes) were CONCLUSION: Excessive epithelial nerve growth in provoked vesti- used to identify and locate mucosal areas with increased density of bulodynia is associated with increased B-cell infiltration and the presence lymphocytes and the presence of germinal centers (ie, signs of immune of germinal centers. This supports the fundamental role of immune acti- activation). B-cell activation index was used to describe the overall in- vation in provoked vestibulodynia. tensity of B-cell infiltration. RESULTS: We found more protein gene product 9.5epositive intra- Key words: germinal center, immune activation, immunohistochem- epithelial fibers in vestibulodynia than in the control samples (6.3/mm istry, inflammation, nerve fibers, NGF, NF2F11, PGP9.5, vestibulodynia, [range, 0.0e15.8] vs 2.0/mm [range, 0.0e12.0]; P¼.006). Neuron vulvar pain, vulvar vestibulitis, vulvodynia

rovoked vestibulodynia (PVD), Recently, we demonstrated the presence endings.8 Thus, it is important to study P which also is referred to as localized of secondary lymphoid tissue, which is the interrelation between immune acti- provoked vulvodynia, manifests as allo- the vestibule-associated lymphoid tissue vation and nerves in PVD. Previous dynia (severe pain by touch) of the (VALT) in the vestibular mucosa, and studies have shown increased density of vulvar vestibular mucosa in the absence showed that VALT becomes activated in nerves in the vestibular mucosa in PVD of any other disease or identifiable PVD. We showed higher numbers of B and increased expression of transient cause.1 cells in PVD than in control samples but receptor potential V1 (TRPV1) channels Histopathologic investigation of PVD found no difference in the density of T in these nerves,9-12 but no specific data typically reveals increased lymphocytic cells between the groups.4 An exagger- on the density of intraepithelial nerve infiltrates in the vestibular mucosa.2,3 ated immunoinflammatory response fibers (IENF) or expression of NGF exist. and dysregulation of inflammation seem We wanted to find out whether the to be present in PVD.5-7 The close rela- density and localization of IENFs and the Cite this article as: Tommola P, Unkila-Kallio L, Paetau tion between immune and neuronal expression of NGF are related to im- A, et al. Immune activation enhances epithelial nerve systems can activate neuroinflammatory mune activation in the vestibular growth in provoked vestibulodynia. Am J Obstet Gynecol processes and lead to sensitization of mucosal tissue in PVD. In addition to the 2016;215:768.e1-8. nerve fibers. Immune cells produce standard neural marker, the protein gene 0002-9378/$36.00 nerve growth factor (NGF), which may product 9.5 (PGP9.5), we used a specific ª 2016 Elsevier Inc. All rights reserved. fi 13 fi http://dx.doi.org/10.1016/j.ajog.2016.07.037 induce nerve sprouting and enhanced marker for neuro laments. To de ne signaling of the nociceptive nerve the sites of immune activation, we used 2

768.e1 American Journal of Obstetrics & Gynecology DECEMBER 2016 ajog.org GYNECOLOGY Original Research standard markers, CD20 (B cells) and exclude dermatologic diseases and to comparisons were made between PVD CD3 (T cells). We explored the differ- confirm the quality of the samples. samples and control samples. In PVD ences in the expression of IENFs and Immunohistochemistry for nerve fibers samples, densities of epithelial nerve fi- NGF between PVD and control samples (10-mm sections) and B and T lympho- bers were also compared between areas and in relation to different B-cell and cytes (5-mm sections) was performed at with or without increased B-cell infil- T-cell densities. the Helsinki and Uusimaa Hospital tration. The representative areas of B-cell District Laboratory Services tissue infiltration in each sample were located Material and Methods laboratory. Routine staining procedures with the use of CD20 staining. To reflect Study subjects according to the manufactures’ in- the overall level of B-cell infiltration of The study material consisted of 27 structions were followed (Table). Im- each sample, we used the B-cell activa- archival vestibulectomy specimens from munostaining for NGF (5-mm sections) tion index (BAI). BAI is the calculated posterior vestibulectomy operations. was performed at the Department of sum (0e12) of 3 different parameters The patients were identified in the Hel- Clinical Chemistry, University of Hel- that were analyzed from each sample: sinki University Hospital patient registry sinki (manual staining procedure15; (1) overall density of B cells in the by matching the diagnosis (vulvar Table). epithelium (score, 0e4), (2) overall vestibulitis, vestibulodynia, and vulvo- density of B cells in the stroma (score, dynia) and the surgical procedure (pos- Tissue analyses 0e4), and (3) absence (score, 0) or terior vestibulectomy). Details of patient Immunohistochemical scoring was per- presence (score, 4) of germinal centers.4 recruitment and data collection have formed under light microscopy (Nikon T-cell density was divided in the CD3- been described previously.4,14 A good Eclipse E800; Nikon Instruments Inc, stained samples into 2 categories: “low quality paraffin block of the tissue Melville, NY) at 200 magnification. density” (<50 cells/20 high-power specimen was required. All the included The scoring of each section was based on field) and “high density” (>50 cells/20 patients had a long disease history a consensus of 2 investigators (P.T., A.P., high-power field). Germinal centers were (4.0 years; range, 2e18 years) of PVD. or S.M.) who were blinded to clinical visualized by CD20 and CD3 stainings. The diagnoses for 8 patients were clas- data of the patients. The number of For NGF quantification, 4 types of sified as primary (symptoms already at PGP9.5-positive IENFs was counted to areas from the PVD samples were iden- the first vaginal entry); the diagnoses for calculate the linear density of IENFs tified: (1) areas with increased B-cell 15 patients were classified as secondary (number of nerve fibers /millimeters of infiltration without IENFs, (2) areas (symptoms appearing later after an in- epithelial outer surface). For identifica- without increased B-cell infiltration with terval of painless intercourses), and the tion of individual fibers, we used the IENFs present, (3) areas with both classification for 4 patients was un- criteria that had been validated for the increased B-cell infiltration and IENFs, known. All patients had been refractory diagnostics of small fiber neuropathies.16 and (4) areas lacking both B-cell infil- to conservative treatments. The time Briefly, the fibers were considered to be tration and IENFs. The number of NGF- from the last attempted medical man- separate if there were clearly 2 individual positive immune cells per visual field agement was >6 months. As control parallel fibers and if the distance between (20 high-power field) in 3 of each type subjects, we recruited 15 healthy volun- 2 different perpendicular sections of a of areas in each sample was counted, and teers with no vulvar complaints who stained axon exceeded 5 times the the mean number of positive cells was underwent benign gynecologic surgery. diameter of an axon. Only fibers clearly calculated. In the control samples, NGF- All participants were premenopausal. penetrating into the epithelium through positive cells were evaluated only in the The median age of the patients with PVD the basal membrane were counted as areas with IENFs present because no was 27 years (range, 18e48 years); the IENFs. For neuron specific neurofila- areas with increased B-cell infiltration median age of the control subjects was ment (NF2F11)epositive IENFs only were found. 30 years (range, 24e44 years; P¼.017). A the presence or absence was docu- The data were analyzed by Statistical 4-mm punch biopsy specimen from the mented. The overall density of neural Package for Social Sciences software posterior vestibule at 5 o’clock position fasciculi in the neural plexus region in (version 22; IBM Corporation, Armonk, was obtained from the control subjects. the subepithelial stroma up to the depth NY). We report medians with minimum Both patients and control subjects had of 1.25 mm (diameter of the 20 and maximum and interquartile range provided informed consent. The local high-power field) was scored semi- (IQR, 25e75%) when appropriate for Ethical Committee approved the study. quantitatively for both neural markers. A continuous data. For comparisons, we single number score from 1e3(1¼low used the Mann-Whitney U-test and Tissues density, 2¼moderate density, 3¼high Wilcoxon signed ranks test for contin- All vestibular tissues were embedded density) was given. uous data and c2 analysis or Fisher’s routinely in paraffin after a maximum of Evaluation of the vestibular glands exact test for categoric data. For corre- 24 hours fixation in 10% buffered was also limited to the depth of 1.25 mm. lations, the Spearman’s correlation test formalin. Five-micrometer sections were The glands were identified on the basis was used. A 2-tailed probability value of first stained with hematoxylin-eosin to of typical morphologic condition. All <.05 was considered significant.

DECEMBER 2016 American Journal of Obstetrics & Gynecology 768.e2 Original Research GYNECOLOGY ajog.org

TABLE Immunohistochemistry procedures

Clone; catalog Pretreatment buffer(pH); Dilution; Detection system; number; catalog number; incubation catalog number; Staining Antibody manufacturer manufacturer time/C manufacturer instrument Protein gene Polyclonal No pretreatment 1:1000 EnVision Detection LabVision product 9.5 SystemsPeroxidase/DAB (PGP9.5) RA95101 30 Min/room K5007 temperature Ultra Clone Ltd, Agilent Technologies Wellow, Isle of Wight, England Inc, Santa Clara, CA Neuron specific 2F11 Tris-EDTA (pH 9,0) 1:200 EnVision Detection LabVision neurofilament SystemsPeroxidase/DAB (NF2F11) M0762 S2367 30 Min/room K5007 temperature Agilent Agilent Agilent Technologies Inc Technologies Inc Technologies Inc CD20 L26 CC1 (pH 8,0) Ready to use UltraView Universal Ventana XT DAB Detection Kit 760-2531 24 Min/room 760-500 temperature Roche Diagnostics Roche Diagnostics Ltd Roche Diagnostics Ltd Ltd, Rotkreuz, Switzerland CD3 2GV6 CC1 (pH 8,0) Ready to use UltraView Universal Ventana XT DAB Detection Kit 790-4341 950-124 32 Min/room 760-500 temperature Roche Diagnostics Ltd Roche Diagnostics Ltd Roche Diagnostics Ltd Nerve growth Polyclonal EnVision FLEX Target 0.5 mg/mL MACH 4 Universal Manual factor (NGF) Retrieval Solution AP Polymer Kit, BioCare (pH 6,1) Medical, Concord, CA sc-548 K8005 Overnight/4C M4U536 Santa Cruz Agilent Biocare Medical Inc, Biotechnology Technologies Inc Concord, CA Inc, Santa Cruz, CA

Tommola et al. Epithelial nerve growth in provoked vestibulodynia. Am J Obstet Gynecol 2016.

Results samples (56.2%). The density of NF2F11-positive IENFs typically Intraepithelial nerve fibers PGP9.5-positive IENFs was significantly occurred as solitary fibers or in clusters Because PVD may be related to increased higher in PVD (6.3/mm [range, of 3e8 fibers (Figure 1, C) and were pain sensitivity, we looked at individual 0.0e15.8]; IQR, 4.4e9.2) than in con- found in 17 of the 27 PVD cases (63%) nerve fibers in the vestibular epithelium. trol samples (2.0/mm [range, 0.0e12.0]; and in none of the control cases Both in PVD samples and control IQR, 0.0e4.3; P¼.006; Figure 1, A and (P<.001). NF2F11-positive IENFs were samples, PGP9.5-positive IENFs were B). No significant difference was found as common in primary and secondary expressed typically in clusters. Thus, in the density of IENFs between 8 pri- PVD (P¼.627). such areas were studied for PGP9.5- mary PVD cases (7.5/mm [range, positive IENFs. PGP9.5-positive IENFs 3.3e15.8]; IQR, 5.3e8.3) and 15 sec- IENFs in relation to B-cell infiltrates were found in 24 of the 27 PVD samples ondary PVD cases (5.0/mm [range, Our previous study showed increased (88.9%) and in 8 of the 15 control 0.0e12.4]; IQR, 2.5e9.2; P¼.332). density of B lymphocytes in the vestibular

768.e3 American Journal of Obstetrics & Gynecology DECEMBER 2016 ajog.org GYNECOLOGY Original Research

FIGURE 1 and the BAI score was found (Spear- ’ ¼ 2¼ Intraepithelial nerve fibers in the vulvar vestibular mucosa man s Rho, 0.400; P .004; R 0.128). NF2F11-positive IENFs were not asso- ciated with the presence or absence of germinal centers. However, the BAI scores of the samples with NF2F11- positive IENFs were higher (5.0 [range, 1.0e9.0]; IQR, 4.0e6.0) than the BAI scores of the samples without fibers (2.0 [range, 1.0e9.0]; IQR, 1.0e3.0; P¼.005). No differences were found in the densities of PGP9.5-positive fibers between the low-density and high-density T-cell groups (6.8/mm [range, 0.0e15.8] and 4.6/mm [range, 0.0e12.4], respectively; P¼.112) or in the presence of NF2F11-positive fibers (P¼.080).

Glandular epithelium and nerve fibers in relation to B lymphocytes Vestibular glands typically occur in the subepithelial layer of the vulvar vestib- ular mucosa.17 In the PGP9.5-stained PVD samples, 14 regions with glan- dular epithelium were identified. Of these, 9 were in areas with increased lymphocytic infiltration that showed a glandular epithelial nerve fiber density of 25.0/mm (range, 3.1e48.0; IQR, 11.3e32.0; Figure 2, A). Five regions were in areas without increased numbers of lymphocytes. Here the glands showed fi fi Intraepithelial nerve fibers (white arrows) A, in provoked vestibulodynia in a mucosal area without signi cantly lower epithelial nerve ber e B-lymphocyte infiltration, B, in a control (stained by protein gene product 9.5), C, in provoked density of 2.0/mm (range, 0.0 17.0; vestibulodynia (stained by neuron specific neurofilament), and D, in provoked vestibulodynia in a IQR, 0.0e16.5; P¼.013). In the mucosal area with B-lymphocyte infiltration (protein gene product 9.5). E and F show the protein control samples, only 1 area of glandular gene product 9.5estained intraepithelial nerve fibers from panel A in a larger magnification. His- epithelium was identified with an tologic sections were counterstained with hematoxylin and photomicrographed with a 20 objective epithelial nerve fiber density of 2.0/mm, (Nikon Eclipse E800; Nikon Instruments Inc., Melville, NY). which was located in an area with no Tommola et al. Epithelial nerve growth in provoked vestibulodynia. Am J Obstet Gynecol 2016. lymphocytic infiltration. In the NF2F11- stained PVD samples, 16 glandular re- mucosa of patients with PVD.4 The the local lymphoid tissue, which we have gions were identified, 12 in areas with density of PGP9.5-positive IENFs was termed as VALT.4 Infiltrating lympho- lymphocytes and 4 in areas without. greater in the areas with increased B-cell cytes were primarily B cells that were Again, epithelial nerve fibers were pre- infiltration (5.3/mm; range, 0.0e23.3; stained by the CD20 marker. T cells were sent more commonly in glands that were IQR, 3.0e9.4) than in the areas with no B distributed more evenly across the sam- surrounded by lymphocytic infiltrates cells (4.0/mm [range, 0.0e11.3]; IQR, ples and did not form clusters. The (11/12) than in glands not surrounded 1.0e8.0; P¼.057; Figure 1, D). In control density of PGP9.5-positive IENFs was by lymphocytic infiltrates (2/4; P¼.053, samples, no areas with increased B-cell significantly higher in samples with Fisher’s exact test). infiltration were found. germinal centers (6.1/mm [range, 4.3e15.8]; IQR, 5.0e9.4) than in sam- Neural fasciculi in the subepithelial IENFs in relation to signs of immune ples without germinal centers (3.0/mm stroma in relation to signs of activation [range, 0.0e13.4]; IQR, 0.0e8.4; immune activation Germinal centers indicate immune P¼.020). A positive correlation between The densities of the neural fasciculi in activation and are the key component of the density of PGP9.5-positive IENFs the subepithelial stroma by PGP9.5 or

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FIGURE 2 vestibular mucosa was greater in PVD Nerve fibers in the vestibular glandular epithelium than in control subjects. Recently, we demonstrated the existence of the VALT4 and showed evidence of immune acti- vation in PVD by identifying germinal centers and higher densities of B cells in PVD than in control subjects. We now show that the epithelial neuro- proliferation is associated with VALTand takes place around the areas with B-cell infiltration. Our results suggest that im- mune activation explains the neuro- proliferation and may contribute to the altered pain sensation in PVD. Germinal centers emerge in the mu- cosa as a sign of immune activation. Although germinal centers mostly consist of B cells, T cells are also present in the follicular area and have important functional roles as helper cells that stimulate the immune responses.20 Density of T cells did not show effect on neuroproliferation. This is in line with our earlier study in which we, un- like others,3,21 found no difference in T-cell density between patients and Glandular epithelial nerve fibers in provoked vestibulodynia in vestibular mucosal areas with control subjects. Further studies are B-lymphocyte infiltration A and B, immunostaining for protein gene product 9.5, C and D, immu- needed to dissect the different T-cell nostaining for neuron specific neurofilament. Histologic sections were counterstained with hema- fi toxylin and photomicrographed with a 20 objective. subgroups in relation to nerve bers and development of germinal centers. Tommola et al. Epithelial nerve growth in provoked vestibulodynia. Am J Obstet Gynecol 2016. PGP9.5 is a well-established neuron- specific marker that detects the thinnest NF2F11 staining did not differ between of wide variation, there was no signifi- unmyelinated sensory C fibers, which PVD and control samples (P¼.110 and cant difference between primary and are <1 mm in thickness.22 PGP9.5 is the .498, respectively) or between primary secondary PVD in the density of NGF- gold standard biomarker for IENFs. and secondary PVD (P¼.289 and .657, positive cells (40.0 [range, 1.0e47.0] However, it is a pan-neuronal marker respectively). No association between and 20.0 [range, 3.0e102.0], respec- and, besides the small fibers, also stains neural tissue density and signs of im- tively; P¼.256). Similarly, densities of subepithelial nerve bundles. Previous mune activation was found (data not these cells in areas with IENFs did not studies on PVD have shown an overall shown). differ between PVD and control samples abundance of neural tissue in the (20.0 [range, 1.0e102.0] vs 17.5 [range, vestibular mucosa of patients with PVD NGF-positive immune cells in 0.0e 68.0], respectively; P¼.906; with no quantification of the IENF relation to lymphocyte infiltrates Figure 3). The numbers of NGF-positive density.3,11,23 Thus, by calculation of the and IENFs immune cells did not differ in relation to linear density of IENFs, the validated NGF is essential in the development of T-cell density either (25.0 [range, method of evaluating IENFs, we were peripheral nervous system by promoting 1.0e102.0] for the low-density samples able to demonstrate an actual increase in growth and survival of neural cells.18 In and 17.0 [range, 0.0e60.0] for the high- the IENFs in PVD. Furthermore, we mature tissue, it has important roles density samples; P¼.300). found that only the expressions of IENFs both in acute nociception and chronic differed between PVD and control pain.19 In the PGP9.5-stained PVD Comment samples and between inflammatory and samples, the number of NGF-positive We conducted a thorough analysis of noninflammatory areas; the overall immune cells was higher in the areas nerve fibers in the vestibular mucosa of density of neural tissue in the stroma with B-cell infiltrates and IENFs (20.0; patients with PVD and healthy control (detected by PGP9.5 and NF2F11) was range, 1.0e102.0) than in the areas subjects by using 2 different neural comparable. This finding disagreed with lacking both B-cell infiltrates and IENFs markers, PGP9.5 and NF2F11. We a previous study that showed neural (4.0 [range, 0.0e24.0]; P<.001). Because showed that the density of IENFs in the hyperplasia by PGP9.5 in PVD

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FIGURE 3 PGP9.5 and NF2F11 stainings. In fi Nerve growth factor positive immune cells in the vestibular mucosa agreement with our nding of IENFs, the density of nerve fibers was higher in the glandular epithelium of glands with surrounding B-cell infiltrates than in area without B cells. These are the first data on the nerve supply of the vestibular glands. The glandular innervation may also be a relevant factor in the sensitized pain perception in PVD. Recent research has revealed that neuropathic pain because of different diseases indicates different somatosen- sory profiles.35 By quantitative sensory testing, it is possible to distinguish be- Immunostaining for nerve growth factorepositive immune cells (red)inA, provoked vestibulodynia tween disorders with irritable noci- and B, control. Histologic sections were counterstained with hematoxylin and photomicrographed with a 20 objective. ceptors that show positive (ie, sensory gain) sensory signs and disorders with Tommola et al. Epithelial nerve growth in provoked vestibulodynia. Am J Obstet Gynecol 2016. nonirritable nociceptors that show negative (ie, sensory loss) sensory signs.36 compared with control samples9 and nerve fibers that are detected by the Many studies that have compared pa- with studies that used S-100 stain- NF2F11 antibody might be myelinated tients with PVD with pain-free control ing.3,23,24 However, the S-100 antigen is A-delta fibers that signal a faster mode of subjects have shown lower tactile, pain, not specific to neural tissue but may also sharp pain. It is important to note that and thermal thresholds and higher stain structures of dendritic cells.25 no NF2F11-positive epithelial nerve fi- suprathreshold magnitude estimations Dendritic cells are numerous in the bers were found in the control samples. for heat in the vulvar vestibule in stroma and epithelium of vestibular The unmyelinated C-fibers and thinly PVD.37,38 Only findings that have sug- mucosa, which may cause misinterpre- myelinated A-delta fibers together gested sensory gain have been reported.39 tation.4 We did not find any difference in constitute the small caliber nerve fibers. This might well be a result of increased the IENF density or in the density of These fibers transmit cold, warm, and density of irritable nociceptors. subepithelial neural tissue between pri- mechanical nociceptive stimuli.28 Many Increased density and sensitivity of mary and secondary PVD, contrary to neuropathic pain states (such as diabetic IENFs in PVD might be due to the up- previous reports that have suggested neuropathy, drug-induced neuropathies, regulation of NGF because of a previ- predominance of the overall neural tis- and the burning mouth syndrome) are ous inflammatory process and immune sue in primary PVD.3,24 characterized by decreased density of activation. In addition to our finding of NF2F11 detects neuronal axons by these small nerve fibers.29,30 Decreased epithelial neuroproliferation and its as- labeling the neurofilaments of the cyto- density of these IENFs in a skin biopsy is sociation with immune activation, skeleton. Neurofilament proteins are the hallmark of the diagnosis of small others have shown increased density of expressed exclusively in neurons and are fiber neuropathies.31 Our current the TRPV1 in PVD.12 NGF induces essential for radial growth, axonal caliber finding of a high density of intra- nerve sprouting and contributes to the maintenance, and myelination of the epithelial small fibers in PVD suggests generation of inflammatory hypersensi- axons.26 In mature neurons, the neuro- that PVD is not a small fiber neuropathy. tivity and allodynia.40-42 Inflammatory filaments are composed of 3 subunits To our knowledge, the only 2 other ex- and immune cells are major sources of with different molecular weights. Phos- amples with increased pain sensitivity NGF.19 In a recent study on experi- phorylated forms of the subunits are and increased density of IENFs that have mental chronic prostatitis in mice, expressed by axons; the non- been documented in the literature are Schwartz et al43 showed elevated levels of phosphorylated forms represent somal rectal hypersensitivity disorder and dry NGF in the prostate tissue and up- and dendritic proteins.27 Our study is eye disorder.32,33 Little is known about regulation of TRPV1 in the dorsal root the first to show NF2F11 expression the distribution of nerve fibers in the ganglion neurons. We found an in the vestibular mucosal tissue. We used genital mucosal tissue of healthy women. increased number of NGF-positive im- the monoclonal 2F11 antibody, which We, like others,2,34 demonstrated mune cells in the mucosal areas with detects the light molecular weight the histopathologic characteristic of B-cell infiltration and IENFs in PVD but phosphorylated form of the subunit (the increased lymphocytic inflammation, could not show any difference in the NFL subunit, 70 kDa), that is known to especially around the vestibular glands in number of NGF-positive cells between be crucial in the myelination process of PVD. We also demonstrated nerve fibers PVD and control samples. However, the axons.26 Thus, it is possible that these in the glandular epithelium by both IENFs were present in only some of the

DECEMBER 2016 American Journal of Obstetrics & Gynecology 768.e6 Original Research GYNECOLOGY ajog.org control samples, which may have caused evaluation of the C-fibers (numbers per proinflammatory immune response in women bias. Furthermore, without analyzing the length unit, millimeters) was not with vulvar vestibulitis syndrome. Am J Obstet Gynecol 2002;186:696-700. actual tissue level differences of NGF, no compromised because of the sample size 8. Donnerer J, Schuligoi R, Stein C. Increased conclusions about the role of NGF in difference. Dual staining to reveal the content and transport of substance P and PVD pain generation can be drawn. It TRPV1 channels in the C-fibers would calcitonin gene-related peptide in sensory may be a question of the amount of NGF have been useful. The detection of nerves innervating inflamed tissue: evidence for produced and not the number of cells myelin structures in the NF2F11- a regulatory function of nerve growth factor fi fi in vivo. Neuroscience 1992;49:693-8. producing it. Also, NGF might have been positive bers could have con rmed 9. fi Bornstein J, Cohen Y, Zarfati D, Sela S, up-regulated already in an earlier phase the A-delta nature of the bers. Ophir E. Involvement of heparanase in the of the disease and could no longer be We provide new information to the pathogenesis of localized vulvodynia. Int J verified in these samples that represent a question of the key mechanisms of Gynecol Pathol 2008;27:136-41. late phase of the condition. NGF recently allodynia in PVD. NGF may be up- 10. Halperin R, Zehavi S, Vaknin Z, Ben-Ami I, Pansky M, Schneider D. The major histopatho- has been linked to many other syn- regulated because of the activation of logic characteristics in the vulvar vestibulitis syn- dromes that indicate chronic and VALTand in turn induces sprouting and drome. Gynecol Obstet Invest 2005;59:75-9. neuropathic pain; research that targets sensitization of the nociceptive nerve fi- 11. Bohm-Starke N, Hilliges M, Falconer C, blocking NGF is ongoing.19 Encouraging bers, which suggests a key role to the Rylander E. Increased intraepithelial innervation results on the novel protein kinase in- interplay between immune and neural in women with vulvar vestibulitis syndrome. Gynecol Obstet Invest 1998;46:256-60. hibitor dilmapimod in reducing cyto- systems. Future research that will focus 12. Tympanidis P, Casula MA, Yiangou Y, kine production of the immune cells on mechanisms in PVD might lead ul- Terenghi G, Dowd P, Anand P. Increased 44 have been reported. Thus, as 1 poten- timately to the development of new vanilloid receptor VR1 innervation in vulvodynia. tial drug candidate, the protein kinase therapeutic interventions. n Eur J Pain 2004;8:129-33. inhibitor could inhibit neurogenic 13. Diepholder HM, Schwechheimer K, Mohadjer M, Knoth R, Volk B. A clinicopathologic inflammation in PVD. Acknowledgments fi and immunomorphologic study of 13 cases of Our study is the rst to report the We thank Kristiina Nokelainen and senior sci- ganglioglioma. Cancer 1991;68:2192-201. linear density of IENFs in PVD and is the entist Kati Räsänen from the Medicum, Depart- 14. Tommola P, Unkila-Kallio L, Paavonen J. first to show the expressions of NF2F11- ment of Clinical Chemistry, University of Helsinki, Long-term well-being after surgical or conser- Helsinki, Finland, and Mia Kero from the positive, possibly A-delta fibers, and vative treatment of severe vulvar vestibulitis. Department of Pathology, University of Helsinki Acta Obstet Gynecol Scand 2012;91:1086-93. innervation of the vestibular glands. A and Helsinki University Hospital, Finland, for 15. Rasanen K, Lehtinen E, Nokelainen K, et al. further strength is the higher number of their expertise in the immunohistochemistry Interleukin-6 increases expression of serine proven cases and control cases than in procedures. protease inhibitor Kazal type 1 through STAT3 in most previous tissue studies on colorectal adenocarcinoma. Mol Carcinog 2015. PVD.3,11,45 The 3-year age difference References http://dx.doi.org/10.1002/mc.22447. 1. 16. between cases and control cases, all being Bornstein J, Goldstein AT, Stockdale CK, Koskinen M, Hietaharju A, Kylaniemi M, et al. et al. 2015 ISSVD, ISSWSH and IPPS A quantitative method for the assessment of premenopausal, is unlikely to play a role. consensus terminology and classification of intraepidermal nerve fibers in small-fiber neu- In line with many previous studies on persistent vulvar pain and vulvodynia. Obstet ropathy. J Neurol 2005;252:789-94. vestibular tissue, our challenge was the Gynecol 2016;127:745-51. 17. Ferris DG, Cox JT, O’Connor DM, size difference between the PVD and 2. Pyka RE, Wilkinson EJ, Friedrich EG Jr, Wright VC, Foerster J. Anatomy and histology of the normal female lower genital tract. In: Modern control samples. To minimize the bias Croker BP. The histopathology of vulvar vestibulitis syndrome. Int J Gynecol Pathol 1988;7:249-57. colposcopy, textbook and atlas, 2nd ed. and to secure appropriate comparison, 3. Goetsch MF, Morgan TK, Korcheva VB, Li H, Dubuque, IA: Kendall/Hunt; 2004:28-35. the control biopsy samples were taken Peters D, Leclair CM. Histologic and receptor 18. Skaper SD. The biology of neurotrophins, exactly from the 5 o’clock position of the analysis of primary and secondary vestibulody- signaling pathways, and functional peptide mi- posterior vestibule. This is the most nia and controls: a prospective study. Am J metics of neurotrophins and their receptors. CNS Neurol Disord Drug Targets 2008;7:46-62. representative area of pain in PVD that Obstet Gynecol 2010;202:614.e1-8. 4. Tommola P, Butzow R, Unkila-Kallio L, 19. Mantyh PW, Koltzenburg M, Mendell LM, has been indicated by studies on vulvar Paavonen J, Meri S. Activation of vestibule- Tive L, Shelton DL. Antagonism of nerve growth pain mapping and studies on the effect of associated lymphoid tissue in localized pro- factor-TrkA signaling and the relief of pain. surgical treatment of PVD.46,47 In addi- voked vulvodynia. Am J Obstet Gynecol Anesthesiology 2011;115:189-204. tion, the depth of all analyses was limited 2015;212:476.e1-8. 20. Brandtzaeg P, Kiyono H, Pabst R, 5. to 1.25 mm according to the depth of Foster DC, Hasday JD. Elevated tissue levels Russell MW. Terminology: nomenclature of of interleukin-1 beta and tumor necrosis factor- mucosa-associated lymphoid tissue. Mucosal control biopsy samples to minimize the alpha in vulvar vestibulitis. Obstet Gynecol Immunol 2008;1:31-7. sample size bias. However, the size dif- 1997;89:291-6. 21. Leclair CM, Leeborg NJ, Jacobson- ference may have biased the evaluation 6. Foster DC, Falsetta ML, Woeller CF, et al. Dunlop E, Goetsch MF, Morgan TK. CD4-posi- of the focally distributed NF2F11 fibers. Site-specific mesenchymal control of inflamma- tive T-cell recruitment in primary-provoked The lack of gland tissue in the control tory pain to yeast challenge in vulvodynia- localized vulvodynia: potential insights into dis- afflicted and pain-free women. Pain 2015;156: ease triggers. J Low Genit Tract Dis 2014;18: samples probably was due to the small 386-96. 195-201. size of the biopsy samples. Most impor- 7. Gerber S, Bongiovanni AM, Ledger WJ, 22. Wilson PO, Barber PC, Hamid QA, et al. tantly, however, the linear density Witkin SS. Defective regulation of the The immunolocalization of protein gene product

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Acta Obstet possible pitfalls. Pathology 2011;43:239-47. 37. Pukall CF, Binik YM, Khalife S, Amsel R, Gynecol Scand 2011;90:1225-31. 26. Gentil BJ, Tibshirani M, Durham HD. Neuro- Abbott FV. Vestibular tactile and pain thresholds filament dynamics and involvement in neurolog- in women with vulvar vestibulitis syndrome. Pain Author and article information ical disorders. Cell Tissue Res 2015;360:609-20. 2002;96:163-75. 27. Laser-Azogui A, Kornreich M, Malka- 38. Lowenstein L, Vardi Y,Deutsch M,et al. Vulvar From the Departments of Obstetrics and Gynecology Gibor E, Beck R. Neurofilament assembly and vestibulitis severity: assessment by sensory and (Drs Tommola, Unkila-Kallio, and Paavonen), Pathology function during neuronal development. Curr pain testing modalities. Pain 2004;107:47-53. (Dr Paetau), Bacteriology and Immunology, Haartman Opin Cell Biol 2015;32:92-101. 39. Farmer MA, Maykut CA, Huberman JS, et al. Institute (Dr Meri), and the Pain Clinic, Anesthesiology, 28. Kandel ER, Schwartz JH, Jessell TM. Prin- Psychophysical properties of female genital Intensive Care, and Pain Medicine (Dr Kalso), University of ciples of neural science. 4th ed. New York, NY: sensation. Pain 2013;154:2277-86. Helsinki and Helsinki University Hospital, and the McGraw-Hill; 2000:472. 40. Woolf CJ, Safieh-Garabedian B, Ma QP, Research Programs Unit, Program of Immunobiology 29. Lauria G. Small fibre neuropathies. Curr Opin Crilly P, Winter J. Nerve growth factor contributes (Dr Meri), University of Helsinki, Helsinki, Finland. Neurol 2005;18:591-7. to the generation of inflammatory sensory hy- Received April 22, 2016; revised June 19, 2016; 30. Lauria G, Majorana A, Borgna M, et al. Tri- persensitivity. Neuroscience 1994;62:327-31. accepted July 11, 2016. geminal small-fiber sensory neuropathy causes 41. Eskander MA, Ruparel S, Green DP, et al. Supported by grants from the Finnish Medical Foun- burning mouth syndrome. Pain 2005;115:332-7. Persistent nociception triggered by nerve dation and Helsinki University Hospital Research Funds 31. Lauria G, Hsieh ST, Johansson O, et al. growth factor (NGF) is mediated by TRPV1 and (grant nos. TYH2013308 and TYH2013340). European federation of neurological Societies/ oxidative mechanisms. J Neurosci 2015;35: The authors report no conflict of interest. Peripheral nerve society guideline on the use of 8593-603. Preliminary results were presented at the XXIII World skin biopsy in the diagnosis of small fiber neu- 42. Lin CL, Heron P, Hamann SR, Smith GM. Congress of the International Society for the Study of ropathy: report of a joint task force of the Euro- Functional distinction between NGF-mediated Vulvovaginal Disease, New York, USA, July 27-29, 2015, pean Federation Of Neurological Societies and plasticity and regeneration of nociceptive at the XXII World Congress on Controversies in Obstetrics, the Peripheral Nerve Society. Eur J Neurol axons within the spinal cord. Neuroscience Gynecology and Infertility, Sept. 17-19, 2015, Budapest, 2010;17:903-12, e44-9. 2014;272:76-87. Hungary, and at the IX European Conference of the 32. Chan CL, Facer P, Davis JB, et al. Sensory 43. Schwartz ES, Xie A, La JH, Gebhart GF. European Society for Infectious Diseases in Obstetrics fibres expressing capsaicin receptor TRPV1 in Nociceptive and inflammatory mediator upre- and Gynecology, Riga, Latvia, Oct. 29-Nov. 1, 2015. patients with rectal hypersensitivity and faecal gulation in a mouse model of chronic prostatitis. Corresponding author: Pa¨ivi Tommola, MD. paivi. urgency. Lancet 2003;361:385-91. Pain 2015;156:1537-44. tommola@kolumbus.fi

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