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Psychophysical evidence of nociceptor sensitization in vulvar vestibulitis syndrome
Pain 94 (2001) 177–183 www.elsevier.com/locate/pain
Psychophysical evidence of nociceptor sensitization in vulvar vestibulitis syndrome Nina Bohm-Starke a,*, Marita Hilliges b, Gunilla Brodda-Jansen c, Eva Rylander a, Erik Torebjo¨rk d a
Division of Obstetrics and Gynaecology, Karolinska Institutet Danderyd Hospital, S-182 88 Danderyd, Sweden b Division of Basic Oral Science, Karolinska Institutet, Huddinge, Sweden c Department of Rehabilitation Medicine, Karolinska Hospital, Stockholm, Sweden d Department of Clinical Neurophysiology, Uppsala University, Uppsala, Sweden Received 31 January 2001; received in revised form 19 April 2001; accepted 18 May 2001
Abstract Vulvar vestibulitis syndrome (VVS) is a long lasting disorder of superficial dyspareunia in young women. Quantitative sensory testing, including mechanical and temperature pain thresholds and warm/cold difference limen (WCL), was performed in the vestibular mucosa in 22 women (mean age 25.0 years) with vestibulitis and 20 control subjects (mean age 25.6 years). The tests were carried out on days 7–11 of the menstrual cycle. Patients had allodynia to mechanical testing with von Frey filaments, 14.3 ^ 3.1 mN in the symptomatic posterior area as compared with 158 ^ 33.5 mN in healthy subjects, P , 0:0001. The pain threshold to heat was 38.6 ^ 0.68C in patients and 43.8 ^ 0.88C in controls, P , 0:0001. In addition, pain threshold to cold was 21.6 ^ 1.28C in patients whereas cooling down to 68C was usually not painful in controls. WCL was 4.9 ^ 0.58C in patients and 9.6 ^ 1.58C in healthy subjects, P , 0:01. The results are compatible with the hypothesis that patients with VVS have an increased innervation and/or sensitization of thermoreceptors and nociceptors in their vestibular mucosa. q 2001 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. Keywords: Vulvar vestibulitis; Quantitative sensory test; Nociception; Hyperalgesia; Sensitization; C-fibres
1. Introduction Women with vulvar vestibulitis syndrome (VVS), are referred to vulvar clinics because of superficial dyspareunia. VVS is a long lasting disorder of pain on distension of the vaginal introitus. Any attempt at vaginal entry is painful, and the majority of these women are unable to have vaginal intercourse. Clinical examination of the vulvar vestibule shows tender areas with various degrees of erythema in the mucosa (Fig. 1). The areas most often affected are the mucosa around the Bartholin glands duct openings and the posterior part of the vestibule (Friedrich, 1987). The hymen is also frequently involved, with tenderness and thickening of the posterior part (Davis and Hutchison, 1999). The aetiology of vulvar vestibulitis is not yet clarified, but is thought to be multifactorial. Several etiological theories have been discussed, such as genital infections with candida and human papilloma virus (Baggish and Miklos, 1995; Bergeron et al., 1997; Davis and Hutchison, 1999). Frequent use of various topical treatments may irritate the mucosa * Corresponding author. Tel.: 146-8-655-5000; fax: 146-8-622-5833. E-mail address: [email protected] (N. Bohm-Starke).
(Marinoff and Turner, 1992) and the influence of oral contraceptives in the development of VVS has yet to be investigated. During the last few years, we have observed an increase in the number of patients with VVS being referred to us. This increase could partly be due to a rising tendency to seek help for this disorder that severely affects the sexual life of patients, but we believe that it also reflects a true increase in the incidence of VVS in the population. The vulvar vestibule is derived from the endoderm of the urogenital sinus. At Hart’s line, at the inner aspects of labia minora, the endodermal tissue meets the ectoderm covering the outer part of the vulva (Friedrich, 1983). The vestibular innervation originates from the pudendal nerve (S2–S4), containing somatic motor efferents and sensory afferents as well as autonomic nerve fibres from the inferior hypogastric plexus and caudal sympathetic chain ganglia (Wesselman et al., 1997). Although the vulvar vestibule is by definition visceral tissue, it is considered to have nonvisceral innervation (Cervero, 1994); thus sensations to touch, temperature and pain are similar to sensations evoked in the skin. On the other hand, the vagina is part of the internal genitalia, the visceral components of the female reproductive system. In the vaginal mucosa, intraepithelial
0304-3959/01/$20.00 q 2001 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. PII: S 0304-395 9(01)00352-9
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nerves are only present in the distal part close to the introitus (Hilliges et al., 1995). The principal symptom of VVS is allodynia evoked by touch and pressure in the vestibular mucosa, without any spontaneous background pain. Despite patients’ complaints of pain, there are only a few investigations focusing on possible affections of the peripheral nerve endings in these women. Morphological studies of the neural content in biopsies from the affected area have revealed structural changes in the innervation pattern with sprouting between epithelial cells (Westrom and Willen, 1998). An increase in superficial (intraepithelial) free nerve endings has been reported (BohmStarke et al., 1998). Furthermore, investigation of neuropeptide content in these intraepithelial nerve endings demonstrated an immunoreactivity to calcitonin gene-related peptide (CGRP) (Bohm-Starke et al., 1999). To our knowledge, putative changes in function of the sensory nerve endings in the vestibular mucosa have not been reported in women with VVS. In the present study we have investigated somatosensory functions as tested in the vestibular mucosa in women with VVS in comparison to healthy controls by using standardized quantitative sensory tests (QST). We wanted to characterize the sensory thresholds in an attempt to elucidate
possible pathophysiological mechanisms in this painful condition.
2. Materials and methods 2.1. Subjects Forty-two women were included in the study, of whom 22 fulfilled Friedrich’s criteria of VVS (Friedrich, 1987). These criteria include: (1) severe pain on vestibular touch or attempted vaginal entry, (2) tenderness to pressure localized within the vestibule and (3) physical findings confined to vestibular erythema of varying degrees. The patients were all recruited from the vulvar clinic at Danderyd Hospital. The control subjects consisted of 20 age-matched university students and hospital personnel. The women included were all healthy with no systemic disorders that might have influenced their sensory perception. None were using oral contraceptives and all had regular menstrual cycles. The characteristics of the two groups are summarized in Table 1. All women gave their informed consent and the study was approved by the Local Ethics Committee of the Karolinska Hospital.
Fig. 1. Photograph of patient with vulvar vestibulitis with characteristic erythema in the posterior part of the vestibule (arrows).
N. Bohm-Starke et al. / Pain 94 (2001) 177–183 Table 1 Clinical data of patients and control subjects Clinical variables
Patients ðn ¼ 22Þ
Control subjects ðn ¼ 20Þ
Age (years) Pregnancies Duration of symptoms (years) Previous candida infections (.3) Smoking (.10/day) b
25.0 (19–34) a 3/22 6.0 (2–13) a 13/22
25.6 (21–30) a 2/20 – 4/20
a b
0/22
1/20
Values are given as mean and range. More than 10 cigarettes/day.
2.2. Experimental design Nineteen patients and 20 control subjects underwent a full QST programme of the mucosa around the vaginal introitus. The QST included perceptual thresholds for warmth and cold. In addition, pain thresholds were obtained for heat, cold, punctate mechanical stimulation, vibration up to 50 Hz and distension of the mucosa. Three additional patients with VVS and six of the control subjects were tested for vibratory evoked pain thresholds up to a frequency of 120 Hz. Apart from the method used in distension of the mucosa,
Fig. 2. Schematic illustration of the investigated areas. Area A was situated in the vicinity of the urethral opening and area B was close to the Bartholin’s gland ductal opening. All tests were performed on the right side. Area A was stimulated first.
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the QST was performed in two different areas of the vulvar vestibule, A and B. Area A was tested in the mucosa in the upper part of the vestibule close to the urethral orifice (Fig. 2). This area is usually not reported as painful and served as a reference area for each subject (Eva et al., 1999). Area B was tested in the mucosa in the vicinity of the Bartholin’s gland duct opening (Fig. 2). All patients reported allodynia to touch and pressure in this area. Areas A and B were tested ipsilaterally on the right side in each subject. The tests were carried out on days 7–11 of the menstrual cycle to avoid interference with ovulation and menstruation. The subjects were lying in the lithotomy position in a room with temperatures of 24–268C for 15 min prior to the investigation. All investigations were carried out by the same examiner (N.B.-S.). The examiner was not blinded to the VVS vs. control status of the women being tested. This was not possible because the signs and symptoms of VVS were so pronounced. The tests were carried out in the order described below. The entire testing programme took about 45 min. 2.2.1. Punctate mechanical stimulation von Frey filaments, calibrated in a logarithmic scale from 1.2 to 575 mN, were used for punctate mechanical stimulation of areas A and B. They were hand-held and applied perpendicularly two or three times each on the mucosal surface. The procedure was carried out in a standardized way, starting with the thinnest filament. Each stimulus had a duration of approximately 2–3 s. The subjects reported verbally the first sensation of pain and the von Frey filament eliciting pain on two out of three applications was defined as pain threshold to be used for statistical analysis. 2.2.2. Thermal stimulation Thresholds for perceived warmth, cold, heat pain and cold pain were determined using the Marstock method (Somedic AB, Stockholm, Sweden). An area of 7 £ 15 mm 2 of the vestibular mucosa was warmed or cooled using a thermal stimulator with semiconductor junctions operating on the Peltier principal (Fruhstorfer et al., 1976). The applied temperature changes were measured as the interface temperature between mucosal surface and thermode. The thresholds were determined as the mean of values obtained in response to three to five stimuli. Starting from an adapting temperature of 318C, the rate of temperature change was 0.58C/s for thresholds for warmth and cold, and 18C/s for heat pain and cold pain thresholds. The temperature range tested was 6–508C. A training session was carried out in all subjects in non-affected skin in the thenar eminence or on the thigh before the vestibular mucosa was investigated. The contact plate, covered with a thin plastic film for sanitary reasons, was lightly applied to the mucosal surface in areas A and B. In the primary session the subjects were instructed to signal the first sensation of warmth and cold,
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changing the direction of thermal stimulation by pressing a button. The average of five stimuli was defined as the warm/ cold difference limen (WCL). Secondly, the pain threshold for heat and cold were determined. The subjects were instructed to press the button when the first sensation of pain occurred. Pain thresholds were determined as the average of three stimuli. It is important to note that temperature and heat pain thresholds were first determined in area A and then in area B. Cold pain thresholds were then determined in area A and finally in area B, in order to avoid paradoxical heat sensations. 2.2.3. Vibration Vibratory evoked pain was tested by a hand-held vibrating stimulator (TVR Model, HV-13D, Heiwa Electronic Industrial Co. Ltd, Japan). A plastic rounded probe of 5 mm diameter with a vibrating amplitude of 2 mm was connected to the stimulator. The probe was applied perpendicularly to the mucosa. The frequency was gradually increased from 0 to 50 Hz in both areas A and B, and the subjects reported verbally if they felt pain. The maximum of 50 Hz was chosen due to lack of standard data reported for the sensitivity to vibration in mucosal tissue. Area A is in close proximity to the pubic bone and vibration of higher frequencies might result in noxious stimuli of the periostium instead of perceived pain in the vestibular mucosa. A group of three extra women with vestibulitis and six of the control subjects were tested in both areas with frequencies ranging from 0 to a maximum of 120 Hz. 2.2.4. Distension Dilating the vaginal introitus in women with vestibulitis causes pain. In order to measure the painful sensation to distension of the tissue, a small and soft rubber balloon (Naturlatex, B. Braun Petzold GmbH, Germany) was placed in the vaginal introitus. The balloon was connected via a plastic tube to a pressure gauge calibrated in mmHg (SENSE Lab, CHAMP, Somedic AB, Ho¨ rby, Sweden). The balloon was gently filled with air through a manually operated pump, dilating the vaginal introitus to a pressure of 0–73 mmHg. The subjects were instructed to report verbally when the first sensation of pain appeared and the balloon was immediately emptied and withdrawn. The procedure was repeated two to three times, with an interval of approximately 2 min between distensions. A mean value was calculated and expressed in mmHg. 2.2.5. Statistical methods Analyses of variance (ANOVA) were used. The results of the punctate mechanical and thermal stimulation were first analyzed by two-way ANOVA with repeated measurements, comparing areas A and B within the various groups. Secondly, one-way ANOVA with post hoc comparisons of means, Tukey HSD test, was performed to compare the obtained thresholds in areas A and B between the different groups. Kruskal–Wallis ANOVA by ranks was used to
analyze the distension interruption thresholds. The significant difference in the number of patients and controls responding with pain to cold stimulation and distension was analyzed by Fischer’s exact test. 3. Results 3.1. Punctate mechanical stimulation All 19 patients were more sensitive, with a lower pain threshold in the symptomatic area B around the Bartholin’s gland duct opening (14.3 ^ 3.1 mN, mean ^ SEM), compared to area A (59.4 ^ 9.3 mN), when tested with von Frey filaments, P , 0:001. No significant difference in pain thresholds in area A (210 ^ 42.7 mN) and B (158 ^ 33.5 mN) was observed in the control group. The patients demonstrated a significant hypersensitivity to mechanical stimulation in both areas A and B, compared to the control group. This difference was most pronounced in area B, P , 0:0001 (Fig. 3). 3.2. Thermotest The differences in thresholds for warmth and cold detection (WCL) were investigated in areas A and B. The patients showed a significant difference between area A (7.2 ^ 0.88C) and area B (4.9 ^ 0.58C), with a decrease in WCL in area B, P , 0:05 (Table 2). In the patient group, this area of the mucosa also demonstrated a decreased WCL compared to the same area in the control group (9.6 ^ 1.58C), P , 0:01 (Fig. 4). There was no significant difference for temperature detection in area A between patients (7.2 ^ 0.88C) and controls (9.3 ^ 1.18C). Differences in thresholds for heat pain in areas A and B within the different groups were found in patients only, area B (38.6 ^ 0.68C) being more sensitive than area A (40.8 ^ 0.68C), P , 0:001 (Table 2). Thresholds for heat pain in the controls were in area A (43.1 ^ 0.88C) and in area B (43.8 ^ 0.8), ns. When comparing areas A and B between patients and controls, lower thresholds for heat
Fig. 3. Pain thresholds (mN, mean ^ SEM) to punctuate stimuli in areas A (A) and B (B). Filled symbols represent patients ðn ¼ 19Þ and open symbols represent control subjects ðn ¼ 20Þ.
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Table 2 Thermotest results a Test
Subjects
Pain in area A
Mean temperature ^ SEM (range) in area A (8C)
Pain in area B
Mean temperature ^ SEM (range) in area B (8C)
WCL WCL CP CP HP HP
P C P C P C
– – 3/19 1/20 19/19 20/20
7.2 ^ 0.8 (2.2–16.6) 9.3 ^ 1.1 (3.0–18.9) 19 ^ 4.6 (13–28) 12.0 40.8 ^ 0.6 (36.5–45.6) 43.1 ^ 0.8 (35.1–49.1)
– – 9/19 0/20 19/19 20/20
4.9 ^ 0.5 (2.2–11.5) 9.6 ^ 1.5 (4.1–29.7) 21.6 ^ 1.2 (17.5–26) – 38.6 ^ 0.6 (35.9–45.1) 43.8 ^ 0.8 (36.4–50)
a
WCL, warm/cold difference limen; CP, cold pain; HP, heat pain; P, patients ðn ¼ 19Þ; C, control subjects ðn ¼ 20Þ.
pain were observed in both areas in the patient group, with P , 0:05 in A and P , 0:0001 in B (Fig. 5). During the investigation of thresholds for cold pain, three patients (out of 19) reported painful sensations in area A at a mean temperature of 19 ^ 4.68C, and nine patients (out of 19) in area B at a mean temperature of 21.6 ^ 1.28C, while only one subject (out of 20) in the control group experienced pain at 128C in area A (Table 2). The number of patients reporting cold pain in both areas (12 out of 19), was significantly higher than in the controls (one out of 20), P ¼ 0:02. The sensation of the evoked pain during cold stimulation varied. Several patients reported a dull and burning sensation rather than icy cold. 3.3. Vibration When the probe was first applied to the mucosa at a low tapping frequency of 1–3 Hz, the stimuli evoked pain in some of the patients. The pain disappeared at frequencies above 3 Hz. Only one patient refused vibration because of pain at 30 Hz in area A and 20 Hz in area B. None of the control subjects experienced pain on vibration up to a frequency of 50 Hz. In the additional testing of frequencies up to 120 Hz, no pain was reported in three patients. Two of the control subjects experienced pain in area A at 100 and 120 Hz, respectively, but no pain was reported in area B in
Fig. 4. WCL (8C, mean ^ SEM) in areas A (A) and B (B). Filled symbols represent patients ðn ¼ 19Þ and open symbols represent control subjects ðn ¼ 20Þ.
the control group. In conclusion, pain was generally not induced by high-frequency vibratory stimulation in any of the groups examined. 3.4. Distension The mean pressure at which the distension was interrupted in patients (41.5 ^ 2.6 mmHg) was significantly lower than in control subjects (62.5 ^ 1.1 mmHg), P,0.001, (Fig. 6). All patients (19 out of 19) interrupted distension of the vaginal introitus due to pain, whereas only two control subjects (out of 20) reported pain. Another six (out of 20) of the control subjects reported various degrees of discomfort during the distension procedure. In the remaining group of 12 control subjects, the distension was not painful or unpleasant but was interrupted due to difficulties in keeping the inflated balloon in the vaginal introitus. 4. Discussion Women with VVS suffer from mechanical allodynia and hyperalgesia in the area around the vaginal introitus (Davis and Hutchison, 1999). Psychophysical studies on tenderness
Fig. 5. Heat pain thresholds (8C, mean ^ SEM) in areas A (A) and B (B). Filled symbols represent patients ðn ¼ 19Þ and open symbols represent control subjects ðn ¼ 20Þ.
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Fig. 6. Distension interruption thresholds (mmHg) in patients (VVS) ðn ¼ 19Þ and control subjects (C) ðn ¼ 20Þ. Black symbols represent subjects reporting pain, grey symbols various degrees of discomfort and open symbols no pain or discomfort.
in this area are rare (Eva et al., 1999). Moreover, there are no published data of normal values for temperature or pain thresholds to sensory stimulations in this area. In our study, thresholds for different sensory stimuli have been investigated in VVS affected mucosa in patients and in corresponding regions in healthy subjects. Evidence for somatosensory dysfunctions, involving several modalities, was found in the posterior part of the vestibule, the most often affected area in VVS. In addition, the results showed sensory abnormalities also in the anterior vestibular mucosa in women with vestibulitis, in apparently normal looking mucosa. The test stimuli used were short lasting and did not exceed pain thresholds. It seems unlikely, therefore, that the observed sensory abnormalities would be caused by sensitization of nociceptors by the test procedure itself. One of the diagnostic criteria of VVS is various degrees of erythema in the most sensitive part of the vestibular mucosa (Fig. 1) (Friedrich, 1987). This erythema could be consistent with a release of CGRP from normally mechanoinsensitive C-nociceptors, which can cause vasodilatation and axon reflex flare even at a very low level of activity (Schmelz et al., 2000a). However, these nociceptors should be ‘silent’ and not spontaneously active unless they are sensitized (Schmidt et al., 1995; Schmelz et al., 2000b). The allodynia evoked by stretch and punctate stimuli and heat hyperalgesia could be consistent with sensitization of both polymodal C-mechano-heat nociceptors (Torebjork et al., 1984) and normally mechano-insensitive C-nociceptors (Schmidt et al., 1995; Schmelz et al., 2000b), leading to temporal summation of the afferent input. In addition, an increase in the number of nociceptive endings would allow for an increased spatial summation of impulses, which could lead to a decrease in the pain threshold. Indeed, morpholo-
gical studies have shown proliferation and sprouting between epithelial cells (Westrom and Willen, 1998) and an increase in superficial (intraepithelial) free nerve endings in VVS (Bohm-Starke et al., 1998). Furthermore, these very thin, intraepithelial nerve endings showed immunoreactivity to CGRP (Bohm-Starke et al., 1999). Not only were pain thresholds lower, but also the detection of non-painful thermal stimuli was actually better in the painful region B, as compared to mucosa in healthy subjects. Again, this could possibly be due to an increase in the number of very thin intraepithelial nerve endings, leading to increased spatial summation of the afferent input. The increase in cold pain sensitivity could also be explained by such peripheral mechanisms, although little is known about sensitization and morphological changes of cold nociceptors in human skin. However, it is known that a block of impulse conduction in Ab- and d-fibres will unmask a C-fibre pain which gives a burning sensation instead of cold at temperatures as high as 22–238C in healthy human subjects (Wahren et al., 1989; Yarnitsky and Ochoa, 1990). Normally, cold pain would be felt around 5–158C. We have no reason to believe that our patients had loss of A-fibres. Rather, their increased and qualitatively different dull or burning pain sensation in response to cold stimuli could be due to sensitization and morphological changes in the C-fibres. Increased pain sensitivity for punctate mechanical and heat stimuli was found also in area A. This again would be consistent with peripheral sensitization of nociceptors. One could also imagine that the phenomenon might represent central sensitization in an area of secondary hyperalgesia surrounding the injury (LaMotte et al., 1991, 1992; Kilo et al., 1994). This might apply for punctate stimulation, which may persist for several hours after the ongoing background pain has subsided (LaMotte et al., 1991). However, two pieces of evidence contradict the theory of a present central sensitization in the present study. Allodynia was generally not present in our patients vibrated in their most painful area B. Instead, there was pain upon application of the vibratory probe on the mucosa and tapping at low frequencies of 1–3 Hz, suggesting activation of peripherally sensitized C-nociceptors which can follow such low frequencies but fatigue at higher frequencies (Torebjork and Hallin, 1974). The other piece of evidence is the hyperalgesia to heat in area A. Heat hyperalgesia has not been shown in the secondary zone in several investigations (LaMotte et al., 1991; Ali et al., 1996), although it has been claimed by others (Serra et al., 1998) to occur as a peripheral phenomenon evoked by heat stimuli clearly above the pain threshold. In essence, our results are in line with the idea that the sensory abnormalities found in our patients are best explained by peripheral noxious mechanisms involving sensitization and/or proliferation of various types of C-nociceptors. The present results have important clinical implications. First of all, the striking sensory abnormalities cannot be
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explained on the basis of psychological factors as suggested by some authors (de Jong et al., 1995). Instead, our evidence shows that the pain mechanisms are best explained by nociceptive rather than neurogenic mechanisms. This would mean that treatment should be directed towards the very periphery either by neurotoxic agents (Rumsfield and Dennis, 1991; Simone et al., 1998; Nolano et al., 1999), which have the potency of destroying superficial nociceptive nerve endings, or by reduction of their hyperexcitability by suitable ion channel blockers. Our findings are also compatible with the favourable outcome of surgery in this region, removing the affected mucosa, including the peripheral part of the primary afferent neuron (Woodruff et al., 1981; Friedrich, 1987; Mann et al., 1992; Davis and Hutchison, 1999). Acknowledgements This study was supported by grants from Fo¨ renade Liv Mutual Group Life Insurance Company, Stockholm, Sweden, and the Swedish Medical Research Council Project No. 5206. References Ali Z, Meyer RA, Campbell JN. Secondary hyperalgesia to mechanical but not heat stimuli following a capsaicin injection in hairy skin. Pain 1996;68:401–411. Baggish MS, Miklos JR. Vulvar pain syndrome: a review. Obstet Gynecol Surv 1995;50:618–627. Bergeron S, Binik YM, Khalife S, Pagidas K. Vulvar vestibulitis syndrome: a critical review. Clin J Pain 1997;13:27–42. Bohm-Starke N, Hilliges M, Falconer C, Rylander E. Increased intraepithelial innervation in women with vulvar vestibulitis syndrome. Gynecol Obstet Invest 1998;46:256–260. Bohm-Starke N, Hilliges M, Falconer C, Rylander E. Neurochemical characterization of the vestibular nerves in women with vulvar vestibulitis syndrome. Gynecol Obstet Invest 1999;48:270–275. Cervero F. Sensory innervation of the viscera: peripheral basis of visceral pain. Physiol Rev 1994;74:95–138. Davis GD, Hutchison CV. Clinical management of vulvodynia. Clin Obstet Gynecol 1999;42:221–233. Eva LJ, Reid WM, MacLean AB, Morrison GD. Assessment of response to treatment in vulvar vestibulitis syndrome by means of the vulvar algesiometer. Am J Obstet Gynecol 1999;181:99–102. Friedrich Jr. EG. The vulvar vestibule. J Reprod Med 1983;11:773–777. Friedrich Jr. EG. Vulvar vestibulitis syndrome. J Reprod Med 1987;32:110– 114. Fruhstorfer H, Lindblom U, Schmidt WC. Method for quantitative estimation of thermal thresholds in patients. J Neurol Neurosurg Psychiatry 1976;39:1071–1075. Hilliges M, Falconer C, Ekman Ordeberg G, Johansson O. Innervation of
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Psychophysical evidence of nociceptor sensitization in vulvar vestibulitis syndrome Nina Bohm-Starke a,*, Marita Hilliges b, Gunilla Brodda-Jansen c, Eva Rylander a, Erik Torebjo¨rk d a
Division of Obstetrics and Gynaecology, Karolinska Institutet Danderyd Hospital, S-182 88 Danderyd, Sweden b Division of Basic Oral Science, Karolinska Institutet, Huddinge, Sweden c Department of Rehabilitation Medicine, Karolinska Hospital, Stockholm, Sweden d Department of Clinical Neurophysiology, Uppsala University, Uppsala, Sweden Received 31 January 2001; received in revised form 19 April 2001; accepted 18 May 2001
Abstract Vulvar vestibulitis syndrome (VVS) is a long lasting disorder of superficial dyspareunia in young women. Quantitative sensory testing, including mechanical and temperature pain thresholds and warm/cold difference limen (WCL), was performed in the vestibular mucosa in 22 women (mean age 25.0 years) with vestibulitis and 20 control subjects (mean age 25.6 years). The tests were carried out on days 7–11 of the menstrual cycle. Patients had allodynia to mechanical testing with von Frey filaments, 14.3 ^ 3.1 mN in the symptomatic posterior area as compared with 158 ^ 33.5 mN in healthy subjects, P , 0:0001. The pain threshold to heat was 38.6 ^ 0.68C in patients and 43.8 ^ 0.88C in controls, P , 0:0001. In addition, pain threshold to cold was 21.6 ^ 1.28C in patients whereas cooling down to 68C was usually not painful in controls. WCL was 4.9 ^ 0.58C in patients and 9.6 ^ 1.58C in healthy subjects, P , 0:01. The results are compatible with the hypothesis that patients with VVS have an increased innervation and/or sensitization of thermoreceptors and nociceptors in their vestibular mucosa. q 2001 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. Keywords: Vulvar vestibulitis; Quantitative sensory test; Nociception; Hyperalgesia; Sensitization; C-fibres
1. Introduction Women with vulvar vestibulitis syndrome (VVS), are referred to vulvar clinics because of superficial dyspareunia. VVS is a long lasting disorder of pain on distension of the vaginal introitus. Any attempt at vaginal entry is painful, and the majority of these women are unable to have vaginal intercourse. Clinical examination of the vulvar vestibule shows tender areas with various degrees of erythema in the mucosa (Fig. 1). The areas most often affected are the mucosa around the Bartholin glands duct openings and the posterior part of the vestibule (Friedrich, 1987). The hymen is also frequently involved, with tenderness and thickening of the posterior part (Davis and Hutchison, 1999). The aetiology of vulvar vestibulitis is not yet clarified, but is thought to be multifactorial. Several etiological theories have been discussed, such as genital infections with candida and human papilloma virus (Baggish and Miklos, 1995; Bergeron et al., 1997; Davis and Hutchison, 1999). Frequent use of various topical treatments may irritate the mucosa * Corresponding author. Tel.: 146-8-655-5000; fax: 146-8-622-5833. E-mail address: [email protected] (N. Bohm-Starke).
(Marinoff and Turner, 1992) and the influence of oral contraceptives in the development of VVS has yet to be investigated. During the last few years, we have observed an increase in the number of patients with VVS being referred to us. This increase could partly be due to a rising tendency to seek help for this disorder that severely affects the sexual life of patients, but we believe that it also reflects a true increase in the incidence of VVS in the population. The vulvar vestibule is derived from the endoderm of the urogenital sinus. At Hart’s line, at the inner aspects of labia minora, the endodermal tissue meets the ectoderm covering the outer part of the vulva (Friedrich, 1983). The vestibular innervation originates from the pudendal nerve (S2–S4), containing somatic motor efferents and sensory afferents as well as autonomic nerve fibres from the inferior hypogastric plexus and caudal sympathetic chain ganglia (Wesselman et al., 1997). Although the vulvar vestibule is by definition visceral tissue, it is considered to have nonvisceral innervation (Cervero, 1994); thus sensations to touch, temperature and pain are similar to sensations evoked in the skin. On the other hand, the vagina is part of the internal genitalia, the visceral components of the female reproductive system. In the vaginal mucosa, intraepithelial
0304-3959/01/$20.00 q 2001 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. PII: S 0304-395 9(01)00352-9
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nerves are only present in the distal part close to the introitus (Hilliges et al., 1995). The principal symptom of VVS is allodynia evoked by touch and pressure in the vestibular mucosa, without any spontaneous background pain. Despite patients’ complaints of pain, there are only a few investigations focusing on possible affections of the peripheral nerve endings in these women. Morphological studies of the neural content in biopsies from the affected area have revealed structural changes in the innervation pattern with sprouting between epithelial cells (Westrom and Willen, 1998). An increase in superficial (intraepithelial) free nerve endings has been reported (BohmStarke et al., 1998). Furthermore, investigation of neuropeptide content in these intraepithelial nerve endings demonstrated an immunoreactivity to calcitonin gene-related peptide (CGRP) (Bohm-Starke et al., 1999). To our knowledge, putative changes in function of the sensory nerve endings in the vestibular mucosa have not been reported in women with VVS. In the present study we have investigated somatosensory functions as tested in the vestibular mucosa in women with VVS in comparison to healthy controls by using standardized quantitative sensory tests (QST). We wanted to characterize the sensory thresholds in an attempt to elucidate
possible pathophysiological mechanisms in this painful condition.
2. Materials and methods 2.1. Subjects Forty-two women were included in the study, of whom 22 fulfilled Friedrich’s criteria of VVS (Friedrich, 1987). These criteria include: (1) severe pain on vestibular touch or attempted vaginal entry, (2) tenderness to pressure localized within the vestibule and (3) physical findings confined to vestibular erythema of varying degrees. The patients were all recruited from the vulvar clinic at Danderyd Hospital. The control subjects consisted of 20 age-matched university students and hospital personnel. The women included were all healthy with no systemic disorders that might have influenced their sensory perception. None were using oral contraceptives and all had regular menstrual cycles. The characteristics of the two groups are summarized in Table 1. All women gave their informed consent and the study was approved by the Local Ethics Committee of the Karolinska Hospital.
Fig. 1. Photograph of patient with vulvar vestibulitis with characteristic erythema in the posterior part of the vestibule (arrows).
N. Bohm-Starke et al. / Pain 94 (2001) 177–183 Table 1 Clinical data of patients and control subjects Clinical variables
Patients ðn ¼ 22Þ
Control subjects ðn ¼ 20Þ
Age (years) Pregnancies Duration of symptoms (years) Previous candida infections (.3) Smoking (.10/day) b
25.0 (19–34) a 3/22 6.0 (2–13) a 13/22
25.6 (21–30) a 2/20 – 4/20
a b
0/22
1/20
Values are given as mean and range. More than 10 cigarettes/day.
2.2. Experimental design Nineteen patients and 20 control subjects underwent a full QST programme of the mucosa around the vaginal introitus. The QST included perceptual thresholds for warmth and cold. In addition, pain thresholds were obtained for heat, cold, punctate mechanical stimulation, vibration up to 50 Hz and distension of the mucosa. Three additional patients with VVS and six of the control subjects were tested for vibratory evoked pain thresholds up to a frequency of 120 Hz. Apart from the method used in distension of the mucosa,
Fig. 2. Schematic illustration of the investigated areas. Area A was situated in the vicinity of the urethral opening and area B was close to the Bartholin’s gland ductal opening. All tests were performed on the right side. Area A was stimulated first.
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the QST was performed in two different areas of the vulvar vestibule, A and B. Area A was tested in the mucosa in the upper part of the vestibule close to the urethral orifice (Fig. 2). This area is usually not reported as painful and served as a reference area for each subject (Eva et al., 1999). Area B was tested in the mucosa in the vicinity of the Bartholin’s gland duct opening (Fig. 2). All patients reported allodynia to touch and pressure in this area. Areas A and B were tested ipsilaterally on the right side in each subject. The tests were carried out on days 7–11 of the menstrual cycle to avoid interference with ovulation and menstruation. The subjects were lying in the lithotomy position in a room with temperatures of 24–268C for 15 min prior to the investigation. All investigations were carried out by the same examiner (N.B.-S.). The examiner was not blinded to the VVS vs. control status of the women being tested. This was not possible because the signs and symptoms of VVS were so pronounced. The tests were carried out in the order described below. The entire testing programme took about 45 min. 2.2.1. Punctate mechanical stimulation von Frey filaments, calibrated in a logarithmic scale from 1.2 to 575 mN, were used for punctate mechanical stimulation of areas A and B. They were hand-held and applied perpendicularly two or three times each on the mucosal surface. The procedure was carried out in a standardized way, starting with the thinnest filament. Each stimulus had a duration of approximately 2–3 s. The subjects reported verbally the first sensation of pain and the von Frey filament eliciting pain on two out of three applications was defined as pain threshold to be used for statistical analysis. 2.2.2. Thermal stimulation Thresholds for perceived warmth, cold, heat pain and cold pain were determined using the Marstock method (Somedic AB, Stockholm, Sweden). An area of 7 £ 15 mm 2 of the vestibular mucosa was warmed or cooled using a thermal stimulator with semiconductor junctions operating on the Peltier principal (Fruhstorfer et al., 1976). The applied temperature changes were measured as the interface temperature between mucosal surface and thermode. The thresholds were determined as the mean of values obtained in response to three to five stimuli. Starting from an adapting temperature of 318C, the rate of temperature change was 0.58C/s for thresholds for warmth and cold, and 18C/s for heat pain and cold pain thresholds. The temperature range tested was 6–508C. A training session was carried out in all subjects in non-affected skin in the thenar eminence or on the thigh before the vestibular mucosa was investigated. The contact plate, covered with a thin plastic film for sanitary reasons, was lightly applied to the mucosal surface in areas A and B. In the primary session the subjects were instructed to signal the first sensation of warmth and cold,
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changing the direction of thermal stimulation by pressing a button. The average of five stimuli was defined as the warm/ cold difference limen (WCL). Secondly, the pain threshold for heat and cold were determined. The subjects were instructed to press the button when the first sensation of pain occurred. Pain thresholds were determined as the average of three stimuli. It is important to note that temperature and heat pain thresholds were first determined in area A and then in area B. Cold pain thresholds were then determined in area A and finally in area B, in order to avoid paradoxical heat sensations. 2.2.3. Vibration Vibratory evoked pain was tested by a hand-held vibrating stimulator (TVR Model, HV-13D, Heiwa Electronic Industrial Co. Ltd, Japan). A plastic rounded probe of 5 mm diameter with a vibrating amplitude of 2 mm was connected to the stimulator. The probe was applied perpendicularly to the mucosa. The frequency was gradually increased from 0 to 50 Hz in both areas A and B, and the subjects reported verbally if they felt pain. The maximum of 50 Hz was chosen due to lack of standard data reported for the sensitivity to vibration in mucosal tissue. Area A is in close proximity to the pubic bone and vibration of higher frequencies might result in noxious stimuli of the periostium instead of perceived pain in the vestibular mucosa. A group of three extra women with vestibulitis and six of the control subjects were tested in both areas with frequencies ranging from 0 to a maximum of 120 Hz. 2.2.4. Distension Dilating the vaginal introitus in women with vestibulitis causes pain. In order to measure the painful sensation to distension of the tissue, a small and soft rubber balloon (Naturlatex, B. Braun Petzold GmbH, Germany) was placed in the vaginal introitus. The balloon was connected via a plastic tube to a pressure gauge calibrated in mmHg (SENSE Lab, CHAMP, Somedic AB, Ho¨ rby, Sweden). The balloon was gently filled with air through a manually operated pump, dilating the vaginal introitus to a pressure of 0–73 mmHg. The subjects were instructed to report verbally when the first sensation of pain appeared and the balloon was immediately emptied and withdrawn. The procedure was repeated two to three times, with an interval of approximately 2 min between distensions. A mean value was calculated and expressed in mmHg. 2.2.5. Statistical methods Analyses of variance (ANOVA) were used. The results of the punctate mechanical and thermal stimulation were first analyzed by two-way ANOVA with repeated measurements, comparing areas A and B within the various groups. Secondly, one-way ANOVA with post hoc comparisons of means, Tukey HSD test, was performed to compare the obtained thresholds in areas A and B between the different groups. Kruskal–Wallis ANOVA by ranks was used to
analyze the distension interruption thresholds. The significant difference in the number of patients and controls responding with pain to cold stimulation and distension was analyzed by Fischer’s exact test. 3. Results 3.1. Punctate mechanical stimulation All 19 patients were more sensitive, with a lower pain threshold in the symptomatic area B around the Bartholin’s gland duct opening (14.3 ^ 3.1 mN, mean ^ SEM), compared to area A (59.4 ^ 9.3 mN), when tested with von Frey filaments, P , 0:001. No significant difference in pain thresholds in area A (210 ^ 42.7 mN) and B (158 ^ 33.5 mN) was observed in the control group. The patients demonstrated a significant hypersensitivity to mechanical stimulation in both areas A and B, compared to the control group. This difference was most pronounced in area B, P , 0:0001 (Fig. 3). 3.2. Thermotest The differences in thresholds for warmth and cold detection (WCL) were investigated in areas A and B. The patients showed a significant difference between area A (7.2 ^ 0.88C) and area B (4.9 ^ 0.58C), with a decrease in WCL in area B, P , 0:05 (Table 2). In the patient group, this area of the mucosa also demonstrated a decreased WCL compared to the same area in the control group (9.6 ^ 1.58C), P , 0:01 (Fig. 4). There was no significant difference for temperature detection in area A between patients (7.2 ^ 0.88C) and controls (9.3 ^ 1.18C). Differences in thresholds for heat pain in areas A and B within the different groups were found in patients only, area B (38.6 ^ 0.68C) being more sensitive than area A (40.8 ^ 0.68C), P , 0:001 (Table 2). Thresholds for heat pain in the controls were in area A (43.1 ^ 0.88C) and in area B (43.8 ^ 0.8), ns. When comparing areas A and B between patients and controls, lower thresholds for heat
Fig. 3. Pain thresholds (mN, mean ^ SEM) to punctuate stimuli in areas A (A) and B (B). Filled symbols represent patients ðn ¼ 19Þ and open symbols represent control subjects ðn ¼ 20Þ.
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Table 2 Thermotest results a Test
Subjects
Pain in area A
Mean temperature ^ SEM (range) in area A (8C)
Pain in area B
Mean temperature ^ SEM (range) in area B (8C)
WCL WCL CP CP HP HP
P C P C P C
– – 3/19 1/20 19/19 20/20
7.2 ^ 0.8 (2.2–16.6) 9.3 ^ 1.1 (3.0–18.9) 19 ^ 4.6 (13–28) 12.0 40.8 ^ 0.6 (36.5–45.6) 43.1 ^ 0.8 (35.1–49.1)
– – 9/19 0/20 19/19 20/20
4.9 ^ 0.5 (2.2–11.5) 9.6 ^ 1.5 (4.1–29.7) 21.6 ^ 1.2 (17.5–26) – 38.6 ^ 0.6 (35.9–45.1) 43.8 ^ 0.8 (36.4–50)
a
WCL, warm/cold difference limen; CP, cold pain; HP, heat pain; P, patients ðn ¼ 19Þ; C, control subjects ðn ¼ 20Þ.
pain were observed in both areas in the patient group, with P , 0:05 in A and P , 0:0001 in B (Fig. 5). During the investigation of thresholds for cold pain, three patients (out of 19) reported painful sensations in area A at a mean temperature of 19 ^ 4.68C, and nine patients (out of 19) in area B at a mean temperature of 21.6 ^ 1.28C, while only one subject (out of 20) in the control group experienced pain at 128C in area A (Table 2). The number of patients reporting cold pain in both areas (12 out of 19), was significantly higher than in the controls (one out of 20), P ¼ 0:02. The sensation of the evoked pain during cold stimulation varied. Several patients reported a dull and burning sensation rather than icy cold. 3.3. Vibration When the probe was first applied to the mucosa at a low tapping frequency of 1–3 Hz, the stimuli evoked pain in some of the patients. The pain disappeared at frequencies above 3 Hz. Only one patient refused vibration because of pain at 30 Hz in area A and 20 Hz in area B. None of the control subjects experienced pain on vibration up to a frequency of 50 Hz. In the additional testing of frequencies up to 120 Hz, no pain was reported in three patients. Two of the control subjects experienced pain in area A at 100 and 120 Hz, respectively, but no pain was reported in area B in
Fig. 4. WCL (8C, mean ^ SEM) in areas A (A) and B (B). Filled symbols represent patients ðn ¼ 19Þ and open symbols represent control subjects ðn ¼ 20Þ.
the control group. In conclusion, pain was generally not induced by high-frequency vibratory stimulation in any of the groups examined. 3.4. Distension The mean pressure at which the distension was interrupted in patients (41.5 ^ 2.6 mmHg) was significantly lower than in control subjects (62.5 ^ 1.1 mmHg), P,0.001, (Fig. 6). All patients (19 out of 19) interrupted distension of the vaginal introitus due to pain, whereas only two control subjects (out of 20) reported pain. Another six (out of 20) of the control subjects reported various degrees of discomfort during the distension procedure. In the remaining group of 12 control subjects, the distension was not painful or unpleasant but was interrupted due to difficulties in keeping the inflated balloon in the vaginal introitus. 4. Discussion Women with VVS suffer from mechanical allodynia and hyperalgesia in the area around the vaginal introitus (Davis and Hutchison, 1999). Psychophysical studies on tenderness
Fig. 5. Heat pain thresholds (8C, mean ^ SEM) in areas A (A) and B (B). Filled symbols represent patients ðn ¼ 19Þ and open symbols represent control subjects ðn ¼ 20Þ.
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Fig. 6. Distension interruption thresholds (mmHg) in patients (VVS) ðn ¼ 19Þ and control subjects (C) ðn ¼ 20Þ. Black symbols represent subjects reporting pain, grey symbols various degrees of discomfort and open symbols no pain or discomfort.
in this area are rare (Eva et al., 1999). Moreover, there are no published data of normal values for temperature or pain thresholds to sensory stimulations in this area. In our study, thresholds for different sensory stimuli have been investigated in VVS affected mucosa in patients and in corresponding regions in healthy subjects. Evidence for somatosensory dysfunctions, involving several modalities, was found in the posterior part of the vestibule, the most often affected area in VVS. In addition, the results showed sensory abnormalities also in the anterior vestibular mucosa in women with vestibulitis, in apparently normal looking mucosa. The test stimuli used were short lasting and did not exceed pain thresholds. It seems unlikely, therefore, that the observed sensory abnormalities would be caused by sensitization of nociceptors by the test procedure itself. One of the diagnostic criteria of VVS is various degrees of erythema in the most sensitive part of the vestibular mucosa (Fig. 1) (Friedrich, 1987). This erythema could be consistent with a release of CGRP from normally mechanoinsensitive C-nociceptors, which can cause vasodilatation and axon reflex flare even at a very low level of activity (Schmelz et al., 2000a). However, these nociceptors should be ‘silent’ and not spontaneously active unless they are sensitized (Schmidt et al., 1995; Schmelz et al., 2000b). The allodynia evoked by stretch and punctate stimuli and heat hyperalgesia could be consistent with sensitization of both polymodal C-mechano-heat nociceptors (Torebjork et al., 1984) and normally mechano-insensitive C-nociceptors (Schmidt et al., 1995; Schmelz et al., 2000b), leading to temporal summation of the afferent input. In addition, an increase in the number of nociceptive endings would allow for an increased spatial summation of impulses, which could lead to a decrease in the pain threshold. Indeed, morpholo-
gical studies have shown proliferation and sprouting between epithelial cells (Westrom and Willen, 1998) and an increase in superficial (intraepithelial) free nerve endings in VVS (Bohm-Starke et al., 1998). Furthermore, these very thin, intraepithelial nerve endings showed immunoreactivity to CGRP (Bohm-Starke et al., 1999). Not only were pain thresholds lower, but also the detection of non-painful thermal stimuli was actually better in the painful region B, as compared to mucosa in healthy subjects. Again, this could possibly be due to an increase in the number of very thin intraepithelial nerve endings, leading to increased spatial summation of the afferent input. The increase in cold pain sensitivity could also be explained by such peripheral mechanisms, although little is known about sensitization and morphological changes of cold nociceptors in human skin. However, it is known that a block of impulse conduction in Ab- and d-fibres will unmask a C-fibre pain which gives a burning sensation instead of cold at temperatures as high as 22–238C in healthy human subjects (Wahren et al., 1989; Yarnitsky and Ochoa, 1990). Normally, cold pain would be felt around 5–158C. We have no reason to believe that our patients had loss of A-fibres. Rather, their increased and qualitatively different dull or burning pain sensation in response to cold stimuli could be due to sensitization and morphological changes in the C-fibres. Increased pain sensitivity for punctate mechanical and heat stimuli was found also in area A. This again would be consistent with peripheral sensitization of nociceptors. One could also imagine that the phenomenon might represent central sensitization in an area of secondary hyperalgesia surrounding the injury (LaMotte et al., 1991, 1992; Kilo et al., 1994). This might apply for punctate stimulation, which may persist for several hours after the ongoing background pain has subsided (LaMotte et al., 1991). However, two pieces of evidence contradict the theory of a present central sensitization in the present study. Allodynia was generally not present in our patients vibrated in their most painful area B. Instead, there was pain upon application of the vibratory probe on the mucosa and tapping at low frequencies of 1–3 Hz, suggesting activation of peripherally sensitized C-nociceptors which can follow such low frequencies but fatigue at higher frequencies (Torebjork and Hallin, 1974). The other piece of evidence is the hyperalgesia to heat in area A. Heat hyperalgesia has not been shown in the secondary zone in several investigations (LaMotte et al., 1991; Ali et al., 1996), although it has been claimed by others (Serra et al., 1998) to occur as a peripheral phenomenon evoked by heat stimuli clearly above the pain threshold. In essence, our results are in line with the idea that the sensory abnormalities found in our patients are best explained by peripheral noxious mechanisms involving sensitization and/or proliferation of various types of C-nociceptors. The present results have important clinical implications. First of all, the striking sensory abnormalities cannot be
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