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ORIGINAL RESEARCH |
From the Karolinska Institutet Danderyd Hospital, Division of Obstetrics and Gynecology, Danderyd; Karolinska Institutet, Division of Basic Oral Sciences, Huddinge; and AstraZeneca, Pathology Unit, Safety Assessment, Södertälje, Sweden.
Address reprint requests to: Nina Bohm-Starke, MD, Division of Obstetrics and Gynecology, Karolinska Institutet Danderyd Hospital, Danderyd, S-182 88, Sweden; E-mail: nina.bohm-starke{at}kvk.ds.sll.se.
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ABSTRACT |
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METHODS: Laser Doppler perfusion imaging was used to map the superficial blood flow in the vestibular mucosa in 20 women with vestibulitis and in 21 healthy control subjects. A possible correlation between perfusion values and graded erythema (1–5) around the vaginal introitus was analyzed. Changes in microvascular density in the posterior part of the mucosa were investigated in sections from ten patients and ten controls by a computer-assisted image-processing program. Induced vasoconstriction of terminal arterioles in the same posterior area was also studied.
RESULTS: Significant increases in perfusion values were registered in the posterior parts of the vestibular mucosa in patients compared with controls. The highest blood flow was registered in the posterior fourchette. The most pronounced erythema was also located in the posterior vestibule in the patients. However, there was no significant correlation between perfusion values and degree of erythema in the same individual. The microvascular density or the ability of vestibular arterioles to constrict did not differ between patients and controls.
CONCLUSION: Women with vestibulitis have an increased superficial blood flow and erythema in the posterior parts of the vestibular mucosa. The increased perfusion, most probably caused by a neurogenic vasodilatation contributes to, but does not fully explain the erythema. Atrophic changes of the surface epithelium should also be considered in the evaluation of an erythema.
The three diagnostic criteria of vulvar vestibulitis syndrome, 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, were stipulated by Friedrich in 1987.1 Friedrich’s criteria are widely used and have served as inclusion criteria for scientific studies. The most painful areas in vestibulitis are the mucosa around the Bartholin’s glands ductal openings and in the posterior part of the vestibule, including the hymen.2–5 It is in these areas where the erythema usually is observed even though it may also be present at the minor vestibular glands along the hymen.
One possible explanation of the erythema could be changes in the mucosal vascular perfusion. Cutaneous blood flow is mainly regulated by the diameter of contractile arterioles branching from subcapillary arterial network of smaller vessels.6 The diameter of the small arterioles is normally regulated by metabolic influence from the adjacent parenchymal cells, variations in temperature, and by neurophysiologic mechanisms.7 Stimulation of sympathetic nerve fibers containing noradrenaline as a transmitter, sometimes costored with neuropeptide Y, results in vasoconstriction. On the other hand, stimulation of parasympathetic nerve fibers is reported to have little effect on blood vessels.8 Furthermore, sensory neurons also have an important influence on the vascular regulation. The release of neuropeptides from C fibers in skin gives rise to an axon reflex causing vasodilatation and increased blood flow.9 The vulvar vestibule is of endodermal visceral origin,10 but is considered to have a nonvisceral innervation.11 We, therefore, assume that the neurophysiologic regulation of the local blood flow in the vestibular mucosa is similar to that of the skin.
Erythema might also be caused by neovascularization resulting in an enhanced microvascular content in the tissue. Angiogenesis with growth of new capillaries from a preexisiting network is regulated by the activation of specific growth factors from an appropriate stimulus.
This process is essential for reproduction, development, and repair, and is also present during angiogenic and neoplastic diseases.12
The aim of the present study was to evaluate vascular changes as a possible underlying cause of mucosal erythema in women with vulvar vestibulitis compared with healthy controls. The superficial blood perfusion was analyzed and correlated to the graded erythema around the vaginal introitus. We also wanted to ascertain whether the microvascular density was altered in the most symptomatic part of the vestibular mucosa in women with vestibulitis, and whether induced vasoconstriction of terminal arterioles in the same area differed from women with no vulvar pain.
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MATERIALS AND METHODS |
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All 30 patients were recruited from the vulvar open care unit at Danderyd Hospital and fulfilled Friedrich’s criteria of vulvar vestibulitis syndrome.1 When the diagnosis of vulvar vestibulitis was established, they were voluntarily asked to participate in the studies. Most of the patients included had earlier been treated with topical and oral medications mainly for recurrent candida infections (Table 1
). At the time of the investigations, no topical or systemic medication was used, and surgery for vulvar vestibulitis had not been performed. In addition, all patients examined by laser Doppler perfusion imaging had regular menstrual cycles, and none used oral contraceptives.
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All women were examined by the same gynecologist (NBS), and genital infections were ruled out. Cultures for sexually transmitted diseases and candida were obtained as well as wet preps to exclude bacterial vaginosis. Any woman with an ongoing genital infection was excluded. The participants gave their informed consent, and the study was approved by the Local Ethics Committee of the Karolinska Hospital.
Evaluation of mucosal erythema and superficial blood flow was carried out on days 7–11 of the menstrual cycle in a room with a temperature of 24–26C. The subjects were resting in lithotomy position for 15 minutes before the investigation to adjust to the environment, thereby reducing sympathetic vasoconstrictor tone. No smoking 2 hours before or coitus within 24 hours before the investigation were allowed. The vestibular mucosa around the vaginal introitus was first inspected, and tissue erythema was graded from 1 to 5, grade 1 representing a pale mucosa and grade 5 a pronounced erythema. All investigations were carried out by the same examiner (NBS).
Tissue perfusion was mapped with a laser Doppler perfusion imager (LDPIwin2.0, Lisca AB, Linköping, Sweden). Labia minora were gently folded apart. The computer-controlled optical scanner, positioned 20 cm above the tissue, directed the laser beam over the vestibulum. The whole vestibular mucosa was scanned moving the laser beam step by step in a rectangular pattern over an area of 5 x 6 cm. The total number of sites measured was 3000. The scanning procedure of about 3 minutes was performed twice with an interval of 5 minutes.
In the presence of moving blood cells, a fraction of light is Doppler shifted, detected and converted into an electrical signal for further processing. The output signal of the processor relates linearly to tissue perfusion and is given as mean perfusion values (V).15 This output signal is sampled and stored by a personal computer (Dell Desktop computer, KMC1A, Stockholm, Sweden). From the collected perfusion values, a color-coded image is generated and presented on a monitor. To facilitate identification of different structures in the scanned area, a black and white photograph is also generated. In the display image, further image processing and data analyses can be made.16
Perfusion analyses were carried out in seven specific areas of the mucosa, located from anterior to posterior of the vestibule (Figure 1). In the anterior vestibulum, lateral to the urethral orifice, an area A was analyzed on the right and left side, comprising of 200–300 measurement sites on each side. Area B represented the anterior and C the posterior part of the hymenal ring around the vaginal introitus, where the number of measurement sites was estimated to be 500 in each part. Approximately 200–300 measurement sites covered area D, representing the areas around the Bartholin’s glands ductal openings on the right and left side (Figure 1). Furthermore, the largest area (E), covering the posterior fourchette, was analyzed in 700–1000 measurement sites. Areas A and B in the anterior part of the vestibulum were reported less painful than the posterior areas (C–E) in patients.17 Comparisons of perfusion were made between areas A and D and areas B and C.
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The microvascular density of the vestibular mucosa was analyzed in previously obtained biopsies from ten women with vestibulitis and ten control subjects.13,14 Punch biopsies of 6 mm were obtained from the area around the right Bartholin’s gland ductal opening (area D, right side), after infiltration with local anesthetics without vasoconstrictor. The biopsies were fixed in 4% paraformaldehyde and paraffin embedded. For the present study, six uniform and randomly selected 4–5 µm thin sections from each biopsy were prepared for immunohistochemistry and incubated for 1 hour in room temperature with factor VIII primary antibody (rabbit antihuman, Dako, Glostrup, Denmark) in 4% swine serum, diluted 1:200 in phosphate buffer. After careful rinsing in Trissaline buffer solution, the biotin-conjugated secondary antibody (goat antirabbit, Dako, Glostrup, Denmark), diluted 1:300, was added for 30 minutes. The bound antibodies were detected by using a standard avidin-biotin-peroxidase system with 3.3' diaminobenzidine tetrahydrochloride as chromogen. The sections were then processed by an interactive computer-assisted image processing program (using a SUN SparcStation 20, Sun Microsystem Computer Corp., CA; MicroGOP 2000s, Context Vision, Linköping, Sweden). After image enhancement procedures, the image was thresholded, and the resulting binary image gave an accurate representation of the immunostained vessels. Subsequently, a stereologic cycloid grid for estimating vessel surface volume was superimposed on the image. The program counted the number of intercept points between cycloids and vessel profiles from which the surface of blood vessels per tissue volume was calculated as mm2/mm3.18,19
The observed mucosal erythema in patients and controls was first divided in two groups, grade 1–3 and grade 4–5. The 
2 test was then used to analyze any differences between patients and controls. The perfusion values of the laser Doppler scanning procedures for each analyzed area were calculated and used for statistical analyses and presented as mean values ± standard error of mean. For areas A and D, right and left side, the mean values were summarized and presented as only areas A and D in the statistics. The results were first analyzed by two-way analysis of variance with repeated measurements, comparing areas A and D and B and C within the various groups. One-way analysis of variance, with post hoc comparisons of means, Tukey honestly significant difference test, was then calculated to compare the perfusion values in areas A–E between the patient group and the control subjects. The correlation between blood flow and observed erythema in each subject was tested by Spearman rank correlation coefficient. The results of the stereologic measurements were analyzed by Mann-Whitney U test for small samples.
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RESULTS |
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). The women included reported no earlier trauma to the pelvic region that might have caused injury to the pudendal nerve. Only two patients and one control subject had gone through vaginal deliveries. During these deliveries, no episiotomies were carried out or any complications reported. Further clinical data of the two groups are summarized in Table 1.
The mucosa around the vaginal introitus revealed a significant difference in erythema as graded from 1 to 5 between patients and controls (P = .01). The graded erythema was 3.0 (3.0–5.0), median and quartiles, in the patients and 2.0 (1.5–2.0) in the control subjects. There was an evident overlap of the two groups (Figure 2). In the patients, the erythema was mostly located in the posterior part of the mucosa and around the Bartholin’s glands ductal openings. However, when erythema was present in the control subjects, it was more often diffusely located around the vaginal introitus.
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). In the anterior vestibulum represented by area A, there was no difference in perfusion (P = .4) between the patients (1.38 ± 0.06 V) and the control group (1.31 ± 0.06 V). However, in all other investigated areas (B–E), significant differences in perfusion between patients and controls were found.
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).
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). No perfusion differences between areas A and D were observed in the control group. After infiltration of noradrenaline in area D, right side, in four patients and four control subjects, the scanned perfusion decreased. The mucosa immediately turned pale at the injection site, and the mean perfusion decrease was 49% (1.14 ± 0.15 V to 0.58 ± 0.07 V) in the patients and 33% (1.41 ± 0.32 V to 0.94 ± 0.11 V) in the control group. The values of blood perfusion and observed erythema in the same subject did not correlate significantly.
The factor VIII stained sections used for stereologic measurements revealed abundant vascularization of small caliber vessels, consisting mainly of capillaries in all tissue sections in both groups. The results of the microvascular quantification in the vestibular mucosa were almost identical in the two groups, 78.1 ± 13.2 mm2/ mm3 (mean value ± standard error of mean) in patients and 76.5 ± 8.1 mm2/mm3 in the control subjects. A previous histopathologic evaluation of the biopsies, after routine staining, revealed a minor-to-moderate infiltration of lymphocytes in the subepithelial layers of the mucosa in both patients and controls.
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DISCUSSION |
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). There was evidence of perfusion differences in various parts of the mucosa and between the two groups. Our results show a significant increase in blood flow in the most painful part of the vestibular mucosa in the patients.
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Vasodilatation may also be neurogenically induced. Neurophysiologic mechanisms of vascular regulation consist of both generalized reflexes and local axon reflexes. General vasoconstriction can be evoked by cooling, mental stress, and arousal. Warming causes vasodilatation with an increased cutaneous blood flow because of the inhibition of vasoconstrictor activity and via active vasodilator mechanisms. Localized axon reflexes may be evoked by mechanical or chemical stimulation of unmyelinated afferent neurons (C fibers) in skin.29 Antidromic stimulation of sensory C fibers with the release of vasoactive neuropeptides initiates an axon reflex flare with local vasodilatation and increased blood flow.30,31 In a recent study using quantitative sensory tests, we obtained clear evidence of sensory abnormalities in the vestibular mucosa in women with vestibulitis. Hyperalgesia and lowered pain thresholds to punctate mechanical and heat stimuli were observed in the vestibulum as a whole, but most pronounced in the posterior part. These results suggest peripheral sensitization and/or proliferation of various types of C nociceptors in the mucosa.32 In one previous morphologic study, proliferation and sprouting of nerve fibers in the mucosa were found.33 We have observed an increase in free nerve endings in the mucosal epithelium in women with vestibulitis.13 We also found that these nerve endings contain calcitonin generelated peptide,14 a neuropeptide present in sensory nerves with direct vasodilator action on cutaneous arterioles. Calcitonin generelated peptide is implicated in neurogenic inflammation and is considered to be the major mediator of neurogenic vasodilatation in skin.9 These results, indicating somatosensory dysfunction of the peripheral nerves, may also explain our present observation of increased blood flow in the vestibular mucosa.
The various degree of mucosal erythema in vestibulitis is the most controversial and questioned diagnostic criterion among clinicians because the vestibular mucosa may have a reddish appearance even under normal conditions in women with no provocative pain in this region. However, in this study, the intensity of the observed erythema around the vaginal introitus was significantly more pronounced in the patient group. On the other hand, values of increased blood flow and the degree of erythema observed in each subject did not correlate significantly. This finding suggests that the intensity of the erythema does not reflect only the level of perfusion. Individual variations of epithelial thickness may also have an influence. In evaluating an erythema, factors that might induce atrophic changes of the surface epithelium should be considered. Increased perfusion in deeper mucosal blood vessels, not detectable by laser Doppler perfusion imaging, may also contribute to the erythema. One should also have in mind that mere inspection of the mucosal color is a subjective method with an enhanced risk of errors.
Changes in the microvascular density of the mucosa caused by neovascularization, for instance, might also enhance local blood flow. Neovascularization is present in inflammatory conditions such as rheumatoid and psoriatic arthritis. The vascular growth in these conditions is believed to be initiated by the action of synovial cytokines.34 Neovascularization in painful erythematous areas of vestibulitis has been reported.35 It was suggested that the angiogenesis is caused by deposits of fibrinogen and inflammatory products in the tissue. Our results do not provide evidence for increased capillary growth in the subepithelial part of the vestibular mucosa in women with vestibulitis.
In the present study, noradrenaline was injected in the vestibular mucosa to evaluate the vasomotor response in the vestibular arterioles. We chose to study area D, one of the most painful areas in patients. The release of noradrenaline from sympathetic nerves induces local vasoconstriction of arterioles via 
- and ß-adrenoreceptors.8 A major decrease of blood flow in the same area was observed in both patients and control subjects, showing an intact ability of the arterioles to constrict in response to adequate stimuli.
The etiology of vulvar vestibulitis is not yet clarified, but is believed to be multifactorial. Several trigger mechanisms have been discussed, such as recurrent candida vulvitis and human papilloma viral infection.2,3 Indeed, our patients did report previous candida infections more often than the controls (Table 1). Frequent use of topical treatment irritating the mucosa36 and psychosexual factors may also contribute to the development of vestibulitis.37 One major problem in solving the etiologic enigma of vestibulitis is the time interval between onset of pain and the first consultation. The mean duration of symptoms in this study was 4.8 years. The triggering process of vestibulitis may indeed have induced an inflammatory process initiating neurophysiologic changes including proliferation and sensitization of C fiber nociceptors.13,14,32,33,38 The inflammation might then subside, but continuous irritation of the hyperalgesic mucosa could maintain the peripheral sensitization of nociceptors and also explain the vasodilatation with increased blood flow and erythema of the most painful parts in the mucosa as demonstrated in this study.
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Footnotes |
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The authors acknowledge Mikael Norman, MD, PhD, Astrid Lindgren Children’s Hospital, Stockholm, Sweden, for valuable comments on the manuscript.
Received April 13, 2001. Received in revised form July 17, 2001. Accepted July 26, 2001.
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REFERENCES |
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