Objective
Women with vestibulodynia exhibit increased pain sensitivity to contact with the vaginal vestibule as well as with vaginal penetration. The mechanism(s) responsible for this effect remains incompletely defined. Based on reports of a possible role for proteases in induction of pain, we compared levels of proteases and protease inhibitors in vaginal secretions from women with vestibulodynia and controls.
Study Design
Vaginal secretions from 76 women with vestibulodynia and from 41 control women were assayed by an enzyme-linked immunosorbent assay for the protease inhibitors, secretory leukocyte protease inhibitor (SLPI) and human epididymis protein-4 (HE-4), and the proteases, kallikrein-5 and cathepsins B and S. Concentrations between subjects and controls were compared and levels related to clinical and demographic variables.
Results
Concentrations of HE-4 and SLPI were markedly reduced in vaginal samples from women with vestibulodynia compared with controls ( P ≤ .006). All other compounds were similar in both groups. HE-4 ( P = .0195) and SLPI ( P = .0033) were lower in women with secondary, but not primary, vestibulodynia than in controls. Subjects who had constant vulvar pain had lower levels of HE-4 and SLPI than did healthy control women ( P ≤ .006) or women who experienced vulvar pain only during sexual intercourse ( P ≤ .0191). There were no associations between HE-4 or SLPI levels and event associated with symptom onset, duration of symptoms, age, number of lifetime sexual partners, or age at sex initiation.
Conclusion
Insufficient vaginal protease inhibitor production may contribute to increased pain sensitivity in an undefined subset of women with secondary vestibulodynia who experience constant vulvar pain.
Vestibulodynia, otherwise known as vulvar vestibulitis syndrome, is a clinically defined disorder characterized by an inability to have pain-free vaginal penetration over a prolonged period of time in the absence of a detectable infectious, neurological, hormonal, or other physical explanation. Symptoms may have begun at the first act of sexual intercourse or tampon insertion (primary vestibulodynia) or after a period of pain-free vaginal penetration (secondary vestibulodynia).
Approximately 50% of women with vestibulodynia can associate a specific event with the onset of symptoms: most commonly sexual intercourse with a specific partner, a vulvovaginal infection (predominantly Candida albicans ), childbirth, or a physical or psychological trauma. Studies have identified increased inflammation and the presence of proinflammatory cytokines, elevated mast cell levels, or a proliferation of nerve fibers in the vestibule of women with vestibulodynia. However, the mechanism(s) responsible for a pain response to normally innocuous stimuli (allodynia) and an exaggerated pain response to a mild pain stimulus (hyperalgesia) remains undetermined.
Recent studies have revealed the existence of a previously unknown nociceptive pain induction pathway. Selective proteolytic cleavage of a receptor, identified as protease-activated receptor 2 (PAR 2), on the surface of sensory afferent neuronal cells unmasks a cryptic sequence on the membrane and results in the induction of long-lasting allodynia and hyperalgesia. The possible involvement of this mechanism in pain sensitization in cancer patients has been recently evaluated. Injection of protease-containing supernatants from in vitro-cultured cancer cell lines into rats and mice elicited PAR 2 activation and the induction of a marked increase in pain sensitivity. This effect was inhibited if the culture supernatants were first incubated with a serine protease inhibitor.
To evaluate whether this protease-induced mechanism might contribute to allodynia and hyperalgesia in women with vestibulodynia, we evaluated vaginal secretions from patients and controls for concentrations of proteases and protease inhibitors. Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor produced by neutrophils, macrophages, and epithelial cells and is present in the female genital tract. Human epididymis protein 4 (HE-4) is an inhibitor of serine, aspartyl, and cysteine proteases and is also present in vaginal secretions. Kallikrein 5 is a serine protease, whereas cathepsin B and cathepsin S are cysteine proteases. All are present in vaginal secretions.
Materials and Methods
The study group consisted of 76 women with strictly defined vestibulodynia: severe pain in the vulvar vestibule upon light touch with a cotton swab, pain during tampon insertion or sexual intercourse, variable degrees of vestibular erythema, and symptom duration for at least 3 consecutive months. Controls were 41 sexually active women with no history of vestibulodynia and no current vaginal disorder.
Women with a fungal or bacterial infection, dermatological disorder, hormonal or neuronal abnormalities, undergoing hormonal or corticosteroid treatment, who were pregnant, or had a history of malignancy were excluded from both groups. Subjects completed a detailed questionnaire with the aid of their physician about present symptoms, medical history, and genital tract pain tolerance. The study was approved by the Institutional Review Board of Weill Cornell Medical College, and written informed consent was obtained from all participants.
Samples were collected by inserting a cotton swab against the posterior vaginal wall for 5 seconds during a speculum examination and then shaking the contents into 1 mL of sterile phosphate-buffered saline. The mixture was immediately centrifuged and the supernatants stored in aliquots at –80°C until tested.
Concentrations of protease inhibitors and proteases in the vaginal samples were measured by commercial enzyme-linked immunosorbent assay kits. Values were converted to nanograms per milliliter by reference to a standard curve that was generated with each assay. A subset of samples was reassayed on a different day to validate reliability. Interassay variability was <10%. The source and lower limit of sensitivity of each kit is as follows: HE-4 (Antibodies-Online, Atlanta, GA; 38 pg/mL), SLPI (R&D Systems, Minneapolis, MN; 25 pg/mL), kallikrein 5 (R&D Systems; 78 pg/mL), cathepsin B (Antibodies-Online; 5 pg/mL), and cathepsin S (R&D Systems; 15.6 pg/mL).
Differences between subjects and controls in each assay and between different groups of subjects were analyzed by the Mann-Whitney test because the values were not normally distributed. Correlations between clinical variables and protease inhibitor concentrations were analyzed by the Spearman rank correlation test. A value of P < .05 was considered significant.
Results
Characteristics of the study subjects are summarized in Table 1 . The ages and race/ethnic breakdown of the women with vestibulodynia and controls were comparable. Among the women with vestibulodynia, 22% had symptoms at their first attempt at vaginal penetration (primary vestibulodynia), whereas 78% developed symptoms following a period of time of pain-free penetration. The duration of symptoms ranged from 3 months to 3 years, the mean age of first sexual intercourse was 19.7 years, the mean number of sexual partners was 5, and none were smokers.
| Variables | Vestibulodynia | Controls |
|---|---|---|
| Number studied | 76 | 41 |
| Mean age, y (SD) | 35.4 (11.0) | 35.7 (11.6) |
| Race/ethnicity | ||
| White | 77.9% | 82.3% |
| Asian | 4.4% | 0% |
| Black | 2.9% | 2.4% |
| Hispanic | 1.5% | 4.8% |
| Unknown | 13.3% | 10.5% |
| Primary vestibulodynia | 22.0% | |
| Secondary vestibulodynia | 78.0% | |
| Median symptom duration, mo | 24.0 (3-360) mo | |
| Mean age at first intercourse, y (SD) | 19.7 (3.4) | ND |
| Median number of sexual partners | 5.0 (0-17) | ND |
| Current smoker | 0 | 0 |
The median concentration of protease inhibitors and proteases in vaginal secretions from women with vestibulodynia and controls is shown in Table 2 . Only the levels of the 2 protease inhibitors, HE-4 ( P = .0019) and SLPI ( P = .0064), were significantly decreased in patients as compared with concentrations in controls.
| Compound | Vestibulodynia (ng/mL), median (range) | Controls (ng/mL), median (range) | P value |
|---|---|---|---|
| HE-4 | 0.4 (<0.04–4.4) | 1.2 (<0.04–5.6) | .0019 |
| SLPI | 29.5 (<0.03–672) | 101 (1.3–769) | .0064 |
| Kallikrein 5 | 4.2 (<0.01–68.9) | 7.4 (0.4–20.3) | .5108 |
| Cathepsin B | 18.4 (0.5–760) | 19.3 (0.8–65.8) | .8042 |
| Cathepsin S | 1.8 (0.1–12.8) | 3.9 (0.1–11.9) | .2751 |
In marked contrast, the levels of the 3 proteases were comparable in both groups. Differences in HE-4 and SLPI levels between women with primary or secondary vestibulodynia are shown in Table 3 . Only in the women with secondary vestibulodynia were HE-4 ( P = .0195) and SLPI ( P = .0033) levels significantly reduced relative to concentrations present in the control women. The reduction in SLPI concentration between women with primary vestibulodynia and controls did not reach statistical significance ( P = .0922).
| Inhibitor | Primary | Secondary | Controls |
|---|---|---|---|
| HE-4, ng/mL | 1.2 (<0.04-4.4) | 0.4 (<0.04-139) a | 1.2 (<0.04-5.6) |
| SLPI, ng/mL | 43.1 (1.4-640) | 23.6 (0.5-670) b | 101 (1.3-769) |
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