Leclair, Catherine M. MD; Goetsch, Martha F. MD, MPH; Li, Hong MS; Morgan, Terry K. MD, PhD
Departments of Obstetrics and Gynecology, Pathology, and Public Health & Preventative Medicine, Oregon Health & Science University, Portland, Oregon.
Corresponding author: Catherine M. Leclair, MD, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, UHN 50, Portland, OR 97239; e-mail: email@example.comAbstract.
Funded by the National Vulvodynia Association. Dr. Morgan's contribution was also funded by the Office of Research on Women's Health and the National Institute of Child Health and Human Development, Oregon BIRCWH HD043488-08.
Presented to the Pacific Coast Obstetric and Gynecologic Society, October 3–7, 2012, Newport Beach, California.
Financial Disclosure The authors did not report any potential conflicts of interest.
OBJECTIVE: To assess whether premenopausal and postmenopausal vestibulodynia have different histologic features.
METHODS: We conducted a retrospective analysis of vestibulectomy specimens from 21 women with postmenopausal vestibulodynia and compared them with 88 premenopausal patients (42 primary, 46 secondary). Women with primary vestibulodynia experienced pain at first introital touch and women with secondary vestibulodynia experienced pain after an interval of painless intercourse. Clinical records established the type of vestibulodynia, duration of symptoms, and hormone status. Tissues were stained for inflammation, nerves, mast cells, estrogen receptor α, and progesterone receptor. Histologic findings in the postmenopausal patients were compared with primary and secondary premenopausal patients using proportional odds logistic regression and analysis of variance.
RESULTS: Seventy-one percent (15/21) of postmenopausal women reported vestibular dyspareunia related to a drop in estrogen either with menopause (13/21) or previously, postpartum (2/21). Eighty-six percent (18/21) of postmenopausal patients were using local or systemic estrogen but pain persisted. Compared with premenopausal primary and secondary vestibular biopsies, postmenopausal tissues had more lymphocytes (unadjusted odds ratio [OR] 9.0, 95% confidence interval [CI] 2.8–33.3; adjusted OR for parity and duration of symptoms 9.1, 95% CI 2.6–31.9; unadjusted OR 6.2, 95% CI 1.9–20.0; adjusted OR 6.6, 95% CI 2.0–21.9, respectively) and mast cells (mean 36 compared with 28 and 36 compared with 26, respectively). There was significantly less neural hyperplasia and progesterone receptor expression in postmenopausal biopsies compared with primary cases but less progesterone receptor and similar neural hyperplasia compared with premenopausal secondary cases. Estrogen receptor α did not vary among groups.
CONCLUSION: Premenopausal and postmenopausal vestibulodynia share histologic features of neurogenic inflammation but differ strikingly in degree. When estrogen supplement does not alleviate symptoms of postmenopausal dyspareunia, vestibulodynia should be considered.
LEVEL OF EVIDENCE: II
Vestibulodynia is a localized pain condition of the vulvar vestibule and is defined as vulvar discomfort, most often described as burning pain, occurring in the absence of relevant visible findings or a clinically identifiable disorder.1 This condition affects approximately 8–15% of women.2–4 Although vestibulodynia is thought to be a leading cause of dyspareunia,5 this condition has been traditionally associated only with premenopausal women. Dyspareunia has been poorly described in menopause, perhaps as a result of assumptions that pain relates simply to tissue aging and hormonal changes.
Addressing dyspareunia and sexual concerns is important throughout a woman's lifetime. Studies indicate that women continue to stay sexually active through the menopause transition and share concerns of dyspareunia and sexual dysfunction similar to their younger counterparts.6,7 Because vulvovaginal hypoestrogenism contributes to poor lubrication and difficult arousal,7–9 many menopausal women treat these symptoms with estrogen supplementation or artificial lubricants, yet when dyspareunia persists, vestibulodynia in this population is often not considered. Persistence of vulvar pain after estrogen support in menopausal women merits evaluation for causes beyond atrophy, including vestibulodynia.
Our vulvar research group has analyzed 109 (88 premenopausal and 21 postmenopausal) archived surgical tissues from patients with vestibulodynia who underwent a vestibulectomy between 2002 and 2008. We recently reported on the histopathologic profile of the 88 premenopausal women with primary and secondary vestibulodynia.10 Primary patients were those who had always noted pain with penetration and secondary patients first had no dyspareunia but then acquired the symptom. Both types of vestibulodynia had histologic features of neurogenic inflammation, which is characterized by abundant lymphocytes, mast cells, and neural hyperplasia or hypertrophy.10 However, there were also significant differences between these two subtypes. Vestibulectomy specimens from women with premenopausal primary vestibulodynia had statistically more neural hyperplasia than did tissue from patients with premenopausal secondary vestibulodynia who in turn had greater lymphocytic infiltration.
In this retrospective analysis, we now describe the histopathologic findings in 21 postmenopausal women with vestibulodynia. We compare these findings with the 88 premenopausal cases from our prior report.10 By comparing these three groups (premenopausal primary, premenopausal secondary, postmenopausal), our objective was to assess whether premenopausal and postmenopausal vestibulodynia have different histologic features that may provide insights into the underlying pathophysiology of this common and difficult condition. Our hypothesis was that vestibule tissues from menopausal women with secondary vestibulodynia would be characterized by neurogenic inflammation similar to premenopausal women with secondary vestibulodynia.
MATERIALS AND METHODS
Using an institutional review board-approved protocol (#3613), we identified 109 archived vestibulectomy specimens from surgeries performed at Oregon Health & Science University from 2002 to 2008. Twenty-one cases were from postmenopausal women diagnosed with secondary vestibulodynia. Eighty-eight cases were from premenopausal women (42 primary and 46 secondary) who have already been described.10 All archived tissue specimens were from patients seen in the Oregon Health & Science University Program in Vulvar Health who met Friedrich criteria for vestibulodynia and had no other identifiable reason for dyspareunia (eg, vulvar dermatosis, infectious vaginitis).11 All patients had reported severe or moderate dyspareunia and had undergone a superficial, localized vestibulectomy.12 Chart review was performed independently by two of the authors (M.F.G. and C.M.L.) to verify patient age, parity, postmenopausal status, primary compared with secondary vestibulodynia classification, hormone therapy (type, delivery, and dose), duration of symptoms, and duration since menopause. Menopause was defined as amenorrhea for at least 12 months or surgical removal of both ovaries. All 109 tissues were stained and analyzed together.
All specimens were obtained from superficial localized vestibulectomies in which the hymen, and occasionally 1 cm of distal vagina, was used for closure.12 Routine paraffin-embedded histologic sections (3–5 micrometers were stained for hematoxylin and eosin to grade chronic inflammation (lymphocytes).10,13 Serial sections were also immunostained for S100 (nerves), CD117 (mast cells), presence of progesterone receptor, and presence of estrogen receptor α.10,13 Notably, estrogen receptor β and epidermal nerve twig staining with protein gene product 9.5 were not informative in our prior study13 and were therefore not used in this analysis. Staining quality and specificity were confirmed using appropriate controls (eg, nerves for S100 and breast tissue for hormone receptors.
Histologic features were scored by two pathologists blinded to clinical and demographic data. Scoring was performed as described in our prior studies where this method was shown to be highly reproducible.10,13 There was good to excellent agreement between pathologists scoring histologic sections for inflammation, neural hyperplasia, and hormone receptors (kappa statistic ranged from .63 to greater than .8). Briefly, chronic inflammation (lymphocytes) was scored as none (0), mild (1+), moderate (2+), or severe (3+). S100-positive nerves were scored as none (0), mild (1+), moderate (2+), or severe (3+) hypertrophy or hyperplasia. Estrogen and progesterone receptor nuclear immunostaining was scored similar to routine breast pathology using a 0–3+ scale: 0=negative; 1+=less than 10%; 2+=11–50%; 3+=51–100% of basal cells. CD117-positive mast cells were quantified as the average number of mast cells per high-power microscopic field (40× objective).
Statistical analyses of the data were performed with SAS statistical software. Patients' demographic and clinical characteristics among study groups were compared using χ2 for parity and GLIMMIX (generalized linear mixed model) procedure for age and duration of symptoms to accommodate nonnormal distribution.14 Median and interquartile range were presented because age, years of symptoms, and years since menopause were skewed (Table 1). As a result of the small sample size, both proportional odds logistic regression models15 for four-category ordinal outcome and binary exact logistic regression models16 for collapsed two-category outcome were used to examine the histopathologic outcome. The proportional odds logistic regression models take advantage of the ordinal nature of the outcomes and are more powerful than binary logistic regression for a given sample size. Results from proportional odds logistic regression models are presented. Both unadjusted and adjusted proportional odds ratios (ORs) and 95% confidence intervals (CIs) with consideration of parity and natural logistic logarithmic transformed duration of symptoms were also estimated (Table 2). Data for mast cell count were normally distributed. The difference of mast cells was determined among groups using analysis of variance; mean and standard error of mean were presented. Two-sided P values were reported; P<.05 was considered statistically significant.
Our sample size had the power to detect a difference based on numbers from our prior premenopausal study.10,13 Sample size analyses used 80% power with a significance level of .05 for all calculations. A 1:2 allocation ratio was used to compare the available 21 postmenopausal samples with 42 premenopausal primary vestibulodynia samples and 46 premenopausal secondary vestibulodynia samples. Neuroinflammation is the hallmark of tissue changes in vestibulodynia and represents our main outcome. Based on the finding of moderate to strong staining for neural hyperplasia and inflammation in 52–76% of premenopausal groups, our sample size can detect a 30–33.5% difference in neural hyperplasia and inflammation using the two-sided z test. Based on rates of positive staining for estrogen receptor α and progesterone receptor in 70–88% of premenopausal tissues, our sample size can detect a 27–32% difference between the premenopausal primary or secondary group and postmenopausal group using the one-sided z test with pooled variance. According to the mean of mast cell counts of 26 or 28 for premenopausal groups, the same sample achieves the proposed study power and significance level to detect a mean difference of 8 between one of the premenopausal groups and the postmenopausal group with a standard deviation of 10 and the two-sided t test.
All 21 postmenopausal patients had secondary vestibulodynia. Demographic information including median age, years since menopause, symptom duration, and parity is listed in Table 1. Seventy-one percent (15/21) of women reported vestibular dyspareunia with a drop in estrogen, although in some patients, this was before menopause. Fifty-two percent of the postmenopausal patients (11/21) developed dyspareunia as one of their initial symptoms at the time of menopause transition. Two had noted onset of dyspareunia when postpartum; it never resolved and they presented during menopause. Two had developed premenopausal dyspareunia and noted worsening dyspareunia with menopause. Six patients (29%) had symptoms that seemed independent in timing from the loss of estrogen. At the time of surgery, 17 of 21 of these patients were using systemic hormone therapy, oral or transdermal, and one was using local estrogen, totaling 86% (18/21) on supplemental estrogen. Ten women (48%) were using systemic progestins for endometrial protection. Three patients were not using any hormones as a result of breast cancer or personal choice (14%).
Histologic features and mast cell counts among groups are presented in Table 2, Figure 1, and Figure 2. Multiple histologic features were different in the postmenopausal patients compared with our previously described premenopausal series.10 Compared with premenopausal primary and secondary biopsies, tissues from postmenopausal patients showed significantly more chronic inflammation (Table 2) (unadjusted OR 9.0, 95% CI 2.8–33.3; adjusted OR for parity and duration of symptoms 9.1, 95% CI 2.6–31.9; unadjusted OR 6.2, 95% CI 1.9–20.0; adjusted OR 6.6, 95% CI 2.0–21.9, respectively) and greater mast cell infiltration (Fig. 2). Mast cell counts were significantly higher in the menopausal group than the primary premenopausal (36 compared with 28, P=.005) or secondary premenopausal (36 compared with 26, P<.001) group. Results remained the same after adjusting for parity and duration of symptoms (P=.01 and P=.03, respectively). There was less neural hyperplasia and progesterone receptor expression in postmenopausal biopsies compared with primary cases and less progesterone receptor but similar neural hyperplasia compared with premenopausal secondary vestibulodynia (Fig. 1). When the results were analyzed excluding the women not using estrogen supplementation (n=3), there was no change in the results. The similar histologic features of women using estrogen (n=18) and women not using estrogen (n=3) are graphically displayed in Figure 3.
We report the histopathologic features of postmenopausal vestibulodynia. Compared with premenopausal primary and secondary vestibular biopsies, postmenopausal tissues had more lymphocytes and mast cells (Figs. 1 and 2, respectively). There was less neural hyperplasia in postmenopausal biopsies compared with primary premenopausal vestibulodynia (Table 2). Premenopausal and postmenopausal vestibulodynia share histologic features of neurogenic inflammation but differ in degree.
Although most research in this field has focused on the premenopausal population, dyspareunia is also an important consideration in postmenopausal women. A recent literature review reports the prevalence of postmenopausal dyspareunia ranging from 2% to 29%17 and another study found that vulvodynia remains significant at 8% from adulthood until age 70 years.18 Kao et al19 reported that 95% of menopausal women with dyspareunia also had localized provoked pain in the vestibule despite the use of hormone supplements in 31% of patients. Because postmenopausal women continue to stay sexually active, dyspareunia, and specifically vestibulodynia, are of clinical and scientific importance.
Marked neurogenic inflammation is a frequently identified histologic characteristic of vestibulodynia.10,13,20–23 We have previously reported significant histologic differences in patients with premenopausal primary vestibulodynia compared with premenopausal secondary vestibulodynia.10 In this retrospective analysis of postmenopausal tissue specimens, neurogenic inflammation was again seen. However, this tissue showed more severe chronic inflammation (lymphocytes) than either premenopausal group and less neural hyperplasia than their premenopausal primary vestibulodynia counterparts. The duration of symptoms did not correlate with increased inflammation, because the group with the least inflammation was the group with the longer duration of symptoms (primary premenopausal). Estrogen receptor expression was not statistically different between all groups, perhaps as a result of varied supplemental hormone regimens used by patients in all groups.
Falling estrogen levels may trigger the development of vestibulodynia. Although the mechanism of vestibulodynia remains unclear, understanding the change in hormonal environment as it correlates to the development of these histologic patterns may help researchers understand the pathophysiology of subtypes of vestibulodynia. The majority (71%) of the patients in our menopausal cohort noted the onset or worsening of pain related to loss of estrogen, either at the time of the menopause transition or with prior postpartum estrogen loss. Twenty-two percent of the premenopausal secondary sufferers reported the advent of pain in the postpartum period. We suspect there is a relationship between hormonal signaling in the vestibule of these women and the neurogenic inflammation characteristic of vestibulodynia. Menopausal estrogen supplementation achieves only low levels of estrogen when compared with the range of physiologic circulating estradiol in premenopausal women. The low estrogen milieu was associated with the greatest lymphocyte and mast cell infiltrates of all groups, perhaps indicating that these cell types respond most strongly to low estrogen levels, stimulating a neural hyperplastic response in the vestibule. When estrogen supplements do not alleviate symptoms of dyspareunia, they may be too late or inadequate to stop the mechanism of tissue changes. The inflammatory response may continue to stimulate hyperplasia and hypertrophy of the nerves, leading to vestibulodynia.
This hypothesis is supported by published studies by Smith and colleagues,24 who reported the association of low estrogen levels and nerve proliferation in vaginas of rats and in the upper genital tracts of mice.25 At the nadir of estrogen levels in estrus cycles, nerves proliferate, and then these nerves retreat when estrogen is highest. This pattern repeats with cyclic plasticity. If the human vestibule shows a similar programming, this may be a phenomenon that explains menopausal vestibulodynia. Despite the common description of estrogen supplementation as “estrogen replacement,” it is meant to be low-dose rather than “replacement” therapy, and tissue levels in the vestibule may be not achieve a threshold that prevents or treats vestibule sensitivity. No one has published histologic analyses of pre-estrogen and postestrogen vestibule biopsies in cases of menopausal dyspareunia, especially because estrogen therapy corrects dyspareunia in many postmenopausal women. However, there may be an estrogen threshold that varies from individual to individual for the presumed nerve changes to recede. As Kao suggests, the common concept of vulvovaginal atrophy is not an adequate construct to explain the findings of vestibular pain.17,26 In 95% of her postmenopausal cohort with vestibular pain and dyspareunia, women used a variety of estrogen supplements and atrophy varied but vestibular pain persisted.19
This study describes the histologic profile of vestibulodynia in a menopausal population and includes representative patients in categories of premenopausal primary vestibulodynia, premenopausal secondary vestibulodynia, and now menopausal vestibulodynia. One limitation of this retrospective study is the ability to draw conclusions from these comparisons of small subgroups. The ability to perform an ad hoc power analysis is limited because no single histologic feature defines vestibulodynia. Another limitation is lack of uniformity of clinical histories and hormone use. Only three women were not on estrogen, but when our data were analyzed excluding this group, results did not vary. The study is descriptive and cannot prove causation.
A diagnosis of vestibulodynia should be considered in menopausal as well as premenopausal women presenting with dyspareunia. Vestibulodynia is a condition that affects women of all ages. Although supporting the menopausal patient with adequate estrogen either locally or systemically is an important clinical consideration, it may not be effective in alleviating vestibular dyspareunia. A better understanding of the relationship between hormone signaling and the pathogenesis of neurogenic inflammation is needed.
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