Catechol-O-methyltransferase gene polymorphism and vulvar pain in women with vulvodynia.

BACKGROUND: The underlying causes of vulvar pain in women with vulvodynia remain poorly understood. Catechol-O-methyltransferase, an enzyme that metabolizes catecholamines, is a neuromodulator that is involved with perception and sensitivity to pain. The catechol-O-methyltransferase gene is polymorphic, and a single nucleotide polymorphism is associated with low activity and heightened pain sensitivity. The variant allele that encodes this polymorphism commonly is called the "L allele" because of its low enzyme activity as opposed to the normal H (high activity) allele. OBJECTIVE: The methionine-containing catechol-O-methyltransferase protein coded by the L allele results in elevated catecholamine levels, reduced inactivation of the dopaminergic and adrenergic systems, and increased sensitivity to pain. This polymorphism not only may decrease the pain threshold in response to acute pain but also may facilitate the development of chronic pain. Therefore, the objective of our study was to assess whether a variation in the catechol-O-methyltransferase genotype is involved in increased pain sensitivity in women with vulvodynia. STUDY DESIGN: We conducted a prospective cohort study. METHODS: Buccal swabs were collected from 167 white women with vulvodynia and 107 control subjects; the DNA was tested for a single nucleotide polymorphism at position 158 (rs4680) in the catechol-O-methyltransferase gene. RESULTS: Women with vulvodynia had a marginally increased, yet not significant, prevalence of the catechol-O-methyltransferase genotype that is associated with high activity of the coded protein: 32.9% in the women with vulvodynia, as opposed to 21.5% in the control subjects (odds ratio, 1.80; 95% confidence interval, 1.02-3.15). Subgrouping the cases based on pain frequency revealed that the elevated occurrence of this catechol-O-methyltransferase genotype was present in 40.6% of the subset of women who experienced pain only with sexual intercourse vs only 21.5% of control subjects (odds ratio, 2.50; 95% confidence interval, 1.27-4.93). Also, women with primary vulvodynia had a significantly higher prevalence of the H allele than did the control subjects (62.9% vs 48.1%; odds ratio, 1.82; 95% confidence interval, 1.05-3.17). CONCLUSION: Increased pain sensitivity in women with vulvodynia is not due to a genetically determined low catechol-O-methyltransferase enzyme activity. Other mechanisms may account for alterations in catechol-O-methyltransferase activity in women with pain that is limited to intercourse or primary vulvodynia that contributes to pain sensitivity.

Vestibulodynia: A Multifactorial Syndrome: A Case Report.

BACKGROUND: Vulvodynia is a difficult-to-treat, chronic, multifactorial malady that drastically lowers the quality of life of afflicted patients. CASE: A 68-year-old woman, who had been treated successfully for vulvodynia years before with medication, returned with a recurrence of vulvodynia symptoms that this time did not respond to treatment. She now had biopsy-confirmed lichen sclerosis and was found to have markedly elevated serum testosterone levels. An imaging study detected an ovarian lesion that, on removal, proved to be afibrothecoma. Postoperatively the testosterone rapidly dropped to normal levels. What was unexpected and unusual was that the vulvar pain disappeared and the lichen sclerosis markedly regressed. CONCLUSION: This case demonstrates a hormonal trigger for the development of vulvodynia.

Neutrophil Gelatinase-Associated Lipocalin Concentration in Vaginal Fluid: Relation to Bacterial Vaginosis and Vulvovaginal Candidiasis.

OBJECTIVE: Neutrophil gelatinase-associated lipocalin (NGAL) is a component of innate immunity that prevents iron uptake by microorganisms. We evaluated whether NGAL was present in vaginal fluid and whether concentrations were altered in women with bacterial vaginosis (BV) or vulvovaginal candidiasis (VVC). METHODS: Vaginal secretions from 52 women with VVC, 43 with BV, and 77 healthy controls were assayed by enzyme-linked immunosorbent assay for NGAL and for concentrations of L-lactic acid. RESULTS: The median concentration of NGAL in vaginal fluid was significantly higher in control women (561 pg/mL) than in women with BV (402 pg/mL; P = .0116) and lower in women with VVC (741 pg/mL; P = .0017). Median lactic acid levels were similar in controls (0.11 mmol/L) and women with VVC (0.13 mmol/L) and were lower in women with BV (0.02 mmol/L; P < .0001). The NGAL and lactic acid concentrations were highly correlated (P < .0001). CONCLUSION: A decrease in Lactobacilli and/or lactic acid plus the absence of leukocytes results in lower vaginal NGAL levels that might facilitate the growth of bacteria associated with BV.

Female genital cutting: confronting cultural challenges and health complications across the lifespan.

Female genital cutting affects over 140 million women worldwide. Prevalent in certain countries of Africa and the Middle East, the practice continues among immigrants to industrialized countries. Female genital cutting is a deeply rooted tradition that confers honor on a woman and her family, yet also a traumatic experience that creates significant dermatological, gynecological, obstetric and infectious disease complications. Little is known about postmenopausal health in cut women. The international community views this practice as a human rights violation. In addition to genital health complications, the medical community must confront an understudied concern of what happens as this population ages. These challenges must be addressed to provide optimal care to women affected by female genital cutting.

Decreased concentration of protease inhibitors: possible contributors to allodynia and hyperalgesia in women with vestibulodynia.

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.

The bacterial microbiome in paired vaginal and vestibular samples from women with vulvar vestibulitis syndrome.

Composition of the bacterial microbiome in the vagina and vestibule from 30 women with vulvar vestibulitis syndrome (VVS) and 15 healthy controls were compared by pyrosequencing 16S rRNA gene amplicons. Vaginal concentrations of interleukin (IL)-1ß were determined by ELISA. Questionnaires elicited clinical and symptom data. Eighteen genera were detected in vaginal samples, and 23 genera were identified in vestibule samples, from women with VVS. The genera at both sites and the mean number of genera in subjects with VVS were largely similar to those in control subjects. However, differences were noted including higher proportions of Streptococcus and Enterococcus in women with VVS. Furthermore, Lactobacillus iners was more frequently identified in women with VVS while L. crispatus was more frequent in the control women. The dominant bacterial genera in the vagina closely paralleled the dominant genera present in the corresponding vestibular sample in both groups, leading us to postulate that vaginal secretions are an important source of bacteria present on the vestibule. Vaginal IL-1ß levels were similar and varied depending on the dominant bacteria. We conclude in this pilot study that no major differences are apparent in the vagina and vestibule between women with or without VVS, except for an increased prevalence of Streptococcus and L. iners in some women with VVS.

Human epididymis protein 4 and secretory leukocyte protease inhibitor in vaginal fluid: relation to vaginal components and bacterial composition.

Human epididymis protein 4 (HE4) is a protease inhibitor and a recently identified serum biomarker for ovarian cancer. Properties of HE4 in the genital tract of healthy women have not been evaluated. We evaluated associations between HE4 and a second vaginal protease inhibitor, secretory leukocyte protease inhibitor (SLPI), with vaginal concentrations of innate immune mediators or proteases and with the types of vaginal bacterial communities. Vaginal secretions were collected from 18 healthy reproductive age women and assayed by enzyme-linked immunosorbent assay for concentrations of HE4, SLPI, kallikrein 5, cathepsin B, interleukin 1ß (IL-1), IL-1 receptor antagonist (IL-1 ra), mannose-binding lectin (MBL), the inducible 70-kDa heat shock protein, and matrix metalloproteinase (MMP)-8. The species composition of vaginal bacterial communities in 16 women was characterized by sequencing amplicons derived from 16S bacterial ribosomal RNA genes. Correlations between any 2 assays were analyzed by the Spearman rank correlation tests. Differences in the concentrations of HE4 and SLPI, and between soluble components and vaginal community types, were analyzed by the Mann-Whitney U tests. Vaginal HE4 concentrations, but not SLPI levels, were positively correlated with levels of IL-1ß (P = .0152), IL-1ra (P = .0061), MBL (P = .0100), and MMP-8 (P = .0315). The median vaginal HE4 level, as well as concentrations of MBL, IL-1ß, IL-1ra, and MMP-8, was highest when Gardnerella vaginalis dominated a vaginal community. The association between HE4, elevated levels of proteases, immune mediators and high proportions of G vaginalis strongly suggests that HE4 is a component of the proinflammatory immune response in the female genital tract.

Influence of vaginal bacteria and D- and L-lactic acid isomers on vaginal extracellular matrix metalloproteinase inducer: implications for protection against upper genital tract infections.

UNLABELLED: We evaluated levels of vaginal extracellular matrix metalloproteinase inducer (EMMPRIN) and matrix metalloproteinase (MMP-8) in vaginal secretions in relation to the composition of vaginal bacterial communities and D- and L-lactic acid levels. The composition of vaginal bacterial communities in 46 women was determined by pyrosequencing the V1 to V3 region of 16S rRNA genes. Lactobacilli were dominant in 71.3% of the women, followed by Gardnerella (17.4%), Streptococcus (8.7%), and Enterococcus (2.2%). Of the lactobacillus-dominated communities, 51.5% were dominated by Lactobacillus crispatus, 36.4% by Lactobacillus iners, and 6.1% each by Lactobacillus gasseri and Lactobacillus jensenii. Concentrations of L-lactic acid were slightly higher in lactobacillus-dominated vaginal samples, but most differences were not statistically significant. D-Lactic acid levels were higher in samples containing L. crispatus than in those with L. iners (P<0.0001) or Gardnerella (P=0.0002). The relative proportion of D-lactic acid in vaginal communities dominated by species of lactobacilli was in concordance with the proportions found in axenic cultures of the various species grown in vitro. Levels of L-lactic acid (P<0.0001) and the ratio of L-lactic acid to D-lactic acid (P=0.0060), but not concentrations of D-lactic acid, were also correlated with EMMPRIN concentrations. Moreover, vaginal concentrations of EMMPRIN and MMP-8 levels were highly correlated (P<0.0001). Taken together, the data suggest the relative proportion of L- to D-lactic acid isomers in the vagina may influence the extent of local EMMPRIN production and subsequent induction of MMP-8. The expression of these proteins may help determine the ability of bacteria to transverse the cervix and initiate upper genital tract infections. IMPORTANCE: A large proportion of preterm births (>50%) result from infections caused by bacteria originating in the vagina, which requires that they traverse the cervix. Factors that influence susceptibility to these infections are not well understood; however, there is evidence that matrix metalloproteinase (MMP-8) is known to alter the integrity of the cervix. In this work, we show that concentrations of vaginal extracellular matrix metalloproteinase inducer (EMMPRIN) are influenced by members of the vaginal microbial community and concentrations of D- or L-lactic acid isomers in vaginal secretions. Elevated levels of D-lactic acid and the ratio of D- to L-lactic acid influence EMMPRIN concentrations as well as MMP-8 levels. Thus, isomers of lactic acid may function as signaling molecules that alter host gene expression and influence risk of infection-related preterm birth.

Assessing menstrual tampon irritation using the "Behind-The-Knee" test.

INTRODUCTION: Colposcopic inspection of the vagina is a routine component of the safety assessment of intravaginal products. However, colposcopic findings occur frequently in healthy women, raising questions about their relevance to intravaginal product safety. Practical disadvantages limit the utility of colposcopy for evaluating menstrual tampons, among them the presence of background microtrauma, the inability to assess effects during menstruation, and, importantly, the question of whether post hoc assessments are sufficiently sensitive to detect small inflammatory changes. The Behind-The-Knee (BTK) test is an alternative for evaluating inflammatory and tissue dryness effects of physical articles by their repeated application to the popliteal fossa under an elastic bandage. It enables concurrent parallel comparisons of experimental and control articles over time and substantially increases the sensitivity of detecting small changes in tissue inflammation. MATERIAL AND METHODS: With the protocol, uncompressed experimental and control tampons yielded comparable relative and absolute erythema scores (after overnight recovery) as did colposcopic assessment of the lower genital tract 3-48 h after menstrual use. Scoring erythema in the BTK test immediately after product removal increased the level of visually discernible inflammation sixfold. In a study of commercial menstrual pads, subclinical inflammation visualized with cross-polarized light correlated with the frequency of subjective reports of discomfort during the test and discriminated the relative tolerability of the two products determined by market surveillance, providing added confidence in the predictive value of the test. CONCLUSION: We believe the BTK test can be a valuable alternative to colposcopy for assessing inflammation and dryness associated with menstrual tampons.

Complexities of the uniquely human vagina.

The vaginal microbiome in healthy women changes over short periods of time, differs among individuals, and varies in its response to sexual intercourse.

Influence of lactic acid on endogenous and viral RNA-induced immune mediator production by vaginal epithelial cells.

OBJECTIVE: To evaluate the influence of lactic acid on immune mediator release from vaginal epithelial cells. METHODS: The human vaginal epithelial cell line, VK2/E6E7, was cultured in the presence or absence of physiological concentrations of lactic acid, and in the presence or absence of the viral Toll-like receptor 3 agonist, poly (inosinic acid:cytidylic acid). Supernatants were assayed by enzyme-linked immunosorbent assay (ELISA) for interleukin (IL)-1ß, IL-6, IL-8, IL-23, transforming growth factor (TGF)-ß and secretory leukocyte protease inhibitor. RESULTS: Vaginal epithelial cells spontaneously released IL-1ß (25.9 pg/mL), IL-8 (1.0 ng/mL), TGF-ß (175 pg/mL), and secretory leukocyte protease inhibitor (33.8 ng/mL). Only TGF-ß production was marginally enhanced (49%) by addition of lactic acid alone. Poly (inosinic acid:cytidylic acid) by itself stimulated the release of IL-6 (305 pg/mL) and enhanced IL-8 production (2.8 ng/mL). The combination of poly (inosinic acid:cytidylic acid) and lactic acid markedly increased IL-8 production (5.0 ng/mL) and induced the release of IL-1ß (96.2 pg/mL). The poly (inosinic acid:cytidylic acid)-mediated lactic acid effect on IL-1ß and IL-8 release was abrogated when the lactic acid was neutralized or if acetic acid was substituted for lactic acid. CONCLUSION: Lactic acid enhances the release of selective mediators from vaginal epithelial cells and stimulates antiviral immune responses.

Can the behind-the-knee clinical test be used to evaluate the mechanical and chemical irritation potential for products intended for contact with mucous membranes?

BACKGROUND: The behind-the-knee (BTK) clinical test system is being used increasingly to replace in-use clinical studies in the evaluation of potential irritation effects of consumer products. The objectives of these studies were to determine if the BTK test could adequately evaluate the potential to cause irritation for feminine protection products intended for mucous membrane contact, and confirm that the BTK test would not underestimate the irritant potential that may occur when products such as tampons come into contact with mucous membranes. MATERIALS AND METHODS: Two identical tampon products were tested in in-use and BTK clinical studies. In the BTK clinical test, sites were scored daily 30-60 min after sample removal (PM scores with no recovery) and prior to application of the next sample (after 18 h of recovery). In the in-use clinical study, 6 sites (labia minora, introitus, lower and middle vaginal walls, upper vagina and cervix) were graded separately for erythema using colposcopy after use of each product for an entire menstrual cycle. RESULTS: In the in-use clinical study, the labia minora were most susceptible to irritation effects with mean erythema scores (± SE) of 0.36 (± 0.05) and 0.50 (± 0.06) for the experimental and control products, respectively. All other sites produced a mean erythema ≤0.26. In the BTK clinical test, the postbaseline averages for erythema for the experimental and control products were 0.19 (± 0.04) and 0.20 (± 0.04) for the AM scores (with recovery), and 1. 25 (± 0.05) and 1. 27 (± 0.06) for the PM scores (no recovery). CONCLUSIONS: Preliminary results showed that the BTK clinical test may be used to evaluate potential irritation effects of products that contact mucous membranes. In a first comparison of 2 tampon products in both the in-use and BTK clinical studies, the BTK protocol produced erythema reactions at the test sites that were similar to or severer than those observed on mucosal sites in the in-use study. Overall, the BTK clinical test showed a higher sensitivity, rapid turnaround time, higher flexibility and easier implementation. Thus, making the BTK clinical test a more useful tool for both safety testing and claim support.

New irritation test method: behind the knee and mucosa.

The epithelium of the lower genital tract, a highly sensitive sensory area where a host of sexual, reproductive and health functions are performed, is a unique anatomical site. It is very responsive to both internal and external stimuli, with a reactive area function of returning to an asymptomatic resting state. Commercial products intended for use in this area must be demonstrated to have minimal inflammatory responses. To determine this, the potential response of the lower genital tract epithelium to such products has to be evaluated. Direct assessment of the vagina is limited, because of its interior location. Behind-the-knee testing offers many advantages. The test products can be compared to commercial products approved by the Food and Drug Administration at the same time, and the use of cross-polarization light permits subsurface evaluation of the extent of inflammation, a more exact measure than surface recording alone.

Lactic acid stimulates interleukin-23 production by peripheral blood mononuclear cells exposed to bacterial lipopolysaccharide.

Lactic acid is the predominant acid present in the vagina. We evaluated the consequences of lactic acid, at physiological levels present in the vagina, on cytokine responses of peripheral blood mononuclear cells (PBMCs) obtained from 10 individuals in the presence or absence of bacterial lipopolysaccharide. Preincubation of PBMCs in 15 mM lactic acid before the addition of lipopolysaccharide resulted in a 246% mean increase in interleukin-23 (IL-23) secretion over that released in the presence of lipopolysaccharide alone (P=0.0068). The lipopolysaccharide-induced production of tumor necrosis factor-α, IL-6, IL-10 and IL-12 was unaffected by lactic acid. IL-23 stimulation was not observed if the lactic acid was neutralized before its addition to the culture medium or if hydrochloric acid was substituted for lactic acid. In the absence of lipopolysaccharide, lactic acid did not stimulate the production of IL-23 or any of the other cytokines. The increase in IL-23 production was proportional to the lactic acid concentration over a 15-60 mM range. We conclude that at body sites characterized by lactic acid accumulation, such as in the human vagina, exposure to gram-negative bacteria results in selective IL-23 production, leading to a subsequent preferential stimulation of the Th17 T lymphocyte pathway.

Should all oocyte donors receive prophylactic antibiotics for retrieval?

In a retrospective study comparing 526 oocyte donors who received prophylactic antibiotics for oocyte retrieval with a comparable group of 625 who did not, the incidence of infection after retrieval was reduced from 0.4% to 0 in the group receiving antibiotics. Donors take risks but have no medical indication for the procedures that they undergo; our data suggest that prophylactic antibiotics at retrieval should be considered to minimize the risk of infection.

Hyaluronan in vaginal secretions: association with recurrent vulvovaginal candidiasis.

OBJECTIVE: We evaluated whether vaginal concentrations of hyaluronan were altered in women with recurrent vulvovaginal candidiasis (RVVC). STUDY DESIGN: Lavage samples from 17 women with acute RVVC, 27 women who were receiving a maintenance antifungal regimen, and 24 control women were tested for hyaluronan and interleukin (IL)-6, IL-12, and IL-23 by enzyme-linked immunosorbent assay. RESULTS: Median vaginal hyaluronan concentrations were 33.8 ng/mL (range, 21.6-66.3 ng/mL) in women with acute RVVC, 15.0 ng/mL (range, 11.2-50.6 ng/mL) in women who were receiving maintenance therapy, and 4.2 ng/mL (range, 3.6-12.0 ng/mL) in control subjects (P

Investigation of the sensitivity of a cross-polarized light visualization system to detect subclinical erythema and dryness in women with vulvovaginitis.

OBJECTIVE: An enhanced visualization technique using polarized light (Syris v600 enhanced visualization system; Syris Scientific LLC, Gray, ME) detects surface and subsurface ( approximately 1 mm depth) inflammation. We sought to compare the Syris v600 system with unaided visual inspection and colposcopy of the female genitalia. STUDY DESIGN: Erythema and dryness of the vulva, introitus, vagina, and cervix were visualized and scored by each method in patients with and without vulvitis. RESULTS: Subsurface visualization was more sensitive in detecting genital erythema and dryness at all sites whether or not symptoms were present. Subsurface inflammation of the introitus, vagina, and cervix only was detected uniquely in women with vulvar vestibulitis syndrome (VVS). A subset of women presenting with VVS exhibited subclinical inflammation of the vulva vestibule and vagina (designated VVS/lichen sclerosus subgroup). CONCLUSION: Enhanced visualization of the genital epithelial subsurface with cross-polarized light may assist in diagnosing subclinical inflammation in vulvar conditions heretofore characterized as sensory syndromes.

Polymorphism in a gene coding for the inflammasome component NALP3 and recurrent vulvovaginal candidiasis in women with vulvar vestibulitis syndrome.

OBJECTIVE: Patients with vulvar vestibulitis syndrome (VVS) and control subjects were tested for a polymorphism in the gene coding for the NALP3 component of inflammasomes, cytoplasmic structures regulating interleukin (IL)-1beta production. STUDY DESIGN: DNA from 143 women with VVS and 182 control women were tested for a length polymorphism in intron 4 of the gene (CIAS1) that codes for NALP3. Vestibular tissue was examined for NALP3 expression. Whole blood cultures were tested for Candida albicans-induced IL-1beta production. RESULTS: The allele 12 frequency was higher in control subjects than in the patients with VVS (P = .02). Among patients with VVS and a self-reported history of recurrent vulvovaginal candidiasis (RVVC), the allele 7 frequency was 43.9% as compared with 30.8% in patients with no history of RVVC and 26.9% in control women (P = .035 vs other patients and .001 vs control subjects). NALP3 was identified in vestibular tissue. C albicans-induced IL-1beta production was reduced in samples from women with the 7,7 genotype (P = .030). CONCLUSION: Polymorphism in the CIAS1 gene may play a central role in the triggering of VVS in a subset of patients.