DOI 10.1007/s10517-020-04771-3 646 Bulletin of Experimental Biology and Medicine, Vol. 168, No. 5, March, 2020 IMMUNOLOGY AND MICROBIOLOGY Differential Expression of Local Immune Response Genes in the Vagina: Implication for the Diagnosis of Vaginal Infections O. V. Budilovskaya1,2, E. V. Shipitsina1,2, E. V. Spasibova1,2, N. A. Pereverzeva1, N. E. Vorob’eva1, N. D. Tsypurdeeva1, A. N. Grigoryev1, and A. M. Savicheva1,2 Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 168, No. 11, pp. 588-592, November, 2019 Original article submitted July 24, 2019 Transcription profiles of genes of local immune response were determined in the vagina of women with bacterial vaginosis, aerobic vaginitis, and vulvovaginal candidosis for detec- tion of the most specific immune markers for these vaginal infections. Laboratory diagnosis of the vaginal infections was performed microscopically; the inflammatory reaction in the vagina (leukorrhea) was defined as the presence of >10 white blood cells per field of view. Transcription profiles of IL1b, IL10, IL18, TNFα, TLR4, GATA3, and CD68 were determined using reverse-transcription quantitative real-time PCR. The strongest predictors of aerobic vaginitis were increased levels of IL1b and IL10 mRNA. Bacterial vaginosis was strongly associated with reduced levels of IL18 and GATA3 mRNA. Increased levels of IL1b and TLR4 transcripts showed significant discriminatory power for vulvovaginal candidosis and leukorrhea. The results of this study suggest differential expression of local immune response genes in the vagina of women with different vaginal infections. Detection of specific immune markers in the vagina using reverse-transcriptase PCR could supplement PCR detection of abnormal vaginal microflora for the diagnosis of vaginal infections. Key Words: vaginal infections; inflammation markers; transcription profiles; reverse tran- scriptase; quantitative reverse transcription PCR Inflammatory and non-inflammatory vaginal infec- BV (microbial imbalance in the vaginal micro- tions, in particular bacterial vaginosis (BV), aerobic biota) is characterized by a significant depletion of vaginitis (AV), and vulvovaginal candidosis (VVC) normal Lactobacillus-dominated microbiota and ex- caused by opportunistic microorganisms are very com- cessive growth of predominantly anaerobic organisms mon in premenopausal women and account for most (e.g. Gardnerella vaginalis, Prevotella spp., Atopo- cases of vaginal discharge [7,11,15]. Along with psy- bium vaginae, and Mobiluncus spp.), which leads to chosexual problems, these infections can lead to gyne- an increase in vaginal pH and malodorous discharge. cological and obstetrical complications [1,13]. Decreased level of lactobacilli and elevated pH are also characteristic of AV, but aerobic microorganisms, like E. coli, group B streptococci and Staphylococ- 1D.O. Ott Research Institute of Obstetrics, Gynecology and Reproduc- tology; 2St. Petersburg State Pediatric Medical University, St. Peters- cus aureus predominate in this condition. Clinical burg, Russia. Address for correspondence: o.budilovskaya@gmail. presentation of AV includes purulent discharge, some com. O. V. Budilovskaya degree of atrophy, and vaginitis. VVC manifested as 0007 -4888/20/1685-0646 © 2020 Springer Science+Business Media, LLC O. V. Budilovskaya, E. V. Shipitsina, et al. 647 curdy-white discharge, vulval and vaginal soreness/ temperature, the samples were immediately tested or itching, and erythema and in ~90% women is caused otherwise stored frozen until testing at -20°C for up by excessive growth of Candida albicans (remaining to 3 months. BV was diagnosed by assessing bacterial cases are caused by other species of yeast-like fungi, morphotypes in Gram-stained vaginal preparations us- e.g. Candida glabrata) [7]. ing the Nugent scoring system [6]. Vaginal microflora Many symptoms and signs of the vaginal infec- was classified as normal (a score of 0 to 3), interme- tions are unspecific and can accompany other condi- diate (4 to 6), or BV (7 to 10). AV was diagnosed using tions e.g. vulval dermatoses of allergic reactions. Mi- AV scores [6] that combine information about bacterial croscopy of vaginal discharge is now the main method microflora, leukocyte count, fraction of toxic leuko- of laboratory diagnostics of these conditions [13]. In cytes and parabasal cells: 0-2 (no AV), 3-4 (mild AV), recent years, PCR-based methods for detection of ab- 5-6 (moderate AV), or 7-10 (severe AV). The diagnosis normal vaginal microflora characteristic of BV and of VVC was based on a combination of clinical signs AV were developed [9,12,14]. Evaluation of mRNA (vaginal discharge, vulval soreness/itching and ery- levels of local immune response markers in vaginal thema, dysuria) and microscopic findings (presence samples obtained for PCR characterization of vaginal of pseudohyphae/mycelia). Leukorrhea was defined as microflora could supplement microbiological patterns the presence of ≥10 white blood cells per high-power of vaginal infections [2]. field at ×1000 on microscopic examination. Our aim was to determine transcription profiles of Transcription profiles of local immune response local immune response genes in the vagina with the genes were determined using an mRNA Profiling focus on discriminatory ability of the immune markers kit (ImmunoKvanteks; DNA Technology). The kit is of different vaginal infections. based on quantification of IL1b, IL10, IL18, TNFa, TLR4, GATA3, and CD68 mRNA using reverse-tran- MATERIALS AND METHODS scription quantitative real-time PCR. The genes were selected from 24 candidate genes of innate immune The study participants were women of reproductive response based on their level of transcription (mRNA age examined in a gynecological clinic in St. Peters- concentration >103 copies/ml) in vaginal fluid of 310 burg in March 2017 to November 2018. The main non-pregnant women of reproductive age [4]. The ref- complaints were vulvovaginal symptoms. During exa- erence transcript used for normalization was of beta- mination, the following vulvovaginal signs and symp- 2-microglobulin mRNA. The analysis was performed toms were revealed: abnormal vaginal discharge, vul- in accordance with manufacturer’s instructions. val soreness/itching and erythema, malodor, dysuria, Statistical analysis was performed with the use of dyspareunia, purulent or mucopurulent endocervical MedCalc Statistical Software 18.11 (MedCalc Soft- exudate, cervical petechiae, and abdominal pain. The ware bvba; https://www.medcalc.org; 2018). All con- exclusion criteria were pregnancy, treatment with oral tinuous variables (age, mRNA levels) were first tested or topical antimicrobials and topical probiotics within for normal distribution using Shapiro—Wilk test, and 4 weeks prior to enrollment, detection of agents of after being characterized as not normally distributed, sexually transmitted infections associated with cervi- were analyzed using Kruskal—Wallis test followed by citis (Chlamydia trachomatis, Neisseria gonor rhoeae, post-hoc pairwise comparisons using Conover’s test. Trichomonas vaginalis, Mycoplasma genitalium, and To assess the discriminatory ability of the immune HSV), signs of cervicitis, diagnosed or suspected pel- markers for the vaginal infections and leukorrhea, vic inflammatory diseases, the use of an intrauterine ROC-curves were plotted and classification perfor- device or contraceptives delivered directly to the vagi- mance characteristics (areas under curves, Youden’s nal mucosa. The study was approved by the Ethical index, and optimum cut-off values) were computed. Committee of the D.O. Ott Research Institute of Ob- All tests for significance were two-sided, and the dif- stetrics, Gynecology, and Reproductology (protocol ferences were statistically significant at p<0.05. No. 78/2016). Informed consent was obtained from all individual participants included in the study. RESULTS From each woman, two vaginal samples were col- lected using Dacron swabs. One sample was trans- Of 137 examined, 6 women were excluded (two were ferred to a slide, stained after Gram, and examined positive for HSV, one had signs of cervicitis, and under a light microscope. The other swab was used for three had abdominal pain). Vaginal infections were molecular testing: the swab was placed in a tube with diagnosed in 37 of 131 enrolled women (28.2%). AV 0.5 ml transportation medium (DNA Technology), (mild to severe forms), BV, and VVC as isolated in- thoroughly stirred, pressed against the tube wall, and fections were detected in 14 (10.7%), 9 (6.9%), and 7 discarded. After transportation within 2-8 h at ambient women (5.3%), respectively. Combination of AV and 648 Bulletin of Experimental Biology and Medicine, Vol. 168, No. 5, March, 2020 IMMUNOLOGY AND MICROBIOLOGY Fig. 1. mRNA levels (log-transformed number of copies/ml) of selected genes of local immune response in the vagina of women with AV, BV, and VVC in comparison with the control group. *p<0.05, **p<0.01, ***p<0.001 in comparison with the control group. BV was found in 4 women (3.1%), BV and VVC in samples, but for IL1b the difference did not reach sta- one woman (0.8%), AV, BV and VVC in 2 women tistical significance. The levels of IL18 mRNA were (1.5%). This, the overall rates of AV, BV, and VVC significantly lower in women with BV as compared to were 15.3% (N=20), 12.2% (N=16), and 7.6% (N=10), control women, whereas no difference was observed respectively. In 94 women, no vaginal infections were when AV or VVC samples were compared