Vaginal Lavage Research Articles

Prevalence of bacterial vaginosis, sexually transmitted infections and their association with HPV infections in asymptomatic women attending antenatal care in Ethiopia.

Sexually transmitted infections (STIs) and human papillomavirus (HPV) infections are common among women of reproductive age and can lead to infertility, adverse pregnancy outcomes, neonatal infections and cervical cancer. In countries with limited medical coverage, untreated infections contribute to high morbidity. This study aimed to expand the current knowledge on the prevalence of bacterial vaginosis (BV) and STIs in pregnant Ethiopian women and assess the association of these conditions with HPV infections. Socio-demographic data and vaginal lavage samples were collected from 779 asymptomatic women aged 18 to 45 years (median age, 25.9 years) attending antenatal care in seven centres across Ethiopia. Multiplex polymerase chain reaction was used to test for BV, Chlamydia trachomatis, Trichomonas vaginalis, Neisseria gonorrhoeae, herpes simplex virus types 1 and 2 (HSV-1/2), Mycoplasma, Ureaplasma, Candida species and HPV. Overall, 26.8% (95% confidence interval (CI): 23.7-29.9) of women tested positive for BV or one of the following STIs: C. trachomatis, T. vaginalis, N. gonorrhoeae, Mycoplasma genitalium, HSV-1/2 or Ureaplasma urealyticum. Additionally, 22.1% tested positive for at least one high-risk HPV type. Chlamydia trachomatis and HSV-2 were significantly more common among women who were positive for HPV and high-risk HPV. This study reveals a high prevalence of asymptomatic pregnant women who are positive for BV, STIs or HPV, putting them at risk of adverse pregnancy outcomes, secondary infertility or cervical cancer in a country with limited medical coverage. Screening and treating these women could be crucial in reducing morbidity.

Prevalence of human papillomaviruses in self-collected samples among women attending antenatal care in Ethiopia: a cross-sectional study.

Cervical cancer is the second most commonly diagnosed cancer in women in Ethiopia. However, data are limited on the prevalence of human papillomavirus (HPV) genotypes. Self-sampled vaginal lavages were obtained consecutively from 783 women attending 7 health facilities across Ethiopia. Genotype prevalence was assessed by Multiplex-Papillomavirus-Genotyping which detects and individually identifies 51 genotypes and 3 subtypes. Genotype-specific prevalence was described and associations with known risk factors were analysed. The overall HPV prevalence (age range 18-45) was 33.1% (95% confidence interval (CI) 29.8-36.4). The prevalence of HPV was different in the rural and urban population with 17.6% (95%CI 11.6-23.7) and 36.8% (95%CI 33.1-40.6) (p < 0.001 chi-square test), respectively. The most common high-risk types were HPV 16 (6.6%), followed by HPV 52 (4.3%), 51 and 39 (both 2.9%). Urban women compared to rural women had a higher risk of being HPV positive (odds ratio 2.36 (95% CI 1.47-3.79; p < 0.001). Age at sexual debut ≤15 years and polygamous husband (in urban women) also increased the risk of being HPV positive nearly two-fold. The high prevalence of hr-HPV in Ethiopian women in the reproductive age group shows the need for screening programs. The nonavalent HPV vaccine covers the most prevalent hr-HPV genotypes as found in this study and can therefore be used effectively. Since antenatal care is the best-utilised health service, implementing self-sampled vaginal lavage could be an opportunity for screening in this age group. Screening algorithms and triage still need to be defined to avoid over-treatment in these women.

Nomogram-based risk assessment for emergency cervical cerclage failure in patients with cervical insufficiency

BackgroundCervical insufficiency can lead to preterm birth and neonatal mortality. Emergency cervical cerclage is a surgical intervention aimed at preventing preterm birth in patients with cervical insufficiency. However, some patients may experience cerclage failure. This study aimed to identify the risk factors associated with cerclage failure and develop a predictive nomogram model for patients with cervical insufficiency undergoing emergency cervical cerclage. MethodsData of 200 patients who underwent emergency cervical cerclage for cervical insufficiency were retrospectively analyzed. Patients were categorized into successful and failed groups based on their ability to take the infant home. Univariate and multivariate logistic regression analyses were performed to identify risk factors for cerclage failure. A nomogram model was developed based on multivariate logistic regression results, and its performance was assessed using receiver operating characteristic curves, calibration plots, and decision curve analysis (DCA). ResultsUnivariate logistic regression analysis identified 11 potential risk factors for cerclage failure, including the presence of polycystic ovary syndrome (PCOS), vaginitis, cervical dilation, preoperative C-reactive protein, routine vaginal lavage after cervical cerclage, delivery, gestational age, extended days, chorioamnionitis, intrauterine infection, cervical laceration, and premature rupture of membranes. Multivariate logistic regression analysis revealed that PCOS, cervical dilation after cervical cerclage were independent risk factors for cerclage failure while routine vaginal lavage was a protective factor against failure. The nomogram predictive model demonstrated an area under the curve value of 0.975, indicating excellent discriminatory ability. The calibration plot showed good consistency between the nomogram predictions and actual observations. DCA demonstrated the strong clinical applicability of the nomogram. ConclusionsThis study successfully identified risk factors associated with emergency cervical cerclage failure in patients with cervical insufficiency and developed a predictive nomogram model. This model can assist clinicians in making informed decisions and accurately predicting the risk of cerclage failure in these patients.

A non-antibiotic antimicrobial drug, a biological bacteriostatic agent, is useful for treating aerobic vaginitis, bacterial vaginosis, and vulvovaginal candidiasis.

Vaginitis is a common infection in women, with approximately 75% of women experiencing at least one episode during their lifetime. Although antimicrobial agents are widely used to treat vaginitis, recurrent vaginitis occurs in some patients. Resistance to these agents is the major cause of recurrent vaginitis. Therefore, there is an urgent need to develop novel drugs. We investigated the efficacy of a new biological bacteriostatic agent (BBA), composed of lysozyme, phytoalexin, chitosan oligosaccharide, sinensetin, 18β/20α-glycyrrhizin, and betaine, against vaginitis using in vitro and in vivo studies. First, we evaluated the antibacterial effects of BBA against 13 microbial strains commonly present in aerobic vaginitis, bacterial vaginosis, vulvovaginal candidiasis, and healthy vaginas. Second, we assessed the safety of various doses of BBA administered orally for 4 weeks in female mice. Third, we examined the in vivo anti-proliferative and anti-inflammatory effects of BBA in Candida albicans-, Candida glabrata-, and Gardnerella-induced vaginitis models. Finally, we evaluated the anti-vaginitis effect of a BBA gel prepared with 0.5% (w/v) ammonium acryloyldimethyltaurate/Vp copolymer. BBA effectively suppressed the growth of the main causative pathogens of vaginitis in vitro. BBA, either undiluted or diluted two-fold, inhibited all microorganisms cultured for 8 h. No obvious organ damage was detected when BBA was administered to mice. Both BBA alone and 70% BBA in a gel formulation effectively inhibited the proliferation of C. albicans, C. glabrata, and Gardnerella in vaginal lavage samples and alleviated tissue inflammation in mice with vaginitis. The 70% BBA gel performed better than BBA alone at treating vaginitis in mice infected with Gardnerella vaginalis. BBA alone and a 70% BBA gel inhibited the growth of pathogens and effectively alleviated inflammation caused by C. albicans, C. glabrata, and G. vaginalis.

Transcriptomics Reveals Effect of Pulsatilla Decoction Butanol Extract in Alleviating Vulvovaginal Candidiasis by Inhibiting Neutrophil Chemotaxis and Activation via TLR4 Signaling.

The Pulsatilla decoction is a well-known herbal remedy used in clinical settings for treating vulvovaginal candidiasis (VVC). However, the specific mechanism that makes it effective is still unclear. Recent studies have shown that in cases of VVC, neutrophils recruited to the vagina, influenced by heparan sulfate (HS), do not successfully engulf Candida albicans (C. albicans). Instead, they release many inflammatory factors that cause damage to the vaginal mucosa. This study aims to understand the molecular mechanism by which the n-butanol extract of Pulsatilla decoction (BEPD) treats VVC through transcriptomics. High-performance liquid chromatography was used to identify the primary active components of BEPD. A VVC mouse model was induced using an estrogen-dependent method and the mice were treated daily with BEPD (20 mg/kg, 40 mg/kg, and 80 mg/kg) for seven days. The vaginal lavage fluid of the mice was analyzed for various experimental indices, including fungal morphology, fungal burden, degree of neutrophil infiltration, and cytokines. Various assessments were then performed on mouse vaginal tissues, including pathological assessment, immunohistochemistry, immunofluorescence, Western blot (WB), quantitative real-time PCR, and transcriptome assays. Our results showed that BEPD reduced vaginal redness and swelling, decreased white discharge, inhibited C. albicans hyphae formation, reduced neutrophil infiltration and fungal burden, and attenuated vaginal tissue damage compared with the VVC model group. The high-dose BEPD group even restored the damaged vaginal tissue to normal levels. The medium- and high-dose groups of BEPD also significantly reduced the levels of IL-1β, IL-6, TNF-α, and LDH. Additionally, transcriptomic results showed that BEPD regulated several chemokine (CXCL1, CXCL3, and CXCL5) and S100 alarmin (S100A8 and S100A9) genes, suggesting that BEPD may treat VVC by affecting chemokine- and alarmin-mediated neutrophil chemotaxis. Finally, we verified that BEPD protects the vaginal mucosa of VVC mice by inhibiting neutrophil recruitment and chemotaxis in an animal model of VVC via the TLR4/MyD88/NF-κB pathway. This study provides further evidence to elucidate the mechanism of BEPD treatment of VVC.

312 In vivo electrophysiology sex differences in the locus coeruleus of wild type F344 rats

OBJECTIVES/GOALS: Women are twice as susceptible to ailments like anxiety, PTSD, and Alzheimer’s disease compared to men. The locus coeruleus (LC), the primary source of noradrenaline for the brain is implicated in these disorders, however physiological sex differences have never been assessed in the LC. METHODS/STUDY POPULATION: To address this gap, In vivo electrophysiology under anesthesia was used to measure single unit activity of noradrenergic LC firing patterns in 4-month-old wild-type Fischer male and female rats. Recordings measured neuronal activity under basal conditions and in response to a footshock stimulation which elicits burst firing in LC neurons. Single unit activity is sorted via automatic valley seeking scan PCA, additional manual sorting is done via line and template method. Analysis is done extracting interspike interval (ISI) and firing rate of single units, additional analysis is done to quantify properties of bursting patterns (burst duration, spikes per burst, interburst interval, etc…). RESULTS/ANTICIPATED RESULTS: This data shows that during LC burst firing, females have longer interspike intervals compared to males, supporting the inhibitory effect of E2 on LC firing. Additionally, females have significantly different waveform patterns than males, indicating possible differences in intrinsic properties, but further supporting sexually distinct physiology of the LC . Because female rats have been estrous cycle tracked via vaginal lavage, stratification into estrus groups and further analysis may uncover differences within females. These data suggest that estrogen acts as a potent neuromodulator of noradrenergic LC neurons, providing valuable insights into the physiology of this brain region. DISCUSSION/SIGNIFICANCE: This study is the first exploration of LC physiological sex differences. This work offers insights into a critical brain region implicated in many diseases, and may pave the way for future therapeutic approaches, particularly for women, who are at a higher risk of neurological disease developing.

N-3-Methylbutyl-benzisoselenazol-3(2H)-one Exerts Antifungal Activity In Vitro and in a Mouse Model of Vulvovaginal Candidiasis.

In the present work, we evaluated the antifungal activities of two novel ebselen analogs, N-allyl-benzisoselenazol-3(2H)-one (N-allyl-bs) and N-3-methylbutylbenzisoselenazol-3(2H)-one (N-3mb-bs). Colorimetric and turbidity assays were performed to determine the minimum inhibitory concentration (MIC) of these compounds in S1 (fluconazole-sensitive) and S2 (fluconazole-resistant) strains of C. albicans. N-3mb-bs was more active than the N-allyl-bs compound. It is noteworthy that the concentration of N-3mb-bs observed to inhibit fungal growth by 50% (18.2 µM) was similar to the concentration observed to inhibit the activity of the yeast plasma membrane H+-ATPase (Pma1p) by 50% (19.6 µM). We next implemented a mouse model of vulvovaginal candidiasis (VVC) using the S1 strain and examined the mouse and yeast proteins present in the vaginal lavage fluid using proteomics. The yeast proteins detected were predominately glycolytic enzymes or virulence factors associated with C. albicans while the mouse proteins present in the lavage fluid included eosinophil peroxidase, desmocollin-1, and gasdermin-A. We then utilized the N-3mb-bs compound (12.5 mg/kg) in the mouse VVC model and observed that it significantly reduced the vaginal fungal burden, histopathological changes in vagina tissue, and expression of myeloperoxidase (MPO). All in all, the present work has identified a potentially promising drug candidate for VVC treatment.

Impaired neutrophil extracellular trap-forming capacity contributes to susceptibility to chronic vaginitis in a mouse model of vulvovaginal candidiasis.

Vulvovaginal candidiasis (VVC), caused by Candida albicans, is characterized by aberrant inflammation by polymorphonuclear neutrophils (PMNs) in the vaginal lumen. Data from the established murine model shows that despite potent antifungal properties, PMNs fail to clear C. albicans due to local heparan sulfate that inhibits the interaction between PMNs and C. albicans, resulting in chronic vaginal immunopathology. To understand the role of neutrophil extracellular traps (NETs) in defense against C. albicans at the vaginal mucosa, we investigated the NET-forming capacity of PMNs in chronic VVC-susceptible (CVVC-S/C3H) and -resistant (CVVC-R/CD-1) mouse strains. Immunofluorescence revealed the formation of NETs (release of DNA with PMN-derived antimicrobial proteins) in PMN-C. albicans cocultures using vaginal conditioned medium (VCM) generated from CVVC-R/CD-1 mice, similar to NET-inducing positive controls. Under these NETotic conditions, PMNs released high levels of double-stranded DNA bound with NET-associated proteins, concomitant with substantial C. albicans killing activity. In contrast, PMN-C. albicans cocultures in VCM from CVVC-S/C3H mice lacked NET formation together with reduced antifungal activity. Similar results were observed in vivo: active NET-C. albicans interaction followed by fungal clearance in inoculated CVVC-R/CD-1 mice, and sustained high vaginal fungal burden and no evidence of NETs in inoculated CVVC-S/C3H mice. Furthermore, the level of Ki67 expression, a putative NETotic PMN marker, was significantly reduced in vaginal lavage fluid from CVVC-S/C3H mice compared to CVVC-R/CD-1 mice. Finally, scanning electron microscopy revealed that PMNs in CVVC-R, but not CVVC-S, conditions exhibited NETs in direct contact with C. albicans hyphae in vitro and in vivo. These results suggest that VVC-associated immunopathology includes impaired NET-mediated antifungal activity.

Cationic Nanoemulsion-Encapsulated Retinoic Acid as an Adjuvant to Promote OVA-Specific Systemic and Mucosal Responses.

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen uptake and then promotes antigen-specific immune responses. In recent years, retinoic acid (RA) has received increasing attention due to its effect in activating the mucosal immune response; however, in order to use RA as a mucosal adjuvant, it is necessary to solve the problem of its dissolution, loading, and delivery. Here, we describe a cationic nanoemulsion-encapsulated retinoic acid (CNE-RA) delivery system composed of the cationic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOTAP), retinoic acid, squalene as the oil phase, polysorbate 80 as surfactant, and sorbitan trioleate 85 as co-surfactant. Its physical and chemical properties were characterized using dynamic light scattering and a spectrophotometer. Immunization of mice with the mixture of antigen (ovalbumin, OVA) and CNE-RA significantly elevated the levels of anti-OVA secretory immunoglobulin A (sIgA) in vaginal lavage fluid and the small intestinal lavage fluid of mice compared with OVA alone. This protocol describes a detailed method for the preparation, characterization, and evaluation of the adjuvant effect of CNE-RA.

Long-term Recordings of Arcuate Nucleus Kisspeptin Neurons Across the Mouse Estrous Cycle.

The arcuate nucleus kisspeptin (ARNKISS) neurons represent the GnRH pulse generator that likely drives pulsatile gonadotropin secretion in all mammals. Using an improved GCaMP fiber photometry system enabling long-term continuous recordings, we aimed to establish a definitive profile of ARNKISS neuronal activity across the murine estrous cycle. As noted previously, a substantial reduction in the frequency of ARNKISS neuron synchronization events (SEs) occurs on late proestrus and extends into estrus. The SE amplitude remains constant throughout the cycle. During metestrus, we unexpectedly detected many multipeak SEs where many SEs occurred rapidly, within 160 seconds of each other. By applying a machine learning-based, k-means clustering analysis, we were further able to detect substantial within-stage variability in the patterns of pulse generator activity. Estrous cycle-dependent changes in SE activity occurred around the time of lights on and off. We also find that a mild stressor such as vaginal lavage reduces ARNKISS neuron SE frequency for up to 3 hours. These observations provide a comprehensive account of ARNKISS neuron activity across the estrous cycle, highlight a new pattern of multipeak SE activity, and introduce a new k-means clustering approach for analyzing ARNKISS neuron population behavior.

SAT371 Gavaging Adult Female Rats with Rothia sp. Leads to Profound Disruption of the Estrous Cycles and Metabolic Consequences

Abstract Disclosure: R. Santos: None. G. Parodi: None. G. Leite: None. W. Morales: None. S.R. Weitsman: None. G. Barlow: None. I. Rivera: None. M. Sanchez: None. D. Flor: None. L. Choi: None. S.L. Gonzales: None. F. Faria: None. M. Villanueva-Millan: None. M. Pimentel: None. R. Mathur: None. Polycystic ovary syndrome (PCOS) is a major endocrine disorder affecting ∼12% of reproductive age women. It has diverse clinical manifestations, including hyperandrogenism, ovarian dysfunction and enlargement, infertility, insulin resistance, and others. The role of the gut microbiome in PCOS is poorly understood. We previously found higher testosterone (T) levels in PCOS stool samples vs. controls and identified bacterial candidates that correlated with testosterone levels. Here, we further investigate the role of one candidate microbe, Rothia sp., in the development of PCOS-like phenotypes. Methods: The presence of androgen biosynthesis associated genes in Rothia sp. was confirmed by RT-PCR. Rothia sp. was cultured in M9 minimal medium plus cholesterol. T production in the supernatant was measured by LC-MS/MS. Next, reproductive-age female rats were gavaged with 106 CFU/mL Rothia or with PBS (controls) daily for 2 weeks. H&amp;E stained slides from daily vaginal lavages were used to assess estrous cycles. Serum T and progesterone (P) levels were measured by LC-MS/MS, and insulin levels by ELISA. Ovaries, uteri, and cardiac blood were harvested. Results: Genes encoding proteins associated with T biosynthesis (cytochrome P450, 3-beta hydroxysteroid dehydrogenase/isomerase family protein and NAD(P)H steroid dehydrogenase-like protein) were detected in Rothia sp. T production in Rothia sp. cultures was confirmed, with levels from 3.2±0.01 pg/mL after 48 hrs to 8.9±0.02 pg/mL after 6 days. T was not detected in negative controls (P&amp;lt;0.001). In gavaged rats, analysis of vaginal slides (controls n=10; Rothia n=10) revealed a disruption of estrous cycles in Rothia rats, with 68% of days in luteal phase (metestrus &amp; diestrus) vs. 46% in controls (P=0.001). The predominance of luteal phase days in Rothia rats was driven by longer time in diestrus (1.96 vs 1.67 days in controls, P=0.04), and shorter time in estrus (1.24 vs 1.91 days in controls, P=0.001). Peak P and T levels occurred in diestrus in both groups. Rothia rats spent more days in diestrus, and the cumulative exposure times to P (P=0.046) and T (P=0.036) in the diestrus phase were significantly higher in Rothia rats vs controls. Final serum insulin levels were higher in Rothia rats (1.8±0.5 ng/dL) vs controls (1.0±0.1 ng/dL; P=0.07). Rothia rats also had heavier ovaries vs controls (0.128±0.007g vs 0.097±0.01g; P=0.07), and higher uterus weights (0.48±0.04g vs. 0.36±0.04g; P=0.04). Conclusion: Here we show that a specific Rothia sp. can produce testosterone in vitro. Gavaging female rats with this microbe results in PCOS-like phenotypes, including higher insulin levels, enlarged ovaries, significant disruption of reproductive cycles and longer exposure to peak progesterone and testosterone. This longer exposure to peak progesterone and testosterone may result in reproductive and metabolic sequelae that warrant further investigation. Presentation Date: Saturday, June 17, 2023

Female genital schistosomiasis, human papilloma virus infection, and cervical cancer in rural Madagascar: a cross sectional study

BackgroundWomen’s health in resource-limited settings can benefit from the integrated management of high-burden diseases, such as female genital schistosomiasis (FGS) and human papilloma virus (HPV)-related cervical cancer. In schistosomiasis-endemic countries such as Madagascar, data on FGS and HPV prevalence are lacking as well as preventive measures for both conditions. This study aims to estimate the prevalence of FGS and HPV in rural Madagascar, and to examine associated risk factors to identify opportunities for improving women’s health.MethodsAfter initial community outreach activities, interested women aged 18–49 years were recruited consecutively in 2021 at three primary health care centers in the district of Marovoay. FGS was detected by colposcopy. Colposcopy images were double-blind reviewed by two independent specialists. A Luminex bead-based assay was performed on cervical vaginal lavage specimens for HPV typing. Crude (CPR) and adjusted prevalence ratios (APR) of associations between selected factors and FGS and HPV positivity were estimated using univariable and multivariable binary Poisson regression with 95% confidence intervals (CIs).ResultsAmong 500 women enrolled, 302 had complete information on FGS and HPV diagnosis, and were thus eligible for analysis. Within the sample, 189 (62.6%, 95% CI: 56.9–68.1) cases of FGS were detected. A total of 129 women (42.7%, 95% CI: 37.1–48.5) tested positive for HPV. In total, 80 women (26.5%, 95% CI: 21.6–31.8]) tested positive for both conditions. No association was observed between FGS and HPV positivity, while previous pregnancy (APR = 0.65, 95% CI: 0.43–0.78) and older age (APR = 0.59, 95% CI: 0.42–0.81) are showing a negative association with HPV infection compared to no previous pregnancy and younger age groups.ConclusionsThe results of the study show that FGS and HPV are highly prevalent in rural Madagascar. The concurrent prevalence of these two conditions requires urgent adaptations of public health strategies to improve women’s health, such as integrated services at primary level of care.Graphical

Age-related sex differences in tongue strength and muscle morphometry in a rat model

ObjectiveTo investigate potential effects of sex on voluntary tongue strength, evoked twitch and tetanic tension, speed of contraction, and muscle fiber cross-sectional area in the muscles of the rat tongue. Additionally, we aimed to determine whether estrous cycle stage impacts any of the dependent variables as a pilot investigation into the use of female rats in a model of tongue exercise and aging. DesignFischer 344-Brown Norway male and female rats in two age groups (16 middle-aged, 16 young-adult) were trained to use a tongue force operandum. Tongue muscle contraction, myosin heavy chain (MyHC) composition, and cross section area of the genioglossus and styloglossus muscles were examined. Vaginal lavage determined estrous cycle stage of the female rats daily. ResultsThe female group had significantly lower evoked twitch and tetanic tension, longer contraction times, and a smaller proportion of MyHC type IIa and MyHC type IIx in the styloglossus muscle. There was no significant sex effect in maximal voluntary tongue force (MVTF) despite a significant weight difference between the male and female groups. There were no significant age or sex effects in the genioglossus. Estrous cycle stage did not have a significant effect on any of the dependent variables. ConclusionsSex and age both have a significant effect on tongue muscle structure and physiology. While the female group showed reduced contraction speed and maximal twitch and tetanic tension relative to the male group, differences in muscle morphology appeared to vary by muscle.

Comparing the therapeutic potentials of Lactobacillus johnsonii vs. Lactobacillus acidophilus against vulvovaginal candidiasis in female rats: an in vivo study.

Vulvovaginal candidiasis (VVC) is a highly prevalent illness affecting women globally. Lactobacilli, which make up the majority of healthy vaginal microbiota (VMB), serve as a powerful barrier against infections. Probiotic therapy has been recommended for the treatment or prevention of VVC. To compare the in vivo therapeutic effects of Lactobacillus johnsonii (B-2178) vs. Lactobacillus acidophilus (LA-5®) on VVC in a rat model, particularly highlighting the immune response of the host vaginal epithelium. In total, 30 female Sprague-Dawley rats were divided into 5 groups; Group 1: no intervention, Group 2: ovariectomy group, while animals in Groups 3-5 were subjected to ovariectomy and an intravaginal inoculation of Candida albicans (C. albicans) to establish VVC. The animals in Groups 4 and 5 received intravaginal lactobacilli treatment with L. acidophilus (LA-5®) and L. johnsonii (B-2178) strains, respectively, for 7 days. C. albicans load was measured in a vaginal lavage 1, 3, and 7 days after the stoppage of the treatment. Histological, morphometric, and immunohistochemical studies of the vaginal tissues were done. IFN-γ, IL-4, and IL-17 were measured in the vaginal tissue. Both L. johnsonii and L. acidophilus significantly reduced C. albicans vaginal load (250 ± 77.46 and 133.33 ± 40.82 CFU/mL) compared to the count before treatment in both groups (4,850 ± 1419.51 and 4966.67 ± 852.45 CFU/mL) even after 7 days of stoppage of lactobacilli treatment. A statistically significant reduction of the pro-inflammatory cytokines IL-17 and IFN-γ was reported in both treated groups compared to the infected untreated group. L. johnsonii has a significant effect on the reduction of hyphae formation of C. albicans as well as the nuclear factor kappa B (NF-κB) immunostaining density of vaginal tissue compared to L. acidophilus. Moreover, treatment with L. johnsonii significantly minimized the epithelium damage triggered by C. albicans infection and restored normal vaginal architecture as evidenced by the histologic and morphometric studies when compared to L. acidophilus. Through maintaining an immune tolerant state in the vaginal epithelium and ameliorating the undesirable uncontrolled inflammatory response in the vaginal tissue, L. johnsonii (B-2178) has the potential to be utilized alone or in combination with other lactobacilli species in probiotic clinical trials to treat or prevent VVC.

The Mechanism of IL-17 Regulating Neutrophils Participating in Host Immunity of RVVC Mice.

Vulvovaginal candidiasis (VVC) and recurrent vulvovaginal candidiasis (RVVC) are the most common lower genital tract infections in reproductive women. In recent years, the research on its pathogenesis mainly focuses on vaginal local immunity and IL-17 as key factors in adaptive immunity, attracting much attention. However, the role of IL-17 in local immunity in VVC and RVVC is poorly understood. At the same time, neutrophils are considered the most effective way to control and eliminate candidal infection and have a controversial role in VVC and RVVC. In this study, we built a mouse RVVC model. After analyzing the vaginal lavage solution of RVVC mice with an inflammatory factor antibody chip and ELISA, we found that IL-17 may play a protective role in RVVC. The experiment of constructing RVVC mice with different concentrations of IL-17 using halofuginone and comparing the vaginal fungi load and vaginal epithelial damage verified that IL-17 had a protective effect in RVVC. In addition, in vitro and in vivo studies found that IL-17 can promote neutrophil apoptosis and recruit neutrophils in the vagina. The neutrophils in the vagina can secrete IL-17 in an autocrine manner. These two may be why IL-17 plays a protective role in RVVC. In summary, the study suggests that IL-17-mediated regulation of neutrophil function is involved in host immune response to RVVC, which helps us to further understand the potential mechanism of IL-17 in RVVC.

Longdan Xiegan decoction ameliorates vulvovaginal candidiasis by inhibiting the NLRP3 inflammasome via the Toll-like receptor /MyD88 pathway

Ethnopharmacological relevanceLongdan Xiegan decoction (LXD) is a standardized herbal prescription originally documented in the “Medical Formula Collection” by the eminent physician Wang Ang during the Qing dynasty. It has been used extensively to treat vulvovaginal candidiasis (VVC). However, despite its effectiveness, the mechanism of action remains unknown. Aim of the studyTo elucidate the mechanism by which LXD relieves VVC via the Toll-like receptor/MyD88 pathway and activation of the NLRP3 inflammasome. Materials and methodsFemale Kunming mice (n = 96) were randomly divided into six groups: control, VVC model, LXD (10/20/40 mL/kg), and positive drug fluconazole. Mice were vaginally administered Candida albicans (C. albicans) solution (20 μL; 1 × 108 colony-forming units/mL), suspended for 5 min, and observed daily for changes in their condition. Continuous dilution was used to determine the number of colony-forming units. Gram, periodic acid-Schiff, Papanicolaou, and hematoxylin and eosin staining were used to determine the extent of infection. Enzyme-linked immunosorbent assay(ELISA) was used to determine the levels of proinflammatory cytokines IL-1β and IL-18. TLR2, TLR4, MyD88, NF-κB, NLRP3, ASC, and caspase-1 protein expression were determined using western blotting. ResultsC. albicans infection destroyed the integrity of the vaginal mucosa, increased fungal burden and the influx of neutrophils into the vaginal cavity, and promoted the secretion of proinflammatory cytokines. C. albicans stimulated the expression of TLR2, TLR4, MyD88, NF-κB, NLRP3, ASC, and caspase-1 in vaginal tissue. Fungal burden, hyphal formation, and C. albicans adhesion were reduced in the 20 and 40 mL/kg LXD groups. Hematoxylin and eosin staining showed that inflammation was reduced and the stratum corneum had recovered in the 20 and 40 mL/kg LXD groups. LXD (20 and 40 mL/kg) significantly reduced IL-1β, IL-18 levels and the number of neutrophils in vaginal lavage and decreased TLR2, TLR4, MyD88, NF-κB, NLRP3, ASC, and caspase-1 expression. ConclusionsThis study systematically demonstrated the therapeutic effect of LXD on protein expression and pathological conditions in VVC mice. The results showed that LXD could eliminate the invasion of vaginal hyphae in mice, reduce the recruitment of neutrophils, and reduce the expression of TLR/MyD88 pathway-related proteins and NLRP3 inflammasome. The above results clearly indicate that LXD may profoundly regulate NLRP3 inflammasome through the TLR/MyD88 pathway and play a therapeutic role in VVC.

Global Cerebral Ischemia Induces Short-Term Changes to Estrous Cycle Regularity and Alters Hypothalamic-Pituitary-Gonadal Axis Function

Global cerebral ischemia (GCI), which mimics cardiac arrest in humans, induces numerous functional alterations throughout the brain, but its impact on the hypothalamic-pituitary-gonadal (HPG) axis remains largely unknown. Importantly, the HPG axis modulates sex hormones and neuropeptides, such as estrogen and kisspeptin, as well as estrous cycle regularity. The goals of this study were to determine if estrous cycle regularity is affected by GCI and to characterize HPG axis functionality post-ischemia. Adult female Wistar rats underwent vaginal lavage for 2 weeks to confirm estrous cycle regularity before being subjected to GCI for 10 minutes or sham surgery. Estrous cycle regularity was monitored for 4 weeks following surgery, after which rats were euthanized. Immunofluorescence was used to detect kisspeptin receptors (Kiss1) and estrogen receptors alpha (ERα) and beta (ERβ) expression in the arcuate nucleus (ARC) of the hypothalamus, as well as the CA1 and dentate gyrus (DG) of the hippocampus. Following surgery, all sham subjects maintained a regular estrous cycle, whereas two thirds of ischemic rats presented a disrupted cycle following GCI (persistent diestrus, average length 13 days), before resuming regular cycling. T-test revealed a significant increase in Kiss1 receptors in the ARC of GCI rodents, as well as a trend for higher ERα expression in the CA1 of ischemic rats. Overall, our results highlight that while estrous cycle disruptions resolve within 2 weeks after the ischemic event, HPG axis dysregulation persists up to one month after GCI, hinting at possible long-term impacts on the reproductive axis.

Vaginal carriage of Haemophilus influenzae in a non-pregnant reproductive-age population

BackgroundHaemophilus influenzae (Hi) is an emerging cause of early onset neonatal sepsis, but mechanisms of transmission are not well understood. We aimed to determine the prevalence of vaginal carriage of Hi in reproductive age women and to examine behavioral and demographic characteristics associated with its carriage.MethodsWe performed a secondary analysis of stored vaginal lavage specimens from a prospective cohort study of nonpregnant reproductive-age women. After extraction of bacterial genomic DNA, samples were tested for the presence of the gene encoding Haemophilus protein d (hpd) by quantitative real-time polymerase chain reaction (PCR) using validated primers and probe. PCR for the V3-V4 region of the 16 S rRNA gene (positive control) assessed sample quality. Samples with cycle threshold (CT) value < 35 were defined as positive. Sanger sequencing confirmed the presence of hpd. Behavioral and demographic characteristics associated with vaginal carriage of Hi were examined.Results415 samples were available. 315 (75.9%) had sufficient bacterial DNA and were included. 14 (4.4%) were positive for hpd. There were no demographic or behavioral differences between the women with Hi vaginal carriage and those without. There was no difference in history of bacterial vaginosis, vaginal microbiome community state type, or presence of Group B Streptococcus in women with and without vaginal carriage of Hi.ConclusionHi was present in vaginal lavage specimens of 4.4% of this cohort. Hi presence was unrelated to clinical or demographic characteristics, though the relatively small number of positive samples may have limited power to detect such differences.

Interactions between vaginal local cytokine IL-2 and high-risk human papillomavirus infection with cervical intraepithelial neoplasia in a Chinese population-based study.

Although interleukin-2 (IL-2) has long been associated with cancer development, its roles in the development of cervical cancer remains unclear. Few studies examined the associations between IL-2 and high-risk human papillomavirus (HPV) with risk of cervical intraepithelial neoplasia (CIN). We aimed to assess the association of IL-2 and high-risk HPV infection with risk of CIN as well as their interactions on the risk of CIN. We performed a cross-sectional analysis of screening data in 2285 women aged 19-65 years who participated in an ongoing community-based cohort of 40,000 women in Shanxi, China in 2014-2015. Both categorical and spline analyses were used to evaluation the association between IL-2 in the local vaginal fluids and prevalence of CIN. In addition, 1503 controls were followed up until January 31, 2019), the nested case-control study design was adopted to evaluate the association of vaginal lavage IL-2 levels and the risk of CIN progression. After adjusting for potential confounders, IL-2 levels were statistically inversely associated with prevalence of CIN (the 1st versus 4th quartile IL-2 levels: the respective odds ratio [OR] and 95% confidence intervals [CI] was: = 1.75 [1.37, 2.23] for CIN, 1.32 [1.01, 1.73] for CIN I, and 3.53 [2.26, 5.52] for CIN II/III). Increased IL-2 levels were inversely associated with prevalence of CIN (P-overall<0.01, P-nonlinearity<0.01 for CIN; P-overall<0.01, P-nonlinearity = 0.01 for CIN I; P-overall <0.01, P-nonlinearity = 0.62 for CIN II/III). The highest prevalence of CIN was observed in women with high-risk HPV, who also had the lowest IL-2 levels (P-interaction < 0.01). Nested case-control study observed an inverse association between IL-2 levels and risk of CIN progression (OR=3.43, [1.17, 10.03]). IL-2 levels in the local vaginal fluids were inversely associated with the risk of CIN in Chinese women either with or without high-risk HPV infection.

Newly Discovered Antimicrobial Peptide Scyampcin44-63 from Scylla paramamosain Exhibits a Multitargeted Candidacidal Mechanism In Vitro and Is Effective in a Murine Model of Vaginal Candidiasis.

The emergence of azole-resistant and biofilm-forming Candida spp. contributes to the constantly increasing incidence of vulvovaginal candidiasis. It is imperative to explore new antifungal drugs or potential substituents, such as antimicrobial peptides, to alleviate the serious crisis caused by resistant fungi. In this study, a novel antimicrobial peptide named Scyampcin44-63 was identified in the mud crab Scylla paramamosain. Scyampcin44-63 exhibited broad-spectrum antimicrobial activity against bacteria and fungi, was particularly effective against planktonic and biofilm cells of Candida albicans, and exhibited no cytotoxicity to mammalian cells (HaCaT and RAW264.7) or mouse erythrocytes. Transcriptomic analysis revealed four potential candidacidal modes of Scyampcin44-63, including promotion of apoptosis and autophagy and inhibition of ergosterol biosynthesis and the cell cycle. Further study showed that Scyampcin44-63 caused damage to the plasma membrane and induced apoptosis and cell cycle arrest at G2/M in C. albicans. Scanning and transmission electron microscopy demonstrated that Scyampcin44-63-treated C. albicans cells were deformed with vacuolar expansion and destruction of organelles. In addition, C. albicans cells pretreated with the autophagy inhibitor 3-methyladenine significantly delayed the candidacidal effect of Scyampcin44-63, suggesting that Scyampcin44-63 might contribute to autophagic cell death. In a murine model of vulvovaginal candidiasis, the fungal burden of vaginal lavage was significantly decreased after treatment with Scyampcin44-63.