Cervicovaginal Fluid Research Articles

The use of quantitative fetal fibronectin for the prediction of preterm birth in women with exposed fetal membranes undergoing emergency cervical cerclage

ObjectiveEmergency cervical cerclage is often considered a controversial salvage measure for those pregnancies at high risk of mid-trimester loss or early preterm birth. To determine the efficacy and benefit of emergency cerclage insertion, we assessed the predictive value of quantitative fetal fibronectin (fFN) concentration in cervicovaginal fluid for spontaneous preterm birth in women with exposed fetal membranes prior to cerclage insertion. Study designThis was a retrospective observational study from St Thomas’ Hospital, London of a cohort of women with singleton pregnancy and exposed fetal membranes presenting between 18 and 23+6 weeks of gestation (n = 35), in the period 2015-2018. fFN concentrations in cervicovaginal fluid were measured quantitatively at presentation in all women within 24 h prior to cerclage insertion. Results35 eligible women with exposed fetal membranes who underwent an emergency cervical cerclage were identified. The median gestational age was 20 + 0 weeks (18 + 6−21 + 2) at presentation and 29 + 3 weeks (23 + 3−38 + 2) at delivery, with a median time from emergency cerclage to delivery of 65.5 days (17–126.5). In total, 12 women (34 %) delivered within 28 days of emergency cerclage insertion. 60 % of women with an fFN level above 500 ng/mL delivered within 28 days of cerclage insertion, and they all delivered before 37 weeks gestation. No women with fFN levels below 10 ng/mL delivered within 28 days and 75 % of these women delivered at term. Concentration of quantitative fFN at presentation correlated negatively with time to delivery (Spearman's rs = −0.52, p = 0.0016). Mann–Whitney U analysis demonstrated a significant difference in the distribution of qfFN concentrations in women who delivered preterm compared to those who did not, both within 28 days from testing (p = 0.0048) and <37 weeks (p = 0.006). ConclusionQuantitative fFN has a role in predicting spontaneous preterm birth even in women with exposed fetal membranes undergoing emergency cervical cerclage. Given the serious risks associated with cervical cerclage surgery, qfFN could be used to counsel these patients at high risk of preterm delivery, as well as assisting in the decision to insert a cerclage.

Cervical Length Estimation and Cervicovaginal Fluid for Placental &amp;lt;i&amp;gt;α&amp;lt;/i&amp;gt;-Microglobulin 1 Testing to Screen Women Had Threatened Preterm Labor for Time till Spontaneous Labor

Objectives: Evaluation of diagnostic performance of rapid testing of cervico-vaginal fluid (CVF) for fetal fibronectin (FFN) and placental α-microglobulin 1 (PAMG-1) as screening of women presented by threatened preterm labor (TPTL) with intact membranes for duration till getting spontaneous labor (SL). Patients & Methods: 37 women presenting with TPTL underwent CVF samplings before digital examination and then underwent transvaginal ultrasonography for estimation of cervical length (CL). All women received fluid and anxiolytic therapy and if uterine contractions persisted, all had received tocolytic therapy with oral nifedipine or intravenous magnesium sulphate according to requirements. Incidence of SL within <48 hr, 2 - 7 and 7 - 14 days was recorded and related to CL and result of FFN and PAMG-1 tests. Results: Incidence of SL was 13.5%, 35.2% and 51.3% within 48-hr, 2 - 7 and 7 - 14 days, respectively. Duration till labor after sampling was positively correlated with CL, while was negatively correlated with positive FFN and PAMG-1 tests. Positive FFN test had high specificity, while positive PAMG-1 test had high sensitivity for labor within 7 days. Regression analysis defined short CL and positive PAMG-1 test as significant predictors for short duration till SL. ROC curve analysis defined short cervix and positive PAMG-1 test as significant predictors for labor within 48-hr and within 2 - 7 days respectively and combined negative PAMG-1 test and CL of 20 - 25 mm were significant predictors for labor within 7 - 14 days. Conclusion: PAMG-1 test had high specificity, if positive, for predicting SL and high NPP, if negative, for excluding labor within 7 days, so it can be used as rapid adjuvant to clinical evaluation to help management decision-making. Moreover, PAMG-1 test is recommended screening test for being easy-to-use bedside test, provides rapid results, can be used after vaginal exam and coitus and does not require a speculum examination or specialized equipment to analyze results.

Cervicovaginal fluid proteomic analysis to identify potential biomarkers for preterm birth

Spontaneous preterm birth is a leading cause of neonatal morbidity and mortality. Early identification of at-risk women by reliable screening tests could reduce the spontaneous preterm birth rate, but conventional methods such as obstetrical history and maternal cervical length screening identify only a minority of spontaneous preterm birth cases. Cervicovaginal fluid might prove to be a useful, readily available biological fluid for identifying spontaneous preterm birth biomarkers. The objective of the study was to identify cervicovaginal fluid biomarkers of early spontaneous preterm birth in a high-risk cohort of pregnant women with a history of spontaneous preterm birth using targeted and shotgun proteomic analyses. A nested case control study (cases were spontaneous preterm birth <34 weeks in the current pregnancy; controls were spontaneous labor and delivery at 39-41 weeks) was performed using cervicovaginal fluid samples collected at 3 study visits (100/7 to 186/7 weeks, 190/7 to 236/7 weeks, and 280/7 to 316/7 weeks). All participants had a history of at least 1 prior spontaneous preterm birth. Targeted proteomic analysis was performed using a stable isotope-labeled proteome derived from endocervical and vaginal mucosal cells. This served as a standard to quantitate candidate protein levels in individual cervicovaginal fluid samples from the second and third study visits using liquid chromatography-multiple reaction monitoring mass spectrometry. The ratio of endogenous peptide area/stable isotope-labeled proteome-derived peptide area was used to measure levels of 42 peptides in 22 proteins. To maximize biomarker discovery in the cervicovaginal fluid samples, shotgun proteomic analysis also was performed utilizing liquid chromatography and ion trap mass spectrometry. A validation study was performed in second-trimester cervicovaginal fluid samples from an independent study group (12 spontaneous preterm birth cases, 19 term delivery controls) using enzyme-linked immunosorbent assay for 5 proteins expressed at higher levels in spontaneous preterm birth cases compared with controls in targeted or shotgun proteomic analyses. For targeted proteomics, cervicovaginal fluid samples from 33 cases and 32 controls at 190/7 to 236/7 weeks and 16 cases and 14 controls at 280/7 to 316/7 weeks from the same pregnancies were analyzed. When samples were compared between cases and controls, the relative abundance of 5 proteins was greater (P= .02-.05) in cases at both visits, while the relative abundance of 1 protein was lower (P= .03) in cases at both visits. For shotgun proteomics analyses, cervicovaginal fluid samples were pooled for 9 spontaneous preterm birth cases and 9 term delivery controls at each study visit. Shotgun proteomics yielded 28 proteins that were detected at levels >2 times higher and 1 protein that was detected at a level <0.5 times lower in spontaneous preterm birth cases compared with controls at all 3 study visits. Validation enzyme-linked immunosorbent assay for 5 proteins that were detected at higher levels in cervicovaginal fluid samples from spontaneous preterm birth cases compared with term delivery controls in proteomics analyses did not demonstrate statistically significant differences between spontaneous preterm birth cases and controls. Potential biomarkers of spontaneous preterm birth were identified by targeted and shotgun proteomics analyses in cervicovaginal fluid samples from high-risk, asymptomatic women. Many of the proteins detected at higher levels in cervicovaginal fluid samples from spontaneous preterm birth cases are extracellular matrix proteins and/or regulate cell membrane physiology. These proteins have substantial biological interest, but validation enzyme-linked immunosorbent assay for 5 of these proteins did not yield clinically useful biomarkers for spontaneous preterm birth.

A 40-Month Follow-Up of Ebola Virus Disease Survivors in Guinea (PostEbogui) Reveals Long-Term Detection of Ebola Viral Ribonucleic Acid in Semen and Breast Milk.

BackgroundWith the increasing frequency and impact of Ebola virus disease (EVD) outbreaks illustrated by recent epidemics, a good understanding of the extent of viral persistance or ribonucleic acid (RNA) detection in body fluids from survivors is urgently needed.MethodsEbola viral RNA shedding was studied with molecular assays in semen (n = 1368), urine (n = 1875), cervicovaginal fluid (n = 549), saliva (n = 900), breast milk (n = 168), and feces (n = 558) from EVD survivors in Guinea (PostEbogui cohort, n = 802) at a regular base period until 40 months after inclusion.ResultsTwenty-seven of 277 (9.8%) male survivors tested positive for Ebola RNA in at least 1 semen sample. The probability of remaining positive for Ebola RNA in semen was estimated at 93.02% and 60.12% after 3 and 6 months. Viral RNA in semen was more frequent in patients with eye pain (P = .036), joint pain (P = .047), and higher antibody levels to Ebola virus antigens (nucleoprotein [P = .001], glycoprotein [P = .05], and viral protein-40 [P = .05]). Ebola RNA was only rarely detected in the following body fluids from EVD survivors: saliva (1 of 454), urine (2 of 593), breast milk (2 of 168), cervicovaginal secretions (0 of 273), and feces (0 of 330). Ribonucleic acid was detected in breast milk 1 month after delivery but 500 days after discharge of Ebola treatment unit (ETU) in 1 woman who became pregnant 7 months after discharge from the ETU.ConclusionsThe frequency and potential long-term presence of viral RNA in semen confirmed that systematic prevention measures in male survivors are required. Our observation in breast milk suggests that our knowledge on viral reservoir in immune-privileged sites and its impact are still incomplete.

Metabolomic biomarkers in cervicovaginal fluid for detecting endometrial cancer through nuclear magnetic resonance spectroscopy

Endometrial cancer (EC) is one of the most common gynecologic neoplasms in developed countries but lacks screening biomarkers. We aim to identify and validate metabolomic biomarkers in cervicovaginal fluid (CVF) for detecting EC through nuclear magnetic resonance (NMR) spectroscopy. We screened 100 women with suspicion of EC and benign gynecological conditions, and randomized them into the training and independent testing datasets using a 5:1 study design. CVF samples were analyzed using a 600-MHz NMR spectrometer equipped with a cryoprobe. Four machine learning algorithms-support vector machine (SVM), partial least squares discriminant analysis (PLS-DA), random forest (RF), and logistic regression (LR), were applied to develop the model for identifying metabolomic biomarkers in cervicovaginal fluid for EC detection. A total of 54 women were eligible for the final analysis, with 21 EC and 33 non-EC. From 29 identified metabolites in cervicovaginal fluid samples, the top-ranking metabolites chosen through SVM, RF and PLS-DA which existed in independent metabolic pathways, i.e. phosphocholine, malate, and asparagine, were selected to build the prediction model. The SVM, PLS-DA, RF, and LR methods all yielded area under the curve values between 0.88 and 0.92 in the training dataset. In the testing dataset, the SVM and RF methods yielded the highest accuracy of 0.78 and the specificity of 0.75 and 0.80, respectively. Phosphocholine, asparagine, and malate from cervicovaginal fluid, which were identified and independently validated through models built using machine learning algorithms, are promising metabolomic biomarkers for the detection of EC using NMR spectroscopy.

Cervico-vaginal placental α-macroglobulin-1 combined with cervical length for the prediction of preterm birth in women with threatened preterm labor.

Preterm birth is a major cause of neonatal morbidity and mortality. There is an urgent need to accurately predict imminent delivery to enable necessary interventions such as tocolytic, glucocorticoid, and magnesium sulfate administration. We aimed to evaluate placental α-macroglobulin-1 as a new diagnostic marker in the prediction of preterm birth. We performed a prospective observational trial in women with intact membranes between 24+0 and 36+6 weeks of gestation. We included both women with and without threatened preterm labor symptoms. We evaluated the test performance of placental α-macroglobulin-1 measurements in cervicovaginal fluid regarding three different presentation-to-delivery intervals: ≤2, ≤7, ≤14days. In addition, we calculated placental α-macroglobulin-1 performance in combination with other prognostic factors such as ultrasonographic cervical length measurements. We included 126 women in the study. We detected high specificity (97%-98%) and negative predictive value (89%-97%) for placental α-macroglobulin-1 at all time intervals. We assessed placental α-macroglobulin-1 in combination with cervical length measurements (≤15mm) in the sub-group of women presenting with threatened preterm labor symptoms (n=63) and detected high positive predictive values (100%) for 7- and 14-day presentation-to-delivery intervals. Our study provides evidence that placental α-macroglobulin-1 testing in cervicovaginal fluid, in combination with cervical length measurements, accurately predicts preterm birth in women with preterm labor symptoms. This novel test combination may be used clinically to triage women presenting with threatened preterm labor, avoiding overtreatment and unnecessary hospitalizations.

Evaluation of early progesterone use in high-risk women to prevent pre-term birth (PTB) predicted with a high level of Cervico-Vaginal Fluid (CVF) trappin2/elafin protein

Evaluation of early progesterone use in high-risk women to prevent pre-term birth (PTB) predicted with a high level of Cervico-Vaginal Fluid (CVF) trappin2/elafin protein

Infection/inflammation-associated preterm delivery within 14 days of presentation with symptoms of preterm labour: A multivariate predictive model.

Multi-marker tests hold promise for identifying symptomatic women at risk of imminent preterm delivery (PTD, <37 week’s gestation). This study sought to determine the relationship of inflammatory mediators and metabolites in cervicovaginal fluid (CVF) with spontaneous PTD (sPTD) and delivery within 14 days of presentation with symptoms of preterm labour (PTL). CVF samples from 94 (preterm = 19, term = 75) singleton women with symptoms of PTL studied between 19+0–36+6 weeks’ gestation were analysed for cytokines/chemokines by multiplexed bead-based immunoassay, while metabolites were quantified by enzyme-based spectrophotometry in a subset of 61 women (preterm = 16, term = 45). Prevalence of targeted vaginal bacterial species was determined for 70 women (preterm = 14, term = 66) by PCR. Overall, 10 women delivered within 14 days of sampling. Predictive capacities of individual biomarkers and cytokine-metabolite combinations for sPTD and delivery within 14 days of sampling were analysed by logistic regression models and area under the receiver operating characteristic curve. Fusobacterium sp., Mubiluncus mulieris and Mycoplasma hominis were detected in more preterm-delivered than term women (P<0.0001), while, M. curtisii was found in more term-delivered than preterm women (P<0.0001). RANTES (0.91, 0.65–1.0), IL-6 (0.79, 0.67–0.88), and Acetate/Glutamate ratio (0.74, 0.61–0.85) were associated with delivery within 14 days of sampling (AUC, 95% CI). There were significant correlations between cytokines and metabolites, and several cytokine-metabolite combinations were associated with sPTD or delivery within 14 days of sampling (e.g. L/D-lactate ratio+Acetate/Glutamate ratio+IL-6: 0.84, 0.67–0.94). Symptomatic women destined to deliver preterm and within 14 days of sampling express significantly higher pro-inflammatory mediators at mid to late gestation. In this cohort, IL-6, Acetate/Glutamate ratio and RANTES were associated with delivery within 14 days of sampling, consistent with their roles in modulating infection-inflammation-associated preterm labour in women presenting with symptoms of preterm birth. Replication of these observations in larger cohorts of women could show potential clinical utility.

IL-15 as an Immune Modulator in Schistosomiasis

Abstract Introduction Schistosomiasis affects approximately 200,000,000 people globally, with 93% of these cases in sub-Saharan Africa. Women who have been infected with Schistosoma haematobium have four times the risk of contracting HIV due to a change in their cervical mucosal immunity. The mechanism for this altered cervical mucosal immunity was not understood until a recent study published by Downs et al, which showed IL-15 levels were significantly lower in cervicovaginal lavage fluid samples of women with Schistosoma haematobium. Schistosoma mansoni, known to cause intestinal schistosomiasis, has also shown the potential to increase the risk of contracting HIV. The objective for this study is to see if IL-15 levels in peripheral blood are significantly different between patients positive for S mansoni and negative controls. Methods Enzyme-linked immunosorbent assays (BD Biosciences, San Jose, CA) for IL-15 were performed on plasma collected from 84 patients in villages in sub-Saharan Africa. These samples were collected at screening, 3-month follow-up, 6-month follow-up, and 9-month follow-up appointments. Results Of 249 plasma samples tested, 20 had detectable IL-15 levels ranging from 8.329 pg/mL to above the upper limit of detection of 500 pg/mL. While the patients that had detectable IL-15 in the peripheral blood were half S mansoni negative and half S mansoni positive, when an S mansoni–positive patient was positive for IL-15, it was across all timepoints. Conclusion The role of IL-15 as an immune modulator can explain the elevated levels in S mansoni–positive patients. All patients in the study were tested for significant comorbidities, with no correlation seen within the negative controls. Further testing is needed to investigate the role of IL-15 in the alteration of localized areas of infection with both Schistosoma mansoni and Schistosoma haematobium. More samples are currently being collected from the patient population in Tanzania, and future studies are planned.

Label-free cervicovaginal fluid proteome profiling reflects the cervix neoplastic transformation.

Cervicovaginal fluid (CVF) is a valuable source of clinical information about the female reproductive tract in both nonpregnant and pregnant women. The aim of this study is to specify the CVF proteome at different stages of cervix neoplastic transformation by label-free quantitation approach based on liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The proteome composition of CVF from 40 women of reproductive age with human papillomavirus (HPV)-associated cervix neoplastic transformation (low-grade squamous intraepithelial lesion [LSIL], high-grade squamous intraepithelial lesion [HSIL], and CANCER) was investigated. Hierarchical clustering and principal component analysis (PCA) of the proteomic data obtained by a label-free quantitation approach show the distribution of the sample set between four major clusters (no intraepithelial lesion or malignancy [NILM], LSIL, HSIL and CANCER) depending on the form of cervical lesion. Multisample ANOVA with subsequent Welch's t test resulted in 117 that changed significantly across the four clinical stages, including 27 proteins significantly changed in cervical cancer. Some of them were indicated as promising biomarkers previously (ACTN4, VTN, ANXA1, CAP1, ANXA2, and MUC5B). CVF proteomic data from the discovery stage were analyzed by the partial least squares-discriminant analysis (PLS-DA) method to build a statistical model, allowing to differentiate severe dysplasia (HSIL and CANCER) from the mild/normal stage (NILM and LSIL), and receiver operating characteristic (ROC) area under the curve (AUC) were obtained on an independent set of 33 samples. The sensitivity of the model was 77%, and the specificity was 94%; AUC was equal to 0.87. CVF proteome proved to be reflect the stage of cervical epithelium neoplastic process.

Tenofovir disoproxil fumarate intravaginal ring for HIV pre-exposure prophylaxis in sexually active women: a phase 1, single-blind, randomised, controlled trial

An intravaginal ring that releases the tenofovir prodrug, tenofovir disoproxil fumarate, provided 100% protection in macaques against simian HIV and was safe in a 14-day clinical trial in sexually abstinent women. We aimed to assess the safety and pharmacokinetics of this intravaginal ring over 90 days in sexually active women. We did a phase 1, single-blind, randomised, placebo-controlled trial to assess safety, pharmacokinetics, and acceptability of a tenofovir disoproxil fumarate intravaginal ring used continuously with monthly ring changes for 3 months. Sexually active women who were HIV negative were randomly assigned (3:1) to a tenofovir disoproxil fumarate ring or placebo ring. Primary safety endpoint was the proportion of women who had grade 2 or higher genitourinary adverse events judged related to study product and any grade 2 or higher adverse event as defined by the Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events. We quantified tenofovir disoproxil fumarate and tenofovir concentrations in cervicovaginal fluid, tenofovir in plasma, and tenofovir diphosphate, the active metabolite, in cervical tissue and dried blood spots 1 month after each ring insertion. We compared changes over time in cervicovaginal fluid cytokine and chemokine concentrations and vaginal microbiota. The study was electively stopped early and is registered with ClinicalTrials.gov, number NCT02762617. Between Feb 24 and July 20, 2017, 17 women were enrolled before study termination. 12 were assigned to receive the tenofovir disoproxil fumarate ring and five were assigned to receive the placebo ring. Two participants in the tenofovir disoproxil fumarate ring group completed 3 months of continuous ring use; eight were asked to discontinue ring use early because of ulcerations (grade 1) near the ring; in the remaining two women, rings were electively removed by study staff on day 20 and day 23. Ulcers were detected a mean of 32 days after ring use (range 23-56). Four of eight participants with ulcers were symptomatic with vaginal discharge; four had ulcers identified when examined; three had two ulcers; all ulcers resolved after ring removal. No participants in the placebo group developed ulcers. No grade 2 product-related adverse events were reported in either group and four non-product-related grade 2 adverse events were reported in the tenofovir disoproxil fumarate ring group. Cervicovaginal fluid tenofovir concentrations did not differ at day 14 (p=0·14) comparing the eight patients who did (median 1·0 × 105 ng/mL [IQR 9·1 × 104-1·1 × 105]) with the four who did not (6·0 × 104 ng/mL [5·6 × 104-1·1 × 105]) develop ulcers. No significant changes in vaginal microbiota were detected in either group. Concentrations of multiple inflammatory cytokines and chemokines were significantly higher at days 14 and 28 compared with baseline in the tenofovir disoproxil fumarate ring group but not the placebo group. Future studies are needed to establish whether the unanticipated finding of ulcerations is specific to this tenofovir disoproxil fumarate ring or generalisable to other sustained topical release formulations of tenofovir or its prodrugs. National Institutes of Health.

CERVICOVAGINAL FLUID CYTOKINES' LEVELS IN PRETERM BIRTH HIGH-RISK WOMEN COULD BE USED AS EARLY PREDICTORS FOR RECURRENT PRETERM BIRTH

Background: Differences in cytokine distribution across different biological fluids; maternal plasma, cervicovaginal fluid (CVF), and amniotic fluid are evident throughout normal pregnancy, but profiles of numerous cytokines varied across trimesters in women delivering term versus preterm in both CVF and serum. Objective: To evaluate cytokine profile in cervicovaginal fluid (CVF) of women had previous preterm birth (PTB) and the effect imposed by cerclage on these levels. Patients and Methods: Seventy study women with previous PTB and currently had cercical length (CL) of 25 mm were studied. At the 24th gestational week (GW; T1), all women had CL estimation and CVF sampling. At time of labor or removal of the suture (T2), study women had CL re-estimation and CVF re-sampling. CVF levels of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 were ELISA estimated. Shirodkar cervical cerclage (CC) was performed within 4 days after T1 sampling. Outcomes included difference in T1-CVF cytokines' levels between study and control women, effect of cerclage on CL and CVF cytokines' levels and the value of T1-CVF cytokines' levels as predictors of pregnancy duration. Results: T1-CVF levels were significantly higher in study than control women and to their T2- levels. T1- and T2-CL in study women was significantly shorter than T1-CL in controls with significant difference between T1- and T2-CL. Percentage of T2-CL decrease was negatively correlated with the percentage of decrease of T2-CVF levels. Duration of pregnancy of study women was negatively correlated with percentage of CL shortening, while was positively correlated with percentage of decrease of T2-CVF levels of MCP-1 and TNF-α. Statistical analyses defined high T1-CVF levels of MCP-1, IL-β and TNF-α as significant early predictor for PTB. Conclusion: High CVF cytokines' levels were associated with increased risk of PTB. Cerclage worked beyond its mechanical action through reduction of CVF cytokines' levels. High CVF cytokines' levels at 24th GW, especially high levels of MCP-1 and TNF-α, may help to predict PTB.

Novel site-specific detection of ovarian cancer using cell-free DNA (cfDNA) modeling

Objective: Despite advancements in ovarian cancer (OC) therapy, the largest impact for improving OC mortality lies within the discovery of screening tests that detect early-stage disease or identify patients at risk for developing OC. The objective of this study was to develop a cell-free DNA (cfDNA) molecular signature to detect OC utilizing site-specific collection of cervicovaginal fluids (CVF).

Avirulins, a Novel Class of HIV-1 Reverse Transcriptase Inhibitors Effective in the Female Reproductive Tract Mucosa.

While extensive research efforts have decreased human immunodeficiency virus (HIV) transmissions and mortalities, new challenges have arisen in the fight to eradicate HIV. Drug resistance to antiretroviral therapy threatens infected individuals, while the prevalence of heterosexual transmission creates an urgent need for therapies effective in the female reproductive tract (FRT) mucosa. We screened a library of 2095 small molecule compounds comprising a unique chemical space, purchased from Asinex Corporation, for antiviral activity against human immunodeficiency virus type 1 (HIV-1) strain BaL and identified several molecular representatives of a unique class of HIV-1 inhibitors, which we termed “Avirulins.” We determined that Avirulins were active against clinical isolates of HIV-1 from genetically variant subtypes, several of which have reduced sensitivity to other antivirals. Avirulins displayed specific dose-dependent inhibition of the HIV-1 drug target, reverse transcriptase (RT). Avirulins were effective against several nucleoside RT-inhibitor resistant strains of HIV-1, as well as one nonnucleoside RT-inhibitor resistant strain containing a 106A mutation, suggesting a noncompetitive mechanism of action. Drugs, which are damaging to the FRT, can increase the risk of HIV-1 transmission. We therefore explored the cytotoxicity of Avirulins against epithelial cells derived from the FRT and found no significant toxicity, even at the highest concentrations tested. Importantly, Avirulin antiviral activity was not diminished in human cervico–vaginal fluid, suggesting retained potency in the milieu of the FRT. Based on these promising results, Avirulins should be valuable chemical scaffolds for development into next-generation treatments and preventatives that target HIV-1.

Vaginal pH measured in vivo: lactobacilli determine pH and lactic acid concentration

BackgroundLactic acid (protonated lactate) has broad antimicrobial activity. Vaginal lactobacilli produce lactic acid, and are known to confer protection against reproductive tract infections when they are predominant in the vaginal microbiota. Using novel ex vivo methods, we showed that cervicovaginal fluid (CVF) from women with a predominantly lactobacilli-morphotype microbiota contains significantly more lactic acid than previously thought, sufficient to inactivate reproductive tract pathogens.Here, we measured vaginal pH in vivo in 20 women with a predominantly lactobacilli-morphotype (low Nugent score) microbiota. We also investigated the in vitro production of protons (as hydrogen ions) and lactate by vaginal lactobacilli.ResultsThe average vaginal pH in these women was 3.80 ± 0.20, and the average lactate concentration was 0.79% ± 0.22% w/v, with pH and lactate concentration tightly correlated for each sample. In vitro, lactobacilli cultured from these CVF samples reached an average pH of 3.92 ± 0.22, but the average lactate concentration was only 0.14% ± 0.06% w/v, approximately five-fold less than in the corresponding CVF samples. When the pH of the cultures was raised, lactate and hydrogen ion production resumed, indicating that production of lactate and hydrogen ions by vaginal lactobacilli is limited primarily by their sensitivity to hydrogen ion concentration (low pH) not lactate concentration.ConclusionsSome vaginal lactobacilli cultures have a lower limiting pH than others, and limiting pHs in vitro showed good correlation with pHs measured in vivo. The limiting pH of the lactobacilli predominant in a woman’s vaginal microbiota seems critical in determining the concentration of antimicrobial lactic acid protecting her.

Evaluation of additive effect of quantitative fetal fibronectin to cervical length for prediction of spontaneous preterm birth among asymptomatic high-risk women

Objective: To determine if quantitative fetal fibronectin (qFFN) in addition to transvaginal ultrasound (TVU) cervical length (CL) measurement between 180/7 and 236/7 weeks would be predictive of spontaneous preterm birth (sPTB) at <350/7 weeks among asymptomatic high-risk women as defined by those with prior sPTB > 200/7 weeks.Material and methods: A prospective observational study of asymptomatic women with singleton gestations between 180/7 and 236/7weeks and one or more prior SPTB was performed. Women at their anatomy scan who opted into universal CL screening were enrolled. At enrollment, a vaginal speculum exam was performed to collect cervico-vaginal fluid from the posterior fornix using fetal fibronectin (FFN) swab. These women were then followed until delivery. Women with multiple gestations, rupture of membranes, vaginal bleeding, intercourse, or vaginal exam within 48 h of enrollment were excluded. Physicians were blinded to the qFFN levels, but the CL measurements were made available. The primary outcome was sPTB < 350/7 weeks.Results: Of the 105 asymptomatic women with prior sPTB who were prospectively enrolled, 19 (18.1%) had recurrent sPTB < 370/7 weeks. None of the sPTB were iatrogenic. Using receiver-operating characteristic curves, qFFN ≥ 10 ng/mL had the highest sensitivity with subsequent lowest false negative rate, while FFN ≥ 50 ng/mL was identified as being the best balance of sensitivity and false positive rate for predicting sPTB < 350/7 weeks. As compared with CL ≤ 25 mm alone, with the use of CL ≤ 25 mm or qFFN ≥ 50 ng/mL as screening criteria for prediction of SPTB < 350/7 weeks, sensitivity improved from 18.2 to 63.6%, specificity decreased from 96.8 to 82.1%, positive predictive value (PPV) decreased from 40.0 to 29.2%, negative predictive value (NPV) marginally improved from 91.1 to 95.1%.Conclusion: In women with singleton gestations with prior SPTB, qFFN can be used as an adjunct to triage patients who are found to have a shortened cervix. Sensitivity and NPVs improved with the addition of qFFN to TVU CL screening alone in women with singleton gestations with prior SPTB. However, specificity and PPVs decreased.

Evaluation of the cervicovaginal environment in asymptomatic Human T-cell lymphotropic virus type 1 infected women

IntroductionHuman T-cell lymphotropic virus type 1 (HTLV-1) is sexually transmitted and causes persistent infection. This virus induces activation of the immune system and production of inflammatory cytokines. This study aimed to assess the cytokine profile and cytopathological findings in the cervicovaginal fluid of asymptomatic HTLV-1-infected women. MethodsHTLV-1-infected and uninfected women were selected at the Centro de Atendimento ao Portador de HTLV in Salvador-Brazil. None of the included HTLV-1-infected women reported any HTLV-1-associated diseases. All volunteers underwent gynecological examination to collect cervicovaginal fluid. Cytokine quantification was performed using the Cytometric Bead Array (CBA) Human Th1/Th2/Th17 kit. Light microscopy was used to evaluate cervicovaginal cytopathology. In addition, proviral load in cervicovaginal fluid and peripheral blood was measured by real-time quantitative polymerase chain reaction. Results112 women (63 HTLV-1-infected and 49 uninfected) were evaluated. No differences were found with respect to cytopathological cervicovaginal findings between the groups. IL-2, TNF, IL-4, IL-10, and IL-17 levels were significantly higher in cervicovaginal fluid of the HTLV-1-infected women than in uninfected women (p<0.05). Conversely, IFN-γ was found to be lower in the HTLV-1-infected women (p<0.001) compared to uninfected individuals. Cervicovaginal proviral load was detectable in 53% of the HTLV-1-infected women and was found to be consistently lower than the proviral load in peripheral blood. ConclusionsHTLV-1 infection induces immune activation in cervicovaginal environment, characterized by elevated concentrations of Th1, Th2, and IL17 in the cervicovaginal fluid.

The diagnostic value of cervicovaginal and serum ferritin levels in relation to spontaneous preterm delivery

Objective The objective of the study was to detect the diagnostic value of cervicovaginal and serum ferritin levels in 20–24 weeks of gestation in the prediction of spontaneous preterm delivery in singleton pregnancies with no well-known risk factors of preterm delivery. Background Preterm delivery is one of the most important health problems in the world that may lead to severe short-term and long-term medical and developmental problems in infants. Patients and methods Venous blood and cervicovaginal fluid samples are drawn from 100 women with singleton pregnancies within 20–24 weeks of gestation at the Department of Obstetric and Gynecologies, El Mehalla Hospital and then they were followed up and the term of delivery were noted. Results Spontaneous preterm delivery in singleton pregnancies with no well-known risk factors is associated with increased levels of cervicovaginal and serum ferritin when measured within 20–24 weeks of gestation and there is a strong positive correlation between both serum and cervicovaginal ferritin levels measuring 0.83. However, the only significant predictor is serum ferritin as an increase by one unit of its value increases the preterm risk by 1.277. About 85% of preterm deliveries can be predicted by serum ferritin. Conclusion It has been observed that 20–24 weeks of gestation serum ferritin level is a significant predictor of preterm delivery in singleton normal pregnancies.

Proteomic pattern of cervico-vaginal fluid (CVF) in an ovarian cancer diagnosis - pilot study.

High grade serous ovarian cancer (HGSC) is the most common type of ovarian cancer and is responsible for about 90% of ovarian cancer deaths. The diagnostic tests currently used do not increase the detection rates for ovarian cancer. There is a great necessity to develop new and non-invasive diagnostic tests for ovarian cancer (OC). Cervico-vaginal fluid (CVF) seems to be a potential and valuable source of biomarkers for genital tract diseases including ovarian cancer. The aim of our pilot study was to undertake a preliminary proteomic analysis of CVF derived from ovarian cancer patients and to compare these with results from a control group. We analysed and compared samples from a group of ovarian cancer patients and a control group of healthy patients. The study used MALDI-TOF coupled with nanoLC and ClinProTools software for MS, MS/MS spectra collection and proteomic analysis. We identified 404 different proteins in the OC group and 417 proteins in the control group. 239 of the proteins were found to be common to both study groups, 165 proteins were unique to the OC subjects, and 178 proteins were unique to the control subjects. We selected three proteins as the OC markers with the greatest potential: cysteine-rich secretory protein 3, fibronectin and Ly6/PLAUR domain-containing protein 3. The proteins we selected seem to possess great potential as markers for the screening and early detection of OC, especially in non-invasive and low-cost diagnostic tests. However, our findings require more advanced and validated proteomic analysis to confirm the suitability of the selected proteins in everyday medical diagnoses.

461: Novel protein biomarker combinations that predict preterm birth

Preterm birth (PTB) remains a substantial cause of perinatal mortality and morbidity. Existing methods of preterm birth prediction and current biomarkers fail to identify the majority of asymptomatic women who will deliver prematurely. We propose that select protein biomarkers detected in the cervicovaginal fluid (CVF) are reflective of spontaneous preterm birth. Our aim was to develop and validate an immunoassay-based test composed of a combination of novel protein biomarkers with potential to predict preterm birth (<37 weeks) from mid-trimester. : Samples of CVF from asymptomatic pregnant women were collected between 19 and 26 weeks of gestation and subsequent pregnancy outcomes were recorded including gestation at delivery. The majority of these women were considered to be at risk of preterm birth, including women with a past history of spontaneous preterm birth, previous cervical surgery and twin pregnancy. Exclusion criteria were cervical cerclage and progesterone therapy. CVF samples were obtained from women attending their routine antenatal care. The study included a total of 90 samples from 76 women, of whom 28 spontaneously delivered <37 weeks and 48 delivered at term. The samples were split into a discovery cohort of 35 term and 22 preterm cases and a validation cohort of 13 term and 6 preterm cases. A total of 10 biomarkers were screened by immunoassay and assessed for their ability to predict PTB based on a unique algorithm of biomarker sub-panels. The optimal multivariate model based on specific biological pathways, such as inflammation, hormonal and epithelial-mesenchymal transition, produced mini biomarker panel tests. When combined these achieved a sensitivity of 81% and specificity of 91% for the initial discovery cohort. These results were confirmed in the validation cohort. These results once re-validated in a larger cohort, have the potential of generating a novel test that would predict preterm birth weeks or even months prior to any clinical presentation for risk of preterm birth. Furthermore, the specific pathways represented by these biomarkers may suggest options for future preventative treatments for preterm birth.