Sialidase Activity in the Cervicovaginal Fluid Is Associated With Changes in Bacterial Components of Lactobacillus-Deprived Microbiota

IntroductionSialidase activity in the cervicovaginal fluid (CVF) is associated with microscopic findings of bacterial vaginosis (BV). Sequencing of bacterial 16S rRNA gene in vaginal samples has revealed that the majority of microscopic BV cases fit into vaginal community-state type IV (CST IV), which was recently named “molecular-BV.” Bacterial vaginosis-associated bacterial species, such as Gardnerella spp., may act as sources of CVF sialidases. These hydrolases lead to impairment of local immunity and enable bacterial adhesion to epithelial and biofilm formation. However, the impact of CVL sialidase on microbiota components and diversity remains unknown.ObjectiveTo assess if CVF sialidase activity is associated with changes in bacterial components of CST IV.MethodsOne hundred forty women were cross-sectionally enrolled. The presence of molecular-BV (CST IV) was assessed by V3–V4 16S rRNA sequencing (Illumina). Fluorometric assays were performed using 2-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUAN) for measuring sialidase activity in CVF samples. Linear discriminant analysis effect size (LEfSe) was performed to identify the differently enriched bacterial taxa in molecular-BV according to the status of CVF sialidase activity.ResultsForty-four participants (31.4%) had molecular-BV, of which 30 (68.2%) had sialidase activity at detectable levels. A total of 24 bacterial taxa were enriched in the presence of sialidase activity, while just two taxa were enriched in sialidase-negative samples.ConclusionSialidase activity in molecular-BV is associated with changes in bacterial components of the local microbiome. This association should be further investigated, since it may result in diminished local defenses against pathogens.

Ureaplasma and Prevotella Colonization with Lactobacillus Abundance During Pregnancy Facilitates Term Birth

Ureaplasma and Prevotella infections are well-known bacteria associated with preterm birth. However, with the development of metagenome sequencing techniques, it has been found that not all Ureaplasma and Prevotella colonizations cause preterm birth. The purpose of this study was to determine the association between Ureaplasma and Prevotella colonization with the induction of preterm birth even in the presence of Lactobacillus. In this matched case-control study, a total of 203 pregnant Korean women were selected and their cervicovaginal fluid samples were collected during mid-pregnancy. The microbiome profiles of the cervicovaginal fluid were analyzed using 16S rRNA gene amplification. Sequencing data were processed using QIIME1.9.1. Statistical analyses were performed using R software, and microbiome analysis was performed using the MicrobiomeAnalyst and Calypso software. A positive correlation between Ureaplasma and other genera was highly related to preterm birth, but interestingly, there was a negative correlation with Lactobacillus and term birth, with the same pattern observed with Prevotella. Ureaplasma and Prevotella colonization with Lactobacillus abundance during pregnancy facilitates term birth, although Ureaplasma and Prevotella are associated with preterm birth. Balanced colonization between Lactobacillus and Ureaplasma and Prevotella is important to prevent preterm birth.

Significance of analysis of lipid extracts in cervical canal secretion for diagnosing of placenta-associated complications of pregnancy

Omics technologies hold great potential as the basis for development of the new diagnostic approaches in obstetrics. Cervicovaginal fluid (CVF) as part of the mother-placenta-fetus system can be used to diagnose obstetric complications. This study aimed to identify the features of lipid composition of the cervical canal secretion peculiar to Intrauterine Growth Restriction (IUGR) and preeclampsia (PE). We took CVF samples from 57 pregnant women and subjected them to an in-depth clinical-anamnestic and mass-spectrometric analysis. Lipid extracts of CVF were analyzed with a liquid chromatography system coupled with a mass analyzer. As a result, we identified 239 lipid compounds. In case of 17 lipids, mathematical analysis revealed significant differences between samples from women with normal pregnancy indicator values (normal group) and patients from the IUGR group (p < 0.05). As for the normal group and PE group patients, there were significant differences identified for 3 lipids (p < 0.05). Comparison of samples from the PE and IUGR groups yielded statistically significant differences in levels of two lipids (p < 0.05). Mainly, the lipids were oxylipins, sphingomyelins, triglycerides, and cardiolipins. The developed diagnostic model had the sensitivity of 0.81 and specificity of 0.91 (cut-off level — 0.50; AUC — 0.85). The data obtained are valuable in the context of development of the new methods of diagnosing placentaassociated complications of pregnancy and for understanding new mechanisms of pathogenesis of these complications.

Complement activation fragments in cervicovaginal fluid are associated with intra-amniotic infection/inflammation and spontaneous preterm birth in women with preterm premature rupture of membranes

Objective: We sought to determine whether the levels of complement and other inflammatory and angiogenic mediators in cervicovaginal fluid (CVF) are independently associated with intra-amniotic infection and/or inflammation (IAI) and imminent spontaneous preterm birth (SPTB, ≤48 hours of sampling) in women with preterm premature rupture of membranes (PPROM). Study design: This was a retrospective study consisting of 85 singleton pregnant women with PPROM at 20+0 to 33+6 weeks. Amniotic fluid (AF) obtained via amniocentesis was cultured and assayed for interleukin-6. CVF samples collected at the time of amniocentesis were assayed for complement C3a, C4a, and C5a, HSP70, M-CSF, M-CSF-R, S100 A8, S100 A9, thrombospondin-2, VEGF, and VEGFR-1 by ELISA. Results: Multivariate logistic regression analyses revealed that elevated CVF concentrations of complement C3a, 4a and 5a were significantly associated with an increased risk of IAI and imminent SPTB, whereas those of M-CSF were associated with IAI, but not imminent SPTB (P=0.063), after adjustment for baseline covariates (e.g., gestational age at sampling). However, univariate and multivariate analyses showed that the CVF concentrations of angiogenic (thrombospondin-2, VEGF, and VEGFR-1) and inflammatory (HSP70, M-CSF-R, S100 A8, and S100 A9) proteins were not associated with either IAI or imminent SPTB. Conclusions: In women with PPROM, elevated CVF concentrations of complement C3a, C4a, and C5a are independently related to an increased risk of IAI and imminent SPTB. These findings suggest that complement activation in CVF is significantly involved in mechanisms underlying preterm birth and in the host response to IAI in the context of PPROM.

Prediction of Spontaneous Preterm Birth in At-risk Women Using Thrombospondin 1 from Cervicovaginal Fluid: A Prospective Observational Study

Introduction Thrombospondin 1, desmoplakin and stratifin are putative biomarkers for the prediction of preterm birth. This study aimed to validate the predictive capability of these biomarkers in patients at risk of preterm birth. Materials and Methods We included 109 women with symptoms of threatened spontaneous preterm birth between weeks 20 0/7 and 31 6/7 of gestation. Inclusion criteria were uterine contractions, cervical length of less than 25 mm, or a personal history of spontaneous preterm birth. Multiple gestations were also included. Samples of cervicovaginal fluid were taken before performing a digital examination and transvaginal ultrasound. Levels of cervicovaginal thrombospondin 1, desmoplakin and stratifin were quantified by enzyme-linked immunosorbent assays. The primary endpoint was spontaneous preterm birth before 34 + 0 weeks of gestation. Results Sixteen women (14.7%) delivered before 34 + 0 weeks. Median levels of thrombospondin 1 were higher in samples where birth occurred before 34 weeks vs. ≥ 34 weeks of gestation (4904 vs. 469 pg/mL, p < 0.001). Receiver operator characteristics analysis resulted in an area under the curve of 0.86 (p < 0.0001). At an optimal cut-off value of 2163 pg/mL, sensitivity, specificity, positive predictive value and negative predictive value were 0.94, 0.77, 0.42 and 0.99, respectively, with an adjusted odds ratio of 32.9 (95% CI: 3.1 – 345, p = 0.004). Multiple gestation, cervical length, and preterm labor had no impact on the results. Survival analysis revealed a predictive period of more than eight weeks. Levels of desmoplakin and stratifin did not differ between groups. Conclusion Thrombospondin 1 allowed long-term risk estimation of spontaneous preterm birth.

A Combination of Cervicovaginal Fluid Glutamate, Acetate and D-Lactate Identified Asymptomatic Low-Risk Women Destined to Deliver Preterm: a Prospective Cohort Study

Due to the modest predictive capacities and limited clinical application of transvaginal ultrasonographic cervical length (CL) and quantitative fetal fibronectin (qfFN) in pregnant women at low risk of preterm birth (PTB), we sought to determine the utility of cervicovaginal fluid (CVF) metabolites (by-products of host-microbial metabolism) for prediction of spontaneous PTB in asymptomatic low-risk women at mid-gestation. This was a prospective sub-cohort study from the ECCLIPPx study cohort. CVF from asymptomatic singleton women (20–22 weeks, n = 168) without a prior history of PTB were analysed for metabolites by enzyme-based spectrophotometry. CL, vaginal pH and qfFN were also measured. Correlation and predictive analyses were performed by Spearman’s correlation, and binary logistic regression and area under receiver operating characteristic curve (AUC), respectively. Of the 168 women enrolled, only CVF samples from 135 (80.4%) women were analysed. There were 6/135 (4.4%) spontaneous PTB (sPTBs), with two of these pregnancies ending ≤ 28 weeks’ gestation. Individually (AUC, 95% CI), only glutamate (0.72, 0.64–0.80) and CL (0.69, 0.60–0.77) were predictive of PTB. However, five multivariable models that more accurately predicted sPTB were also identified, i.e. a combination of: glutamate, acetate and D-lactate (GAD, 0.82, 0.74–0.89); CL and qfFN only (0.78, 0.70–0.85); CL, qfFN, glutamate and acetate (0.88, 0.81–0.93); CL, qfFN and GAD (0.94, 0.88–0.98); and GAD and pH (0.86, 0.79–0.92). Correlations between CL, pH and qfFN and metabolites were also observed. In this cohort, a midtrimester combination of CVF glutamate, acetate and D-lactate predicted preterm birth more accurately than individual metabolites, cervical length and fetal fibronectin with a very low false-positive rate and high positive predictive value. Further testing in populations with higher preterm birth rates is required.

Placental α-microglobulin-1 in cervicovaginal fluid and cervical length to predict preterm birth by Thai women with symptoms of labor

Background Presence of placental α microglobulin-1 (PAMG-1) in cervicovaginal fluid is a bedside test to predict preterm delivery. Objective To determine whether the accuracy of a positive PAMG-1 test result to predict preterm birth within 7 days and 14 days in our hospital setting can be improved by adding cervical length. Methods We recruited 180 pregnant women who attended the labor ward of Siriraj Hospital, Thailand, from 2016 to 2018 for this prospective observational study of diagnostic accuracy. We used data from 161 women who met inclusion criteria including symptoms of preterm labor between 200/7 and 366/7 weeks’ gestation without ruptured membranes and with cervical dilatation <3 cm and effacement <80%. Presence of PAMG-1 in cervicovaginal fluid was tested using a PartoSure kit, cervical length was measured by transvaginal ultrasound, and the time to spontaneous delivery was calculated. Results Pregnant women with labor pain who had cervical length <30 mm (45/161; 28%) went into delivery within 7 days, and women with a cervical length <15 mm (11/14; 79%) went into delivery within 7 days. When the PAMG-1 test result was positive and cervical length was ≤15 mm, the positive predictive value (PPV) was 83%; and when cervical length was ≤30 mm the PPV was 69%. The optimal cut off from receiver operating characteristic curve analysis showed that a cervical length <25 mm and PAMG-1 positive result has a PPV of 80% to predict preterm birth within 7 days and 90% within 14 days. The area under the curve (95% confidence interval) for a positive PAMG-1 result and cervical length ≤25 mm to predict preterm birth <7 days was 0.61 (0.50, 0.73) and <14 days was 0.60 (0.49, 0.70). Conclusions Cervical length ranging 15–30 mm combined with a positive PAMG-1 test result has a high accuracy to predict imminent spontaneous delivery within 7 days by women with preterm labor and cervical dilatation <3 cm in clinical practice.

Cervicovaginal COVID-19 Positivity: A Pilot Study

Purpose: ACE 2 RNA expression has been detected in organs of the female reproductive tract, suggesting that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could potentially infect female reproductive organs. In this study, we investigated the presence of SARS-CoV-2 virus in the cervicovaginal fluid.Materials and Methods: Our study included 31 female patients aged 18–65 years. The presence of SARS-COV-2 RNA was investigated by RT-PCR in two separate cervicovaginal swab samples collected from patients 14 days apart. Viral RNA was extracted using viral nucleic acid buffer (vNAT) solution, and SARS-COV-2 RNA was analyzed using Bio-speedy SARS-CoV-2 RT-qPCR kits in Bio-Rad CFX96 TouchTM device.Results: The first and second cervical swab samples were collected from 22 of 31 patients 14 days apart. The first cervical swab sample was collected from 9 patients; however, the second swab sample could not be collected after 14 days. SARS-COV-2 RNA result was negative in 100% of a total of 53 cervicovaginal swab samples collected. Moreover, the SARS-COV-2 RNA result was negative in the nasopharyngeal swab of babies after delivery in three pregnant women.Conclusion: Negative SARS-COV-2 RNA results in cervicovaginal swab samples suggest that there is no sexual transmission of COVID-19 and no vertical transmission during pregnancy. However, the number of studies conducted on this subject and the sample size examined are still insufficient.

Serum and Cervicovaginal Fluid Antibody Profiling in Herpes Simplex Virus (HSV) Seronegative Recipients of the HSV529 Vaccine

Previous HSV2 vaccines have not prevented genital herpes. Concerns have been raised about the choice of antigen, the type of antibody induced by the vaccine, and whether antibody is present in the genital tract where infection occurs. We reported results of a trial of an HSV2 replication-defective vaccine, HSV529, that induced serum neutralizing antibody responses in 78% of HSV1 -/HSV2 - vaccine recipients. Here we show that HSV1 -/HSV2 - vaccine recipients developed antibodies to epitopes of several viral proteins; however, fewer antibody epitopes were detected in vaccine recipients compared with naturally infected persons. HSV529 induced antibodies that mediated HSV2-specific NK cell activation. Depletion of gD-binding antibody from sera reduced neutralizing titers by 62% and NK cell activation by 81%. HSV2 gD antibody was detected in cervicovaginal fluid at about one-third the level of that in serum. A vaccine that induces potent serum antibodies transported to the genital tract might reduce HSV genital infection.