Predictive potential of various plasma inflammation-, angiogenesis-, and extracellular matrix remodeling-associated mediators for intra-amniotic inflammation and/or microbial invasion of the amniotic cavity in preterm labor.

PURPOSE: To determine whether various inflammatory-, angiogenic/anti-angiogenic-, and extracellular matrix remodeling-associated proteins in plasma, alone or in combination with conventional blood-based markers, can predict intra-amniotic inflammation and/or microbial invasion of the amniotic cavity (IAI/MIAC) in women with spontaneous preterm labor (PTL). METHODS: A total of 193 singleton pregnant women with PTL (23-33 weeks) were included in this retrospective cohort study. Plasma samples were obtained at the time of amniocentesis. Amniotic fluid (AF) was cultured for microorganism detection and consequent MIAC diagnosis. IL-6 levels were determined in AF and used to identify IAI (AF IL-6 ≥ 2.6 ng/mL). Endostatin, haptoglobin, IGFBP-2/3, LBP, M-CSF, MMP-2/8, pentraxin 3, PlGF, S100A8/A9, and VEGFR-1 levels were assayed in plasma samples by ELISA. CRP levels and neutrophil-to-lymphocyte ratio (NLR) were measured. RESULTS: Plasma LBP, MMP-8, and S100A8/A9 levels, CRP levels, and NLR were significantly higher, and plasma IGFBP-2 and MMP-2 levels were significantly lower in women with IAI/MIAC than in those without this condition, whereas no baseline variables differed significantly between the two groups. Using a stepwise regression analysis, a noninvasive prediction model for IAI/MIAC was developed, which included plasma LBP, MMP-2, and MMP-8 levels (area under the curve [AUC], 0.785). The AUC for this prediction model was significantly or borderline greater than that of any single factor included in the model. CONCLUSIONS: IGFBP-2, LBP, MMP-2, MMP-8, and S100A8/A9 may represent valuable plasma biomarkers for predicting IAI/MIAC in women with PTL. Combination of LBP, MMP-2, and MMP-8 expression data can significantly improve the predictive potential for IAI/MIAC.

Whole-genome sequencing-based phylogeny, antibiotic resistance, and invasive phenotype of Escherichia coli strains colonizing the cervix of women in preterm labor.

BACKGROUND: Escherichia coli is a major neonatal pathogen and the leading cause of early-onset sepsis in preterm newborns. Maternal E. coli strains are transmitted to the newborn causing invasive neonatal disease. However, there is a lack of data regarding the phenotypic and genotypic characterization of E. coli strains colonizing pregnant women during labor. METHODS: This prospective study performed at the University of Oklahoma Medical Center (OUHSC) from March 2014 to December 2015, aimed to investigate the colonization rate, and the phylogeny, antibiotic resistance traits, and invasive properties of E. coli strains colonizing the cervix of fifty pregnant women diagnosed with preterm labor (PTL). Molecular analyses including bacterial whole-genome sequencing (WGS), were performed to examine phylogenetic relationships among the colonizing strains and compare them with WGS data of representative invasive neonatal E. coli isolates. Phenotypic and genotypic antibiotic resistance traits were investigated. The bacteria's ability to invade epithelial cells in vitro was determined. RESULTS: We recruited fifty women in PTL. Cervical samples yielded E. coli in 12 % (n=6). The mean gestational age was 32.5 (SD±3.19) weeks. None delivered an infant with E. coli disease. Phenotypic and genotypic antibiotic resistance testing did not overall demonstrate extensive drug resistance traits among the cervical E. coli isolates, however, one isolate was multi-drug resistant. The isolates belonged to five different phylogroups, and WGS analyses assigned each to individual multi-locus sequence types. Single nucleotide polymorphism-based comparisons of cervical E. coli strains with six representative neonatal E. coli bacteremia isolates demonstrated that only half of the cervical E. coli isolates were phylogenetically related to these neonatal invasive strains. Moreover, WGS comparisons showed that each cervical E. coli isolate had distinct genomic regions that were not shared with neonatal E. coli isolates. Cervical and neonatal E. coli isolates that were most closely related at the phylogenetic level had similar invasion capacity into intestinal epithelial cells. In contrast, phylogenetically dissimilar cervical E. coli strains were the least invasive among all isolates. CONCLUSIONS: This pilot study showed that a minority of women in PTL were colonized in the cervix with E. coli, and colonizing strains were not phylogenetically uniformly representative of E. coli strains that commonly cause invasive disease in newborns. Larger studies are needed to determine the molecular characteristics of E. coli strains colonizing pregnant women associated with an increased risk of neonatal septicemia.

Relationship between histologic chorioamnionitis and genital tract cultures in pre term labour.

The objective was to determine the relationship of histological chorioamnionitis (HCA) with genital tract cultures in preterm birth. Among two hundred women recruited for the study, 100 were taken as cases with gestational age between ≥28 and <37 weeks and 100 women with gestational age >37 weeks were taken as controls. Vaginal swabs were taken for culture sensitivity and vaginal smears were made for performing whiff test and heat dry gram stained smear was examined for growth of microorganisms. Histopathologic examination of the placenta was done after delivery. 49 cases and 26 controls had evidence of histological chorioamnionitis. A significant difference was observed in relation to the presence of E. coli, presence of clue cells, positive whiff test and occurrence of bacterial vaginosis in subjects with and without histological chorioamnionitis. Thus, we conclude that the presence of histological chorioamnionitis is closely related to the presence of pathogenic microorganisms in the cervicovaginal region.IMPACT STATEMENTWhat is already known on the subject? Histologic chorioamnionitis has been regarded to reflect amniotic fluid infection and there are studies showing an association between histologic chorioamnionitis, amniotic fluid, and subchorionic plate cultures. Nevertheless, studies of the correlation of the cervical swab cultures with intrauterine infection in preterm birth remain inconclusive.What do the results of this study add? Histologic chorioamnionitis is closely related to the presence of pathogenic microorganisms in the cervicovaginal region.What are the implications of these findings for clinical practice and/or further research? High vaginal swab cultures and gram staining of vaginal smear is useful in detecting antenatal patients who are at a higher risk for preterm labour. After detection, early intervention may be done to avoid preterm deliveries in these high-risk pregnancies.

Characterization of vaginal microbiota in women with preterm labor with intra-amniotic inflammation.

This study aims to investigate the relation between vaginal microbiota and exposition to intra-amniotic inflammation (IAI). We conducted a prospective cohort study in women with preterm labor <34 weeks who had undergone amniocentesis to rule out IAI. Vaginal samples were collected after amniocentesis. Women with IAI included those with positive amniotic fluid (AF) for a microorganism identified by specific culture media and Sanger sequencing 16S ribosomal RNA gene and/or high AF interleukin (IL)-6 levels. Vaginal microbiota was characterized by 16S ribosomal RNA gene amplicon sequencing. Specific quantitative PCR targeted to Lactobacillus spp. was also performed. Regression models were used to evaluate associations between vaginal microbiota and exposition to IAI. Concerning our results, 64 women were included. We observed an inverse association between AF IL-6 levels and load of Lactobacillus spp. Depletion in Lactobacillus spp. load was significantly associated with an early gestational age at delivery and a short latency to delivery. Microbial-diversity was found to be a risk factor for the subsequent occurrence of clinical chorioamnionitis. To the contrary, higher Lactobacillus spp. load had a protective role. In conclusion, the study identifies reduced bacterial load of Lactobacillus spp. in women exposed to IAI and found microbial-diversity and Lactobacillus spp. depletion to be associated with a worse perinatal outcome.

Chlamydia trachomatis screening in preterm labor: A systematic review and meta-analysis.

OBJECTIVE: Spontaneous preterm labor (PTL) is responsible for approximately half of all preterm births with intrauterine infection being an important risk factor for PTL. Chlamydia trachomatis infections have been associated with preterm prelabor rupture of membranes (P-PROM) and preterm birth, but its impact on PTL has not previously been specified. The aim of this study was to evaluate the overall prevalence of Chlamydia trachomatis infections in pregnant women with threatened PTL compared to those not in threatened PTL. STUDY DESIGN: A literature search was performed in electronic databases using combinations of: "Chlamydia", "vaginal cervical infection" and "preterm labor." Cohort and case-controlled studies examining threatened PTL and Chlamydia trachomatis infection demonstrated by culture or NAAT methods at time of diagnosis of threatened labor. The Meta-analyses of Observational Studies in Epidemiology (MOOSE) guidelines for reporting of observational studies for systematic reviews was used. Bias was assessed with the Methodological Index for Non-Randomized Studies (MINORS) score. Meta-analysis was performed using a random effects model. RESULTS: Four studies were identified. A total of 591 women were included, 309 in the threatened PTL, and 282 controls not in threatened PTL. Women presenting in PTL had an increased risk of screening positive for Chlamydia trachomatis compared to the control group (27/308 (9%) vs 3/282 (1%); OR 7.74, 95% CI 2.64-22.71). CONCLUSIONS: The incidence of Chlamydia trachomatis in women with threatened PTL is approximately 9%, and significantly increased compared to asymptomatic controls. Women with threatened PTL should be considered for screening for Chlamydia trachomatis.

Detection of urinary Chlamydia trachomatis, Mycoplasma genitalium and human papilloma virus in the first trimester of pregnancy by PCR method.

BACKGROUND: Miscarriage and preterm delivery are the most important challenges of pregnancy. Different bacterial and viral infection may cause miscarriage and preterm delivery. Among bacterial factors, Mycoplasma genitalium and Chlamydia trachomatis have the most important role and human papilloma virus (HPV) is the leading viral factor in this regard. METHODS: First void urine samples were collected from 119 pregnant women who visited health centers for routine first-trimester screening (12-14 weeks gestation). About 10 ml of the sample was centrifuged at 3000×g for 20 min and 1-2 ml of the sediment was transferred to sterile microfuges and stored at - 20 °C until analysis. DNA extraction was conducted using A101211 kits imported by Pars Tous Biotechnology Company. The following commercial kits, imported by Pars Tous Biotechnology, were used for PCR. RESULTS: There is no significant association between urinary isolation of C. trachomatis and miscarriage (P = 0.93) and there is no significant association between urinary isolation of M. genitalium and miscarriage (P = 0.80). Regarding HPV, since all urine samples were PCR-negative, comparison was not possible. C. trachomatis was isolated from the urine samples of 6.72% of the pregnant women who underwent first-trimester screening in health centers using PCR. Previous studies reported a mean chlamydia isolation rate of 3% from urine specimens collected from pregnant women in general. T test showed no significant difference between the two groups (P = 0.10). Based on present study the mycoplasma isolation rate was 17.65% using PCR. Previous studies reported a mean mycoplasma isolation rate of 10% from urine specimens collected from pregnant women in general. T-test showed a significant difference between the two groups (P = 0.03). DISCUSSION: First void urine samples in pregnant women may be an appropriate sample for detection of C. trachomatis and M. genitalium; however, it is not a good method for HPV isolation therefore vaginal or cervical discharge specimens should be used instead for detection of HPV.

Plasma inflammatory and immune proteins as predictors of intra-amniotic infection and spontaneous preterm delivery in women with preterm labor: a retrospective study.

BACKGROUND: We investigated whether various inflammatory and immune proteins in plasma predict intra-amniotic infection and imminent preterm delivery in women with preterm labor and compared their predictive ability with that of amniotic fluid (AF) interleukin (IL)-6 and serum C-reactive protein (CRP). METHODS: This retrospective cohort study included 173 consecutive women with preterm labor who underwent amniocentesis for diagnosis of infection and/or inflammation in the AF. The AF was cultured, and assayed for IL-6. CRP levels and cervical length by transvaginal ultrasound were measured at the time of amniocentesis. The stored maternal plasma was assayed for IL-6, matrix metalloproteinase (MMP)-9, and complements C3a and C5a using ELISA kits. The primary and secondary outcome criteria were positive AF cultures and spontaneous preterm delivery (SPTD) within 48 h, respectively. Univariate, multivariate, and receiver operating characteristic analysis were used for the statistical analysis. RESULTS: In bivariate analyses, elevated plasma IL-6 level was significantly associated with intra-amniotic infection and imminent preterm delivery, whereas elevated plasma levels of MMP-9, C3a, and C5a were not associated with these two outcomes. On multivariate analyses, an elevated plasma IL-6 level was significantly associated with intra-amniotic infection and imminent preterm delivery after adjusting for confounders, including high serum CRP levels and short cervical length. In predicting intra-amniotic infection, the area under the curve (AUC) was significantly lower for plasma IL-6 than for AF IL-6 but was similar to that for serum CRP. Differences in the AUCs between plasma IL-6, AF IL-6, and serum CRP were not statistically significant in predicting imminent preterm delivery. CONCLUSIONS: Maternal plasma IL-6 independently predicts intra-amniotic infection in women with preterm labor; however, it has worse diagnostic performance than that of AF IL-6 and similar performance to that of serum CRP. To predict imminent preterm delivery, plasma IL-6 had an overall diagnostic performance similar to that of AF IL-6 and serum CRP. Plasma MMP-9, C3a, and C5a levels could not predict intra-amniotic infection or imminent preterm delivery.

The prevalence of abnormal vaginal flora and predictive factors for intrauterine infection in pregnant Korean women with preterm labor.

OBJECTIVE: To investigate the prevalence of abnormal vaginal flora (AVF) and predictive factors for intrauterine infection in pregnant Korean women with preterm labor. MATERIALS AND METHODS: The authors reviewed the medical records of 106 pregnant Korean women with preterm labor admitted to Eulji Medical Center between January 2006 and August 2011. The results of vaginal discharge tests and maternal serum C-reactive protein (CRP) level at admission, placental biopsy, and perinatal outcomes were searched. The prevalence of abnormal vaginal flora was calculated. The perinatal outcomes and predictive factors for intrauterine infections were analyzed based on placental pathology and early-onset neonatal sepsis. RESULTS: The prevalence of abnormal vaginal flora was 75.4%. Ureaplasma urealyticunz (UU), intermediate flora, Candidiasis, bacterial vaginosis, and aerobic bacterial colonization were detected in 40.6%, 38.7%, 17%, 14.2%, and 11.3% of the women, respectively. The frequency of early-onset neonatal sepsis was significantly different between women with aerobic bacterial colonization and those with normal flora (p = 0.008). An elevated maternal serum CRP level was an independent intrauterine infection predictor (odds ratio, 1.918; 95% confidence interval, 1.102-3.338; p = 0.048). CONCLUSION: Aerobic bacterial colonization may predict early-onset neonatal sepsis. An elevated maternal serum CRP level was an independent intrauterine infection predictor based on placental infections and early-onset neonatal sepsis.

Preterm labor and neonatal sepsis caused by intrauterine Helicobacter cinaedi infection.

Helicobacter cinaedi is a rare pathogen but known to cause bacteremia, cellulitis and enterocolitis. Recently, cases of involving various organs are increasingly reported such as endocarditis, meningitis, and kidney cyst infection. We report a case of intrauterine H. cinaedi infection leading preterm birth and neonatal sepsis. A 29-year-old pregnant women who was no underlying disease hospitalized due to threatened preterm labor at 22 weeks of gestation. Clinical findings showed uterine tenderness, fever, leukocytosis and elevated C-reactive protein. H. cinaedi was isolated from amniotic fluid obtained by transabdominal amniocentesis. We diagnosed as intrauterine H. cinaedi infection and administered intravenous ampicillin followed by oxytocin to terminate pregnancy. A live 446 g male infant was delivered. The patient was no signs of infection throughout postpartum course and discharged on post-delivery day 5. The neonate was admitted in neonatal intensive care unit and administered ampicillin and amikacin. H. cinaedi was isolated from umbilical cord blood culture. He has no signs of infection on day 5 but died from uncontrollable hyperglycemia and ketoacidosis on 15 days of age. H. cinaedi can cause intrauterine infection during pregnancy and lead preterm labor and neonatal sepsis.

Maternal and fetal roles in bacterially induced preterm labor in the mouse.

BACKGROUND: The relative roles of the mother and fetus in signaling for labor remain poorly understood. OBJECTIVE: We previously demonstrated using gene knockout (KO) mice that Escherichia coli-induced preterm delivery is completely dependent on MyD88, a toll-like receptor adaptor protein. Here we leveraged this finding to conduct a genetic experiment testing whether the mother, the fetus, or both signal for parturition in bacterially induced labor. STUDY DESIGN: Six different maternal/fetal genotype combinations for MyD88 were studied: wild-type (WT) dams carrying one of the following: (1) WT or (2) MyD88 heterozygous (het) fetuses (generated by mating WT females with WT or MyD88-knockout [KO] males, respectively); (3) WT dams carrying MyD88-KO fetuses (generated by replacing the ovaries of WT females with MyD88-KO ovaries, followed by mating with MyD88-KO males); a similar strategy was used to generate MyD88-KO dams carrying (4) MyD88-KO, (5) MyD88 het, or (6) WT fetuses. On day 14.5 of gestation, mice received intrauterine injections of either 1 × 10(9) killed E coli or sterile medium. Delivery of ≥ 1 fetus within 48 hours was considered preterm. A separate group of similarly treated pregnant mice was euthanized 5 hours after surgery for gene expression and tissue analysis. RESULTS: E coli-induced preterm delivery is dependent on maternal and not fetal genotype: > 95% of WT and < 5% of MyD88-KO dams deliver prematurely, regardless of fetal genotype (P = .0001). In contrast, fetal survival in utero is influenced by fetal genotype: in MyD88-KO dams, in which premature birth rarely occurs, only 81% of WT and 86% of MyD88-heterozygous fetuses were alive 48 hours after surgery compared with 100% of MyD88-KO fetuses (P < .01). Messenger ribonucleic acids for the inflammatory mediators interleukin-1ß, tumor necrosis factor, interleukin-6, and cyclooxygenase-2 were elevated in uterine tissues only in WT mothers treated with E coli and were low or undetectable in the uteri of KO mothers or in animals treated with saline. Serum progesterone levels were lower in KO mothers with WT ovaries than in WT mothers with KO ovaries, but bacterial exposure did not have an impact on these levels. CONCLUSION: In the murine E coli-induced labor model, preterm delivery and uterine expression of inflammatory mediators is determined by the mother and not the fetus and is not attributable to a decline in serum progesterone.

Depletion of polymorphonuclear leukocytes has no effect on preterm delivery in a mouse model of Escherichia coli-induced labor.

OBJECTIVE: The objective of the study was to investigate the role of polymorphonuclear leukocytes (PMNs) in a mouse model of Escherichia coli-induced labor. STUDY DESIGN: Intraperitoneal injection of rabbit antimouse PMN antiserum or control was performed in CD-1 mice 29 hours and 5 hours prior to laparotomy and intrauterine injection of either killed E coli or phosphate-buffered saline on day 14.5 of pregnancy. Preterm delivery was defined as delivery of at least 1 pup within 48 hours. Circulating leukocyte counts were determined manually or by flow cytometry at the time of surgery and 8, 24, and 48 hours afterward. Maternal and fetal tissues were analyzed in a separate group of animals 8 hours after surgery. RESULTS: Pretreatment with anti-PMN antiserum significantly decreased the numbers of circulating leukocytes and the proportion of neutrophils among all leukocytes by 70-80% at surgery and at least 8 hours thereafter. Neutrophil depletion significantly reduced 2 markers of neutrophil activation in the uterus and placenta (neutrophil elastase and myeloperoxidase activity) and neutrophil infiltration into gestational tissues in bacterially treated animals to baseline (control) levels but did not affect preterm birth rates. The large E coli-induced increases in uterine inflammatory markers (interleukin-1ß, tumor necrosis factor, chemokine ligand-5, cyclooxygenase-2) were not affected or were only minimally affected by neutrophil depletion. CONCLUSION: Although PMN antiserum reduces both neutrophil number and activity, it does not diminish sensitivity to bacterially induced delivery or meaningfully alter the expression of inflammatory markers in the mouse model. Preterm birth and inflammation in this model are not likely to depend on neutrophil function.

Cervical microbiota in women with preterm prelabor rupture of membranes.

OBJECTIVE: To analyze the cervical microbiota in women with preterm prelabor rupture of membranes (PPROM) by pyrosequencing and to document associations between cervical microbiota, cervical inflammatory response, microbial invasion of the amniotic cavity (MIAC), histological chorioamnionitis, and intraamniotic infection (IAI). STUDY DESIGN: Sixty-one women with singleton pregnancies complicated by PPROM were included in the study. Specimens of cervical and amniotic fluid were collected on admission. The cervical microbiota was assessed by 16S rRNA gene sequencing by pyrosequencing. Interleukin (IL)-6 concentration in the cervical fluid and amniotic fluid was measured by ELISA and lateral flow immunoassay, respectively. RESULTS: Four bacterial community state types [CST I (Lactobacillus crispatus dominated), CST III (Lactobacillus iners dominated), CST IV-A (non-Lactobacillus bacteria dominated), and CST IV-B (Gardnerella vaginalis and Sneathia sanguinegens dominated)] were observed in the cervical microbiota of women with PPROM. Cervical fluid IL-6 concentrations differed between CSTs (CST I = 145 pg/mL, CST III = 166 pg/mL, CST IV-A = 420 pg/mL, and CST IV-B = 322 pg/mL; p = 0.004). There were also differences in the rates of MIAC, of both MIAC and histological chorioamnionitis, and of IAI among CSTs. No difference in the rate of histological chorioamnionitis was found among CSTs. CONCLUSIONS: The cervical microbiota in PPROM women in this study was characterized by four CSTs. The presence of non-Lactobacillus CSTs was associated with a strong cervical inflammatory response and higher rates of MIAC, both MIAC and histological chorioamnionitis, and IAI representing a PPROM subtype with pronounced inflammation. CST I represents the dominant type of PPROM with a low rate of MIAC, IAI, and the combination of MIAC and histological chorioamnionitis.

Non-invasive prediction of intra-amniotic infection and/or inflammation in patients with cervical insufficiency or an asymptomatic short cervix (≤15 mm).

PURPOSE: To identify non-invasive parameters to predict intra-amniotic infection and/or inflammation (IAI) in patients with cervical insufficiency or an asymptomatic short cervix (≤15 mm). METHODS: This retrospective cohort study included 72 asymptomatic women with cervical insufficiency (n = 54) or an asymptomatic short cervix (n = 18) at 17-28 weeks. Maternal blood was collected for the determination of the C-reactive protein (CRP) level and white blood cell (WBC) count, and sonography was performed to measure the cervical length shortly after amniocentesis. Amniotic fluid (AF) was cultured and interleukin-6 (IL-6) level and WBC count were determined. RESULTS: The prevalence of intra-amniotic inflammation and a positive AF culture was 22.2 % (16/72) and 8.3 % (6/72), respectively. The best cut-off value for AF IL-6 in predicting the presence of intra-amniotic infection was ≥7.6 ng/mL and was used to diagnose the presence of intra-amniotic inflammation. Women with intra-amniotic inflammation, regardless of culture results, were at increased risk for preterm delivery and adverse outcomes compared to women without intra-amniotic inflammation. In multivariable regression, CRP was the only non-invasive variable statistically significantly associated with IAI. Moreover, the area under the curves for the CRP and AF WBC were not significantly different. CONCLUSIONS: In women with cervical insufficiency or a short cervix, the risk for IAI can be predicted fairly and non-invasively by measurements of serum CRP. Overall, this non-invasive parameter appears to have similar accuracy to the AF WBC counts for predicting IAI.

Detection of microbial invasion of the amniotic cavity by analysis of cervicovaginal proteins in women with preterm labor and intact membranes.

OBJECTIVE: Microbial invasion of the amniotic cavity (MIAC) is common in early preterm labor and is associated with maternal and neonatal infectious morbidity. MIAC is usually occult and is reliably detected only with amniocentesis. We sought to develop a noninvasive test to predict MIAC based on protein biomarkers in cervicovaginal fluid (CVF) in a cohort of women with preterm labor (phase 1) and to validate the test in an independent cohort (phase 2). STUDY DESIGN: This was a prospective study of women with preterm labor who had amniocentesis to screen for MIAC. MIAC was defined by positive culture and/or 16S ribosomal DNA results. Nine candidate CVF proteins were analyzed by enzyme-linked immunosorbent assay. Logistic regression was used to identify combinations of up to 3 proteins that could accurately classify the phase 1 cohort (N = 108) into those with or without MIAC. The best models, selected by area under the curve (AUC) of the receiver operating characteristic curve in phase 1, included various combinations of interleukin (IL)-6, chemokine (C-X-C motif) ligand 1 (CXCL1), alpha fetoprotein, and insulin-like growth factor binding protein-1. Model performance was then tested in the phase 2 cohort (N = 306). RESULTS: MIAC was present in 15% of cases in phase 1 and 9% in phase 2. A 3-marker CVF model using IL-6 plus CXCL1 plus insulin-like growth factor binding protein-1 had AUC 0.87 in phase 1 and 0.78 in phase 2. Two-marker models using IL-6 plus CXCL1 or alpha fetoprotein plus CXCL1 performed similarly in phase 2 (AUC 0.78 and 0.75, respectively), but were not superior to CVF IL-6 alone (AUC 0.80). A cutoff value of CVF IL-6 ≥463 pg/mL (which had 81% sensitivity in phase 1) predicted MIAC in phase 2 with sensitivity 79%, specificity 78%, positive predictive value 38%, and negative predictive value 97%. CONCLUSION: High levels of IL-6 in CVF are strongly associated with MIAC. If developed into a bedside test or rapid laboratory assay, cervicovaginal IL-6 might be useful in selecting patients in whom the probability of MIAC is high enough to warrant amniocentesis or transfer to a higher level of care. Such a test might also guide selection of potential subjects for treatment trials.

Systemic and local inflammatory response in women with preterm prelabor rupture of membranes.

OBJECTIVE: To evaluate the inflammatory pattern in maternal circulation, amniotic cavity, cervix and vagina from women with preterm prelabor rupture of membranes (PPROM) considering the occurrence of microbial invasion of the amniotic cavity (MIAC). METHODOLOGY: A prospective study was performed in 58 women with PPROM before 34+0 weeks of gestational age. Twenty-six proteins were analyzed by a multiple immunoassay in samples of amniotic fluid, serum, cervix and vagina. Association of an inflammatory response in the invasive and non-invasive samples with MIAC was investigated. RESULTS: The rate of MIAC was 36.2% (21/58). Both amniotic fluid IL-6 and cervical C-reactive protein (CRP) showed to be independent predictors of MIAC. A cut-off level of cervical CRP≥1836 pg/mL showed a detection rate of 75%, false positive rate of 19% and positive and negative predictive values to predict MIAC of 67% and 87%, respectively. There were no independent biomarkers of MIAC either in the serum or vaginal compartment. CONCLUSION: A cervical inflammatory response mediated by CRP was observed in PPROM women with MIAC. Evaluation of serum or vaginal samples did not add valuable information regarding the outcome evaluated.

A novel molecular microbiologic technique for the rapid diagnosis of microbial invasion of the amniotic cavity and intra-amniotic infection in preterm labor with intact membranes.

PROBLEM: The diagnosis of microbial invasion of the amniotic cavity (MIAC) has been traditionally performed using traditional cultivation techniques, which require growth of microorganisms in the laboratory. Shortcomings of culture methods include the time required (days) for identification of microorganisms, and that many microbes involved in the genesis of human diseases are difficult to culture. A novel technique combines broad-range real-time polymerase chain reaction with electrospray ionization time-of-flight mass spectrometry (PCR/ESI-MS) to identify and quantify genomic material from bacteria and viruses. METHOD OF STUDY: AF samples obtained by transabdominal amniocentesis from 142 women with preterm labor and intact membranes (PTL) were analyzed using cultivation techniques (aerobic, anaerobic, and genital mycoplasmas) as well as PCR/ESI-MS. The prevalence and relative magnitude of intra-amniotic inflammation [AF interleukin 6 (IL-6) concentration ≥ 2.6 ng/mL], acute histologic chorioamnionitis, spontaneous preterm delivery, and perinatal mortality were examined. RESULTS: (i) The prevalence of MIAC in patients with PTL was 7% using standard cultivation techniques and 12% using PCR/ESI-MS; (ii) seven of ten patients with positive AF culture also had positive PCR/ESI-MS [≥17 genome equivalents per PCR reaction well (GE/well)]; (iii) patients with positive PCR/ESI-MS (≥17 GE/well) and negative AF cultures had significantly higher rates of intra-amniotic inflammation and acute histologic chorioamnionitis, a shorter interval to delivery [median (interquartile range-IQR)], and offspring at higher risk of perinatal mortality, than women with both tests negative [90% (9/10) versus 32% (39/122) OR: 5.6; 95% CI: 1.4-22; (P < 0.001); 70% (7/10) versus 35% (39/112); (P = 0.04); 1 (IQR: <1-2) days versus 25 (IQR: 5-51) days; (P = 0.002), respectively]; (iv) there were no significant differences in these outcomes between patients with positive PCR/ESI-MS (≥17 GE/well) who had negative AF cultures and those with positive AF cultures; and (v) PCR/ESI-MS detected genomic material from viruses in two patients (1.4%). CONCLUSION: (i) Rapid diagnosis of intra-amniotic infection is possible using PCR/ESI-MS; (ii) the combined use of biomarkers of inflammation and PCR/ESI-MS allows for the identification of specific bacteria and viruses in women with preterm labor and intra-amniotic infection; and (iii) this approach may allow for administration of timely and specific interventions to reduce morbidity attributed to infection-induced preterm birth.

Regulation of apoptosis and innate immune stimuli in inflammation-induced preterm labor.

An innate immune response is required for successful implantation and placentation. This is regulated, in part, by the a2 isoform of V-ATPase (a2V) and the concurrent infiltration of M1 (inflammatory) and M2 (anti-inflammatory) macrophages to the uterus and placenta. The objective of the present study was to identify the role of a2V during inflammation-induced preterm labor in mice and its relationship to the regulation of apoptosis and innate immune responses. Using a mouse model of infection-induced preterm delivery, gestational tissues were collected 8 h after intrauterine inoculation on day 14.5 of pregnancy with either saline or peptidoglycan (PGN; a TLR 2 agonist) and polyinosinic-polycytidylic acid [poly(I:C); a TLR3 agonist], modeling Gram-positive bacterial and viral infections, respectively. Expression of a2V decreased significantly in the placenta, uterus, and fetal membranes during PGN+poly(I:C)-induced preterm labor. Expression of inducible NO synthase was significantly upregulated in PGN+poly(I:C)-treated placenta and uterus. PGN+poly(I:C) treatment disturbed adherens junction proteins and increased apoptotic cell death via an extrinsic pathway of apoptosis among uterine decidual cells and spongiotrophoblasts. F4/80(+) macrophages were increased and polarization was skewed in PGN+poly(I:C)-treated uterus toward double-positive CD11c(+) (M1) and CD206(+) (M2) cells, which are critical for the clearance of dying cells and rapid resolution of inflammation. Expression of Nlrp3 and activation of caspase-1 were increased in PGN+poly(I:C)-treated uterus, which could induce pyroptosis. These results suggest that the double hit of PGN+poly(I:C) induces preterm labor via reduction of a2V expression and simultaneous activation of apoptosis and inflammatory processes.

A hemolytic pigment of Group B Streptococcus allows bacterial penetration of human placenta.

Microbial infection of the amniotic fluid is a significant cause of fetal injury, preterm birth, and newborn infections. Group B Streptococcus (GBS) is an important human bacterial pathogen associated with preterm birth, fetal injury, and neonatal mortality. Although GBS has been isolated from amniotic fluid of women in preterm labor, mechanisms of in utero infection remain unknown. Previous studies indicated that GBS are unable to invade human amniotic epithelial cells (hAECs), which represent the last barrier to the amniotic cavity and fetus. We show that GBS invades hAECs and strains lacking the hemolysin repressor CovR/S accelerate amniotic barrier failure and penetrate chorioamniotic membranes in a hemolysin-dependent manner. Clinical GBS isolates obtained from women in preterm labor are hyperhemolytic and some are associated with covR/S mutations. We demonstrate for the first time that hemolytic and cytolytic activity of GBS is due to the ornithine rhamnolipid pigment and not due to a pore-forming protein toxin. Our studies emphasize the importance of the hemolytic GBS pigment in ascending infection and fetal injury.

The anti-phospholipid antibody-dependent and independent effects of periodontopathic bacteria on threatened preterm labor and preterm birth.

PURPOSE: Periodontal disease is considered to be a risk factor for threatened preterm labor (TPL) and preterm birth (PB), but pathogenic mechanisms have not yet been elucidated. We hypothesized that infection with periodontopathic bacteria may enhance thrombosis through molecular mimicry with TLRVYK peptides on beta-2 glycoprotein I, a target molecule in anti-phospholipid syndrome. This study aimed to examine the effects of periodontitis on TPL and PB. METHODS: Ninety-five pregnant women (47 TPL and 48 healthy subjects) participated. Periodontal clinical parameters and periodontopathic bacteria were examined. Molecular mimicry between TLRVYK peptides and homologous peptides on the periodontopathic bacteria was examined by enzyme-linked immunosorbent assay (ELISA) using rabbit polyclonal antibodies specific for the respective peptides (SIRVYK on Aggregatibacter actinomycetemcomitans, TLRIYT on Porphyromonus gingivalis, and TLALYK on Treponema denticola). Serum high-sensitivity C-reactive protein, anti-TLRVYK and anti-SIRVYK IgG antibodies were measured using ELISA. RESULTS: Among the rabbit antibodies specific for the bacterial homologous peptides, only anti-SIRVYK IgG antibody reacted with TLRVYK peptides. Multivariable analysis showed that anti-SIRVYK IgG antibody was significantly associated with diagnosis of TPL. Of 95 births, 14 (14.7 %) delivered preterm. The preterm birth rate was higher in the anti-SIRVYK IgG antibody >median group than in the ≤median group. Of the 47 TPL subjects 13 had PB, and ordinal logistic regression analysis revealed that past smoking, presence of P. gingivalis and anti-SIRVYK IgG antibody were significantly correlated with PB. CONCLUSIONS: Infection with P. gingivalis and the antibody response to SIRVYK might be associated with TPL and PB.

Detección de infección por Mycoplasma en las gestantes con riesgo de parto pretérmino / Detection of Mycoplasma infection in pregnants with a risk of preterm delivery

Introducción: el parto pretérmino es el que se produce antes de las 37 sem de gestación; la sepsis vaginal constituye uno de los factores de riesgo predisponentes para este, de ahí que continúe siendo motivo de preocupación para obstetras y neonatólogos. Objetivo: evaluar el uso del Test Mycoplasma System Plus en gestaciones con riesgo de parto pretérmino, así como algunos de sus aspectos clínicos. Métodos: se realizó un estudio retrospectivo descriptivo de enero a septiembre de 2010 en el Hospital Profesor Eusebio Hernández, la muestra estuvo constituida por 88 pacientes ingresadas en el servicio de cuidados especiales perinatales con gestaciones menores de 34 sem a las que se les realizó el test y tuvieron el parto en este centro, los datos fueron recogidos de las historias clínicas e informes de microbiología agrupados en un formulario y procesados mediante estadísticas descriptivas y de distribución de frecuencia. Resultados: el 67 porciento, de las pacientes presentaban infección moderada o severa a ureaplasma mientras que los exudados vaginales simples fueron negativos en un 71,5 porciento, el antimicrobiano más utilizado fue la eritromicina. Conclusiones: la mayoría de las pacientes después del tratamiento adecuado llegaron al término de la gestación, así como presentaban exudados vaginales simple negativos, pero con un alto índice de infección a ureaplasma urealyticum

Introduction: the pre-term labor is produced before the 37 weeks of pregnancy; the vaginal sepsis is one of the predisposing risk factors to it, being a motive of worry for obstetricians and neonatologists. Objective: to assess the use of Test Mycoplasma System Plus in pregnancies with risk of pre-term labor, as well as of its clinical features. Methods: a descriptive and retrospective study was conducted from January to September, 2010 in the Profesor Eusebio Hernández Hospital; sample included 88 patients admitted in the perinatal special care unit with pregnancies under 34 weeks and performing of test who gave birth in this institution; data were collected from the medical records and microbiology reports grouped in a form and processed by descriptive statistics and of frequency distribution. Results: the 6l7 percent of patients had a moderate or severe infection due to ureaplasma where as the vaginal exudates were negatives in the 71.5 percent, the more used antimicrobial agents was the erythromycin. Conclusions: most of patients after an appropriate treatment arrive to a term pregnancy and also had negative simple vaginal exudate but with a high rate of infection due to ureaplasma urelyticum