Questioning the fetal microbiome illustrates pitfalls of low-biomass microbial studies.

Whether the human fetus and the prenatal intrauterine environment (amniotic fluid and placenta) are stably colonized by microbial communities in a healthy pregnancy remains a subject of debate. Here we evaluate recent studies that characterized microbial populations in human fetuses from the perspectives of reproductive biology, microbial ecology, bioinformatics, immunology, clinical microbiology and gnotobiology, and assess possible mechanisms by which the fetus might interact with microorganisms. Our analysis indicates that the detected microbial signals are likely the result of contamination during the clinical procedures to obtain fetal samples or during DNA extraction and DNA sequencing. Furthermore, the existence of live and replicating microbial populations in healthy fetal tissues is not compatible with fundamental concepts of immunology, clinical microbiology and the derivation of germ-free mammals. These conclusions are important to our understanding of human immune development and illustrate common pitfalls in the microbial analyses of many other low-biomass environments. The pursuit of a fetal microbiome serves as a cautionary example of the challenges of sequence-based microbiome studies when biomass is low or absent, and emphasizes the need for a trans-disciplinary approach that goes beyond contamination controls by also incorporating biological, ecological and mechanistic concepts.

Antibiotic treatment reduces the intensity of intraamniotic inflammation in pregnancies with idiopathic vaginal bleeding in the second trimester of pregnancy.

BACKGROUND: Idiopathic bleeding in the second trimester of pregnancy complicates <1% of all pregnancies. This pregnancy complication can be caused by alterations in local hemostasis in the decidua due to infection/inflammation in the choriodecidual niche. This condition is associated with intraamniotic inflammatory complications. Antibiotic therapy effectively reduces the intensity of intraamniotic inflammation in certain pregnancy pathologies. However, whether antibiotic administration can reduce the intensity of the intraamniotic inflammatory response or eradicate microorganisms in patients with idiopathic bleeding during the second trimester of pregnancy remains unclear. OBJECTIVE: This study primarily aimed to determine whether antimicrobial agents can reduce the magnitude of intraamniotic inflammation in patients with idiopathic bleeding in the second trimester of pregnancy by assessing the concentration of interleukin-6 in the amniotic fluid before and after 7 days of antibiotic treatment. The secondary aim was to determine whether treatment with a combination of antibiotics altered the microbial load of Ureaplasma species DNA in amniotic fluid. STUDY DESIGN: This retrospective cohort study included singleton-gestation patients with idiopathic bleeding between 15+0 and 27+6 weeks who underwent transabdominal amniocentesis at the time of admission. Follow-up amniocentesis was performed in a subset of patients unless abortion or delivery occurred earlier. Concentrations of interleukin-6 were measured in the amniotic fluid samples, and the presence of microbial invasion of the amniotic cavity was assessed using culture and molecular microbiological methods. Intraamniotic inflammation was defined as an interleukin-6 concentration ≥3000 pg/mL in the amniotic fluid samples. RESULTS: A total of 36 patients with idiopathic bleeding in the second trimester of pregnancy were included. All the patients underwent initial amniocentesis. Patients with intraamniotic inflammation (n=25) were treated using a combination of antibiotics consisting of intravenous ceftriaxone, intravenous metronidazole, and peroral clarithromycin. The patients without intraamniotic inflammation (n=11) were treated expectantly. In total, 25 patients delivered 7 days after admission. All patients with intraamniotic inflammation at the initial amniocentesis who delivered after 7 days underwent follow-up amniocentesis. Treatment with antibiotics decreased the interleukin-6 concentration in the amniotic fluid at follow-up amniocentesis compared with that at the initial amniocentesis in patients with intraamniotic inflammation (median [interquartile range]: 3457 pg/mL [2493-13,203] vs 19,812 pg/mL [11,973-34,518]; P=.0001). Amniotic fluid samples with Ureaplasma species DNA had a lower microbial load at the time of follow-up amniocentesis compared with the initial amniocentesis (median [interquartile range]: 1.5×105 copies DNA/mL [1.3×105-1.7×105] vs 8.0×107 copies DNA/mL [6.7×106-1.6×108]; P=.02). CONCLUSION: Antibiotic therapy was associated with reduced intraamniotic inflammation in patients with idiopathic bleeding in the second trimester complicated by intraamniotic inflammation. Moreover, antibiotic treatment has been associated with a reduction in the microbial load of Ureaplasma species DNA in the amniotic fluid.

Clinical chorioamnionitis at term: definition, pathogenesis, microbiology, diagnosis, and treatment.

Clinical chorioamnionitis, the most common infection-related diagnosis in labor and delivery units, is an antecedent of puerperal infection and neonatal sepsis. The condition is suspected when intrapartum fever is associated with two other maternal and fetal signs of local or systemic inflammation (eg, maternal tachycardia, uterine tenderness, maternal leukocytosis, malodorous vaginal discharge or amniotic fluid, and fetal tachycardia). Clinical chorioamnionitis is a syndrome caused by intraamniotic infection, sterile intraamniotic inflammation (inflammation without bacteria), or systemic maternal inflammation induced by epidural analgesia. In cases of uncertainty, a definitive diagnosis can be made by analyzing amniotic fluid with methods to detect bacteria (Gram stain, culture, or microbial nucleic acid) and inflammation (white blood cell count, glucose concentration, interleukin-6, interleukin-8, matrix metalloproteinase-8). The most common microorganisms are Ureaplasma species, and polymicrobial infections occur in 70% of cases. The fetal attack rate is low, and the rate of positive neonatal blood cultures ranges between 0.2% and 4%. Intrapartum antibiotic administration is the standard treatment to reduce neonatal sepsis. Treatment with ampicillin and gentamicin have been recommended by professional societies, although other antibiotic regimens, eg, cephalosporins, have been used. Given the importance of Ureaplasma species as a cause of intraamniotic infection, consideration needs to be given to the administration of antimicrobial agents effective against these microorganisms such as azithromycin or clarithromycin. We have used the combination of ceftriaxone, clarithromycin, and metronidazole, which has been shown to eradicate intraamniotic infection with microbiologic studies. Routine testing of neonates born to affected mothers for genital mycoplasmas could improve the detection of neonatal sepsis. Clinical chorioamnionitis is associated with decreased uterine activity, failure to progress in labor, and postpartum hemorrhage; however, clinical chorioamnionitis by itself is not an indication for cesarean delivery. Oxytocin is often administered for labor augmentation, and it is prudent to have uterotonic agents at hand to manage postpartum hemorrhage. Infants born to mothers with clinical chorioamnionitis near term are at risk for early-onset neonatal sepsis and for long-term disability such as cerebral palsy. A frontier is the noninvasive assessment of amniotic fluid to diagnose intraamniotic inflammation with a transcervical amniotic fluid collector and a rapid bedside test for IL-8 for patients with ruptured membranes. This approach promises to improve diagnostic accuracy and to provide a basis for antimicrobial administration.

Angiogenic imbalance in pregnancies with preterm prelabor rupture of membranes between 34 and 37 weeks of gestation.

INTRODUCTION: This study aimed to identify whether microbial invasion of the amniotic cavity and/or intra-amniotic inflammation in women with late preterm prelabor rupture of membranes (PPROM) was associated with changes in concentrations of soluble fms-like tyrosine kinase-1 (sFlt-1), placental growth factor (PlGF) and its ratio in maternal serum, and whether placental features consistent with maternal vascular malperfusion further affect their concentrations. MATERIAL AND METHODS: This historical study included 154 women with singleton pregnancies complicated by PPROM between gestational ages 34+0 and 36+6 weeks. Transabdominal amniocentesis was performed as part of standard clinical management to evaluate the intra-amniotic environment. Women were categorized into two subgroups based on the presence of microorganisms and/or their nucleic acids in amniotic fluid (determined by culturing and molecular biology method) and intra-amniotic inflammation (by amniotic fluid interleukin-6 concentration evaluation): (1) those with the presence of microorganisms and/or inflammation (at least one present) and (2) those with negative amniotic fluid for infection/inflammation (absence of both). Concentrations of sFlt-1 and PlGF were assessed using the Elecsys® sFlt-1 and Elecsys® PlGF immunoassays and converted into multiples of medians. RESULTS: Women with the presence of microorganisms and/or inflammation in amniotic fluid had lower serum concentrations of sFlt-1 and sFlt-1/PlGF ratios and higher concentrations of PlGF compared with those with negative amniotic fluid. (sFlt-1: presence: median 1.0 multiples of the median (MoM), vs negative: median: 1.5 MoM, P = 0.003; PlGF: presence: median 0.7 MoM, vs negative: median 0.4 MoM, P = 0.02; sFlt-1/PlGF: presence: median 8.9 vs negative 25.0, P = 0.001). Higher serum concentrations of sFlt-1 and sFlt-1/PlGF ratios as well as lower concentrations of PlGF were found in the subsets of women with maternal vascular malperfusion than in those without maternal vascular malperfusion. CONCLUSIONS: Among women experiencing late PPROM, angiogenic imbalance in maternal serum is primarily observed in those without both microbial invasion of the amniotic cavity and intra-amniotic inflammation. Additionally, there is an association between angiogenic imbalance and the presence of maternal vascular malperfusion.

Adverse obstetric outcomes in cases of meconium-stained amniotic fluid complicated with intrapartum fever.

PURPOSE: Meconium-stained amniotic fluid (MSAF) often signifies colonization of the amniotic sac by microorganisms. This study investigated additional adverse obstetric outcomes associated with MSAF in deliveries complicated by maternal intrapartum fever (IF). METHODS: This retrospective study included all singleton pregnancies from 2014 to 2020, with intrapartum maternal fever ≥ 38 °C during a trial of labor. In accordance with departmental protocol, all patients received intravenous antibiotic therapy consisting of ampicillin and gentamicin in the absence of allergies to these medications. Subsequent antibiotic therapy was adjusted based on the culture results. Antibiotic treatment was discontinued postpartum after 48 h without fever. Swab cultures were obtained immediately postpartum from both the maternal and fetal sides of the placenta. Maternal and fetal outcomes, along with positive placental cultures, were compared between participants with MSAF&IF and those with clear amniotic fluid &IF (control group). RESULTS: In comparison to the control group (n = 1089), the MSAF&IF group (n = 264) exhibited significantly higher rates of cesarean delivery (CD) (p = 0.001), CD due to non-reassuring fetal heart rate (p = 0.001), and cord pH ≤ 7.1 (p = 0.004). Positive swab cultures from the placental maternal and fetal sides were more prevalent among the MSAF&IF group (23.1% vs. 17.6%, p = 0.041 and 29.2% vs. 22.9%, p = 0.032, respectively). Placental cultures yielding gastrointestinal pathogens and extended spectrum beta-lactamase were notably more common in the MSAF&IF group compared to controls (p = 0.023). However, there was no significant difference between groups regarding the rate of group B streptococcus positive placental cultures. CONCLUSIONS: Women experiencing IF and MSAF during labor face an elevated risk of CD compared to those with IF alone. The presence of MSAF heightens the risk of positive placental cultures, particularly with gastrointestinal and extended spectrum beta-lactamase pathogens.

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.

The Influence of Dental Status and Blood Parameters Characterizing Endogenous Intoxication on the Timing of Childbirth.

Background and Objectives: Epidemiological and microbiological-immunological studies have led to the conclusion that periodontal disease may be a risk factor for preterm birth. The aim of this study was to investigate and identify the relationship of some hematological cellular biomarkers characterizing the chronic oral focus of infection with pregnancy outcomes and their impact on those outcomes. Materials and Methods: Clinical and laboratory tests were conducted on 100 pregnant women, grouped by full-term or preterm births, with the assessment of the following markers: DMF, CPI and PIRI, PHP, microbiological examination of periodontal pockets and amniotic fluid, WBS count, WBCSI, LGI, and NMR. A statistical analysis was carried out with SPSS Statistics version 19.0. Results: Women with preterm labor had higher-grade caries (CSL > 0.3), while women with full-term childbirth had moderate caries (CSL < 0.3). A satisfactory level of oral hygiene efficiency was found in 50% (group 1) and 38.1% (group 2) of the expectant mothers. The periodontal status by the PIRI showed tissue lesions in 20.7% (group 1) and 92.9% (group 2) of the women. The WBCSI was 2.27 ± 0.82 and 2.15 ± 0.68, the NMR was 9.29 ± 5.119 and 11.62 ± 7.78, and the LGI was 3.54 ± 1.1 and 3.73 ± 0.81 in groups 1 and 2, respectively. Comparative analysis of bacterial contamination of the amniotic fluid revealed the predominance of Fusobacterium nucleatum (64.3%), Tannerella forsythia (57.1%), Prevotella intermedia (50%), Porphyromonas gingivalis (57.1%), Staphylococcus aureus (45.2%), and Candida albicans (50%) in women with premature birth. Conclusions: In women with preterm birth, the values of the indices characterizing a chronic oral focus of infection evoke more significant correlations with the timing of delivery, which indicates the significant role of an oral focus of infection. The presence of microbial invasion of amniotic fluid may indicate the role of periodontopathogenic bacteria in pregnant women diagnosed with a risk of preterm birth.

Noninvasive prediction models of intra-amniotic infection in women with preterm labor.

BACKGROUND: Among women with preterm labor, those with intra-amniotic infection present the highest risk of early delivery and the most adverse outcomes. The identification of intra-amniotic infection requires amniocentesis, perceived as too invasive by women and physicians. Noninvasive methods for identifying intra-amniotic infection and/or early delivery are crucial to focus early efforts on high-risk preterm labor women while avoiding unnecessary interventions in low-risk preterm labor women. OBJECTIVE: This study modeled the best performing models, integrating biochemical data with clinical and ultrasound information to predict a composite outcome of intra-amniotic infection and/or spontaneous delivery within 7 days. STUDY DESIGN: From 2015 to 2020, data from a cohort of women, who underwent amniocentesis to rule in or rule out intra-amniotic infection or inflammation, admitted with a diagnosis of preterm labor at <34 weeks of gestation at the Hospital Clinic and Hospital Sant Joan de Déu, Barcelona, Spain, were used. At admission, transvaginal ultrasound was performed, and maternal blood and vaginal samples were collected. Using high-dimensional biology, vaginal proteins (using multiplex immunoassay), amino acids (using high-performance liquid chromatography), and bacteria (using 16S ribosomal RNA gene amplicon sequencing) were explored to predict the composite outcome. We selected ultrasound, maternal blood, and vaginal predictors that could be tested with rapid diagnostic techniques and developed prediction models employing machine learning that was applied in a validation cohort. RESULTS: A cohort of 288 women with preterm labor at <34 weeks of gestation, of which 103 (35%) had a composite outcome of intra-amniotic infection and/or spontaneous delivery within 7 days, were included in this study. The sample was divided into derivation (n=116) and validation (n=172) cohorts. Of note, 4 prediction models were proposed, including ultrasound transvaginal cervical length, maternal C-reactive protein, vaginal interleukin 6 (using an automated immunoanalyzer), vaginal pH (using a pH meter), vaginal lactic acid (using a reflectometer), and vaginal Lactobacillus genus (using quantitative polymerase chain reaction), with areas under the receiving operating characteristic curve ranging from 82.2% (95% confidence interval, ±3.1%) to 85.2% (95% confidence interval, ±3.1%), sensitivities ranging from 76.1% to 85.9%, and specificities ranging from 75.2% to 85.1%. CONCLUSION: The study results have provided proof of principle of how noninvasive methods suitable for point-of-care systems can select high-risk cases among women with preterm labor and might substantially aid in clinical management and outcomes while improving the use of resources and patient experience.

Rapid diagnosis of intra-amniotic infection using nanopore-based sequencing.

OBJECTIVES: Early diagnosis and treatment of intra-amniotic infection is crucial. Rapid pathogen identification allows for a definite diagnosis and enables proper management. We determined whether the 16S amplicon sequencing performed by a nanopore sequencing technique make possible rapid bacterial identification at the species level in intra-amniotic infection. METHODS: Five cases of confirmed intra-amniotic infection, determined by either cultivation or 16S rDNA polymerase chain reaction (PCR) Sanger sequencing, and 10 cases of women who underwent mid-trimester genetic amniocentesis were included. DNA was extracted from amniotic fluid and PCR was performed on the full-length 16S rDNA. Nanopore sequencing was performed. The results derived from nanopore sequencing were compared with those derived from cultivation and Sanger sequencing methods. RESULTS: Bacteria were successfully detected from amniotic fluid using nanopore sequencing in all cases of intra-amniotic infection. Nanopore sequencing identified additional bacterial species and polymicrobial infections. All patients who underwent a mid-trimester amniocentesis had negative cultures, negative 16S PCR Sanger sequencing and nanopore sequencing. Identification of the microorganisms using nanopore sequencing technique at the bacterial species level was achieved within 5-9 h from DNA extraction. CONCLUSIONS: This is the first study demonstrating that the nanopore sequencing technique is capable of rapid diagnosis of intra-amniotic infection using fresh amniotic fluid samples.

Are bacteria, fungi, and archaea present in the midtrimester amniotic fluid?

OBJECTIVES: This study was conducted to determine whether bacteria, fungi, or archaea are detected in the amniotic fluid of patients who underwent midtrimester amniocentesis for clinical indications. METHODS: Amniotic fluid samples from 692 pregnancies were tested by using a combination of culture and end-point polymerase chain reaction (PCR) techniques. Intra-amniotic inflammation was defined as an interleukin-6 concentration >2,935 pg/mL. RESULTS: Microorganisms were detected in 0.3% (2/692) of cases based on cultivation, 1.73% (12/692) based on broad-range end-point PCR, and 2% (14/692) based on the combination of both methods. However, most (13/14) of these cases did not have evidence of intra-amniotic inflammation and delivered at term. Therefore, a positive culture or end-point PCR in most patients appears to have no apparent clinical significance. CONCLUSIONS: Amniotic fluid in the midtrimester of pregnancy generally does not contain bacteria, fungi, or archaea. Interpretation of amniotic fluid culture and molecular microbiologic results is aided by the assessment of the inflammatory state of the amniotic cavity. The presence of microorganisms, as determined by culture or a microbial signal in the absence of intra-amniotic inflammation, appears to be a benign condition.

Intra-amniotic inflammation in the mid-trimester of pregnancy is a risk factor for neuropsychological disorders in childhood.

OBJECTIVES: Intra-amniotic inflammation is a subclinical condition frequently caused by either microbial invasion of the amniotic cavity or sterile inflammatory stimuli, e.g., alarmins. An accumulating body of evidence supports a role for maternal immune activation in the genesis of fetal neuroinflammation and the occurrence of neurodevelopmental disorders such as cerebral palsy, schizophrenia, and autism. The objective of this study was to determine whether fetal exposure to mid-trimester intra-amniotic inflammation is associated with neurodevelopmental disorders in children eight to 12 years of age. METHODS: This is a retrospective case-control study comprising 20 children with evidence of prenatal exposure to intra-amniotic inflammation in the mid-trimester and 20 controls matched for gestational age at amniocentesis and at delivery. Amniotic fluid samples were tested for concentrations of interleukin-6 and C-X-C motif chemokine ligand 10, for bacteria by culture and molecular microbiologic methods as well as by polymerase chain reaction for eight viruses. Neuropsychological testing of children, performed by two experienced psychologists, assessed cognitive and behavioral domains. Neuropsychological dysfunction was defined as the presence of an abnormal score (<2 standard deviations) on at least two cognitive tasks. RESULTS: Neuropsychological dysfunction was present in 45% (9/20) of children exposed to intra-amniotic inflammation but in only 10% (2/20) of those in the control group (p=0.03). The relative risk (RR) of neuropsychological dysfunction conferred by amniotic fluid inflammation remained significant after adjusting for gestational age at delivery [aRR=4.5 (1.07-16.7)]. Of the 11 children diagnosed with neuropsychological dysfunction, nine were delivered at term and eight of them had mothers with intra-amniotic inflammation. Children exposed to intra-amniotic inflammation were found to have abnormalities in neuropsychological tasks evaluating complex skills, e.g., auditory attention, executive functions, and social skills, whereas the domains of reasoning, language, and memory were not affected in the cases and controls. CONCLUSIONS: Asymptomatic sterile intra-amniotic inflammation in the mid-trimester of pregnancy, followed by a term birth, can still confer to the offspring a substantial risk for neurodevelopmental disorders in childhood. Early recognition and treatment of maternal immune activation in pregnancy may be a strategy for the prevention of subsequent neurodevelopmental disorders in offspring.

The search for aliens within us: a review of evidence and theory regarding the foetal microbiome.

The microbiome is believed to be established during the birthing process through exposure to the maternal microbiome and immediate external environment. The absence of a microbiome prior to birth is based on the sterile womb hypothesis, which was formulated at the beginning of the 20th century and is supported primarily by the culture-based approach in microbiological studies.Findings of bacterial presence in products of fertilization such as the placenta, amniotic fluid, foetal membranes, and umbilical cord blood in studies using next-generation DNA sequencing technologies began to challenge the sterile nature of the intrauterine environment during gestation. These studies have been mainly criticized by their approach to contamination and inconclusive evidence of viability. The implications of bacterial presence in utero are far reaching in medicine and basic sciences. If commensal bacteria exist in the foetus, antibiotic therapies in pregnancy particularly for asymptomatic cases will need to be re-evaluated. Experimental studies utilizing gnotobiology may also be impacted by a realignment of theory.This review of existing literature aims to provide insight into the existence of bacteria in utero, specifically the foetal microbiome through analysis of experimental evidence and theoretical concepts, and to suggest approaches that may further provide clarity into this inquiry.

The role of intraamniotic inflammation in threatened midtrimester miscarriage.

BACKGROUND: The assessment and management of patients with threatened midtrimester miscarriage is a clinical challenge because the etiology of this condition is poorly understood. OBJECTIVE: This study aimed to examine the frequency of intraamniotic infection or inflammation and the effect of antibiotics in patients presenting with regular uterine contractions and intact membranes before 20 weeks of gestation. STUDY DESIGN: This retrospective study comprised patients who met the following criteria: (1) singleton gestation, (2) gestational age before 20 weeks, (3) the presence of regular uterine contractions confirmed by a tocodynamometer (8 or more contractions in 60 minutes), (4) intact amniotic membranes, and (5) transabdominal amniocentesis performed for the evaluation of the microbiologic and inflammatory status of the amniotic cavity. Samples of amniotic fluid were cultured for aerobic and anaerobic bacteria and genital mycoplasmas, and polymerase chain reaction was performed to detect Ureaplasma species. Amniotic fluid was tested for white blood cell counts and matrix metalloproteinase-8 concentrations to diagnose intraamniotic inflammation. Patients with intraamniotic inflammation, or intraamniotic infection, were treated with antibiotics (a combination of ceftriaxone, clarithromycin, and metronidazole). Treatment success was defined as the resolution of intraamniotic infection/inflammation at the follow-up amniocentesis or delivery after 34 weeks of gestation. RESULTS: 1) Intraamniotic inflammation was present in 88% (15/17) of patients, whereas infection was detectable in only 2 cases; 2) objective evidence of resolution of intraamniotic inflammation after antibiotic treatment was demonstrated in 100% (4/4) of patients who underwent a follow-up amniocentesis; 3) 30% (5/15) of women receiving antibiotics delivered after 34 weeks of gestation (3 of the 5 patients had a negative follow-up amniocentesis, and 2 of the women were without a follow-up amniocentesis); 4) the overall treatment success of antibiotics was 40% (6/15; 4 cases of objective evidence of resolution of intra-amniotic inflammation and 5 cases of delivery after 34 weeks of gestation). CONCLUSION: The prevalence of intraamniotic inflammation in patients who presented with a threatened midtrimester miscarriage was 88% (15/17), and, in most cases, microorganisms could not be detected. Antibiotic treatment, administered to patients with intraamniotic inflammation, was associated with either objective resolution of intraamniotic inflammation or delivery after 34 weeks of gestation in 40% (6/15) of the cases.

Exploratory and confirmatory analysis to investigate the presence of vaginal metabolome expression of microbial invasion of the amniotic cavity in women with preterm labor using high-performance liquid chromatography.

BACKGROUND: Although the influence of microbial invasion of the amniotic cavity on the development of spontaneous preterm delivery is unquestionable, the use of an invasive procedure to diagnose the status of an infection limits its clinical translation. OBJECTIVE: This study aimed to use exploratory and confirmatory analyses to investigate the presence of vaginal metabolome expression of microbial invasion of the amniotic cavity in women diagnosed as having preterm labor using high-performance liquid chromatography. STUDY DESIGN: In 140 women with singleton pregnancies and a diagnosis of preterm labor at <34 weeks' gestation, we analyzed vaginal amino acid concentrations using high-performance liquid chromatography. Vaginal samples were collected shortly after the amniocentesis performed at admission to rule out microbial invasion of the amniotic cavity. Data were normalized for the median of all the amino acid concentrations evaluated. Microbial invasion of the amniotic cavity was defined as a positive aerobic or anaerobic amniotic fluid culture for the presence of bacteria or yeast or Ureaplasma species or Mycoplasma hominis in the mycoplasma culture or a positive polymerase chain reaction result for 16S rRNA gene sequence. Exploratory analysis was performed in half of the sample and confirmatory analysis in the other half. We compared vaginal amino acid concentrations between women with and without microbial invasion of the amniotic cavity in both cohorts. The area under the curve with 95% confidence interval values were calculated for vaginal amino acids with significant differences. RESULTS: In the exploratory cohort (2014-2015), 17 of 76 women (22.3%) had microbial invasion of the amniotic cavity compared with 14 of 72 (19.4%) in the confirmatory cohort (2016-2017). In the exploratory cohort, we found significantly higher amino acid concentrations of vaginal taurine, lysine, and cysteine and significantly lower concentrations of vaginal glutamate, aspartate, and the aspartate to asparagine ratio. These significant differences were confirmed in the confirmatory cohort. The area under the curve of these vaginal amino acids to predict microbial invasion of the amniotic cavity ranged between 0.72 and 0.79, with cysteine being the amino acid with the best performance with an area under the curve of 0.79 (95% confidence interval, 0.71-0.88). CONCLUSION: We found the vaginal metabolome expression of microbial invasion of the amniotic cavity in women with preterm labor and intact membranes. These findings might open the possibility to develop noninvasive diagnostic tools of microbial invasion of the amniotic cavity with the aim of selecting women who would most likely benefit from an amniocentesis for this indication.

Amniotic fluid interleukin 6 and interleukin 8 are superior predictors of fetal lung injury compared with maternal or fetal plasma cytokines or placental histopathology in a nonhuman primate model.

BACKGROUND: Intra-amniotic infection or inflammation is common in early preterm birth and associated with substantial neonatal lung morbidity owing to fetal exposure to proinflammatory cytokines and infectious organisms. Amniotic fluid interleukin 8, a proinflammatory cytokine, was previously correlated with the development of neonatal bronchopulmonary dysplasia, but whether amniotic fluid cytokines or placental pathology more accurately predicts neonatal lung pathology and morbidity is unknown. We have used a pregnant nonhuman primate model of group B Streptococcus infection to study the pathogenesis of intra-amniotic infection, bacterial invasion of the amniotic cavity and fetus, and microbial-host interactions. In this nonhuman primate model, we have studied the pathogenesis of group B Streptococcus strains with differing potential for virulence, which has resulted in a spectrum of intra-amniotic infection and fetal lung injury that affords the opportunity to study the inflammatory predictors of fetal lung pathology and injury. OBJECTIVE: This study aimed to determine whether fetal lung injury is best predicted by placental histopathology or the cytokine response in amniotic fluid or maternal plasma. STUDY DESIGN: Chronically catheterized pregnant monkeys (Macaca nemestrina, pigtail macaque) at 116 to 125 days gestation (term at 172 days) received a choriodecidual inoculation of saline (n=5), weakly hemolytic group B Streptococcus strain (n=5, low virulence), or hyperhemolytic group B Streptococcus strain (n=5, high virulence). Adverse pregnancy outcomes were defined as either preterm labor, microbial invasion of the amniotic cavity, or development of the fetal inflammatory response syndrome. Amniotic fluid and maternal and fetal plasma samples were collected after inoculation, and proinflammatory cytokines (tumor necrosis factor alpha, interleukin beta, interleukin 6, interleukin 8) were measured by a multiplex assay. Cesarean delivery was performed at the time of preterm labor or within 1 week of inoculation. Fetal necropsy was performed at the time of delivery. Placental pathology was scored in a blinded fashion by a pediatric pathologist, and fetal lung injury was determined by a semiquantitative score from histopathology evaluating inflammatory infiltrate, necrosis, tissue thickening, or collapse scored by a veterinary pathologist. RESULTS: The principal findings in our study are as follows: (1) adverse pregnancy outcomes occurred more frequently in animals receiving hyperhemolytic group B Streptococcus (80% with preterm labor, 80% with fetal inflammatory response syndrome) than in animals receiving weakly hemolytic group B Streptococcus (40% with preterm labor, 20% with fetal inflammatory response syndrome) and in controls (0% preterm labor, 0% fetal inflammatory response syndrome); (2) despite differences in the rate of adverse pregnancy outcomes and fetal inflammatory response syndrome, fetal lung injury scores were similar between animals receiving the weakly hemolytic group B Streptococcus strains and animals receiving the hyperhemolytic group B Streptococcus strains; (3) fetal lung injury score was significantly correlated with peak amniotic fluid cytokines interleukin 6 and interleukin 8 but not tumor necrosis factor alpha or interleukin 1 beta; and (4) fetal lung scores were poorly correlated with maternal and fetal plasma cytokine levels and placental pathology. CONCLUSION: Amniotic fluid interleukin 6 and interleukin 8 levels were superior predictors of fetal lung injury than placental histopathology or maternal plasma cytokines. This evidence supports a role for amniocentesis in the prediction of neonatal lung morbidity owing to intra-amniotic infection, which cannot be provided by cytokine analysis of maternal plasma or placental histopathology.

Bacteria in the amniotic fluid without inflammation: early colonization vs. contamination.

OBJECTIVES: Intra-amniotic infection, defined by the presence of microorganisms in the amniotic cavity, is often accompanied by intra-amniotic inflammation. Occasionally, laboratories report the growth of bacteria or the presence of microbial nucleic acids in amniotic fluid in the absence of intra-amniotic inflammation. This study was conducted to determine the clinical significance of the presence of bacteria in amniotic fluid samples in the absence of intra-amniotic inflammation. METHODS: A retrospective cross-sectional study included 360 patients with preterm labor and intact membranes who underwent transabdominal amniocentesis for evaluation of the microbial state of the amniotic cavity as well as intra-amniotic inflammation. Cultivation techniques were used to isolate microorganisms, and broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) was utilized to detect the nucleic acids of bacteria, viruses, and fungi. RESULTS: Patients whose amniotic fluid samples evinced microorganisms but did not indicate inflammation had a similar perinatal outcome to those without microorganisms or inflammation [amniocentesis-to-delivery interval (p=0.31), spontaneous preterm birth before 34 weeks (p=0.83), acute placental inflammatory lesions (p=1), and composite neonatal morbidity (p=0.8)]. CONCLUSIONS: The isolation of microorganisms from a sample of amniotic fluid in the absence of intra-amniotic inflammation is indicative of a benign condition, which most likely represents contamination of the specimen during the collection procedure or laboratory processing rather than early colonization or infection.

Clinical chorioamnionitis at term X: microbiology, clinical signs, placental pathology, and neonatal bacteremia - implications for clinical care.

OBJECTIVES: Clinical chorioamnionitis at term is considered the most common infection-related diagnosis in labor and delivery units worldwide. The syndrome affects 5-12% of all term pregnancies and is a leading cause of maternal morbidity and mortality as well as neonatal death and sepsis. The objectives of this study were to determine the (1) amniotic fluid microbiology using cultivation and molecular microbiologic techniques; (2) diagnostic accuracy of the clinical criteria used to identify patients with intra-amniotic infection; (3) relationship between acute inflammatory lesions of the placenta (maternal and fetal inflammatory responses) and amniotic fluid microbiology and inflammatory markers; and (4) frequency of neonatal bacteremia. METHODS: This retrospective cross-sectional study included 43 women with the diagnosis of clinical chorioamnionitis at term. The presence of microorganisms in the amniotic cavity was determined through the analysis of amniotic fluid samples by cultivation for aerobes, anaerobes, and genital mycoplasmas. A broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry was also used to detect bacteria, select viruses, and fungi. Intra-amniotic inflammation was defined as an elevated amniotic fluid interleukin-6 (IL-6) concentration ≥2.6 ng/mL. RESULTS: (1) Intra-amniotic infection (defined as the combination of microorganisms detected in amniotic fluid and an elevated IL-6 concentration) was present in 63% (27/43) of cases; (2) the most common microorganisms found in the amniotic fluid samples were Ureaplasma species, followed by Gardnerella vaginalis; (3) sterile intra-amniotic inflammation (elevated IL-6 in amniotic fluid but without detectable microorganisms) was present in 5% (2/43) of cases; (4) 26% of patients with the diagnosis of clinical chorioamnionitis had no evidence of intra-amniotic infection or intra-amniotic inflammation; (5) intra-amniotic infection was more common when the membranes were ruptured than when they were intact (78% [21/27] vs. 38% [6/16]; p=0.01); (6) the traditional criteria for the diagnosis of clinical chorioamnionitis had poor diagnostic performance in identifying proven intra-amniotic infection (overall accuracy, 40-58%); (7) neonatal bacteremia was diagnosed in 4.9% (2/41) of cases; and (8) a fetal inflammatory response defined as the presence of severe acute funisitis was observed in 33% (9/27) of cases. CONCLUSIONS: Clinical chorioamnionitis at term, a syndrome that can result from intra-amniotic infection, was diagnosed in approximately 63% of cases and sterile intra-amniotic inflammation in 5% of cases. However, a substantial number of patients had no evidence of intra-amniotic infection or intra-amniotic inflammation. Evidence of the fetal inflammatory response syndrome was frequently present, but microorganisms were detected in only 4.9% of cases based on cultures of aerobic and anaerobic bacteria in neonatal blood.

Ureaplasma species and preterm birth: current perspectives.

Preterm birth is the leading cause of neonatal morbidity and mortality worldwide, and the human Ureaplasma species are most frequently isolated from the amniotic fluid and placenta in these cases. Ureaplasma colonisation is associated with infertility, stillbirth, histologic chorioamnionitis, and neonatal morbidities, including congenital pneumonia, bronchopulmonary dysplasia, meningitis and perinatal death. The human Ureaplasma spp. are separated into Ureaplasma urealyticum and Ureaplasma parvum with 14 known serotypes. The small genome has several genes, which code for surface proteins; most significantly the Multiple Banded Antigen (MBA) where an antigenic C-terminal domain elicits a host antibody response. Other genes code for various virulence factors such as IgA protease and urease. Ureaplasma spp. infection is diagnosed by culture and polymerase chain reaction (PCR) and commercial assays are available to improve turnaround time. Microbroth dilution assays are routinely used to test antimicrobial susceptibility of clinical Ureaplasma spp. especially against doxycycline, azithromycin, ofloxacin and josamycin. Resistance to macrolides, fluoroquinolones and tetracyclines has been reported. A concise review of Ureaplasma spp. and their role in pregnancy outcomes, especially preterm birth, offers insight into the early diagnosis and appropriate antibiotic therapy to prevent long-term complications of Ureaplasma spp. infections.

Development and validation of a multivariable prediction model of spontaneous preterm delivery and microbial invasion of the amniotic cavity in women with preterm labor.

BACKGROUND: Early spontaneous preterm delivery is often associated with microbial invasion of the amniotic cavity and/or intraamniotic inflammation. OBJECTIVE: The objective of the study was to develop and validate clinically feasible multivariable prediction models of spontaneous delivery within 7 days and microbial invasion of the amniotic cavity in women admitted with diagnose of preterm labor and intact membranes below 34 weeks. STUDY DESIGN: We used data from a cohort of women admitted from 2012 to 2018 with diagnosis of preterm labor below 34 weeks who had undergone amniocentesis to rule out microbial invasion of the amniotic cavity. The main outcome was spontaneous delivery within 7 days from admission. The secondary outcome was microbial invasion of the amniotic cavity, defined by a positive culture and/or 16S ribosomal RNA gene in the amniotic fluid. The sample (n = 358) was divided into derivation (2012-2016) and validation cohorts (2017-2018). Logistic regression models using a stepwise selection of variables were developed for the outcomes evaluated. We explored as predictive variables ultrasound cervical length measurement at admission, maternal C-reactive protein, gestational age, amniotic fluid glucose, and interleukin-6 (expressed as log units). Models were developed in the derivation cohort and applied to the validation cohort and diagnostic performance was calculated. RESULTS: The derivation cohort included 263 women and the validation cohort 95 women. One hundred five of the women (39%, 105 of 268) spontaneously delivered in the following 7 days and 68 (19%, 68 of 358) had microbial invasion of the amniotic cavity. For spontaneous delivery within 7 days after admission, 4 predictors were identified: cervical length at admission, gestational age, amniotic fluid glucose, and interleukin-6. The diagnostic performance of the model was assessed in the validation cohort using the receiver operating characteristic curve and showed an area under curve of 0.86 (95% confidence interval, 0.77-0.95) with a detection rate of spontaneous delivery within 7 days of 87%, a false-positive rate of 33%, a negative predictive value of 80%, and a negative likelihood ratio of 0.1908. For microbial invasion of the amniotic cavity, 2 independent predictors of the amniotic cavity were identified: amniotic fluid glucose and maternal C-reactive protein. The receiver operating characteristic curve and an area under curve in the validation cohort was 0.83 (95% confidence interval, 0.70-0.96) with a detection rate of 76%, a false-positive rate of 8%, a negative predictive value of 93%, and a negative likelihood ratio of 0.2591. CONCLUSION: In women with preterm labor, we propose 2 clinically feasible prediction models to classify as low vs high risk of spontaneous delivery within 7 days and of microbial invasion of the amniotic cavity. The models showed a high diagnostic performance and could be of value to optimize clinical management.

Midtrimester amniotic fluid from healthy pregnancies has no microorganisms using multiple methods of microbiologic inquiry.

BACKGROUND: There is controversy about whether the amniotic fluid contains bacteria. With the use of sequencing-based methods, recent studies report that the amniotic fluid is colonized by microorganisms. However, background-contaminating DNA might lead to false-positive findings when such a low microbial biomass sample is examined. OBJECTIVE: The purpose of this study was to determine whether the midtrimester amniotic fluid of patients who subsequently had normal pregnancy outcomes contains a microbial signature. STUDY DESIGN: In this prospective cohort study, 42 amniotic fluid samples were collected from 37 pregnancies (5 twin and 32 singletons) during genetic amniocentesis in the midtrimester. The subsequent pregnancy outcomes of all the participants were followed. Multiple methods were used to detect the presence of microorganisms in this study, which included cultivation, quantitative real-time polymerase chain reaction, and 16S ribosomal RNA gene sequencing. Multiple positive control samples (n=16) served as quality control samples and included 3 adult fecal samples, 4 vaginal swabs, and 9 artificial bacterial communities that were run in parallel with negative control samples (n=12) that included 4 samples from the hospital operating room and 8 samples from the laboratory, to account for background-contaminating DNA during each step of the experiments. RESULTS: No bacteria under anaerobic or aerobic conditions or genital mycoplasmas were cultured from any of the amniotic fluid samples. Quantitative polymerase chain reaction did not reveal greater copy numbers of 16S ribosomal RNA gene in amniotic fluid samples than in negative control samples. 16S Ribosomal RNA gene sequencing did not indicate a significant difference in the microbial richness or community structures between amniotic fluid and negative control samples. CONCLUSION: With multiple methods of microbiologic inquiry, no microorganisms were identified in the midtrimester amniotic fluid of healthy pregnancies with a normal pregnancy outcome.