Prediction of preeclampsia throughout gestation with maternal characteristics and biophysical and biochemical markers: a longitudinal study.

BACKGROUND: The current approach to predict preeclampsia combines maternal risk factors and evidence from biophysical markers (mean arterial pressure, Doppler velocimetry of the uterine arteries) and maternal blood proteins (placental growth factor, soluble vascular endothelial growth factor receptor-1, pregnancy-associated plasma protein A). Such models require the transformation of biomarker data into multiples of the mean values by using population- and site-specific models. Previous studies have focused on a narrow window in gestation and have not included the maternal blood concentration of soluble endoglin, an important antiangiogenic factor up-regulated in preeclampsia. OBJECTIVE: This study aimed (1) to develop models for the calculation of multiples of the mean values for mean arterial pressure and biochemical markers; (2) to build and assess the predictive models for preeclampsia based on maternal risk factors, the biophysical (mean arterial pressure) and biochemical (placental growth factor, soluble vascular endothelial growth factor receptor-1, and soluble endoglin) markers collected throughout pregnancy; and (3) to evaluate how prediction accuracy is affected by the presence of chronic hypertension and gestational age. STUDY DESIGN: This longitudinal case-cohort study included 1150 pregnant women: women without preeclampsia with (n=49) and without chronic hypertension (n=871) and those who developed preeclampsia (n=166) or superimposed preeclampsia (n=64). Mean arterial pressure and immunoassay-based maternal plasma placental growth factor, soluble vascular endothelial growth factor receptor-1, and soluble endoglin concentrations were available throughout pregnancy (median of 5 observations per patient). A prior-risk model for preeclampsia was established by using Poisson regression based on maternal characteristics and obstetrical history. Next, multiple regression was used to fit biophysical and biochemical marker data as a function of maternal characteristics by using data collected at 8 to 15+6, 16 to 19+6, 20 to 23+6, 24 to 27+6, 28 to 31+6, and 32 to 36+6 week intervals, and observed values were converted into multiples of the mean values. Then, multivariable prediction models for preeclampsia were fit based on the biomarker multiples of the mean data and prior-risk estimates. Separate models were derived for overall, preterm, and term preeclampsia, which were evaluated by receiver operating characteristic curves and sensitivity at fixed false-positive rates. RESULTS: (1) The inclusion of soluble endoglin in prediction models for all preeclampsia, together with the prior-risk estimates, mean arterial pressure, placental growth factor, and soluble vascular endothelial growth factor receptor-1, increased the sensitivity (at a fixed false-positive rate of 10%) for early prediction of superimposed preeclampsia, with the largest increase (from 44% to 54%) noted at 20 to 23+6 weeks (McNemar test, P<.05); (2) combined evidence from prior-risk estimates and biomarkers predicted preterm preeclampsia with a sensitivity (false-positive rate, 10%) of 55%, 48%, 62%, 72%, and 84% at 8 to 15+6, 16 to 19+6, 20 to 23+6, 24 to 27+6, and 28 to 31+6 week intervals, respectively; (3) the sensitivity for term preeclampsia (false-positive rate, 10%) was 36%, 36%, 41%, 43%, 39%, and 51% at 8 to 15+6, 16 to 19+6, 20 to 23+6, 24 to 27+6, 28 to 31+6, and 32 to 36+6 week intervals, respectively; (4) the detection rate for superimposed preeclampsia among women with chronic hypertension was similar to that in women without chronic hypertension, especially earlier in pregnancy, reaching at most 54% at 20 to 23+6 weeks (false-positive rate, 10%); and (5) prediction models performed comparably to the Fetal Medicine Foundation calculators when the same maternal risk factors and biomarkers (mean arterial pressure, placental growth factor, and soluble vascular endothelial growth factor receptor-1 multiples of the mean values) were used as input. CONCLUSION: We introduced prediction models for preeclampsia throughout pregnancy. These models can be useful to identify women at risk during the first trimester who could benefit from aspirin treatment or later in pregnancy to inform patient management. Relative to prediction performance at 8 to 15+6 weeks, there was a substantial improvement in the detection rate for preterm and term preeclampsia by using data collected after 20 and 32 weeks' gestation, respectively. The inclusion of plasma soluble endoglin improves the early prediction of superimposed preeclampsia, which may be valuable when Doppler velocimetry of the uterine arteries is not available.

Toward a new taxonomy of obstetrical disease: improved performance of maternal blood biomarkers for the great obstetrical syndromes when classified according to placental pathology.

BACKGROUND: The major challenge for obstetrics is the prediction and prevention of the great obstetrical syndromes. We propose that defining obstetrical diseases by the combination of clinical presentation and disease mechanisms as inferred by placental pathology will aid in the discovery of biomarkers and add specificity to those already known. OBJECTIVE: To describe the longitudinal profile of placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and the PlGF/sFlt-1 ratio throughout gestation, and to determine whether the association between abnormal biomarker profiles and obstetrical syndromes is strengthened by information derived from placental examination, eg, the presence or absence of placental lesions of maternal vascular malperfusion. STUDY DESIGN: This retrospective case cohort study was based on a parent cohort of 4006 pregnant women enrolled prospectively. The case cohort of 1499 pregnant women included 1000 randomly selected patients from the parent cohort and all additional patients with obstetrical syndromes from the parent cohort. Pregnant women were classified into six groups: 1) term delivery without pregnancy complications (n=540; control); 2) preterm labor and delivery (n=203); 3) preterm premature rupture of the membranes (n=112); 4) preeclampsia (n=230); 5) small-for-gestational-age neonate (n=334); and 6) other pregnancy complications (n=182). Maternal plasma concentrations of PlGF and sFlt-1 were determined by enzyme-linked immunosorbent assays in 7560 longitudinal samples. Placental pathologists, masked to clinical outcomes, diagnosed the presence or absence of placental lesions of maternal vascular malperfusion. Comparisons between mean biomarker concentrations in cases and controls were performed by utilizing longitudinal generalized additive models. Comparisons were made between controls and each obstetrical syndrome with and without subclassifying cases according to the presence or absence of placental lesions of maternal vascular malperfusion. RESULTS: 1) When obstetrical syndromes are classified based on the presence or absence of placental lesions of maternal vascular malperfusion, significant differences in the mean plasma concentrations of PlGF, sFlt-1, and the PlGF/sFlt-1 ratio between cases and controls emerge earlier in gestation; 2) the strength of association between an abnormal PlGF/sFlt-1 ratio and the occurrence of obstetrical syndromes increases when placental lesions of maternal vascular malperfusion are present (adjusted odds ratio [aOR], 13.6 vs 6.7 for preeclampsia; aOR, 8.1 vs 4.4 for small-for-gestational-age neonates; aOR, 5.5 vs 2.1 for preterm premature rupture of the membranes; and aOR, 3.3 vs 2.1 for preterm labor (all P<0.05); and 3) the PlGF/sFlt-1 ratio at 28 to 32 weeks of gestation is abnormal in patients who subsequently delivered due to preterm labor with intact membranes and in those with preterm premature rupture of the membranes if both groups have placental lesions of maternal vascular malperfusion. Such association is not significant in patients with these obstetrical syndromes who do not have placental lesions. CONCLUSION: Classification of obstetrical syndromes according to the presence or absence of placental lesions of maternal vascular malperfusion allows biomarkers to be informative earlier in gestation and enhances the strength of association between biomarkers and clinical outcomes. We propose that a new taxonomy of obstetrical disorders informed by placental pathology will facilitate the discovery and implementation of biomarkers as well as the prediction and prevention of such disorders.

Methods for Monitoring Risk of Hypoxic Damage in Fetal and Neonatal Brains: A Review.

Fetal, perinatal, and neonatal asphyxia are vital health issues for the most vulnerable groups in human beings, including fetuses, newborns, and infants. Severe reduction in oxygen and blood supply to the fetal brain can cause hypoxic-ischemic encephalopathy (HIE), leading to long-term neurological disorders, including mental impairment and cerebral palsy. Such neurological disorders are major healthcare concerns. Therefore, there has been a continuous effort to develop clinically useful diagnostic tools for accurately and quantitatively measuring and monitoring blood and oxygen supply to the fetal and neonatal brain to avoid severe consequences of asphyxia HIE and neonatal encephalopathy. Major diagnostic technologies used for this purpose include fetal heart rate monitoring, fetus scalp blood sampling, ultrasound imaging, magnetic resonance imaging, X-ray computed tomography, and nuclear medicine. In addition, given the limitations and shortcomings of traditional diagnostic methods, emerging technologies such as near-infrared spectroscopy and photoacoustic imaging have also been introduced as stand-alone or complementary solutions to address this critical gap in fetal and neonatal care. This review provides a thorough overview of the traditional and emerging technologies for monitoring fetal and neonatal brain oxygenation status and describes their clinical utility, performance, advantages, and disadvantages.

RNA Sequencing Reveals Distinct Immune Responses in the Chorioamniotic Membranes of Women with Preterm Labor and Microbial or Sterile Intra-amniotic Inflammation.

Preterm labor precedes premature birth, the leading cause of neonatal morbidity and mortality worldwide. Preterm labor can occur in the context of either microbe-associated intra-amniotic inflammation (i.e., intra-amniotic infection) or intra-amniotic inflammation in the absence of detectable microorganisms (i.e., sterile intra-amniotic inflammation). Both intra-amniotic infection and sterile intra-amniotic inflammation trigger local immune responses that have deleterious effects on fetal life. Yet, the extent of such immune responses in the fetal tissues surrounding the amniotic cavity (i.e., the chorioamniotic membranes) is poorly understood. By using RNA sequencing (RNA seq) as a discovery approach, we found that there were significant transcriptomic differences involving host response to pathogens in the chorioamniotic membranes of women with intra-amniotic infection compared to those from women without inflammation. In addition, the sterile or microbial nature of intra-amniotic inflammation was associated with distinct transcriptomic profiles in the chorioamniotic membranes. Moreover, the immune response in the chorioamniotic membranes of women with sterile intra-amniotic inflammation was milder in nature than that induced by microbes and involved the upregulation of alarmins and inflammasome-related molecules. Lastly, the presence of maternal and fetal inflammatory responses in the placenta was associated with the upregulation of immune processes in the chorioamniotic membranes. Collectively, these findings provide insight into the immune responses against microbes or alarmins that take place in the fetal tissues surrounding the amniotic cavity, shedding light on the immunobiology of preterm labor and birth.

Vaginal host immune-microbiome interactions in a cohort of primarily African-American women who ultimately underwent spontaneous preterm birth or delivered at term.

BACKGROUND: Recent studies suggest that alterations in the vaginal microbiome allow for the assessment of the risk for spontaneous preterm birth (PTB), the leading cause of neonatal morbidity and mortality worldwide. However, the associations between the local immune response and the vaginal microbiome are still poorly understood. Herein, we characterize the vaginal host immune-microbiome interactions in women who ultimately underwent PTB and in those who delivered at term. METHODS: Vaginal fluid samples from 52 pregnant women (of whom 18 underwent PTB and 34 delivered at term) were collected between 10 and 32 weeks of gestation in a case-control study. Concentrations of 33 immune mediators were determined using sensitive and specific immunoassays. The previously published 16S rRNA gene sequence and bacterial phylotype data of these subjects were utilized in this study. Linear mixed effects models were utilized to test associations between vaginal immune mediator concentrations and bacterial phylotype relative abundances. RESULTS: 1) In the overall study population, vaginal concentrations of CXCL10, CCL2, CCL3, SLP1 and VEGF negatively correlated with non-Lactobacillus, Community State Type IV (CST IV) members of the vaginal microbiome; 2) CXCL10, in particular, negatively correlated with 15 bacterial phylotypes, most of which are typical members of CST IV, such as Gardnerella vaginalis, Megasphaera spp., and Atopobium vaginae; 3) Gemella spp., also members of CST IV, negatively correlated with vaginal concentrations of VEGF, CCL2, CCL3, SLPI, and CXCL10; 4) when comparing PTB cases to term controls, five soluble immune mediators (CCL26, CCL22, CCL2, CXCL10, and IL-16), especially CCL26, were negatively correlated with five typical members of CST IV: Sneathia sanguinegens, Parvimonas micra, Veillonellaceae, BVAB2, and Gemella spp.; and 5) Sneathia sanguinegens had stronger negative associations with all five soluble immune mediators (CCL26, CCL22, CCL2, CXCL10, and IL-16) in PTB cases than in term controls. CONCLUSIONS: The assessment of vaginal host immune-microbiome interactions revealed that specific soluble immune mediators, mainly CXCL10, negatively correlated with typical members of CST IV of the vaginal microbiome. Sneathia sanguinegens, in particular, had stronger negative associations with different immune mediators, including CXCL10 and CCL26, in women who ultimately underwent PTB compared to those who delivered at term. These findings provide insight into the vaginal host immune-microbiome interactions in normal and complicated pregnancies.

Personalized assessment of cervical length improves prediction of spontaneous preterm birth: a standard and a percentile calculator.

BACKGROUND: A sonographic short cervix (length <25 mm during midgestation) is the most powerful predictor of preterm birth. Current clinical practice assumes that the same cervical length cutoff value should apply to all women when screening for spontaneous preterm birth, yet this approach may be suboptimal. OBJECTIVE: This study aimed to (1) create a customized cervical length standard that considers relevant maternal characteristics and gestational age at sonographic examination and (2) assess whether the customization of cervical length evaluation improves the prediction of spontaneous preterm birth. STUDY DESIGN: This retrospective analysis comprises a cohort of 7826 pregnant women enrolled in a longitudinal protocol between January 2006 and April 2017 at the Detroit Medical Center. Study participants met the following inclusion criteria: singleton pregnancy, ≥1 transvaginal sonographic measurements of the cervix, delivery after 20 weeks of gestation, and available relevant demographics and obstetrical history information. Data from women without a history of preterm birth or cervical surgery who delivered at term without progesterone treatment (N=5188) were used to create a customized standard of cervical length. The prediction of the primary outcome, spontaneous preterm birth at <37 weeks of gestation, was assessed in a subset of pregnancies (N=7336) that excluded cases with induced labor before 37 weeks of gestation. Area under the receiver operating characteristic curve and sensitivity at a fixed false-positive rate were calculated for screening at 20 to 23 6/7, 24 to 27 6/7, 28 to 31 6/7, and 32 to 35 6/7 weeks of gestation in asymptomatic patients. Survival analysis was used to determine which method is better at predicting imminent delivery among symptomatic women. RESULTS: The median cervical length remained fundamentally unchanged until 20 weeks of gestation and subsequently decreased nonlinearly with advancing gestational age among women who delivered at term. The effects of parity and maternal weight and height on the cervical length were dependent on the gestational age at ultrasound examination (interaction, P<.05 for all). Parous women had a longer cervix than nulliparous women, and the difference increased with advancing gestation after adjusting for maternal weight and height. Similarly, maternal weight was nonlinearly associated with a longer cervix, and the effect was greater later in gestation. The sensitivity at a 10% false-positive rate for prediction of spontaneous preterm birth at <37 weeks of gestation by a short cervix ranged from 29% to 40% throughout pregnancy, yet it increased to 50%, 50%, 53%, and 54% at 20 to 23 6/7, 24 to 27 6/7, 28 to 31 6/7, and 32 to 35 6/7 weeks of gestation, respectively, for a low, customized percentile (McNemar test, P<.001 for all). When a cervical length <25 mm was compared to the customized screening at 20 to 23 6/7 weeks of gestation by using a customized percentile cutoff value that ensured the same negative likelihood ratio for both screening methods, the customized approach had a significantly higher (about double) positive likelihood ratio in predicting spontaneous preterm birth at <33, <34, <35, <36, and <37 weeks of gestation. Among symptomatic women, the difference in survival between women with a customized cervical length percentile of ≥10th and those with a customized cervical length percentile of <10th was greater than the difference in survival between women with a cervical length ≥25 mm and those with a cervical length <25 mm. CONCLUSION: Compared to the use of a cervical length <25 mm, a customized cervical length assessment (1) identifies more women at risk of spontaneous preterm birth and (2) improves the distinction between patients at risk for impending preterm birth in those who have an episode of preterm labor.

Fetal T Cell Activation in the Amniotic Cavity during Preterm Labor: A Potential Mechanism for a Subset of Idiopathic Preterm Birth.

Prematurity is the leading cause of perinatal morbidity and mortality worldwide. In most cases, preterm birth is preceded by spontaneous preterm labor, a syndrome that is associated with intra-amniotic inflammation, the most studied etiology. However, the remaining etiologies of preterm labor are poorly understood; therefore, most preterm births are categorized as idiopathic. In this study, we provide evidence showing that the fetal immune system undergoes premature activation in women with preterm labor without intra-amniotic inflammation, providing a potential new mechanism of disease for some cases of idiopathic preterm birth. First, we showed that fetal T cells are a predominant leukocyte population in amniotic fluid during preterm gestations. Interestingly, only fetal CD4+ T cells were increased in amniotic fluid of women who underwent idiopathic preterm labor and birth. This increase in fetal CD4+ T cells was accompanied by elevated amniotic fluid concentrations of T cell cytokines such as IL-2, IL-4, and IL-13, which are produced by these cells upon in vitro stimulation, but was not associated with the prototypical cytokine profile observed in women with intra-amniotic inflammation. Also, we found that cord blood T cells, mainly CD4+ T cells, obtained from women with idiopathic preterm labor and birth displayed enhanced ex vivo activation, which is similar to that observed in women with intra-amniotic inflammation. Finally, we showed that the intra-amniotic administration of activated neonatal CD4+ T cells induces preterm birth in mice. Collectively, these findings provide evidence suggesting that fetal T cell activation is implicated in the pathogenesis of idiopathic preterm labor and birth.

Effector and Activated T Cells Induce Preterm Labor and Birth That Is Prevented by Treatment with Progesterone.

Preterm labor commonly precedes preterm birth, the leading cause of perinatal morbidity and mortality worldwide. Most research has focused on establishing a causal link between innate immune activation and pathological inflammation leading to preterm labor and birth. However, the role of maternal effector/activated T cells in the pathogenesis of preterm labor/birth is poorly understood. In this study, we first demonstrated that effector memory and activated maternal T cells expressing granzyme B and perforin are enriched at the maternal-fetal interface (decidua) of women with spontaneous preterm labor. Next, using a murine model, we reported that prior to inducing preterm birth, in vivo T cell activation caused maternal hypothermia, bradycardia, systemic inflammation, cervical dilation, intra-amniotic inflammation, and fetal growth restriction, all of which are clinical signs associated with preterm labor. In vivo T cell activation also induced B cell cytokine responses, a proinflammatory macrophage polarization, and other inflammatory responses at the maternal-fetal interface and myometrium in the absence of an increased influx of neutrophils. Finally, we showed that treatment with progesterone can serve as a strategy to prevent preterm labor/birth and adverse neonatal outcomes by attenuating the proinflammatory responses at the maternal-fetal interface and cervix induced by T cell activation. Collectively, these findings provide mechanistic evidence showing that effector and activated T cells cause pathological inflammation at the maternal-fetal interface, in the mother, and in the fetus, inducing preterm labor and birth and adverse neonatal outcomes. Such adverse effects can be prevented by treatment with progesterone, a clinically approved strategy.

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.

Disorders of placental villous maturation are present in one-third of cases with spontaneous preterm labor.

OBJECTIVES: Spontaneous preterm labor is an obstetrical syndrome accounting for approximately 65-70% of preterm births, the latter being the most frequent cause of neonatal death and the second most frequent cause of death in children less than five years of age worldwide. The purpose of this study was to determine and compare to uncomplicated pregnancies (1) the frequency of placental disorders of villous maturation in spontaneous preterm labor; (2) the frequency of other placental morphologic characteristics associated with the preterm labor syndrome; and (3) the distribution of these lesions according to gestational age at delivery and their severity. METHODS: A case-control study of singleton pregnant women was conducted that included (1) uncomplicated pregnancies (controls, n=944) and (2) pregnancies with spontaneous preterm labor (cases, n=438). All placentas underwent histopathologic examination. Patients with chronic maternal diseases (e.g., chronic hypertension, diabetes mellitus, renal disease, thyroid disease, asthma, autoimmune disease, and coagulopathies), fetal malformations, chromosomal abnormalities, multifetal gestation, preeclampsia, eclampsia, preterm prelabor rupture of the fetal membranes, gestational hypertension, gestational diabetes mellitus, and HELLP (hemolysis, elevated liver enzymes and low platelet count) syndrome were excluded from the study. RESULTS: Compared to the controls, the most prevalent placental lesions among the cases were the disorders of villous maturation (31.8% [106/333] including delayed villous maturation 18.6% [62/333] vs. 1.4% [6/442], q<0.0001, prevalence ratio 13.7; and accelerated villous maturation 13.2% [44/333] vs. 0% [0/442], q<0.001). Other lesions in decreasing order of prevalence included hypercapillarized villi (15.6% [68/435] vs. 3.5% [33/938], q<0.001, prevalence ratio 4.4); nucleated red blood cells (1.1% [5/437] vs. 0% [0/938], q<0.01); chronic inflammatory lesions (47.9% [210/438] vs. 29.9% [282/944], q<0.0001, prevalence ratio 1.6); fetal inflammatory response (30.1% [132/438] vs. 23.2% [219/944], q<0.05, prevalence ratio 1.3); maternal inflammatory response (45.5% [195/438] vs. 36.1% [341/944], q<0.01, prevalence ratio 1.2); and maternal vascular malperfusion (44.5% [195/438] vs. 35.7% [337/944], q<0.01, prevalence ratio 1.2). Accelerated villous maturation did not show gestational age-dependent association with any other placental lesion while delayed villous maturation showed a gestational age-dependent association with acute placental inflammation (q-value=0.005). CONCLUSIONS: Disorders of villous maturation are present in nearly one-third of the cases of spontaneous preterm labor.

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.

Cellular immune responses in amniotic fluid of women with preterm clinical chorioamnionitis.

OBJECTIVE: Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. Some preterm births are associated with clinical chorioamnionitis; yet, this condition has been poorly investigated. Herein, we characterized the amniotic fluid cellular immune responses in women with preterm clinical chorioamnionitis. METHODS AND SUBJECTS: Amniotic fluid samples were obtained from women with preterm clinical chorioamnionitis and a positive or negative microbiological culture (n = 17). The cellular composition of amniotic fluid was evaluated using fluorescence microscopy, scanning and transmission electron microscopy, and flow cytometry. Women without preterm clinical chorioamnionitis were also examined (n = 10). RESULTS: Amniotic fluid from women with preterm clinical chorioamnionitis and a positive culture had: (1) abundant neutrophils associated with viable and non-viable bacteria, (2) neutrophils performing phagocytosis, (3) neutrophils forming NETs, (4) increased numbers of neutrophils, monocytes/macrophages, and CD4+ T cells, and (5) high expression of IL-1ß by neutrophils and monocytes/macrophages. Amniotic fluid from women with preterm clinical chorioamnionitis and proven infection tended to have fewer monocytes/macrophages and CD4+ T cells compared to those without chorioamnionitis. CONCLUSION: We provide the first morphologic and phenotypic characterization of the cellular immune responses in the amniotic cavity of women with preterm clinical chorioamnionitis, a condition associated with adverse neonatal outcomes.

Placental delayed villous maturation is associated with evidence of chronic fetal hypoxia.

Background Normal development of the human placenta, referred to as villous tree maturation, entails formation of the vasculosyncytial membranes. These structures develop by the approximation of syncytiotrophoblasts with the villous capillary endothelium and constitute the most efficient sites of gaseous exchange in the placenta. Defective maturation of the villous tree can lead to deficient vasculosyncytial membranes, implicated in the high incidence of hypoxic complications. Hypoxia, in turn, can stimulate production of erythropoietin, whereby increased fetal plasma or amniotic fluid concentrations of this hormone reflect fetal hypoxemia. The current study was undertaken to determine whether delayed villous maturation is associated with changes in amniotic fluid erythropoietin concentrations. Methods Placental histologic examination was performed using hematoxylin and eosin. Subsequent to histologic assessment of delayed villous maturation, the diagnosis was confirmed with CD-15 immunohistochemistry. The controls (n = 61) were pregnancies without villous maturation abnormalities, and cases (n = 5) were pregnancies with delayed villous maturation. Amniotic fluid erythropoietin concentrations were measured using a specific immunoassay. Results Concentrations of erythropoietin in the amniotic fluid (1) of controls were less than the limit of detection and (2) of cases with delayed villous maturation were significantly higher than those of controls (P-value = 0.048). Conclusion Delayed villous maturation is associated with higher concentrations of amniotic fluid erythropoietin.

Pregnancy-specific transcriptional changes upon endotoxin exposure in mice.

Objectives Pregnant women are more susceptible to certain infections; however, this increased susceptibility is not fully understood. Herein, systems biology approaches were utilized to elucidate how pregnancy modulates tissue-specific host responses to a bacterial product, endotoxin. Methods Pregnant and non-pregnant mice were injected with endotoxin or saline on 16.5 days post coitum (n=8-11 per group). The uterus, cervix, liver, adrenal gland, kidney, lung, and brain were collected 12 h after injection and transcriptomes were measured using microarrays. Heatmaps and principal component analysis were used for visualization. Differentially expressed genes between groups were assessed using linear models that included interaction terms to determine whether the effect of infection differed with pregnancy status. Pathway analysis was conducted to interpret gene expression changes. Results We report herein a multi-organ atlas of the transcript perturbations in pregnant and non-pregnant mice in response to endotoxin. Pregnancy strongly modified the host responses to endotoxin in the uterus, cervix, and liver. In contrast, pregnancy had a milder effect on the host response to endotoxin in the adrenal gland, lung, and kidney. However, pregnancy did not drastically affect the host response to endotoxin in the brain. Conclusions Pregnancy imprints organ-specific host immune responses upon endotoxin exposure. These findings provide insight into the host-response against microbes during pregnancy.

Cellular immune responses in amniotic fluid of women with a sonographic short cervix.

Objectives A sonographic short cervix is one of the strongest predictors of preterm delivery. However, the cellular immune composition of amniotic fluid in women with a short cervix has not yet been described. Herein, we determined cellular and soluble immune responses in amniotic fluid from pregnant women with a mid-trimester asymptomatic short cervix. Methods Amniotic fluid samples (n=77) were collected from asymptomatic women with a cervical length between 15 and 25 mm (n=36, short cervix) or ≤15 mm (n=41, severely short cervix) diagnosed by ultrasound. Flow cytometry and multiplex measurement of cytokines/chemokines were performed. Results (1) The cellular immune composition of amniotic fluid did not differ between women with a severely short cervix (≤15 mm) and those with a short cervix 15-25 mm; (2) amniotic fluid concentrations of multiple cytokines/chemokines were higher in women with a severely short cervix (≤15 mm) than in those with a short cervix 15-25 mm; (3) the cellular immune composition of amniotic fluid did not differ between women with a severely short cervix (≤15 mm) who ultimately underwent preterm delivery and those who delivered at term; and (4) amniotic fluid concentrations of IL-2, but not other immune mediators, were increased in women with a severely short cervix (≤15 mm) who ultimately delivered preterm compared to those who delivered at term. Conclusions Women with a severely short cervix (≤15 mm) have increased concentrations of pro-inflammatory mediators in the amniotic cavity; yet, these do not translate to changes in the cellular immune response.

Cellular immune responses in amniotic fluid of women with preterm prelabor rupture of membranes.

Background Preterm birth is the leading cause of perinatal morbidity and mortality. Preterm prelabor rupture of membranes (pPROM) occurs in 30% of preterm births; thus, this complication is a major contributor to maternal and neonatal morbidity. However, the cellular immune responses in amniotic fluid of women with pPROM have not been investigated. Methods Amniotic fluid samples were obtained from women with pPROM and a positive (n = 7) or negative (n = 10) microbiological culture. Flow cytometry was performed to evaluate the phenotype and number of amniotic fluid leukocytes. The correlation between amniotic fluid immune cells and an interleukin-6 (IL-6) concentration or a white blood cell (WBC) count in amniotic fluid was calculated. Results Women with pPROM and a positive amniotic fluid culture had (1) a greater number of total leukocytes in amniotic fluid, including neutrophils and monocytes/macrophages and (2) an increased number of total T cells in amniotic fluid, namely CD4+ T cells and CD8+ T cells, but not B cells. The numbers of neutrophils and monocytes/macrophages were positively correlated with IL-6 concentrations and WBC counts in amniotic fluid of women with pPROM. Conclusion Women with pPROM and a positive amniotic fluid culture exhibit a more severe cellular immune response than those with a negative culture, which is associated with well-known markers of intra-amniotic inflammation.

Disorders of placental villous maturation in fetal death.

Objective The aims of this study were to ascertain the frequency of disorders of villous maturation in fetal death and to also delineate other placental histopathologic lesions in fetal death. Methods This was a retrospective observational cohort study of fetal deaths occurring among women between January 2004 and January 2016 at Hutzel Women's Hospital, Detroit, MI, USA. Cases comprised fetuses with death beyond 20 weeks' gestation. Fetal deaths with congenital anomalies and multiple gestations were excluded. Controls included pregnant women without medical/obstetrical complications and delivered singleton, term (37-42 weeks) neonate with 5-min Apgar score ≥7 and birthweight between the 10th and 90th percentiles. Results Ninety-two percent (132/143) of placentas with fetal death showed placental histologic lesions. Fetal deaths were associated with (1) higher frequency of disorders of villous maturation [44.0% (64/143) vs. 1.0% (4/405), P < 0.0001, prevalence ratio, 44.6; delayed villous maturation, 22% (31/143); accelerated villous maturation, 20% (28/143); and maturation arrest, 4% (5/143)]; (2) higher frequency of maternal vascular malperfusion lesions [75.5% (108/143) vs. 35.7% (337/944), P < 0.0001, prevalence ratio, 2.1] and fetal vascular malperfusion lesions [88.1% (126/143) vs. 19.7% (186/944), P < 0.0001, prevalence ratio, 4.5]; (3) higher frequency of placental histologic patterns suggestive of hypoxia [59.0% (85/143) vs. 9.3% (82/942), P < 0.0001, prevalence ratio, 6.8]; and (4) higher frequency of chronic inflammatory lesions [53.1% (76/143) vs. 29.9% (282/944), P < 0.001, prevalence ratio 1.8]. Conclusion This study demonstrates that placentas of women with fetal death were 44 times more likely to present disorders of villous maturation compared to placentas of those with normal pregnancy. This suggests that the burden of placental disorders of villous maturation lesions is substantial.

Microbial burden and inflammasome activation in amniotic fluid of patients with preterm prelabor rupture of membranes.

Background Intra-amniotic inflammation, which is associated with adverse pregnancy outcomes, can occur in the presence or absence of detectable microorganisms, and involves activation of the inflammasome. Intra-amniotic inflammasome activation has been reported in clinical chorioamnionitis at term and preterm labor with intact membranes, but it has not yet been investigated in women with preterm prelabor rupture of membranes (preterm PROM) in the presence/absence of detectable microorganisms. The aim of this study was to determine whether, among women with preterm PROM, there is an association between detectable microorganisms in amniotic fluid and intra-amniotic inflammation, and whether intra-amniotic inflammasome activation correlates with microbial burden. Methods Amniotic fluids from 59 cases of preterm PROM were examined for the presence/absence of microorganisms through culture and 16S ribosomal RNA (rRNA) gene quantitative real-time polymerase chain reaction (qPCR), and concentrations of interleukin-6 (IL-6) and ASC [apoptosis-associated spec-like protein containing a caspase recruitment domain (CARD)], an indicator of inflammasome activation, were determined. Results qPCR identified more microbe-positive amniotic fluids than culture. Greater than 50% of patients with a negative culture and high IL-6 concentration in amniotic fluid yielded a positive qPCR signal. ASC concentrations were greatest in patients with high qPCR signals and elevated IL-6 concentrations in amniotic fluid (i.e. intra-amniotic infection). ASC concentrations tended to increase in patients without detectable microorganisms but yet with elevated IL-6 concentrations (i.e. sterile intra-amniotic inflammation) compared to those without intra-amniotic inflammation. Conclusion qPCR is a valuable complement to microbiological culture for the detection of microorganisms in the amniotic cavity in women with preterm PROM, and microbial burden is associated with the severity of intra-amniotic inflammatory response, including inflammasome activation.

Inhibition of the NLRP3 inflammasome can prevent sterile intra-amniotic inflammation, preterm labor/birth, and adverse neonatal outcomes†.

Sterile intra-amniotic inflammation is commonly observed in patients with spontaneous preterm labor, a syndrome that commonly precedes preterm birth, the leading cause of perinatal morbidity and mortality worldwide. However, the mechanisms leading to sterile intra-amniotic inflammation are poorly understood and no treatment exists for this clinical condition. Herein, we investigated whether the alarmin S100B could induce sterile intra-amniotic inflammation by activating the NLRP3 inflammasome, and whether the inhibition of this pathway could prevent preterm labor/birth and adverse neonatal outcomes. We found that the ultrasound-guided intra-amniotic administration of S100B induced a 50% rate of preterm labor/birth and a high rate of neonatal mortality (59.7%) without altering the fetal and placental weights. Using a multiplex cytokine array and immunoblotting, we reported that S100B caused a proinflammatory response in the amniotic cavity and induced the activation of the NLRP3 inflammasome in the fetal membranes, indicated by the upregulation of the NLRP3 protein and increased release of active caspase-1 and mature IL-1ß. Inhibition of the NLRP3 inflammasome via the specific inhibitor MCC950 prevented preterm labor/birth by 35.7% and reduced neonatal mortality by 26.7%. Yet, inhibition of the NLRP3 inflammasome at term did not drastically obstruct the physiological process of parturition. In conclusion, the data presented herein indicate that the alarmin S100B can induce sterile intra-amniotic inflammation, preterm labor/birth, and adverse neonatal outcomes by activating the NLRP3 inflammasome, which can be prevented by inhibiting such a pathway. These findings provide evidence that sterile intra-amniotic inflammation could be treated by targeting the NLRP3 inflammasome.

The origin of amniotic fluid monocytes/macrophages in women with intra-amniotic inflammation or infection.

Background Monocytes, after neutrophils, are the most abundant white blood cells found in the amniotic cavity of women with intra-amniotic inflammation/infection. However, the origin of such cells has not been fully investigated. Herein, we determined (1) the origin of amniotic fluid monocytes/macrophages from women with intra-amniotic inflammation/infection, (2) the relationship between the origin of amniotic fluid monocytes/macrophages and preterm or term delivery and (3) the localization of monocytes/macrophages in the placental tissues. Methods Amniotic fluid samples (n = 16) were collected from women with suspected intra-amniotic inflammation or infection. Amniotic fluid monocytes/macrophages were purified by fluorescence-activated cell sorting, and DNA fingerprinting was performed. Blinded placental histopathological evaluations were conducted. Immunohistochemistry was performed to detect CD14+ monocytes/macrophages in the placental tissues. Results DNA fingerprinting revealed that (1) 56.25% (9/16) of amniotic fluid samples had mostly fetal monocytes/macrophages, (2) 37.5% (6/16) had predominantly maternal monocytes/macrophages and (3) one sample (6.25% [1/16]) had a mixture of fetal and maternal monocytes/macrophages. (4) Most samples with predominantly fetal monocytes/macrophages were from women who delivered early preterm neonates (77.8% [7/9]), whereas all samples with mostly maternal monocytes/macrophages or a mixture of both were from women who delivered term or late preterm neonates (100% [7/7]). (5) Most of the women included in this study presented acute maternal and fetal inflammatory responses in the placenta (85.7% [12/14]). (6) Women who had mostly fetal monocytes/macrophages in amniotic fluid had abundant CD14+ cells in the umbilical cord and chorionic plate, whereas women with mostly maternal amniotic fluid monocytes/macrophages had abundant CD14+ cells in the chorioamniotic membranes. Conclusion Amniotic fluid monocytes/macrophages can be of either fetal or maternal origin, or a mixture of both, in women with intra-amniotic inflammation or infection. These immune cells could be derived from the fetal and maternal vasculature of the placenta.