Preeclampsia at term: evidence of disease heterogeneity based on the profile of circulating cytokines and angiogenic factors.

BACKGROUND: Intravascular inflammation and an antiangiogenic state have been implicated in the pathophysiology of preeclampsia. On the basis of the profiles of their angiogenic/antiangiogenic factors, women with preeclampsia at term may be classified into 2 subgroups with different characteristics and prevalence of adverse outcomes. This study was undertaken to examine whether these 2 subgroups of preeclampsia at term also show differences in their profiles of intravascular inflammation. OBJECTIVE: This study aimed to determine the plasma profiles of cytokines and chemokines in women with preeclampsia at term who had a normal or an abnormal angiogenic profile. STUDY DESIGN: A nested case-control study was conducted to include women classified into 3 groups: women with an uncomplicated pregnancy (n=213) and women with preeclampsia at term with a normal (n=55) or an abnormal (n=41) angiogenic profile. An abnormal angiogenic profile was defined as a plasma ratio of placental growth factor and soluble fms-like tyrosine kinase-1 multiple of the median <10th percentile for gestational age. Concentrations of cytokines were measured by multiplex immunoassays. RESULTS: Women with preeclampsia at term and an abnormal angiogenic profile showed evidence of the greatest intravascular inflammation among the study groups. These women had higher plasma concentrations of 5 cytokines (interleukin-6, interleukin-8, interleukin-12/interleukin-23p40, interleukin-15, and interleukin-16) and 7 chemokines (eotaxin, eotaxin-3, interferon-γ inducible protein-10, monocyte chemotactic protein-4, macrophage inflammatory protein-1ß, macrophage-derived chemokine, and thymus and activation-regulated chemokine compared to women with an uncomplicated pregnancy. By contrast, women with preeclampsia at term and a normal angiogenic profile, compared to women with an uncomplicated pregnancy, had only a higher plasma concentration of monocyte chemotactic protein-4. A correlation between severity of the antiangiogenic state, blood pressure, and plasma concentrations of a subset of cytokines was observed. CONCLUSION: Term preeclampsia can be classified into 2 clusters. One is characterized by an antiangiogenic state coupled with an excessive inflammatory process, whereas the other has neither of these features. These findings further support the heterogeneity of preeclampsia at term and may explain the distinct clinical outcomes.

Preeclampsia at term can be classified into 2 clusters with different clinical characteristics and outcomes based on angiogenic biomarkers in maternal blood.

BACKGROUND: An antiangiogenic state has emerged as a mechanism of disease in preeclampsia. Angiogenic biomarkers are used in the risk assessment of this syndrome, particularly of early disease. The role of an antiangiogenic state in late preeclampsia is unclear. OBJECTIVE: This study aimed to determine the prevalence, characteristics, and clinical significance of angiogenic/antiangiogenic factor abnormalities in women with preeclampsia stratified according to gestational age at delivery. STUDY DESIGN: Two studies were conducted: (1) a longitudinal nested case-control study comprising women with preeclampsia (n=151) and a control group (n=540); and (2) a case series of patients with preeclampsia (n=452). In patients with preeclampsia, blood was collected at the time of diagnosis. Plasma concentrations of placental growth factor and soluble fms-like tyrosine kinase-1 were determined by enzyme-linked immunosorbent assays. An abnormal angiogenic profile was defined as a plasma ratio of placental growth factor and soluble fms-like tyrosine kinase-1 expressed as a multiple of the median <10th percentile for gestational age based on values derived from the longitudinal study. The proportion of patients diagnosed with preeclampsia who had an abnormal angiogenic profile was determined in the case-series participants and stratified by gestational age at delivery into early (≤34 weeks), intermediate (34.1-36.9 weeks), and term (≥37 weeks) preeclampsia. The demographics, clinical characteristics, and pregnancy outcomes of women with preeclampsia with and without an abnormal angiogenic profile were compared. RESULTS: The prevalence of an abnormal angiogenic profile was higher in preterm than in term preeclampsia (for early, intermediate, and term in the case-control study: 90%, 100%, and 39%; for the case series: 98%, 80%, and 55%, respectively). Women with preeclampsia at term who had an abnormal angiogenic profile were more frequently nulliparous (57% vs 35%), less likely to smoke (14% vs 26%), at greater risk for maternal (14% vs 5%) or neonatal (7% vs 1%) complications, and more often had placental lesions consistent with maternal vascular malperfusion (42% vs 23%; all, P<.05) than those without an abnormal profile. Women with preeclampsia at term who had a normal angiogenic profile had a higher frequency of chronic hypertension (36% vs 21%) and were more likely to have class ≥2 obesity (41% vs 23%) than those with an abnormal profile (both, P<.05). CONCLUSION: Patients with early preeclampsia had an abnormal angiogenic profile in virtually all cases, whereas only 50% of women with preeclampsia at term had such abnormalities. The profile of angiogenic biomarkers can be used to classify patients with preeclampsia at term, on the basis of mechanisms of disease, into 2 clusters, which have different demographics, clinical characteristics, and risks of adverse maternal and neonatal outcomes. These findings provide a simple approach to classify preeclampsia at term and have implications for future clinical care and research.

Perturbations in kinetics of the thrombin generation assay identify women at risk of preeclampsia in the first trimester and provide the rationale for a preventive approach.

BACKGROUND: Activation of the coagulation system and increased thrombin generation have been implicated in the pathophysiology of preeclampsia, and this rationale supports the administration of low-molecular-weight heparin to prevent this syndrome in patients at risk. Yet, randomized trials of this prophylactic measure have yielded contradictory results. A possible explanation is that only a subset of patients with preeclampsia have excessive thrombin generation and would benefit from the administration of low-molecular-weight heparin. Therefore, the key questions are whether and when patients who subsequently develop preeclampsia present evidence of abnormal thrombin generation. OBJECTIVE: This study aimed to determine (1) the kinetics of thrombin generation throughout gestation in women with a normal pregnancy and in those with early and late preeclampsia, and (2) the diagnostic performance of in vivo thrombin generation parameters to predict the development of preeclampsia. STUDY DESIGN: This retrospective, nested case-control study was based on a prospective longitudinal cohort of singleton gestations. Cases comprised women who developed preeclampsia (n=49), and controls consisted of patients with a normal pregnancy (n=45). Preeclampsia was classified into early-onset (n=24) and late-onset (n=25). Longitudinal changes in the parameters of the thrombin generation assay (lag time, time to peak thrombin concentration, peak thrombin concentration, endogenous thrombin generation, and velocity index) throughout gestation were compared between the study groups, and normal pregnancy percentiles were derived from the control group. We tested whether a single parameter or a combination of parameters, derived from the kinetics of thrombin generation, could identify patients who subsequently developed preeclampsia. Time-related parameters <10th percentile were considered short, and concentration-related parameters >90th percentile were considered high. RESULTS: (1) Patients who developed preeclampsia (early- and late-onset) had abnormal thrombin generation kinetics as early as 8 to 16 weeks of pregnancy; (2) patients with a combination of a short lag time and high peak thrombin concentration at 8 to 16 weeks of pregnancy had an odds ratio of 43.87 for the subsequent development of preeclampsia (area under the curve, 0.79; sensitivity, 56.8%; specificity, 92.7%; positive likelihood ratio, 7.76); (3) at 16 to 22 weeks of gestation, patients with a combination of a short lag time and a high velocity index had an odds ratio of 16 for the subsequent development of preeclampsia (area under the curve, 0.78; sensitivity, 62.2%; specificity, 92.5%; positive likelihood ratio, 8.29). CONCLUSION: During early pregnancy, the thrombin generation assay can identify the subset of patients at a greater risk for the development of preeclampsia owing to accelerated and enhanced production of thrombin. This observation provides a rationale for testing the efficacy of low-molecular-weight heparin in this subset of patients. We propose that future research on the efficacy of low-molecular-weight heparin and other interventions targeting the coagulation system to prevent preeclampsia should be focused on patients with abnormal kinetics of thrombin generation.

Meconium-stained amniotic fluid.

Green-stained amniotic fluid, often referred to as meconium-stained amniotic fluid, is present in 5% to 20% of patients in labor and is considered an obstetric hazard. The condition has been attributed to the passage of fetal colonic content (meconium), intraamniotic bleeding with the presence of heme catabolic products, or both. The frequency of green-stained amniotic fluid increases as a function of gestational age, reaching approximately 27% in post-term gestation. Green-stained amniotic fluid during labor has been associated with fetal acidemia (umbilical artery pH <7.00), neonatal respiratory distress, and seizures as well as cerebral palsy. Hypoxia is widely considered a mechanism responsible for fetal defecation and meconium-stained amniotic fluid; however, most fetuses with meconium-stained amniotic fluid do not have fetal acidemia. Intraamniotic infection/inflammation has emerged as an important factor in meconium-stained amniotic fluid in term and preterm gestations, as patients with these conditions have a higher rate of clinical chorioamnionitis and neonatal sepsis. The precise mechanisms linking intraamniotic inflammation to green-stained amniotic fluid have not been determined, but the effects of oxidative stress in heme catabolism have been implicated. Two randomized clinical trials suggest that antibiotic administration decreases the rate of clinical chorioamnionitis in patients with meconium-stained amniotic fluid. A serious complication of meconium-stained amniotic fluid is meconium aspiration syndrome. This condition develops in 5% of cases presenting with meconium-stained amniotic fluid and is a severe complication typical of term newborns. Meconium aspiration syndrome is attributed to the mechanical and chemical effects of aspirated meconium coupled with local and systemic fetal inflammation. Routine naso/oropharyngeal suctioning and tracheal intubation in cases of meconium-stained amniotic fluid have not been shown to be beneficial and are no longer recommended in obstetrical practice. A systematic review of randomized controlled trials suggested that amnioinfusion may decrease the rate of meconium aspiration syndrome. Histologic examination of the fetal membranes for meconium has been invoked in medical legal litigation to time the occurrence of fetal injury. However, inferences have been largely based on the results of in vitro experiments, and extrapolation of such findings to the clinical setting warrants caution. Fetal defecation throughout gestation appears to be a physiologic phenomenon based on ultrasound as well as in observations in animals.

One-third of patients with eclampsia at term do not have an abnormal angiogenic profile.

OBJECTIVES: An abnormal angiogenic profile is present in about one-half of women with preeclampsia at term. Few studies examined the roles of angiogenic biomarkers in eclampsia. The aims of this study were to determine (1) whether the degree of an anti-angiogenic state, reflected by a low placental growth factor (PlGF) to soluble fms-like tyrosine kinase-1 (sFlt-1) ratio, in women with eclampsia differed from that of women with severe preeclampsia; and (2) the prevalence of women who had an abnormal angiogenic profile at the diagnoses of preterm and term eclampsia. METHODS: A cross-sectional study was conducted to include women in the following groups: (1) uncomplicated pregnancy (n=40); (2) severe preeclampsia (n=50); and (3) eclampsia (n=35). Maternal serum concentrations of PlGF and sFlt-1 were determined by immunoassays. RESULTS: Women with preterm, but not term, eclampsia had a more severe anti-angiogenic state than those with severe preeclampsia (lower PlGF and PlGF/sFlt-1 ratio, each p<0.05). However, the difference diminished in magnitude with increasing gestational age (interaction, p=0.005). An abnormal angiogenic profile was present in 95% (19/20) of women with preterm eclampsia but in only 67% (10/15) of women with eclampsia at term. CONCLUSIONS: Angiogenic biomarkers can be used for risk assessment of preterm eclampsia. By contrast, a normal profile of angiogenic biomarkers cannot reliably exclude patients at risk for eclampsia at term. This observation has major clinical implications given that angiogenic biomarkers are frequently used in the triage area as a test to rule out preeclampsia.

Maternal plasma cytokines and the subsequent risk of uterine atony and postpartum hemorrhage.

OBJECTIVES: To determine whether the maternal plasma concentrations of cytokines are higher in pregnant women with postpartum hemorrhage (PPH) compared to pregnant women without PPH. METHODS: A retrospective case-control study included 36 women with PPH and 72 matched controls. Cases and controls were matched for gestational age at delivery, labor status, delivery route, parity, and year of sample collection. Maternal plasma samples were collected up to 3 days prior to delivery. Comparison of the plasma concentrations of 29 cytokines was performed by using linear mixed-effects models and included adjustment for covariates and multiple testing. A false discovery rate adjusted p-value <0.1 was used to infer significance. Random forest models with evaluation by leave-one-out and 9-fold cross-validation were used to assess the combined value of the proteins in predicting PPH. RESULTS: Concentrations of interleukin (IL)-16, IL-6, IL-12/IL-23p40, monocyte chemotactic protein 1 (MCP-1), and IL-1ß were significantly higher in PPH than in the control group. This difference remained significant after adjustment for maternal age, clinical chorioamnionitis, and preeclampsia. Multi-protein random forest proteomics models had moderate cross-validated accuracy for prediction of PPH [area under the ROC curve, 0.69 (0.58-0.81) by leave-one-out cross validation and 0.73 (0.65-0.81) by 9-fold cross-validation], and the inclusion of clinical and demographic information did not increase the prediction performance. CONCLUSIONS: Pregnant women with severe PPH had higher median maternal plasma concentrations of IL-16, IL-6, IL-12/IL-23p40, MCP-1, and IL-1ß than patients without PPH. These cytokines could serve as biomarkers or their pathways may be therapeutic targets.

Molecular subclasses of preeclampsia characterized by a longitudinal maternal proteomics study: distinct biomarkers, disease pathways and options for prevention.

OBJECTIVES: The heterogeneous nature of preeclampsia is a major obstacle to early screening and prevention, and a molecular taxonomy of disease is needed. We have previously identified four subclasses of preeclampsia based on first-trimester plasma proteomic profiles. Herein, we expanded this approach by using a more comprehensive panel of proteins profiled in longitudinal samples. METHODS: Proteomic data collected longitudinally from plasma samples of women who developed preeclampsia (n=109) and of controls (n=90) were available from our previous report on 1,125 proteins. Consensus clustering was performed to identify subgroups of patients with preeclampsia based on data from five gestational-age intervals by using select interval-specific features. Demographic, clinical, and proteomic differences among clusters were determined. Differentially abundant proteins were used to identify cluster-specific perturbed KEGG pathways. RESULTS: Four molecular clusters with different clinical phenotypes were discovered by longitudinal proteomic profiling. Cluster 1 involves metabolic and prothrombotic changes with high rates of early-onset preeclampsia and small-for-gestational-age neonates; Cluster 2 includes maternal anti-fetal rejection mechanisms and recurrent preeclampsia cases; Cluster 3 is associated with extracellular matrix regulation and comprises cases of mostly mild, late-onset preeclampsia; and Cluster 4 is characterized by angiogenic imbalance and a high prevalence of early-onset disease. CONCLUSIONS: This study is an independent validation and further refining of molecular subclasses of preeclampsia identified by a different proteomic platform and study population. The results lay the groundwork for novel diagnostic and personalized tools of prevention.

Further Evidence that an Episode of Premature Labor Is a Pathologic State: Involvement of the Insulin-Like Growth Factor System.

INTRODUCTION: Approximately 47% of women with an episode of preterm labor deliver at term; however, their infants are at greater risk of being small for gestational age and for neurodevelopmental disorders. In these cases, a pathologic insult may disrupt the homeostatic responses sustaining pregnancy. We tested the hypothesis of an involvement of components of the insulin-like growth factor (IGF) system. METHODS: This is a cross-sectional study in which maternal plasma concentrations of pregnancy-associated plasma protease (PAPP)-A, PAPP-A2, insulin-like growth factor-binding protein 1 (IGFBP-1), and IGFBP-4 were determined in the following groups of women: (1) no episodes of preterm labor, term delivery (controls, n = 100); (2) episode of preterm labor, term delivery (n = 50); (3) episode of preterm labor, preterm delivery (n = 100); (4) pregnant women at term not in labor (n = 61); and (5) pregnant women at term in labor (n = 61). Pairwise differences in maternal plasma concentrations of PAPP-A, PAPP-A2, IGFBP-1, and IGFBP-4 among study groups were assessed by fitting linear models on log-transformed data and included adjustment for relevant covariates. Significance of the group coefficient in the linear models was assessed via t-scores, with p < 0.05 deemed a significant result. RESULTS: Compared to controls, (1) women with an episode of premature labor, regardless of a preterm or a term delivery, had higher mean plasma concentrations of PAPP-A2 and IGFBP-1 (each p < 0.05); (2) women with an episode of premature labor who delivered at term also had a higher mean concentration of PAPP-A (p < 0.05); and (3) acute histologic chorioamnionitis and spontaneous labor at term were not associated with significant changes in these analytes. CONCLUSION: An episode of preterm labor involves the IGF system, supporting the view that the premature activation of parturition is a pathologic state, even in those women who delivered at term.

Human Plasma Proteome During Normal Pregnancy.

The human plasma proteome is underexplored despite its potential value for monitoring health and disease. Herein, using a recently developed aptamer-based platform, we profiled 7288 proteins in 528 plasma samples from 91 normal pregnancies (Gene Expression Omnibus identifier GSE206454). The coefficient of variation was <20% for 93% of analytes (median 7%), and a cross-platform correlation for selected key angiogenic and anti-angiogenic proteins was significant. Gestational age was associated with changes in 953 proteins, including highly modulated placenta- and decidua-specific proteins, and they were enriched in biological processes including regulation of growth, angiogenesis, immunity, and inflammation. The abundance of proteins corresponding to RNAs specific to populations of cells previously described by single-cell RNA-Seq analysis of the placenta was highly modulated throughout gestation. Furthermore, machine learning-based prediction of gestational age and of time from sampling to term delivery compared favorably with transcriptomic models (mean absolute error of 2 weeks). These results suggested that the plasma proteome may provide a non-invasive readout of placental cellular dynamics and serve as a blueprint for investigating obstetrical disease.

Transcriptome changes in maternal peripheral blood during term parturition mimic perturbations preceding spontaneous preterm birth†.

The complex physiologic process of parturition includes the onset of labor, which requires the orchestrated stimulation of a common pathway involving uterine contractility, cervical ripening, and chorioamniotic membrane activation. However, the labor-specific processes taking place in these tissues have limited use as predictive biomarkers unless they can be probed in non-invasive samples, such as the peripheral blood. Herein, we utilized a transcriptomic dataset to assess labor-specific changes in the peripheral blood of women who delivered at term. We identified a set of genes that were differentially expressed with labor and enriched for immunological processes, and these gene expression changes were strongly correlated with results from prior studies, providing in silico validation of our findings. We then identified significant correlations between labor-specific transcriptomic changes in the maternal circulation and those detected in the chorioamniotic membranes, myometrium, and cervix of women at term, demonstrating that tissue-specific labor signatures are partly mirrored in the peripheral blood. Finally, we demonstrated a significant overlap between the peripheral blood transcriptomic changes in term parturition and those observed in asymptomatic women, prior to the diagnosis of preterm prelabor rupture of the membranes, who ultimately delivered preterm. Collectively, we provide evidence that the normal process of labor at term is characterized by a unique immunological expression signature, which may serve as a useful tool for assessing labor status and for potentially identifying women at risk for preterm birth.

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.

Pregnancy tailors endotoxin-induced monocyte and neutrophil responses in the maternal circulation.

OBJECTIVE: To comprehensively characterize monocyte and neutrophil responses to E. coli and its product [lipopolysaccharide (LPS) or endotoxin] in vitro during pregnancy. MATERIAL OR SUBJECTS: Peripheral blood was collected from pregnant women during the third trimester (n = 20) and from non-pregnant women (n = 20). METHODS: The number, phagocytic activity, and reactive oxygen species (ROS) production of peripheral monocytes and neutrophils were investigated using flow cytometry. The phenotypes of peripheral monocytes and neutrophils after acute or chronic LPS stimulation were also determined using flow cytometry. Cytokine profiles were quantified for LPS-stimulated peripheral blood mononuclear cells (PBMCs) and a whole blood TruCulture® system using a multiplex immunoassay. RESULTS: Increased number, phagocytic activity, and ROS production capacity of monocytes and neutrophils were found in pregnant compared to non-pregnant women. Additionally, specific subsets of pro-inflammatory monocytes (IL-6+CD14+ or MIP-1α+CD14+ cells) and neutrophils (IL-1ß+CD15+ or MIP-1ß+CD15+ cells) were increased in pregnant women in response to acute LPS stimulation. Moreover, distinct subsets of intermediate-activated monocytes expressing CD142, IL-6, and IL-1RA were increased in pregnant women upon chronic LPS stimulation. Last, pregnant women displayed a different cytokine profile than non-pregnant women in LPS-stimulated PBMCs and in whole blood. CONCLUSIONS: Pregnancy tailors the immune responses of circulating monocytes and neutrophils to endotoxin, a Gram-negative bacterial product.

The role of the placenta in spontaneous preterm labor and delivery with intact membranes.

OBJECTIVES: To determine whether placental vascular pathology and impaired placental exchange due to maturational defects are involved in the etiology of spontaneous preterm labor and delivery in cases without histologic acute chorioamnionitis. METHODS: This was a retrospective, observational study. Cases included pregnancies that resulted in spontaneous preterm labor and delivery (<37 weeks), whereas uncomplicated pregnancies that delivered fetuses at term (≥37-42 weeks of gestation) were selected as controls. Placental histological diagnoses were classified into three groups: lesions of maternal vascular malperfusion, lesions of fetal vascular malperfusion, and placental microvasculopathy, and the frequency of each type of lesion in cases and controls was compared. Moreover, we specifically searched for villous maturational abnormalities in cases and controls. Doppler velocimetry of the umbilical and uterine arteries were performed in a subset of patients. RESULTS: There were 184 cases and 2471 controls, of which 95 and 1178 had Doppler studies, respectively. The frequency of lesions of maternal vascular malperfusion was greater in the placentas of patients with preterm labor than in the control group [14.1% (26/184) vs. 8.8% (217/2471) (p=0.023)]. Disorders of villous maturation were more frequent in the group with preterm labor than in the control group: 41.1% (39/95) [delayed villous maturation in 31.6% (30/95) vs. 2.5% (13/519) in controls and accelerated villous maturation in 9.5% (9/95) vs. none in controls]. CONCLUSIONS: Maturational defects of placental villi were associated with approximately 41% of cases of unexplained spontaneous preterm labor and delivery without acute inflammatory lesions of the placenta and with delivery of appropriate-for-gestational-age fetuses.

Lipidomic analysis of patients with microbial invasion of the amniotic cavity reveals up-regulation of leukotriene B4.

Bioactive lipids derived from the metabolism of polyunsaturated fatty acids are important mediators of the inflammatory response. Labor per se is considered a sterile inflammatory process. Intra-amniotic inflammation (IAI) due to microorganisms (i.e., intra-amniotic infection) or danger signals (i.e., sterile IAI) has been implicated in the pathogenesis of preterm labor and clinical chorioamnionitis at term. Early and accurate diagnosis of microbial invasion of the amniotic cavity (MIAC) requires analysis of amniotic fluid (AF). It is possible that IAI caused by microorganisms is associated with a stereotypic lipidomic profile, and that analysis of AF may help in the identification of patients with this condition. To test this hypothesis, we analyzed the fatty acyl lipidome of AF by liquid chromatography-mass spectrometry from patients in spontaneous labor at term and preterm gestations. We report that the AF concentrations of proinflammatory lipid mediators of the 5-lipoxygenase pathway are significantly higher in MIAC than in cases of sterile IAI. These results suggest that the concentrations of 5-lipoxygenase metabolites of arachidonic acid, 5-hydroxyeicosatetraenoic acid, and leukotriene B4 in particular could serve as potential biomarkers of MIAC. This finding could have important implications for the rapid identification of patients who may benefit from anti-microbial treatment.-Maddipati, K. R., Romero, R., Chaiworapongsa ,T., Chaemsaithong, P., Zhou, S.-L., Xu, Z., Tarca, A. L., Kusanovic, J. P., Gomez, R., Chaiyasit, N., Honn, K. V. Lipidomic analysis of patients with microbial invasion of the amniotic cavity reveals up-regulation of leukotriene B4.

The maternal plasma proteome changes as a function of gestational age in normal pregnancy: a longitudinal study.

OBJECTIVE: Pregnancy is accompanied by dramatic physiological changes in maternal plasma proteins. Characterization of the maternal plasma proteome in normal pregnancy is an essential step for understanding changes to predict pregnancy outcome. The objective of this study was to describe maternal plasma proteins that change in abundance with advancing gestational age and determine biological processes that are perturbed in normal pregnancy. STUDY DESIGN: A longitudinal study included 43 normal pregnancies that had a term delivery of an infant who was appropriate for gestational age without maternal or neonatal complications. For each pregnancy, 3 to 6 maternal plasma samples (median, 5) were profiled to measure the abundance of 1125 proteins using multiplex assays. Linear mixed-effects models with polynomial splines were used to model protein abundance as a function of gestational age, and the significance of the association was inferred via likelihood ratio tests. Proteins considered to be significantly changed were defined as having the following: (1) >1.5-fold change between 8 and 40 weeks of gestation; and (2) a false discovery rate-adjusted value of P < .1. Gene ontology enrichment analysis was used to identify biological processes overrepresented among the proteins that changed with advancing gestation. RESULTS: The following results were found: (1) Ten percent (112 of 1125) of the profiled proteins changed in abundance as a function of gestational age; (2) of the 1125 proteins analyzed, glypican-3, sialic acid-binding immunoglobulin-type lectin-6, placental growth factor, C-C motif-28, carbonic anhydrase 6, prolactin, interleukin-1 receptor 4, dual-specificity mitogen-activated protein kinase 4, and pregnancy-associated plasma protein-A had more than a 5-fold change in abundance across gestation (these 9 proteins are known to be involved in a wide range of both physiological and pathological processes, such as growth regulation, embryogenesis, angiogenesis immunoregulation, inflammation etc); and (3) biological processes associated with protein changes in normal pregnancy included defense response, defense response to bacteria, proteolysis, and leukocyte migration (false discovery rate, 10%). CONCLUSION: The plasma proteome of normal pregnancy demonstrates dramatic changes in both the magnitude of changes and the fraction of the proteins involved. Such information is important to understand the physiology of pregnancy and the development of biomarkers to differentiate normal vs abnormal pregnancy and determine the response to interventions.

The prediction of fetal death with a simple maternal blood test at 20-24 weeks: a role for angiogenic index-1 (PlGF/sVEGFR-1 ratio).

BACKGROUND: Fetal death is an obstetrical syndrome that annually affects 2.4 to 3 million pregnancies worldwide, including more than 20,000 in the United States each year. Currently, there is no test available to identify patients at risk for this pregnancy complication. OBJECTIVE: We sought to determine if maternal plasma concentrations of angiogenic and antiangiogenic factors measured at 24-28 weeks of gestation can predict subsequent fetal death. STUDY DESIGN: A case-cohort study was designed to include 1000 randomly selected subjects and all remaining fetal deaths (cases) from a cohort of 4006 women with a singleton pregnancy, enrolled at 6-22 weeks of gestation, in a pregnancy biomarker cohort study. The placentas of all fetal deaths were histologically examined by pathologists who used a standardized protocol and were blinded to patient outcomes. Placental growth factor, soluble endoglin, and soluble vascular endothelial growth factor receptor-1 concentrations were measured by enzyme-linked immunosorbent assays. Quantiles of the analyte concentrations (or concentration ratios) were estimated as a function of gestational age among women who delivered a live neonate but did not develop preeclampsia or deliver a small-for-gestational-age newborn. A positive test was defined as analyte concentrations (or ratios) <2.5th and 10th centiles (placental growth factor, placental growth factor/soluble vascular endothelial growth factor receptor-1 [angiogenic index-1] and placental growth factor/soluble endoglin) or >90th and 97.5th centiles (soluble vascular endothelial growth factor receptor-1 and soluble endoglin). Inverse probability weighting was used to reflect the parent cohort when estimating the relative risk. RESULTS: There were 11 fetal deaths and 829 controls with samples available for analysis between 24-28 weeks of gestation. Three fetal deaths occurred <28 weeks and 8 occurred ≥28 weeks of gestation. The rate of placental lesions consistent with maternal vascular underperfusion was 33.3% (1/3) among those who had a fetal death <28 weeks and 87.5% (7/8) of those who had this complication ≥28 weeks of gestation. The maternal plasma angiogenic index-1 value was <10th centile in 63.6% (7/11) of the fetal death group and in 11.1% (92/829) of the controls. The angiogenic index-1 value was <2.5th centile in 54.5% (6/11) of the fetal death group and in 3.7% (31/829) of the controls. An angiogenic index-1 value <2.5th centile had the largest positive likelihood ratio for predicting fetal death >24 weeks (14.6; 95% confidence interval, 7.7-27.7) and a relative risk of 29.1 (95% confidence interval, 8.8-97.1), followed by soluble endoglin >97.5th centile and placental growth factor/soluble endoglin <2.5th, both with a positive likelihood ratio of 13.7 (95% confidence interval, 7.3-25.8) and a relative risk of 27.4 (95% confidence interval, 8.2-91.2). Among women without a fetal death whose plasma angiogenic index-1 concentration ratio was <2.5th centile, 61% (19/31) developed preeclampsia or delivered a small-for-gestational-age neonate; when the 10th centile was used as the cut-off, 37% (34/92) of women had these adverse outcomes. CONCLUSION: (1) A maternal plasma angiogenic index-1 value <2.5th centile (0.126) at 24-28 weeks of gestation carries a 29-fold increase in the risk of subsequent fetal death and identifies 55% of subsequent fetal deaths with a false-positive rate of 3.5%; and (2) 61% of women who have a false-positive test result will subsequently experience adverse pregnancy outcomes.

Fetal death: an extreme manifestation of maternal anti-fetal rejection.

OBJECTIVE: The aim of this study was to determine the association between chronic placental inflammation and amniotic fluid (AF) markers of maternal anti-fetal rejection as well as the presence of microorganisms in the AF fluid of patients with fetal death. STUDY DESIGN: This cohort study included 40 patients with fetal death whose placentas were examined for chronic inflammatory lesions and whose AF chemokine ligand (CXCL)10 and interleukin (IL)-6 concentrations were determined by immunoassays. AF was processed for bacteria, mycoplasmas and viruses using cultivation and molecular microbiologic techniques (i.e. PCR-ESI/MS). RESULTS: (1) The most prevalent placental findings were maternal vascular underperfusion (63.2%, 24/38), followed by chronic inflammatory lesions (57.9%, 22/38); (2) chronic chorioamnionitis (18/38) was three times more frequent than villitis of unknown etiology (6/38); (3) an elevated AF CXCL10 concentration (above the 95th centile) was present in 60% of the cases, and a receiver operating characteristics (ROC)-derived cut-off of 2.9 ng/mL had a sensitivity of 73% and a specificity of 75% in the identification of chronic placental inflammatory lesions; (4) only five cases had microbial invasion of the amniotic cavity, and the presence of microorganisms did not correlate with chronic placental inflammation. CONCLUSION: In women with unexplained fetal death, there is an association between elevated AF CXCL10 and chronic placental inflammatory lesions. Therefore, we conclude that a subset of patients with fetal death may have endured a breakdown of maternal-fetal tolerance, which cannot be attributed to microorganisms in the amniotic cavity.

Clinical chorioamnionitis at term: the amniotic fluid fatty acyl lipidome.

Clinical chorioamnionitis at term (TCC) is the most common obstetrical infliction diagnosed in labor and delivery units worldwide and is associated with a substantial increase in maternal and neonatal morbidity and mortality. This obstetrical complication is a heterogeneous condition, as only half of patients have detectable microorganisms in the amniotic cavity. Because bioactive lipids play a key role in the initiation and resolution of an inflammatory response, we aimed to characterize the amniotic fluid lipidome in patients with TCC. We studied the amniotic fluid of patients in the following groups: 1) spontaneous labor at term without clinical chorioamnionitis (TLB) and 2) spontaneous labor at term with clinical chorioamnionitis (TCC). The TCC group was subdivided into a) those with microbial invasion of the amniotic cavity (TCC-MIAC) and b) those without microbial invasion of the amniotic cavity (TCC-noMIAC). The amniotic fluid concentration of proinflammatory lipid mediators did not differ between patients in TLB with TCC. In contrast, concentration of lipids with anti-inflammatory/proresolution properties was significantly lower in all patients with TCC than in those with TLB. These results suggest that while proinflammatory lipid mediators are involved in infection-driven intra-amniotic inflammation, a relative deficiency of anti-inflammatory/proresolution lipid mediator biosynthesis is a characteristic of TCC.

Maternal plasma angiogenic index-1 (placental growth factor/soluble vascular endothelial growth factor receptor-1) is a biomarker for the burden of placental lesions consistent with uteroplacental underperfusion: a longitudinal case-cohort study.

BACKGROUND: Placental lesions consistent with maternal vascular underperfusion (MVU) are thought to be pathogenically linked to preeclampsia, small-for-gestational-age newborns, fetal death, and spontaneous preterm labor and delivery; yet, these lesions cannot be diagnosed antenatally. We previously reported that patients with such conditions and lesions have an abnormal profile of the angiogenic placental growth factor (PlGF) and antiangiogenic factors (eg, soluble vascular endothelial growth factor receptor [sVEGFR]-1). OBJECTIVE: The objectives of this study were to: (1) examine the relationship between the maternal plasma PlGF/sVEGFR-1 concentration ratio (referred to herein as angiogenic index-1) and the burden of histologic placental features consistent with MVU; and (2) test the hypothesis that angiogenic index-1 can identify patients in the midtrimester who are destined to deliver before 34 weeks of gestation with multiple (ie, ≥3) histologic placental features consistent with MVU. STUDY DESIGN: A 2-stage case-cohort sampling strategy was used to select participants from among 4006 women with singleton gestations enrolled from 2006 through 2010 in a longitudinal study. Maternal plasma angiogenic index-1 ratios were determined using enzyme-linked immunosorbent assays. Placentas underwent histologic examination according to standardized protocols by experienced pediatric pathologists who were blinded to clinical diagnoses and pregnancy outcomes. The diagnosis of lesions consistent with MVU was made using criteria proposed by the Perinatal Section of the Society for Pediatric Pathology. Weighted analyses were performed to reflect the parent cohort; "n*" is used to reflect weighted frequencies. RESULTS: (1) Angiogenic index-1 (PlGF/sVEGFR-1) concentration ratios were determined in 7560 plasma samples collected from 1499 study participants; (2) the prevalence of lesions consistent with MVU was 21% (n* = 833.9/3904) and 27% (n* = 11.4/42.7) of women with ≥3 MVU lesions delivered before 34 weeks of gestation; (3) a low angiogenic index-1 (<2.5th quantile for gestational age) in maternal plasma samples obtained within 48 hours of delivery had a sensitivity of 73% (n* = 8.3/11.4; 95% confidence interval [CI], 47-98%), a specificity of 94% (n* = 3130.9/3316.2; 95% CI, 94-95%), a positive likelihood ratio of 12.2, and a negative likelihood ratio of 0.29 in the identification of patients who delivered placentas with ≥3 MVU lesions at <34 weeks; (4) prospectively, at 20-23 weeks of gestation, a maternal plasma concentration of angiogenic index-1 <2.5th quantile identified 70% (n* = 7.2/10.3; 95% CI, 42-98%) of patients who delivered placentas with ≥3 MVU lesions before 34 weeks (specificity, 97% [n* = 2831.3/2918; 95% CI, 96-98%]; positive likelihood ratio, 23; negative likelihood ratio, 0.31); and (5) among women without obstetrical complications who delivered at term, angiogenic index-1 was lower in women with than without placental lesions consistent with MVU (P < .05). CONCLUSION: Maternal plasma angiogenic index-1 (PlGF/sVEGFR-1) is the first biomarker for the burden of placental lesions consistent with MVU. We propose that an accumulation of these lesions in placentas delivered before 34 weeks is a histologic counterpart of an antiangiogenic profile.