Risk-Factor Based Lead Screening and Correlation with Blood Lead Levels in Pregnancy.

OBJECTIVES: Lead exposure has devastating neurologic consequences for children and may begin in utero. The American College of Obstetricians and Gynecologists recommends prenatal lead screening using a risk factor-based approach rather than universal blood testing. The clinical utility of this approach has not been studied. We evaluated a risk-factor based questionnaire to detect elevated blood lead levels in pregnancy. METHODS: We performed a secondary analysis of a cohort of parturients enrolled to evaluate the association of lead with hypertensive disorders of pregnancy. We included participants in this analysis if they had a singleton pregnancy ≥ 34 weeks' gestation with blood lead levels recorded. Participants completed a lead risk factor survey modified for pregnancy. We defined elevated blood lead as ≥ 2 µg/dL, as this was the clinically reportable level. RESULTS: Of 102 participants enrolled in the cohort, 92 had blood lead measured as part of the study. The vast majority (78%) had 1 or more risk factor for elevated lead using the questionnaire yet none had clinical blood lead testing during routine visits. Only two participants (2.2%) had elevated blood lead levels. The questionnaire had high sensitivity but poor specificity for predicting detectable lead levels (sensitivity 100%, specificity 22%). CONCLUSIONS FOR PRACTICE: Prenatal risk-factor based lead screening appears underutilized in practice and does not adequately discriminate between those with and without elevated blood levels. Given the complexity of the risk factor-based approach and underutilization, the benefit and cost-effectiveness of universal lead testing should be further explored.

AP39, a Modulator of Mitochondrial Bioenergetics, Reduces Antiangiogenic Response and Oxidative Stress in Hypoxia-Exposed Trophoblasts: Relevance for Preeclampsia Pathogenesis.

Although the cause of preeclampsia, a pregnancy complication with significant maternal and neonatal morbidity, has not been fully characterized, placental ischemia attributable to impaired spiral artery remodeling and abnormal secretion of antiangiogenic factors are thought to be important in the pathogenesis of the disease. Placental ischemia could impair trophoblast mitochondrial function and energy production, leading to the release of reactive oxygen species (ROS). ROS have been shown to stabilize hypoxia-inducible factor (HIF)-1α, which, in turn, may induce transcription of antiangiogenic factors, soluble fms-like tyrosine kinase 1 (sFLT1), and soluble endoglin in trophoblasts. Herein, we tested whether the angiogenic imbalance and oxidative stress in the preeclamptic placenta may be prevented by improving mitochondrial function. First, to evaluate the cause-effect relationship between mitochondrial function and sFLT1 production, a human trophoblast primary cell culture model was established in which hypoxia induced mitochondrial ROS production and concurrent sFLT1 increase. Second, treatment with AP39, a novel mitochondria-targeted hydrogen sulfide donor, prevented ROS production, reduced HIF-1α protein levels, and diminished sFLT1 production. Finally, AP39, a modulator of mitochondrial bioenergetics enhanced cytochrome c oxidase activity, reversed oxidative stress and antiangiogenic response in hypoxic trophoblasts. These results suggest that placental hypoxia induces ROS production, HIF-1α stabilization, and sFLT1 up-regulation; these pathophysiological alterations can be attenuated by mitochondrial-targeted antioxidants.

Placenta accreta spectrum: biomarker discovery using plasma proteomics.

BACKGROUND: Many cases of placenta accreta spectrum are not diagnosed antenatally, despite identified risk factors and improved imaging methods. Identification of plasma protein biomarkers could further improve the antenatal diagnosis of placenta accreta spectrum . OBJECTIVE: The purpose of this study was to determine if women with placenta accreta spectrum have a distinct plasma protein profile compared with control subjects. STUDY DESIGN: We obtained plasma samples before delivery from 16 participants with placenta accreta spectrum and 10 control subjects with similar gestational ages (35.1 vs 35.5 weeks gestation, respectively). We analyzed plasma samples with an aptamer-based proteomics platform for alterations in 1305 unique proteins. Heat maps of the most differentially expressed proteins (T test, P<.01) were generated with matrix visualization and analysis software. Principal component analysis was performed with the use of all 1305 proteins and the top 21 dysregulated proteins. We then confirmed dysregulated proteins using enzyme-linked immunosorbent assay and report significant differences between placenta accreta spectrum and control cases (Wilcoxon-rank sum test, P<.05). RESULTS: Many of the top 50 proteins that significantly dysregulated in participants with placenta accreta spectrum were inflammatory cytokines, factors that regulate vascular remodeling, and extracellular matrix proteins that regulate invasion. Placenta accreta spectrum, with the use of the top 21 proteins, distinctly separated the placenta accreta spectrum cases from control cases (P<.01). Using enzyme-linked immunosorbent assay, we confirmed 4 proteins that were dysregulated in placenta accreta spectrum compared with control cases: median antithrombin III concentrations (240.4 vs 150.3 mg/mL; P=.002), median plasminogen activator inhibitor 1 concentrations (4.1 vs 7.1 ng/mL; P<.001), soluble Tie2 (13.5 vs 10.4 ng/mL; P=.02), soluble vascular endothelial growth factor receptor 2 (9.0 vs 5.9 ng/mL; P=.003). CONCLUSION: Participants with placenta accreta spectrum had a unique and distinct plasma protein signature.

Epidemiology and Mechanisms of De Novo and Persistent Hypertension in the Postpartum Period.

BACKGROUND: The pathophysiology of hypertension in the immediate postpartum period is unclear. METHODS AND RESULTS: We studied 988 consecutive women admitted to a tertiary medical center for cesarean section of a singleton pregnancy. The angiogenic factors soluble fms-like tyrosine kinase 1 and placental growth factor, both biomarkers associated with preeclampsia, were measured on antepartum blood samples. We then performed multivariable analyses to determine factors associated with the risk of developing postpartum hypertension. Of the 988 women, 184 women (18.6%) developed postpartum hypertension. Of the 184 women, 77 developed de novo hypertension in the postpartum period, and the remainder had a hypertensive disorder of pregnancy in the antepartum period. A higher body mass index and history of diabetes mellitus were associated with the development of postpartum hypertension. The antepartum ratio of soluble fms-like tyrosine kinase 1 to placental growth factor positively correlated with blood pressures in the postpartum period (highest postpartum systolic blood pressure [r=0.29, P<0.001] and diastolic blood pressure [r=0.28, P<0.001]). Moreover, the highest tertile of the antepartum ratio of soluble fms-like tyrosine kinase 1 to placental growth factor was independently associated with postpartum hypertension (de novo hypertensive group: odds ratio, 2.25; 95% confidence interval, 1.19-4.25; P=0.01; in the persistent hypertensive group: odds ratio, 2.61; 95% confidence interval, 1.12-6.05; P=0.02) in multivariable analysis. Women developing postpartum hypertension had longer hospitalizations than those who remained normotensive (6.5±3.5 versus 5.7±3.4 days; P<0.001). CONCLUSIONS: Hypertension in the postpartum period is relatively common and is associated with prolonged hospitalization. Women with postpartum hypertension have clinical risk factors and an antepartum plasma angiogenic profile similar to those found in women with preeclampsia. These data suggest that women with postpartum hypertension may represent a group of women with subclinical or unresolved preeclampsia.

Sequential plasma angiogenic factors levels in women with suspected preeclampsia.

BACKGROUND: Alterations in circulating angiogenic factors are associated with the diagnosis of preeclampsia and correlate with adverse perinatal outcomes during the third trimester. OBJECTIVE: Analysis of the sequential levels of plasma angiogenic factors among patients admitted for evaluation of preeclampsia. STUDY DESIGN: We performed an observational study among women with singleton pregnancies admitted to Beth Israel Deaconess Medical Center, Boston, Massachusetts, for evaluation of preeclampsia at less than 37 weeks of gestation. Plasma samples were collected on admission and daily for the first 3 days and then weekly until delivery. Doppler ultrasound was performed on admission (within 48 hours) and then weekly (within 24 hours of blood collection) to evaluate uteroplacental and umbilical blood flows. Maternal demographics, hospital course, mode of delivery, diagnosis of hypertensive disorder, adverse maternal outcomes (elevated liver function enzymes, low platelet count, pulmonary edema, cerebral hemorrhage, convulsion, acute renal insufficiency, or maternal death), and adverse fetal/neonatal outcomes (small for gestational age, abnormal umbilical artery Doppler, fetal death, and neonatal death) were recorded. Circulating angiogenic factors (soluble fms-like tyrosine kinase and placental growth factor were measured on automated platform in a single batch after delivery and in a blinded fashion. Data are presented as median (25th to 75th centile), mean, or proportions as appropriate. RESULTS: During the study period, data from 100 women were analyzed for the study, and 43 had adverse outcomes. Women with adverse outcomes had lower gestational age of delivery, higher systolic and diastolic blood pressures during hospitalization, and lower birthweight and placental weight (all P < .01). These patients had higher soluble fms-like tyrosine kinase and soluble fms-like tyrosine kinase/placental growth factor ratio on admission and continued to have an increase in levels throughout hospital course. The median (25th to 75th) soluble fms-like tyrosine kinase/placental growth factor ratio among patients with adverse outcomes was 205.9 (72.5, 453.1) versus 47.5 (9.7, 87.0) among women without adverse outcomes (P < .001). The median (25th to 75th) absolute change per day in soluble fms-like tyrosine kinase levels (pg/mL) was 491.0 pg/mL (120.3, 1587.2) among women with adverse outcomes versus 81.3 pg/mL (-177.9, 449.0) among women without adverse outcomes (P = .01). Similarly the absolute change per day for soluble fms-like tyrosine kinase/placental growth factor ratio was 15.1 (1.8, 58.1) versus 2.7 (-0.6, 8.3) between the two groups (P = .004). The mean (range) days from admission to delivery was 6 (0-35) among subjects with soluble fms-like tyrosine kinase/placental growth factor ratio ≥85 and 14 (0-39) below a ratio of 85 (P < .001). The positive predictive value for plasma soluble fms-like tyrosine kinase/placental growth factor ratio ≥85 at admission for indicated delivery within 2 weeks was 91% (83-99%). Admission plasma soluble fms-like tyrosine kinase/placental growth factor ratio positively correlated with pre-delivery uterine artery resistive index (r = 0.35; P = .004). CONCLUSION: Among women admitted for evaluation of preeclampsia, women at risk for adverse pregnancy outcomes have higher soluble fms-like tyrosine kinase/placental growth factor ratio on admission, which continued to rise until delivery. Women with high soluble fms-like tyrosine kinase/placental growth factor ratios delivered sooner than women with low soluble fms-like tyrosine kinase/placental growth factor levels. These data support the hypothesis that targeting angiogenic imbalance in preeclampsia may lead to prolongation of pregnancy.

Ouabain inhibits placental sFlt1 production by repressing HSP27-dependent HIF-1α pathway.

Up-regulation of placental soluble fms-like tyrosine kinase 1 (sFlt1) contributes to the pathogenesis of preeclampsia. To evaluate novel upstream pathways that regulate placental sFlt1 production, we screened a library of natural compounds (n=502) in human placental cell lines. Here, we report 3 compounds in the cardiac glycoside family, ouabain, gitoxigenin, and digitoxin, that inhibit placental sFlt1 production at nanomolar concentrations in vitro. We further characterized ouabain and demonstrated that it inhibits sFlt1 mRNA and protein expression in human placental cytotrophoblasts and explant cultures in a dose- and time-dependent manner. Ouabain down-regulated sFlt1 production by inhibiting hypoxia-inducible factor 1 (HIF-1α) protein expression in the placenta. Furthermore, we found that phosphorylation of heat-shock protein 27 (HSP27) was necessary for ouabain to inhibit HIF-1α translation. In a rat model of pregnancy-induced hypertension, ouabain reduced mean arterial pressure and enhanced placental HSP27 phosphorylation without any adverse effects on pups. Further studies are needed to explore the usefulness of targeting HIF-1α/HSP27 pathway in preeclampsia.

Modeling risk for severe adverse outcomes using angiogenic factor measurements in women with suspected preterm preeclampsia.

INTRODUCTION: Preeclampsia (PE) is a pregnancy-specific syndrome associated with adverse maternal and fetal outcomes. Patient-specific risks based on angiogenic factors might better categorize those who might have a severe adverse outcome. METHODS: Women evaluated for suspected PE at a tertiary hospital (2009-2012) had pregnancy outcomes categorized as 'referent' or 'severe', based solely on maternal/fetal findings. Outcomes that may have been influenced by a PE diagnosis were considered 'unclassified'. Soluble fms-like tyrosine kinase (sFlt1) and placental growth factor (PlGF) were subjected to bivariate discriminant modeling, allowing patient-specific risks to be assigned for severe outcomes. RESULTS: Three hundred twenty-eight singleton pregnancies presented at ≤34.0 weeks' gestation. sFlt1 and PlGF levels were adjusted for gestational age. Risks above 5 : 1 (10-fold over background) occurred in 77% of severe (95% CI 66 to 87%) and 0.7% of referent (95% CI <0.1 to 3.8%) outcomes. Positive likelihood ratios for the modeling and validation datasets were 19 (95% CI 6.2-58) and 15 (95% CI 5.8-40) fold, respectively. CONCLUSIONS: This validated model assigns patient-specific risks of any severe outcome among women attending PE triage. In practice, women with high risks would receive close surveillance with the added potential for reducing unnecessary preterm deliveries among remaining women. © 2015 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd.

Angiogenic factors and the risk of adverse outcomes in women with suspected preeclampsia.

BACKGROUND: An imbalance in circulating angiogenic factors plays a central role in the pathogenesis of preeclampsia. METHODS AND RESULTS: We prospectively studied 616 women who were evaluated for suspected preeclampsia. We measured plasma levels of antiangiogenic soluble fms-like tyrosine kinase 1 (sFlt1) and proangiogenic placental growth factor (PlGF) at presentation and examined for an association between the sFlt1/PlGF ratio and subsequent adverse maternal and perinatal outcomes within 2 weeks. The median sFlt1/PlGF ratio at presentation was elevated in participants who experienced any adverse outcome compared with those who did not (47.0 [25th-75th percentile, 15.5-112.2] versus 10.8 [25th-75th percentile, 4.1-28.6]; P<0.0001). Among those presenting at <34 weeks (n=167), the results were more striking (226.6 [25th-75th percentile, 50.4-547.3] versus 4.5 [25th-75th percentile, 2.0-13.5]; P<0.0001), and the risk was markedly elevated when the highest sFlt1/PlGF ratio tertile was compared with the lowest (odds ratio, 47.8; 95% confidence interval, 14.6-156.6). Among participants presenting at <34 weeks, the addition of sFlt1/PlGF ratio to hypertension and proteinuria significantly improved the prediction for subsequent adverse outcomes (area under the curve, 0.93 for hypertension, proteinuria, and sFlt1/PlGF versus 0.84 for hypertension and proteinuria alone; P=0.001). Delivery occurred within 2 weeks of presentation in 86.0% of women with an sFlt1/PlGF ratio ≥85 compared with 15.8% of women with an sFlt1/PlGF ratio <85 (hazard ratio, 15.2; 95% confidence interval, 8.0-28.7). CONCLUSIONS: In women with suspected preeclampsia presenting at <34 weeks, circulating sFlt1/PlGF ratio predicts adverse outcomes occurring within 2 weeks. The accuracy of this test is substantially better than that of current approaches and may be useful in risk stratification and management. Additional studies are warranted to validate these findings.

Nitroxide-HMP-Protects Human Trophoblast HTR-8/SVneo Cells from H2O2-Induced Oxidative Stress by Reducing the HIF1A Signaling Pathway.

Preeclampsia (PE) is a pregnancy-specific syndrome affecting 5-7% of patients. There is no effective treatment available. Early abnormal placental development is associated with oxidative stress (OS) and a release of reactive oxygen species (ROS) in the placenta. This phenomenon leads to downstream signaling, Hypoxia Inducible Factor 1A (HIF1A) stabilization and transcription of the anti-angiogenic factors soluble fms-like tyrosine kinase 1 (sFLT1) and soluble endoglin (sEng), which are known to cause endothelial and trophoblast dysfunction and cardinal features of PE: hypertension, proteinuria and, in severe cases, eclampsia. We tested whether 3-(Hydroxymethyl)-1-oxy-2,2,5,5-tetramethylpyrrolidine (HMP)-a nitroxide-type antioxidant molecule-can reduce placental OS and mitigate PE symptoms in vitro. We induced OS in human trophoblast (HTR-8/SVneo) cells with hydrogen peroxide (H2O2) and assessed whether modulating cell redox function with HMP reduces cell injury, mitochondrial stress and HIF1A and sFLT1 production. Pre-treatment with HMP reduced mitochondrial-derived ROS production, restored LC3B expression and reduced HIF1A and sFLT1 expression in H2O2-exposed HTR-8/SVneo trophoblast cells. HMP improved the mitochondrial electron chain enzyme activity, indicating that a reduction in OS alleviates mitochondrial stress and also reduces anti-angiogenic responses. In reducing placental trophoblast OS, HMP presents a potential novel therapeutic approach for the treatment of PE. Future investigation is warranted regarding the in vivo use of HMP.

A Novel Dual-Function Redox Modulator Relieves Oxidative Stress and Anti-Angiogenic Response in Placental Villus Explant Exposed to Hypoxia-Relevance for Preeclampsia Therapy.

BACKGROUND: Preeclampsia (PE) is a severe, life-threatening complication during pregnancy (~5-7%), and no causative treatment is available. Early aberrant spiral artery remodeling is associated with placental stress and the release of oxygen radicals and other reactive oxygen species (ROS) in the placenta. This precedes the production of anti-angiogenic factors, which ultimately leads to endothelial and trophoblast damage and the key features of PE. We tested whether a novel dual-function redox modulator-AKT-1005-can effectively reduce placental oxidative stress and alleviate PE symptoms in vitro. METHOD: Isolated human villous explants were exposed to hypoxia and assessed to determine whether improving cell-redox function with AKT-1005 diminished ROS production, mitochondrial stress, production of the transcription factor HIF1A, and downstream anti-angiogenic responses (i.e., sFLT1, sEng production). MitoTEMPO was used as a reference antioxidant. RESULTS: In our villous explant assays, pretreatment with AKT-1005 reduced mitochondrial-derived ROS production, reduced HIF-1A, sFLT1, and sEng protein expression, while increasing VEGF in hypoxia-exposed villous trophoblast cells, with better efficiency than MitoTEMPO. In addition, AKT-1005 improved mitochondrial electron chain enzyme activity in the stressed explant culture. CONCLUSIONS: The redox modulator AKT-1005 has the potential to intervene with oxidative stress and can be efficacious for PE therapy. Future studies are underway to assess the in vivo efficacy of HMP.

Angiogenic factors and prediction for ischemic placental disease in future pregnancies.

OBJECTIVES: Ischemic placental disease (IPD), including preeclampsia, abruption, and fetal growth restriction, often recurs in subsequent pregnancies. Angiogenic factors of placental origin have been implicated in the pathogenesis of preeclampsia, but have not been studied as predictors of IPD in subsequent pregnancies. We hypothesized that elevated angiogenic factors in an index pregnancy would be associated with recurrence of IPD. STUDY DESIGN: We conducted a retrospective cohort study of patients undergoing evaluation for preeclampsia who had angiogenic factors measured in an index pregnancy and experienced a subsequent pregnancy at the same institution. Patients with IPD in the index pregnancy were included. A high ratio of soluble fms-like tyrosine kinase 1 (sFlt1) and placental growth factor (PlGF) was defined as greater than or equal to 85. MAIN OUTCOME MEASURES: The primary outcome was IPD in a subsequent pregnancy. RESULTS: We included 109 patients in the analysis. The sFlt1/PlGF ratio was elevated in 30% of participants. Those with an elevated ratio were more likely to be nulliparous in the index pregnancy, and less likely to have chronic hypertension. The recurrence of IPD in the study was 27%, with a non-significant difference in risk based on a high sFlt-1/P1GF ratio RR 0.58 (95% CI 0.21 - 1.6) compared to a low ratio. CONCLUSIONS: A high sFlt1/P1GF ratio in an index pregnancy is not associated with a higher risk of IPD in a subsequent pregnancy. These data suggest placental angiogenic biomarkers are specific to the pregnancy and not a reflection of maternal predisposition to IPD.

Evaluation of angiogenic factors in the decision to admit women with suspected preeclampsia.

OBJECTIVE: To compare outcomes, specifically development of preeclampsia with severe features (sPE), between angiogenic biomarker-based admission and admission based on routine clinical care. STUDY DESIGN: This secondary analysis of a prospective study evaluated soluble fms-like tyrosine kinase-1 (sFlt1)/placental growth factor (PlGF) ratio in women presenting to triage for preeclampsia evaluation. Biomarkers levels were measured in samples collected from triage and analyzed retrospectively after outcomes were achieved. For this analysis patients would be hypothetically assigned to 'discharged' with a sFlt1/PlGF ratio ≤ 38 and 'admitted' with a sFlt1/PlGF ratio > 85. Development of sPE and other outcomes were then compared using the biomarker and clinical criteria for admission. RESULTS: 459 patients were included in this analysis. Using biomarker criteria, a larger proportion of patients were hypothetically discharged (67.8% vs 51.0%, p < 0.0001). A larger proportion of patients 'admitted' with a high biomarker level developed sPE (69.5% vs 40.9%, p < 0.0001). A sFlt1/PlGF ratio ≤ 38 had a negative predictive value of 96.8% for development of sPE within two weeks. CONCLUSION: Assessment of angiogenic biomarkes that 'discharges' patients with a low sFlt1/PlGF ratio and 'admits' patients with high ratio could result in reduced admissions and increased admission of patients at risk for developing sPE. Randomized trials are needed to determine the effectiveness of angiogenic biomarker use in decision making in a triage setting among women with suspected preeclampsia.

Total Versus Free Placental Growth Factor Levels in the Pathogenesis of Preeclampsia.

Elevated circulating sFLT-1 (soluble fms-like tyrosine kinase) and low levels of its ligand, PlGF (placental growth factor), are key characteristics of preeclampsia. However, it is unclear if the low levels of plasma PlGF noted during preeclampsia are due to decreased placental production of PlGF or due to binding of PlGF by increased circulating sFLT-1. Here, we describe a biochemical procedure to dissociate PlGF-sFLT-1 complex ex vivo and when used in conjunction with an immunoassay platform, demonstrate a method to measure total and free PlGF in human blood samples. Using this method, we noted that plasma free PlGF levels were significantly lower in preeclampsia (N=22) than in nonhypertensive controls (N=24; mean, 314 versus 686 pg/mL, P<0.05), but total PlGF levels were not different (mean, 822 versus 800 pg/mL, P=0.49). In contrast, total sFLT-1 levels were significantly higher in preeclampsia than in nonhypertensive controls (mean, 16 957 versus 3029 pg/mL, P<0.01) and sFLT-1 levels correlated with bound PlGF levels (bound PlGF=total PlGF-free PlGF) in these samples (r2=0.68). We confirmed these findings in an independent cohort of subjects (N=49). Furthermore, we did not detect any difference in PlGF mRNA by quantitative polymerase chain reaction or in PlGF protein expression by immunohistochemistry in preeclamptic placentas when compared with nonhypertensive controls. In contrast, sFLT-1 mRNA and protein levels were upregulated in placentas from women with preeclampsia. Taken together with prior studies, our results provide evidence that decrease in circulating PlGF noted during preeclampsia is largely mediated by excess circulating sFLT-1.

Lead exposure and association with angiogenic factors and hypertensive disorders of pregnancy.

OBJECTIVES: Lead exposure has been associated with hypertensive disorders of pregnancy. Angiogenic factors, including soluble fms-like tyrosine kinase 1 (sFlt1) and placental growth factor (PlGF), are aberrant in preeclampsia, but have not been correlated with lead levels. We evaluated the association of lead exposure with angiogenic factors. STUDY DESIGN: This cross sectional study utilized a convenience sample of singleton pregnancies ≥34 weeks' gestation. Blood lead and angiogenic factors were measured before delivery; bone lead was measured postpartum. We dichotomized bone and blood lead into the top tertile versus the bottom tertiles and used log-binomial regression to assess the association between lead and a high angiogenic ratio. MAIN OUTCOME MEASURES: The outcomes were high sFlt1 to PlGF ratio and development of a hypertensive disorder of pregnancy. RESULTS: We enrolled 102 participants, of whom 98 had at least one lead measurement and an angiogenic factor result. Median bone lead was 3.8 ug/g (2.0 - 6.6) and median blood lead was 0.2 ug/dL (0.2 - 0.4). Incidence of hypertensive disorders of pregnancy was 31%. When comparing the highest tertile of bone lead to the bottom two tertiles, there was no association with a high sFlt1/PlGF ratio or hypertensive disorders of pregnancy. Similar results were observed for the exposure of blood lead. CONCLUSIONS: Lead exposure was not an important contributor to an elevated angiogenic factor ratio or hypertensive disorders of pregnancy in our U.S. POPULATION: However, lead exposure was modest in our population and we cannot exclude a relationship with hypertensive disorders of pregnancy.

Placental sFLT1 is associated with complement activation and syncytiotrophoblast damage in preeclampsia.

The immune complement system protects against pathogens; however, excess activation results in disease like hemolytic uremic syndrome, a clinical imitator of preeclampsia. Vascular endothelial factor (VEGF) protects against aberrant complement activation and is inhibited by soluble fms-like tyrosine kinase-1 (sFLT1) in other organs. We hypothesize that sFLT1 promotes complement-mediated placental damage through VEGF inhibition in preeclampsia. Objective: Quantify placental complement activity and sFLT1 expression in preeclampsia, and the subgroup of preeclampsia with hemolysis elevated liver enzymes low platelets (HELLP) syndrome. Methods: Placental complement activation marker C4d, membrane attack complex (MAC), and sFLT1 expression was quantified using immunofluores cence microscopy. Results: Placentas from 18 controls, 25 preeclampsia, including 6 cases of HELLP syndrome were identified. Placental C4d expression was greater in PE (median 6.4 [IQR: 5.1, 8.3]) compared to controls (4.4 [3.6, 5.5]; p = 0.003). MAC expression was also increased in preeclampsia compared to controls (6.5 [5.8, 8.7]; 5.4 [2.9, 5.9], p = 0.001). Placental sFLT1 expression was also higher in preeclampsia (p <0.0001). C4d and MAC were strongly correlated with sFLT1 levels in the placenta (R = 0.72; p < 0.0001 and R = 0.59; p = 0.01, respectively). Complement and sFLT1 expression was elevated in HELLP compared to preeclampsia without laboratory abnormalities, but this difference did not reach statistical significance. Conclusion: Increased placental complement activation and damage was seen in preeclampsia and correlates with sFLT1 expression. Our findings support the importance of the complement pathway in preeclampsia.

Analytical validation of soluble fms-like tyrosine and placental growth factor assays on B·R·A·H·M·S KRYPTOR Compact Plus automated immunoassay platform.

BACKGROUND: Preeclampsia is one of the leading hypertensive disorders of pregnancy. Angiogenic biomarkers such as anti-angiogenic factor soluble fms-like tyrosine kinase 1 (sFlt1) and pro-angiogenic factor placental growth factor (PlGF) are involved in the pathophysiology of preeclampsia. OBJECTIVE: The aim of this study is to validate the analytical performance of sFlt1 and PlGF on the B·R·A·H·M·S KRYPTOR Compact Plus (ThermoFisher Scientific). STUDY DESIGN: We examined K2-EDTA plasma samples from 50 patients on B·R·A·H·M·S KRYPTOR Compact Plus, an automated immunoassay platform. QC materials were used to assess intra- and inter-precision of the assay. Lower limit of quantitation and interference studies were determined using pooled patient plasma. RESULTS: The sFlt1 and PlGF assays demonstrated an analytical measuring range of 90-69,000 pg/mL and 11-7000 pg/mL, respectively (r2 > 0.99). Lower limit of quantitation (20% CV) was interpolated to be 35 pg/mL for sFlt1 and 10 pg/mL for PlGF. Total precision for both assay displayed CVs of <10%. Interference studies showed that both assays were not significantly affected by hemolysis up to an H-index of 1100 for sFlt1 and 300 for PlGF; L- and I-index of 800 and 80 respectively for both assays. The Passing-Bablok regression analysis for sFlt1/PlGF yielded an equation of y = 1.05x + 0.02, and the Bland Altman analysis showed an average bias of 0.84. CONCLUSION: Plasma levels of sFlt1 and PlGF measured on the B·R·A·H·M·S KRYPTOR Compact Plus platform demonstrate excellent analytical performance and are acceptable as clinical grade assays.

Angiogenic biomarkers in triage and risk for preeclampsia with severe features.

There is an urgent need for biomarkers that can help stratify women with suspected preeclampsia (PE) for the subsequent appearance of PE with severe features (sPE), to improve risk assessment and direct monitoring related to complications of sPE. Elevated levels of circulating anti-angiogenic factors like soluble fms-like tyrosine kinase 1 (sFlt1) and decreased free levels of pro-angiogenic factors such as placental growth factor (PlGF) are associated with adverse outcomes related to preeclampsia (PE). Here, we report in a single-center prospective study (N = 402) that plasma levels of these circulating angiogenic markers predict sPE within two weeks among women presenting with suspected PE in the preterm period (<37 weeks). sFlt1/PlGF ratio of >38 at the triage visit had a positive predictive value (PPV) of 47% and a negative predictive value (NPV) of 98% for the presence of sPE within 2 weeks. Among patients presenting <34 weeks, the PPV for sPE improved to 65% with NPV of 98%. sFlt1/PlGF ratio >85, had a PPV of 59% in all patients and 74% among patients presenting <34 weeks for the presence of sPE within 2 weeks. When we restricted the analysis to hospitalized patients, PPVs were 58% and 63% in all patients and 73% and 77% for patients presenting <34 weeks for plasma sFlt1/PlGF cutoff >38 and >85 respectively. Clinical trials are needed to evaluate whether risk stratification with angiogenic biomarkers in patients with suspected PE will lead to improved maternal and fetal outcomes among women at risk for severe disease.

Diagnostic utility of soluble fms-like tyrosine kinase 1 and soluble endoglin in hypertensive diseases of pregnancy.

OBJECTIVE: The objective of this pilot study was to evaluate the clinical utility of soluble fms-like tyrosine kinase 1 (sFlt 1) and soluble endoglin (sEng) in the differential diagnosis of hypertension in late pregnancy. STUDY DESIGN: We analyzed serum levels of sFlt 1 and sEng in women with gestational hypertension (GHTN; n = 17), chronic hypertension (CHTN; n = 19), preeclampsia (n = 19), and normal pregnancy (n = 20) in the third trimester. We calculated the sensitivity, specificity, and positive and negative likelihood ratio (LR) for each factor in diagnosing preeclampsia. RESULTS: The sensitivity and specificity of sFlt 1 in differentiating preeclampsia from normal pregnancy were 90% and 90%, respectively, and 90% and 95% for sEng. In women with GHTN, they were 79% and 88% for sFlt 1; 84% and 88% for sEng; 90% and 63% for uric acid. In women with CHTN, they were 84% and 95% for sFlt 1; 84% and 79% for sEng; 68%; and 78% for uric acid. The positive LR for preeclampsia was 9 for sFlt 1 and 7 for sEng in women with normal pregnancy; in women with GHTN; 6.7 for sFlt 1 and 7.2 for sEng; in CHTN, 16 for sFlt 1 and 4 for sEng. Serum uric acid had a positive LR of only 2.4 in women with GHTN and 3.1 in women with CHTN. CONCLUSION: Both sFlt 1 and sEng may prove useful in differentiating preeclampsia from other hypertensive diseases of pregnancy. A prospective cohort study should be performed determine the clinical utility of measuring these proteins.

Relationship between hypoxia and downstream pathogenic pathways in preeclampsia.

Defects in angiogenesis and mitochondrial function in the placenta contribute to the pathogenesis of preeclampsia; however upstream regulators of these pathways are not known. It has been argued that placental hypoxia secondary to abnormal spiral artery remodeling may play a causal role in the angiogenic and mitochondrial abnormalities noted in preeclampsia. The aim of this study was to evaluate the relationship between hypoxia-inducible factor-1α (HIF-1α) ¸ a surrogate of hypoxia, and soluble fms-tyrosine kinase 1 (sFlt1), a circulating anti-angiogenic factor, and microRNA 210 (miR-210), a microRNA that regulates mitochondrial function, in human placentas from preeclamptic and non-hypertensive pregnancies. We first confirmed a 2.5-fold upregulation of HIF-1α protein in placentas from preeclampsia when compared to non-hypertensive controls. Consistent with prior studies, we also observed a 10-fold upregulated sFlt1 mRNA and 2-fold upregulated miR-210 in preeclamptic tissue. Interestingly, while sFlt1 mRNA correlated with miR-210 in preeclampsia (R2 = 0.77, p = 0.0004), there were no significant correlations between these molecules and HIF1α expression. We conclude that non-hypoxia pathways may be involved in the abnormal angiogenic and metabolic alterations noted in preeclampsia.

Placental soluble fms-like tyrosine kinase expression in small for gestational age infants and risk for adverse outcomes.

INTRODUCTION: Soluble fms-like tyrosine kinase 1 (sFLT-1) is an anti-angiogenic factor implicated in the pathogenesis of preterm preeclampsia. We evaluated sFLT-1 expression and placental pathology in pregnancies complicated by small for gestational age (SGA) infants (<10th percentile), without evidence of preeclampsia. METHODS: Clinical and histologic data were compared between groups with high or low sFLT-1 expression determined by immunohistochemistry on archived placentas. RESULTS: Nineteen of 69 placentas showed high sFLT-1 expression. The high sFLT-1 group had higher predelivery median systolic blood pressure (BP); 140 (interquartile range (IQR) 133-152) vs. 126 (118-139) mm Hg (p = 0.003), and median diastolic BP; 87 (78-94) vs. 77.5 (71-86) mm Hg (p = 0.02). Abnormal umbilical Doppler abnormalities were more prevalent; 89.5% vs. 46% (p = 0.001). These pregnancies delivered earlier; 31.9 weeks (28.3-34.7 weeks) vs. 37.1 weeks (33.7-38.7 weeks) (p < 0.001), and infants had lower birthweight; 980 grams (520-1545 grams) vs. 2087.5 grams (1455-2340 grams) (p < 0.001). Placental-weight to fetal-weight ratios, a marker of vascular insufficiency, was increased in the high sFlt-1 group: 0.18 (0.14-0.28) vs 0.15 (0.13-0.18), p = 0.03. Placentas with high sFLT-1 showed more decidual vasculopathy; 42.1% vs. 10.0% (p = 0.005), infarction; 36.8% vs. 14.0% (p = 0.048), distal villous hypoplasia; 78.9% vs. 36.0% (p = 0.001), and fetal thrombotic vasculopathy; 47.4% vs. 16.0% (p = 0.01). DISCUSSION: Placental sFLT-1 expression is upregulated in approximately 28% of non-preeclamptic pregnancies complicated by SGA infants. These pregnancies showed increased placental vascular pathology, more umbilical Doppler abnormalities, and earlier delivery with lower birthweight. A subgroup of non-preeclamptic fetal growth restriction with upregulated sFlt-1 expression may share a common pathogenic pathway with preterm preeclampsia. This subgroup is worthy of additional study.