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Maternal plasma concentrations of angiogenic/antiangiogenic factors in the third trimester of pregnancy to identify the patient at risk for stillbirth at or near term and severe late preeclampsia

  • Tinnakorn Chaiworapongsa
    Correspondence
    Correspondence to: Roberto Romero, MD, DMedSci, and Tinnakorn Chaiworapongsa, MD, Perinatology Research Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Wayne State University/Hutzel Women's Hospital, 3990 John R, Box 4, Detroit, MI 48201
    Affiliations
    Perinatology Research Branch, National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, MD, and Detroit, MI

    Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI
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  • Roberto Romero
    Correspondence
    Correspondence to: Roberto Romero, MD, DMedSci, and Tinnakorn Chaiworapongsa, MD, Perinatology Research Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Wayne State University/Hutzel Women's Hospital, 3990 John R, Box 4, Detroit, MI 48201
    Affiliations
    Perinatology Research Branch, National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, MD, and Detroit, MI
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  • Steven J. Korzeniewski
    Affiliations
    Perinatology Research Branch, National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, MD, and Detroit, MI

    Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI
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  • Juan Pedro Kusanovic
    Affiliations
    Department of Obstetrics and Gynecology, Pontificia Universidad Católica de Chile, Center for Research and Innovation in Maternal-Fetal Medicine (CIMAF), Sótero del Río Hospital, Santiago, Chile
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  • Eleazar Soto
    Affiliations
    Perinatology Research Branch, National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, MD, and Detroit, MI

    Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI
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  • Jennifer Lam
    Affiliations
    Perinatology Research Branch, National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, MD, and Detroit, MI

    Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI
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  • Zhong Dong
    Affiliations
    Perinatology Research Branch, National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, MD, and Detroit, MI
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  • Nandor G. Than
    Affiliations
    Perinatology Research Branch, National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, MD, and Detroit, MI

    Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI
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  • Lami Yeo
    Affiliations
    Perinatology Research Branch, National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, MD, and Detroit, MI

    Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI
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  • Edgar Hernandez-Andrade
    Affiliations
    Perinatology Research Branch, National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, MD, and Detroit, MI

    Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI
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  • Agustín Conde-Agudelo
    Affiliations
    Perinatology Research Branch, National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, MD, and Detroit, MI
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  • Sonia S. Hassan
    Affiliations
    Perinatology Research Branch, National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, MD, and Detroit, MI

    Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI
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Published:January 21, 2013DOI:https://doi.org/10.1016/j.ajog.2013.01.016

      Objective

      To determine whether maternal plasma concentrations of placental growth factor (PlGF), soluble endoglin (sEng), and soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) at 30-34 weeks of gestation can identify patients at risk for stillbirth, late preeclampsia, and delivery of small-for-gestational-age (SGA) neonates.

      Study Design

      A prospective cohort study included 1269 singleton pregnant women from whom blood samples were obtained at 30-34 weeks of gestation and who delivered at >34 weeks of gestation. Plasma concentrations of PlGF, sEng, and sVEGFR-1 were determined by enzyme-linked immunosorbent assay.

      Results

      The prevalence of late (>34 weeks of gestation) preeclampsia, severe late preeclampsia, stillbirth, and SGA was 3.2% (n = 40), 1.8% (n = 23), 0.4% (n = 5), and 8.5% (n = 108), respectively. A plasma concentration of PlGF/sEng <0.3 MoM was associated with severe late preeclampsia (adjusted odds ratio, 16); the addition of PlGF/sEng to clinical risk factors increased the area under the receiver-operating characteristic curve from 0.76 to 0.88 (P = .03). The ratio of PlGF/sEng or PlGF/sVEGFR-1 in the third trimester outperformed those obtained in the first or second trimester and uterine artery Doppler velocimetry at 20-25 weeks of gestation for the prediction of severe late preeclampsia (comparison of areas under the receiver-operating characteristic curve; each P ≤ .02). Both PlGF/sEng and PlGF/sVEGFR-1 ratios achieved a sensitivity of 74% with a fixed false-positive rate of 15% for the identification of severe late preeclampsia. A plasma concentration of PlGF/sVEGFR-1 <0.12 MoM at 30-34 weeks of gestation had a sensitivity of 80%, a specificity of 94%, and a likelihood ratio of a positive test of 14 for the identification of subsequent stillbirth. Similar findings (sensitivity 80%; specificity 93%) were observed in a separate case-control study.

      Conclusion

      Risk assessment for stillbirth and severe late preeclampsia in the third trimester is possible with the determination of maternal plasma concentrations of angiogenic and antiangiogenic factors at 30-34 weeks of gestation.

      Key words

      Preeclampsia, a leading cause of maternal and perinatal morbidity/mortality worldwide,
      • Romero R.
      • Lockwood C.
      • Oyarzun E.
      • Hobbins J.C.
      Toxemia: new concepts in an old disease.
      • Redman C.W.
      • Sargent I.L.
      Latest advances in understanding preeclampsia.
      • Berg C.J.
      • Callaghan W.M.
      • Syverson C.
      • Henderson Z.
      Pregnancy-related mortality in the United States, 1998 to 2005.
      • Zhang J.
      • Meikle S.
      • Trumble A.
      Severe maternal morbidity associated with hypertensive disorders in pregnancy in the United States.
      affects 2-8% of all pregnancies and has a complex pathophysiology that may include abnormal physiologic transformation of the spiral arteries,
      • Brosens I.A.
      Morphological changes in the utero-placental bed in pregnancy hypertension.
      • Brosens J.J.
      • Pijnenborg R.
      • Brosens I.A.
      The myometrial junctional zone spiral arteries in normal and abnormal pregnancies: a review of the literature.
      • Pijnenborg R.
      • Anthony J.
      • Davey D.A.
      • et al.
      Placental bed spiral arteries in the hypertensive disorders of pregnancy.
      • Robertson W.B.
      • Brosens I.
      • Dixon G.
      Maternal uterine vascular lesions in the hypertensive complications of pregnancy.
      • Brosens I.
      • Pijnenborg R.
      • Vercruysse L.
      • Romero R.
      The “Great Obstetrical Syndromes” are associated with disorders of deep placentation.
      • Romero R.
      • Kusanovic J.P.
      • Chaiworapongsa T.
      • Hassan S.S.
      Placental bed disorders in preterm labor, preterm PROM, spontaneous abortion and abruptio placentae.
      intravascular inflammation,
      • Sacks G.P.
      • Studena K.
      • Sargent K.
      • Redman C.W.
      Normal pregnancy and preeclampsia both produce inflammatory changes in peripheral blood leukocytes akin to those of sepsis.
      • Redman C.W.
      • Sacks G.P.
      • Sargent I.L.
      Preeclampsia: an excessive maternal inflammatory response to pregnancy.
      • Gervasi M.T.
      • Chaiworapongsa T.
      • Pacora P.
      • et al.
      Phenotypic and metabolic characteristics of monocytes and granulocytes in preeclampsia.
      endothelial cell dysfunction,
      • Friedman S.A.
      • Schiff E.
      • Emeis J.J.
      • Dekker G.A.
      • Sibai B.M.
      Biochemical corroboration of endothelial involvement in severe preeclampsia.
      • Lyall F.
      • Greer I.A.
      The vascular endothelium in normal pregnancy and pre-eclampsia.
      • Roberts J.M.
      • Taylor R.N.
      • Goldfien A.
      Clinical and biochemical evidence of endothelial cell dysfunction in the pregnancy syndrome preeclampsia.
      • Roberts J.M.
      Endothelial dysfunction in preeclampsia.
      • Taylor R.N.
      • de Groot C.J.
      • Cho Y.K.
      • Lim K.H.
      Circulating factors as markers and mediators of endothelial cell dysfunction in preeclampsia.
      • Chaiworapongsa T.
      • Romero R.
      • Yoshimatsu J.
      • et al.
      Soluble adhesion molecule profile in normal pregnancy and pre-eclampsia.
      • Petrozella L.
      • Mahendroo M.
      • Timmons B.
      • Roberts S.
      • McIntire D.
      • Alexander J.M.
      Endothelial microparticles and the antiangiogenic state in preeclampsia and the postpartum period.
      excessive thrombin generation,
      • de Boer K.
      • ten Cate J.W.
      • Sturk A.
      • Borm J.J.
      • Treffers P.E.
      Enhanced thrombin generation in normal and hypertensive pregnancy.
      • Chaiworapongsa T.
      • Yoshimatsu J.
      • Espinoza J.
      • et al.
      Evidence of in vivo generation of thrombin in patients with small-for-gestational-age fetuses and pre-eclampsia.
      • Cadroy Y.
      • Grandjean H.
      • Pichon J.
      • et al.
      Evaluation of six markers of haemostatic system in normal pregnancy and pregnancy complicated by hypertension or pre-eclampsia.
      • Higgins J.R.
      • Walshe J.J.
      • Darling M.R.
      • Norris L.
      • Bonnar J.
      Hemostasis in the uteroplacental and peripheral circulations in normotensive and pre-eclamptic pregnancies.
      • Hayashi M.
      • Inoue T.
      • Hoshimoto K.
      • Negishi H.
      • Ohkura T.
      • Inaba N.
      Characterization of five marker levels of the hemostatic system and endothelial status in normotensive pregnancy and pre-eclampsia.
      oxidative stress,
      • Hubel C.A.
      Oxidative stress in the pathogenesis of preeclampsia.
      • Vaughan J.E.
      • Walsh S.W.
      Oxidative stress reproduces placental abnormalities of preeclampsia.
      • Myatt L.
      • Kossenjans W.
      • Sahay R.
      • Eis A.
      • Brockman D.
      Oxidative stress causes vascular dysfunction in the placenta.
      • Zhou X.
      • Zhang G.Y.
      • Wang J.
      • Lu S.L.
      • Cao J.
      • Sun L.Z.
      A novel bridge between oxidative stress and immunity: the interaction between hydrogen peroxide and human leukocyte antigen G in placental trophoblasts during preeclampsia.
      and/or an antiangiogenic state.
      • Torry D.S.
      • Wang H.S.
      • Wang T.H.
      • Caudle M.R.
      • Torry R.J.
      Preeclampsia is associated with reduced serum levels of placenta growth factor.
      • Tidwell S.C.
      • Ho H.N.
      • Chiu W.H.
      • Torry R.J.
      • Torry D.S.
      Low maternal serum levels of placenta growth factor as an antecedent of clinical preeclampsia.
      • Maynard S.E.
      • Min J.Y.
      • Merchan J.
      • et al.
      Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia.
      • Koga K.
      • Osuga Y.
      • Yoshino O.
      • et al.
      Elevated serum soluble vascular endothelial growth factor receptor 1 (sVEGFR-1) levels in women with preeclampsia.
      • Taylor R.N.
      • Grimwood J.
      • Taylor R.S.
      • McMaster M.T.
      • Fisher S.J.
      • North R.A.
      Longitudinal serum concentrations of placental growth factor: evidence for abnormal placental angiogenesis in pathologic pregnancies.
      • Tsatsaris V.
      • Goffin F.
      • Munaut C.
      • et al.
      Overexpression of the soluble vascular endothelial growth factor receptor in preeclamptic patients: pathophysiological consequences.
      • Chaiworapongsa T.
      • Romero R.
      • Espinoza J.
      • et al.
      Evidence supporting a role for blockade of the vascular endothelial growth factor system in the pathophysiology of preeclampsia: Young Investigator Award.
      • Venkatesha S.
      • Toporsian M.
      • Lam C.
      • et al.
      Soluble endoglin contributes to the pathogenesis of preeclampsia.
      • Weissgerber T.L.
      • Roberts J.M.
      • Jeyabalan A.
      • et al.
      Haptoglobin phenotype, angiogenic factors, and preeclampsia risk.
      For Editors' Commentary, see Contents
      Preeclampsia may be classified as “early” or “late” according to gestational age at diagnosis or delivery. The gestational age cutoff most frequently used is 34 weeks.
      • von Dadelszen P.
      • Magee L.A.
      • Roberts J.M.
      Subclassification of preeclampsia.
      Early preeclampsia is associated with multisystemic involvement,
      • Romero R.
      • Vizoso J.
      • Emamian M.
      • et al.
      Clinical significance of liver dysfunction in pregnancy-induced hypertension.
      • Romero R.
      • Mazor M.
      • Lockwood C.J.
      • et al.
      Clinical significance, prevalence, and natural history of thrombocytopenia in pregnancy-induced hypertension.
      a higher frequency of small-for-gestational-age (SGA) neonates,
      • Long P.A.
      • Abell D.A.
      • Beischer N.A.
      Fetal growth retardation and pre-eclampsia.
      and placental vascular lesions of underperfusion.
      • Ogge G.
      • Chaiworapongsa T.
      • Kusanovic J.P.
      • et al.
      Evidence that placental lesions are more frequent in early-onset than in late-onset preeclampsia.
      Because early preeclampsia is a frequent indication for preterm delivery, the condition is also associated with a higher rate of neonatal morbidity.
      • Sibai B.M.
      Preeclampsia as a cause of preterm and late preterm (near-term) births.
      In contrast, preeclampsia at term is associated with better neonatal outcomes than preterm preeclampsia. Although emphasis has focused on early preeclampsia, most cases of preeclampsia occur at or near term.
      • Sibai B.
      • Dekker G.
      • Kupferminc M.
      Pre-eclampsia.
      • Ales K.L.
      • Charlson M.E.
      Epidemiology of preeclampsia and eclampsia.
      • Tuffnell D.J.
      • Jankowicz D.
      • Lindow S.W.
      • et al.
      Outcomes of severe pre-eclampsia/eclampsia in Yorkshire 1999/2003.
      Consequently, late preeclampsia accounts for a substantial proportion of medically indicated late preterm births
      • Sibai B.M.
      Preeclampsia as a cause of preterm and late preterm (near-term) births.
      • McIntire D.D.
      • Leveno K.J.
      Neonatal mortality and morbidity rates in late preterm births compared with births at term.
      • Lubow J.M.
      • How H.Y.
      • Habli M.
      • Maxwell R.
      • Sibai B.M.
      Indications for delivery and short-term neonatal outcomes in late preterm as compared with term births.
      • Ananth C.V.
      • Vintzileos A.M.
      Maternal-fetal conditions necessitating a medical intervention resulting in preterm birth.
      and severe maternal morbidity, which includes most cases of eclampsia,
      • Kenneth L.
      • Hall D.R.
      • Gebhardt S.
      • Grove D.
      Late onset preeclampsia is not an innocuous condition.
      • Liu S.
      • Joseph K.S.
      • Liston R.M.
      • et al.
      Incidence, risk factors, and associated complications of eclampsia.
      • Duley L.
      • Gulmezoglu A.M.
      • Henderson-Smart D.J.
      • Chou D.
      Magnesium sulphate and other anticonvulsants for women with pre-eclampsia.
      the form of the disease that accounts for most maternal deaths.
      • Moodley J.
      Maternal deaths associated with hypertensive disorders of pregnancy: a population-based study.
      Hence, the identification of predictors of late preeclampsia is a healthcare priority.
      Stillbirth, another obstetrical syndrome
      • Romero R.
      Prenatal medicine: the child is the father of the man.
      • Fretts R.C.
      Etiology and prevention of stillbirth.
      which may or may not be related to preeclampsia, affects >3 million women in the third trimester worldwide each year.
      • Froen J.F.
      • Cacciatore J.
      • McClure E.M.
      • et al.
      Stillbirths: why they matter.
      • Lawn J.E.
      • Yakoob M.Y.
      • Haws R.A.
      • Soomro T.
      • Darmstadt G.L.
      • Bhutta Z.A.
      3.2 million stillbirths: epidemiology and overview of the evidence review.
      • Bhutta Z.A.
      • Yakoob M.Y.
      • Lawn J.E.
      • et al.
      Stillbirths: what difference can we make and at what cost?.
      The circumstances surrounding stillbirth vary depending on socioeconomic conditions.
      • Flenady V.
      • Middleton P.
      • Smith G.C.
      • et al.
      Stillbirths: the way forward in high-income countries.
      • Lawn J.E.
      • Blencowe H.
      • Pattinson R.
      • et al.
      Stillbirths: Where? When? Why? How to make the data count?.
      In high-income countries, stillbirth is associated with fetal growth restriction or placental insufficiency; however, in nearly one-half of the cases, the cause is unknown.
      • Lawn J.E.
      • Blencowe H.
      • Pattinson R.
      • et al.
      Stillbirths: Where? When? Why? How to make the data count?.
      Intrapartum complications, preeclampsia, and infection play a more important role in the cause of stillbirth in low-income countries.
      • McClure E.M.
      • Saleem S.
      • Pasha O.
      • Goldenberg R.L.
      Stillbirth in developing countries: a review of causes, risk factors and prevention strategies.
      • Menezes E.V.
      • Yakoob M.Y.
      • Soomro T.
      • Haws R.A.
      • Darmstadt G.L.
      • Bhutta Z.A.
      Reducing stillbirths: prevention and management of medical disorders and infections during pregnancy.
      • Yakoob M.Y.
      • Lawn J.E.
      • Darmstadt G.L.
      • Bhutta Z.A.
      Stillbirths: epidemiology, evidence, and priorities for action.
      Currently, there is no effective risk assessment tool for the detection of stillbirth at or near term.
      • Flenady V.
      • Middleton P.
      • Smith G.C.
      • et al.
      Stillbirths: the way forward in high-income countries.
      • Smith G.C.
      Predicting antepartum stillbirth.
      • Hui D.
      • Okun N.
      • Murphy K.
      • Kingdom J.
      • Uleryk E.
      • Shah P.S.
      Combinations of maternal serum markers to predict preeclampsia, small for gestational age, and stillbirth: a systematic review.
      An imbalance of angiogenic/antiangiogenic factors has been implicated in the pathophysiologic description of preeclampsia,
      • Reuvekamp A.
      • Velsing-Aarts F.V.
      • Poulina I.E.
      • Capello J.J.
      • Duits A.J.
      Selective deficit of angiogenic growth factors characterises pregnancies complicated by pre-eclampsia.
      • Ahmed A.
      New insights into the etiology of preeclampsia: identification of key elusive factors for the vascular complications.
      • Luttun A.
      • Carmeliet P.
      Soluble VEGF receptor Flt1: the elusive preeclampsia factor discovered?.
      • Bujold E.
      • Romero R.
      • Chaiworapongsa T.
      • et al.
      Evidence supporting that the excess of the sVEGFR-1 concentration in maternal plasma in preeclampsia has a uterine origin.
      • Crispi F.
      • Dominguez C.
      • Llurba E.
      • Martin-Gallan P.
      • Cabero L.
      • Gratacos E.
      Placental angiogenic growth factors and uterine artery Doppler findings for characterization of different subsets in preeclampsia and in isolated intrauterine growth restriction.
      • Chaiworapongsa T.
      • Romero R.
      • Gotsch F.
      • et al.
      Low maternal concentrations of soluble vascular endothelial growth factor receptor-2 in preeclampsia and small for gestational age.
      • Chaiworapongsa T.
      • Romero R.
      • Tarca A.L.
      • et al.
      A decrease in maternal plasma concentrations of sVEGFR-2 precedes the clinical diagnosis of preeclampsia.
      • Benton S.J.
      • Hu Y.
      • Xie F.
      • et al.
      Angiogenic factors as diagnostic tests for preeclampsia: a performance comparison between two commercial immunoassays.
      • Garovic V.D.
      The role of angiogenic factors in the prediction and diagnosis of preeclampsia superimposed on chronic hypertension.
      • Ghosh S.K.
      • Raheja S.
      • Tuli A.
      • Raghunandan C.
      • Agarwal S.
      Serum PLGF as a potential biomarker for predicting the onset of preeclampsia.
      • Chaiworapongsa T.
      • Romero R.
      • Savasan Z.A.
      • et al.
      Maternal plasma concentrations of angiogenic/anti-angiogenic factors are of prognostic value in patients presenting to the obstetrical triage area with the suspicion of preeclampsia.
      • Hagmann H.
      • Thadhani R.
      • Benzing T.
      • Karumanchi S.A.
      • Stepan H.
      The promise of angiogenic markers for the early diagnosis and prediction of preeclampsia.
      • Molvarec A.
      • Szarka A.
      • Walentin S.
      • Szucs E.
      • Nagy B.
      • Rigo Jr, J.
      Circulating angiogenic factors determined by electrochemiluminescence immunoassay in relation to the clinical features and laboratory parameters in women with pre-eclampsia.
      • Rana S.
      • Powe C.E.
      • Salahuddin S.
      • et al.
      Angiogenic factors and the risk of adverse outcomes in women with suspected preeclampsia.
      • Verlohren S.
      • Stepan H.
      • Dechend R.
      Angiogenic growth factors in the diagnosis and prediction of pre-eclampsia.
      • Staff A.C.
      • Braekke K.
      • Johnsen G.M.
      • Karumanchi S.A.
      • Harsem N.K.
      Circulating concentrations of soluble endoglin (CD105) in fetal and maternal serum and in amniotic fluid in preeclampsia.
      • Smith G.C.
      • Wear H.
      The perinatal implications of angiogenic factors.
      • Weed S.
      • Bastek J.A.
      • Anton L.
      • Elovitz M.A.
      • Parry S.
      • Srinivas S.K.
      Examining the correlation between placental and serum placenta growth factor in preeclampsia.
      • Maynard S.E.
      • Karumanchi S.A.
      Angiogenic factors and preeclampsia.
      • Lam C.
      • Lim K.H.
      • Karumanchi S.A.
      Circulating angiogenic factors in the pathogenesis and prediction of preeclampsia.
      • Karumanchi S.A.
      • Maynard S.E.
      • Stillman I.E.
      • Epstein F.H.
      • Sukhatme V.P.
      Preeclampsia: a renal perspective.
      • Levine R.J.
      • Karumanchi S.A.
      Circulating angiogenic factors in preeclampsia.
      pregnancies with SGA neonates,
      • Chaiworapongsa T.
      • Espinoza J.
      • Gotsch F.
      • et al.
      The maternal plasma soluble vascular endothelial growth factor receptor-1 concentration is elevated in SGA and the magnitude of the increase relates to Doppler abnormalities in the maternal and fetal circulation.
      • Crispi F.
      • Llurba E.
      • Dominguez C.
      • Martin-Gallan P.
      • Cabero L.
      • Gratacos E.
      Predictive value of angiogenic factors and uterine artery Doppler for early- versus late-onset pre-eclampsia and intrauterine growth restriction.
      • Asvold B.O.
      • Vatten L.J.
      • Romundstad P.R.
      • Jenum P.A.
      • Karumanchi S.A.
      • Eskild A.
      Angiogenic factors in maternal circulation and the risk of severe fetal growth restriction.
      • Gourvas V.
      • Dalpa E.
      • Konstantinidou A.
      • Vrachnis N.
      • Spandidos D.A.
      • Sifakis S.
      Angiogenic factors in placentas from pregnancies complicated by fetal growth restriction [review].
      stillbirth,
      • Espinoza J.
      • Chaiworapongsa T.
      • Romero R.
      • et al.
      Unexplained fetal death: another anti-angiogenic state.
      • Chaiworapongsa T.
      • Romero R.
      • Kusanovic J.P.
      • et al.
      Unexplained fetal death is associated with increased concentrations of anti-angiogenic factors in amniotic fluid.
      • Chaiworapongsa T.
      • Kusanovic J.P.
      • Savasan Z.A.
      • et al.
      Fetal death: a condition with a dissociation in the concentrations of soluble vascular endothelial growth factor receptor-2 between the maternal and fetal compartments.
      and other obstetrical complications.
      • Bixel K.
      • Silasi M.
      • Zelop C.M.
      • et al.
      Placental origins of angiogenic dysfunction in mirror syndrome.
      • Espinoza J.
      • Romero R.
      • Nien J.K.
      • et al.
      A role of the anti-angiogenic factor sVEGFR-1 in the ‘mirror syndrome’ (Ballantyne's syndrome).
      • Kanter D.
      • Lindheimer M.D.
      • Wang E.
      • et al.
      Angiogenic dysfunction in molar pregnancy.
      • Koga K.
      • Osuga Y.
      • Tajima T.
      • et al.
      Elevated serum soluble fms-like tyrosine kinase 1 (sFlt1) level in women with hydatidiform mole.
      • Kusanovic J.P.
      • Romero R.
      • Espinoza J.
      • et al.
      Twin-to-twin transfusion syndrome: an antiangiogenic state?.
      • Llurba E.
      • Marsal G.
      • Sanchez O.
      • et al.
      Angiogenic and antiangiogenic factors before and after resolution of maternal mirror syndrome.
      • Rana S.
      • Venkatesha S.
      • DePaepe M.
      • Chien E.K.
      • Paglia M.
      • Karumanchi S.A.
      Cytomegalovirus-induced mirror syndrome associated with elevated levels of circulating antiangiogenic factors.
      Changes in the concentrations of the angiogenic factor (placental growth factor [PlGF]) and antiangiogenic factors (soluble vascular endothelial growth factor receptor-1 [sVEGFR-1; also known as soluble fms-like tyrosine kinase-1] and soluble endoglin [sEng]) in maternal circulation precede the clinical diagnosis of preeclampsia,
      • Chaiworapongsa T.
      • Romero R.
      • Kim Y.M.
      • et al.
      Plasma soluble vascular endothelial growth factor receptor-1 concentration is elevated prior to the clinical diagnosis of pre-eclampsia.
      • Moore Simas T.A.
      • Crawford S.L.
      • Solitro M.J.
      • Frost S.C.
      • Meyer B.A.
      • Maynard S.E.
      Angiogenic factors for the prediction of preeclampsia in high-risk women.
      • Erez O.
      • Romero R.
      • Espinoza J.
      • et al.
      The change in concentrations of angiogenic and anti-angiogenic factors in maternal plasma between the first and second trimesters in risk assessment for the subsequent development of preeclampsia and small-for-gestational age.
      • Romero R.
      • Nien J.K.
      • Espinoza J.
      • et al.
      A longitudinal study of angiogenic (placental growth factor) and anti-angiogenic (soluble endoglin and soluble vascular endothelial growth factor receptor-1) factors in normal pregnancy and patients destined to develop preeclampsia and deliver a small for gestational age neonate.
      • Unal E.R.
      • Robinson C.J.
      • Johnson D.D.
      • Chang E.Y.
      Second-trimester angiogenic factors as biomarkers for future-onset preeclampsia.
      • Widmer M.
      • Villar J.
      • Benigni A.
      • Conde-Agudelo A.
      • Karumanchi S.A.
      • Lindheimer M.
      Mapping the theories of preeclampsia and the role of angiogenic factors: a systematic review.
      • Park C.W.
      • Park J.S.
      • Shim S.S.
      • Jun J.K.
      • Yoon B.H.
      • Romero R.
      An elevated maternal plasma, but not amniotic fluid, soluble fms-like tyrosine kinase-1 (sFlt-1) at the time of mid-trimester genetic amniocentesis is a risk factor for preeclampsia.
      SGA,
      • Crispi F.
      • Llurba E.
      • Dominguez C.
      • Martin-Gallan P.
      • Cabero L.
      • Gratacos E.
      Predictive value of angiogenic factors and uterine artery Doppler for early- versus late-onset pre-eclampsia and intrauterine growth restriction.
      • Asvold B.O.
      • Vatten L.J.
      • Romundstad P.R.
      • Jenum P.A.
      • Karumanchi S.A.
      • Eskild A.
      Angiogenic factors in maternal circulation and the risk of severe fetal growth restriction.
      and stillbirth.
      • Romero R.
      • Chaiworapongsa T.
      • Erez O.
      • et al.
      An imbalance between angiogenic and anti-angiogenic factors precedes fetal death in a subset of patients: results of a longitudinal study.
      Most studies that examined the value of these biomarkers, however, have focused on the prediction of preeclampsia during the first
      • Smith G.C.
      • Crossley J.A.
      • Aitken D.A.
      • et al.
      Circulating angiogenic factors in early pregnancy and the risk of preeclampsia, intrauterine growth restriction, spontaneous preterm birth, and stillbirth.
      • Parra-Cordero M.
      • Rodrigo R.
      • Barja P.
      • et al.
      Prediction of early and late pre-eclampsia from maternal characteristics, uterine artery Doppler and markers of vasculogenesis during the first trimester of pregnancy.
      • Di L.G.
      • Ceccarello M.
      • Cecotti V.
      • et al.
      First trimester maternal serum PIGF, free beta-hCG, PAPP-A, PP-13, uterine artery Doppler and maternal history for the prediction of preeclampsia.
      • Poon L.C.
      • Akolekar R.
      • Lachmann R.
      • Beta J.
      • Nicolaides K.H.
      Hypertensive disorders in pregnancy: screening by biophysical and biochemical markers at 11-13 weeks.
      or second trimesters.
      • Park C.W.
      • Park J.S.
      • Shim S.S.
      • Jun J.K.
      • Yoon B.H.
      • Romero R.
      An elevated maternal plasma, but not amniotic fluid, soluble fms-like tyrosine kinase-1 (sFlt-1) at the time of mid-trimester genetic amniocentesis is a risk factor for preeclampsia.
      • Pedrosa A.C.
      • Matias A.
      Screening for pre-eclampsia: a systematic review of tests combining uterine artery Doppler with other markers.
      • Wikstrom A.K.
      • Larsson A.
      • Eriksson U.J.
      • Nash P.
      • Norden-Lindeberg S.
      • Olovsson M.
      Placental growth factor and soluble FMS-like tyrosine kinase-1 in early-onset and late-onset preeclampsia.
      • Robinson C.J.
      • Johnson D.D.
      • Chang E.Y.
      • Armstrong D.M.
      • Wang W.
      Evaluation of placenta growth factor and soluble Fms-like tyrosine kinase 1 receptor levels in mild and severe preeclampsia.
      The results of such studies largely suggest that an imbalance between angiogenic and antiangiogenic factors increases the likelihood of preterm preeclampsia at a higher magnitude than that of term preeclampsia.
      • Chaiworapongsa T.
      • Romero R.
      • Espinoza J.
      • et al.
      Evidence supporting a role for blockade of the vascular endothelial growth factor system in the pathophysiology of preeclampsia: Young Investigator Award.
      • Crispi F.
      • Llurba E.
      • Dominguez C.
      • Martin-Gallan P.
      • Cabero L.
      • Gratacos E.
      Predictive value of angiogenic factors and uterine artery Doppler for early- versus late-onset pre-eclampsia and intrauterine growth restriction.
      • Chaiworapongsa T.
      • Romero R.
      • Kim Y.M.
      • et al.
      Plasma soluble vascular endothelial growth factor receptor-1 concentration is elevated prior to the clinical diagnosis of pre-eclampsia.
      • Parra-Cordero M.
      • Rodrigo R.
      • Barja P.
      • et al.
      Prediction of early and late pre-eclampsia from maternal characteristics, uterine artery Doppler and markers of vasculogenesis during the first trimester of pregnancy.
      • Robinson C.J.
      • Johnson D.D.
      • Chang E.Y.
      • Armstrong D.M.
      • Wang W.
      Evaluation of placenta growth factor and soluble Fms-like tyrosine kinase 1 receptor levels in mild and severe preeclampsia.
      • Levine R.J.
      • Maynard S.E.
      • Qian C.
      • et al.
      Circulating angiogenic factors and the risk of preeclampsia.
      • Kusanovic J.P.
      • Romero R.
      • Chaiworapongsa T.
      • et al.
      A prospective cohort study of the value of maternal plasma concentrations of angiogenic and anti-angiogenic factors in early pregnancy and midtrimester in the identification of patients destined to develop preeclampsia.
      • Levine R.J.
      • Lam C.
      • Qian C.
      • et al.
      Soluble endoglin and other circulating antiangiogenic factors in preeclampsia.
      • Ohkuchi A.
      • Hirashima C.
      • Matsubara S.
      • et al.
      Alterations in placental growth factor levels before and after the onset of preeclampsia are more pronounced in women with early onset severe preeclampsia.
      Yet, not all studies have arrived at the same conclusion.
      • Powers R.W.
      • Jeyabalan A.
      • Clifton R.G.
      • et al.
      Soluble fms-Like tyrosine kinase 1 (sFlt1), endoglin and placental growth factor (PlGF) in preeclampsia among high risk pregnancies.
      • Srinivas S.K.
      • Larkin J.
      • Sammel M.D.
      • et al.
      The use of angiogenic factors in discriminating preeclampsia: are they ready for prime time?.
      • Kleinrouweler C.
      • Wiegerinck M.
      • Ris-Stalpers C.
      • et al.
      Accuracy of circulating placental growth factor, vascular endothelial growth factor, soluble fms-like tyrosine kinase 1 and soluble endoglin in the prediction of pre-eclampsia: a systematic review and meta-analysis.
      Thus far, no cohort study has evaluated the predictive performance of these biomarkers in the third trimester for the identification of patients at risk for stillbirth at or near term or late-onset preeclampsia.
      Recently, a new approach in screening for adverse pregnancy outcomes proposes to focus on the prevention of pregnancy complications at term. Such an approach would identify the more prevalent disease (eg, preeclampsia at term), and predictive models could be applied to low-income settings, where most maternal and perinatal deaths occur.
      • Kenneth L.
      • Hall D.R.
      • Gebhardt S.
      • Grove D.
      Late onset preeclampsia is not an innocuous condition.
      • Smith G.C.
      Researching new methods of screening for adverse pregnancy outcome: lessons from pre-eclampsia.
      The objective of this study was to determine whether maternal plasma concentrations of PlGF, sEng, sVEGFR-1, and their ratios at 30-34 weeks of gestation could be used to identify patients at risk for stillbirth, late preeclampsia, severe late preeclampsia, or delivery of SGA neonates.

      Methods

      Study design

      We first designed a cohort study of women who had a venipuncture at 30-34 weeks of gestation and outcome data to examine the value of PlGF, sVEGFR-1, and sEng in the identification of patients who subsequently experienced late preeclampsia, severe late preeclampsia, stillbirth, and SGA. Subsequent to this cohort study, a case-control study was performed to determine whether these biomarkers and their ratios could identify patients at risk for stillbirth at or near term in a different population.

      Cohort study

      A prospective longitudinal cohort study was conducted between November 2003 and August 2006 to identify biological markers for the prediction of preeclampsia, SGA neonates, and stillbirth. Patients were enrolled in the prenatal clinic of the Sotero del Rio Hospital, a tertiary care center in Santiago, Chile, and followed until delivery. Inclusion criteria were singleton gestation and 6-22 weeks of gestation. Exclusion criteria were (1) preterm labor, preterm prelabor rupture of membranes, preeclampsia, or impaired fetal growth at the time of recruitment; (2) known major fetal anomaly or fetal death; (3) active vaginal bleeding; and (4) serious medical illness (renal insufficiency, congestive heart disease, chronic respiratory insufficiency, or active hepatitis). At enrollment and each subsequent visit, patients underwent a venipuncture for the collection of maternal blood. The protocol consisted of collecting samples every 4 weeks until 24 weeks of gestation and every 2 weeks thereafter until delivery.
      We previously reported the predictive performance of angiogenic/antiangiogenic factors at 6-15 and 20-25 weeks of gestation and uterine artery Doppler velocimetry (UADV) at 20-25 weeks of gestation for preeclampsia in this cohort.
      • Kusanovic J.P.
      • Romero R.
      • Chaiworapongsa T.
      • et al.
      A prospective cohort study of the value of maternal plasma concentrations of angiogenic and anti-angiogenic factors in early pregnancy and midtrimester in the identification of patients destined to develop preeclampsia.
      In summary, 2998 consecutive women were enrolled during the study period; 2495 women had a plasma sample collected in early pregnancy. Of those, an additional plasma sample was obtained in the mid trimester from 1917 women. Subsequently, an additional 204 patients without results of UADV in the second trimester were excluded. Ninety-one patients were lost to follow up; the remaining 1622 patients had been included in a previous article that examined the role of angiogenic/antiangiogenic factors at 6-15 and 20-25 weeks of gestation.
      • Kusanovic J.P.
      • Romero R.
      • Chaiworapongsa T.
      • et al.
      A prospective cohort study of the value of maternal plasma concentrations of angiogenic and anti-angiogenic factors in early pregnancy and midtrimester in the identification of patients destined to develop preeclampsia.
      The current study involved a subset of this cohort that excluded patients who delivered at ≤34 weeks of gestation (n = 27) and those who did not have a plasma sample collected at 30-34 weeks of gestation (n = 326) to examine the role of angiogenic/antiangiogenic factors at 30-34 weeks for the identification of adverse pregnancy outcomes at >34 weeks of gestation.
      All women provided written informed consent before participating in the study. The use of clinical and ultrasound data and the collection and use of maternal blood for research purposes was approved by the institutional review boards of the Sotero del Rio Hospital, Santiago, Chile (an affiliate of the Pontificia Catholic University of Santiago, Chile), and the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services.

      Outcomes of the study

      The outcomes of the study included late preeclampsia, severe late preeclampsia, SGA without preeclampsia, and stillbirth. Preeclampsia was defined as new-onset hypertension that developed at >20 weeks of gestation and proteinuria. Hypertension was defined as systolic ≥140 mm Hg and/or diastolic blood pressure ≥90 mm Hg that was measured at 2 occasions, 4 hours to 1 week apart. Proteinuria was defined as a urine protein of ≥300 mg in a 24-hour urine collection or 2 random urine specimens that were obtained 4 hours to 1 week apart and showed ≥1+ by dipstick or 1 dipstick that demonstrated ≥2+ protein. Late preeclampsia was defined as patients with preeclampsia who delivered at >34 weeks of gestation.
      • von Dadelszen P.
      • Magee L.A.
      • Roberts J.M.
      Subclassification of preeclampsia.
      Severe preeclampsia was diagnosed based on criteria of the American College of Obstetricians and Gynecologists.
      American College of Obstetricians and Gynecologists
      ACOG practice bulletin no. 33: diagnosis and management of preeclampsia and eclampsia.
      SGA was defined as a birthweight of <10th percentile for gestational age according to the Chilean birthweight distribution of a Hispanic population.
      • Gonzalez R.P.
      • Gomez R.M.
      • Castro R.S.
      • et al.
      [A national birth weight distribution curve according to gestational age in Chile from 1993 to 2000].
      Stillbirth was defined as death of a fetus before delivery that was not a consequence of an induced termination of pregnancy (including intrapartum and antepartum stillbirth).
      • MacDorman M.F.
      • Kirmeyer S.
      The challenge of fetal mortality.
      Abnormal UADV was defined as a mean uterine artery Doppler pulsatility index of >1.45.
      • Albaiges G.
      • Missfelder-Lobos H.
      • Lees C.
      • Parra M.
      • Nicolaides K.H.
      One-stage screening for pregnancy complications by color Doppler assessment of the uterine arteries at 23 weeks' gestation.
      • Yu C.K.
      • Khouri O.
      • Onwudiwe N.
      • Spiliopoulos Y.
      • Nicolaides K.H.
      Fetal Medicine Foundation Second-Trimester Screening Group
      Prediction of pre-eclampsia by uterine artery Doppler imaging: relationship to gestational age at delivery and small-for-gestational age.

      Data quality

      Customized case report forms and a perinatal database were generated. Data were extracted from medical records by trained research nurses. To account for misclassification, abstracters were trained, the data collection methods were verified, and data logic was monitored. Cases of uncertainty were resolved by iterative discussion among 3 of the authors. Gestational age at venipuncture and at delivery was based on best obstetrical estimates with the use of the last menstrual period and the earliest fetal biometric parameters, which were performed at ≤20 weeks of gestation in 98.2% of cases.

      Sample collection and immunoassays

      Blood was obtained by venipuncture and collected into tubes that contained ethylenediaminetetraacetic acid. Samples were centrifuged and stored at −70°C. Maternal plasma concentrations of sVEGFR-1, PlGF, and sEng were determined by sensitive and specific immunoassays (R&D Systems, Minneapolis, MN) as previously described.
      • Kusanovic J.P.
      • Romero R.
      • Chaiworapongsa T.
      • et al.
      A prospective cohort study of the value of maternal plasma concentrations of angiogenic and anti-angiogenic factors in early pregnancy and midtrimester in the identification of patients destined to develop preeclampsia.
      The inter- and intra-assay coefficients of variation (CV) were 1.4% and 3.9% for sVEGFR-1, 2.3% and 4.6% for sEng, and 6.02% and 4.8% for PlGF. The sensitivity of the assays was 16.97 pg/mL for sVEGFR-1, 0.08 ng/mL for sEng, and 9.52 pg/mL for PlGF. The laboratory personnel who performed the assays were blinded to the clinical information.

      Statistical analysis

      Differences in distributions of dichotomous and categorical variables were tested with the χ2 test or Fisher exact test where appropriate; continuous parameters were compared by analysis of variance or Friedman's 2-way nonparametric analysis of variance test with Bonferroni correction for multiple comparisons, depending on the distribution of data. Normality was assessed with the Kolmogorov-Smirnov test and visual plot inspection.
      Quantile regression was used to calculate median analyte ratio concentrations (PlGF/sVEGFR-1, PlGF/sEng) that were conditional on gestational age among uncomplicated pregnancies (n = 886).
      • Koenker R.
      • Bassett G.
      Regression quantiles.
      Multiple of the median (MoM) values were calculated for both analyte ratios for each patient. MoM cutoffs were determined based on inspection of receiver operating characteristic curves that were calculated for each outcome (stillbirth, late preeclampsia, severe late preeclampsia, and SGA without preeclampsia). Prognostic logistic regression models were constructed for each outcome. Covariables included in adjusted models were selected on the basis of clinical knowledge. Model reduction was performed additionally based on the plausibility of regression coefficients, association with independent variables, and the magnitude of change in the main-effect parameter estimates.
      • Steyerberg E.W.
      • Eijkemans M.J.
      • Harrell Jr, F.E.
      • Habbema J.D.
      Prognostic modelling with logistic regression analysis: a comparison of selection and estimation methods in small data sets.
      To account for potential model over-fitting, when van Houwelingen and le Cessie's heuristic shrinkage estimator was below 0.85 (indicator of instability), bootstrap estimated linear shrinkage factors and Firth's penalized maximum likelihood estimation were used to calculate conservative estimates that were less likely to be affected by over-fitting.
      • Steyerberg E.W.
      • Eijkemans M.J.
      • Harrell Jr, F.E.
      • Habbema J.D.
      Prognostic modelling with logistic regression analysis: a comparison of selection and estimation methods in small data sets.
      • Harrell Jr, F.E.
      • Lee K.L.
      • Mark D.B.
      Multivariable prognostic models: issues in developing models, evaluating assumptions and adequacy, and measuring and reducing errors.
      Predictive performance metrics were also calculated for each outcome. Paired sample nonparametric statistical techniques were used to compare area under the receiver operating characteristic curves (AUC) of models that were constructed with logistic regression for the identification of selected pregnancy outcomes.
      • DeLong E.R.
      • DeLong D.M.
      • Clarke-Pearson D.L.
      Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach.
      A McNemar's test was also used to test for differences in sensitivity at a fixed false-positive rate of 15%. A 5% threshold for type I error was used to determine statistical significance. Statistical analyses were performed with SAS software (version 9.3; Cary, NC).

      Case-control study for stillbirth

      Participants were identified from a cohort of 5828 singleton pregnancies who were either enrolled in a similar longitudinal protocol to that used in the Chilean cohort or another cross-sectional protocol from 2007-2009 at Hutzel Women's Hospital, Detroit, MI. Stillbirth was defined as death of a fetus before delivery (which was not a consequence of an induced termination of pregnancy).
      • MacDorman M.F.
      • Kirmeyer S.
      The challenge of fetal mortality.
      In the longitudinal study, plasma samples were obtained from the first or early second trimester and at the time of each prenatal visit (scheduled every 4 weeks until 24 weeks and every 2 weeks thereafter until delivery). In the cross-sectional study, patients were enrolled when they presented to the labor and delivery unit with a suspicion of spontaneous preterm labor or medically-indicated preterm birth. Among 31 cases of stillbirth at ≥34 weeks of gestation, 5 cases were included because they had a plasma sample collected at 30-34 weeks of gestation. Control subjects were identified from uncomplicated pregnancies who both delivered an appropriate weight for gestational age neonate at term and had a plasma sample collected at 30-34 weeks of gestation. Control subjects were matched to cases at a ratio of 6 to 1 on gestational age at venipuncture, parity, ethnicity, tobacco use, and body mass index. Maternal plasma concentrations of sVEGFR-1, sEng, and PlGF were determined by sensitive and specific immunoassays (R&D Systems) similar to those used in the Chilean cohort, as described earlier.
      All women provided written informed consent before participating in the study. The use of clinical and ultrasound data and collection and utilization of maternal blood for research purposes was approved by the institutional review boards of the Wayne State University, Detroit, MI, and the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services.
      Differences among cases and control subjects were tested with the use of χ2, Fisher exact, or Mann-Whitney U tests where appropriate. AUC was calculated, and sensitivities and specificities were determined with the use of absolute value thresholds for each biomarker ratio that were derived from the inspection of receiver operating characteristic curves.

      Results

      The cohort study included 1269 pregnant women (Figure 1). The prevalence of late preeclampsia, severe late preeclampsia, stillbirth, and SGA without preeclampsia was 3.2% (n = 40), 1.8% (n = 23); 0.4% (n = 5), and 8.5% (n = 108), respectively. Among 23 patients who received a diagnosis of severe preeclampsia, 6 experienced severe high blood pressure and severe proteinuria; 4 had severe high blood pressure; 4 had severe high blood pressure with severe proteinuria with SGA fetuses; 3 had SGA fetuses; 2 had severe headache with severe proteinuria; 2 had severe proteinuria, 1 had severe high blood pressure, and 1 had severe proteinuria and pulmonary edema. Table 1 displays the demographic and obstetrical characteristics of patients with SGA neonates, preeclampsia, stillbirth, other complications and of patients without any of these complications (uncomplicated pregnancy). There were no significant differences in the mean gestational age at venous sampling or mean duration of sample storage among the 4 groups. The distribution of baseline characteristics did not significantly differ between patients included in the current study compared with the overall cohort (data not shown). Similarly, there were no significant differences in the risk of stillbirth or SGA between the entire cohort and subcohort. However, by design, participants in the subcohort were more likely to deliver at >34 weeks of gestation. Patients in this subcohort had a lower frequency of preeclampsia than those in the entire cohort (3.2% vs 4.8%; P = .03). Three patients who had a diagnosis of gestational hypertension before venipuncture at 30-34 weeks of gestation; however, subsequent preeclampsia did not develop. The median MoM plasma concentration of PlGF/sVEGFR-1 and PlGF/sEng was significantly lower in patients with subsequent stillbirth, preeclampsia, and SGA than in patients without these conditions (P < .05 for each comparison; Table 1).
      Figure thumbnail gr1
      FIGURE 1Flow diagram of patients enrolled in the study
      Chaiworapongsa. Third-trimester screening by angiogenic/antiangiogenic factors for preeclampsia and stillbirth. Am J Obstet Gynecol 2013.
      TABLE 1Demographical and obstetrical characteristics of the study population
      Patient characteristicsUncomplicated pregnancy (n = 886)Pregnancy with small-for-gestational-age neonate (n = 108)Preeclampsia (n = 40)Stillbirth (n = 5)Other complications (n = 230)
      Maternal age, y
      Data are given as mean ± SD;
      26.2 ± 5.926.5 ± 7.123.6 ± 5.4
      Indicated significant difference (P < .05) compared with combined “other + uncomplicated pregnancy” categories;
      27 ± 1028 ± 6.5
      Tobacco use, n (%)93 (10.5)20 (18.50)5 (12.50)028 (12.2)
      Nulliparous, n (%)356 (40.2)48 (44.4)28 (70)
      Indicated significant difference (P < .05) compared with combined “other + uncomplicated pregnancy” categories;
      2 (40)76 (33.0)
      Multiparous with history of preeclampsia, n (%)19 (2.1)1 (0.9)3 (7.5)
      Indicated significant difference (P < .05) compared with combined “other + uncomplicated pregnancy” categories;
      014 (6.1)
      Multiparous without history of preeclampsia, n (%)511 (57.7)59 (54.60)9 (22.50)
      Indicated significant difference (P < .05) compared with combined “other + uncomplicated pregnancy” categories;
      3 (60)140 (60.9)
      Body mass index, kg/m2
      Data are given as mean ± SD;
      24.6 ± 4.224 ± 4.327.4 ± 822.4 ± 1.627.3 ± 6
      Gestational age at venipuncture, wk
      Data are given as mean ± SD;
      32.2 ± 1.132.2 ± 1.132.2 ± 1.232 ± 0.932.2 ± 1.2
      Storage time, y
      Data are given as mean ± SD;
      6.8 ± 0.76.8 ± 0.86.9 ± 1.16.6 ± 0.66.8 ± 0.7
      Gestational age at delivery, k
      Data are given as mean ± SD;
      39.6 ± 1.139.4 ± 1.138.48 ± 1.6
      Indicated significant difference (P < .05) compared with combined “other + uncomplicated pregnancy” categories;
      36.5 ± 2.3
      Indicated significant difference (P < .05) compared with combined “other + uncomplicated pregnancy” categories;
      38.6 ± 1.7
      Birthweight, g
      Data are given as mean ± SD;
      3505 ± 3992710 ± 230
      Indicated significant difference (P < .05) compared with combined “other + uncomplicated pregnancy” categories;
      3096 ± 550
      Indicated significant difference (P < .05) compared with combined “other + uncomplicated pregnancy” categories;
      2896 ± 6423366 ± 521
      Placental growth factor/soluble vascular endothelial growth factor receptor-1 (multiple of the median)
      Data are given as median (interquartile range) that were calculated among uncomplicated pregnancies (n = 886) by quantile regression:
      ,
      Median = 1.8863+ (−0.0508 × gestational week);
      1.00 (0.51–1.83)0.53 (0.21–1.22)
      Indicated significant difference (P < .05) compared with combined “other + uncomplicated pregnancy” categories;
      0.21 (0.08–0.50)
      Indicated significant difference (P < .05) compared with combined “other + uncomplicated pregnancy” categories;
      0.08 (0.07–0.1)
      Indicated significant difference (P < .05) compared with combined “other + uncomplicated pregnancy” categories;
      0.73 (0.33–1.27)
      Placental growth factor/soluble endoglin (multiple of the median)
      Data are given as median (interquartile range) that were calculated among uncomplicated pregnancies (n = 886) by quantile regression:
      ,
      Median = 354.3280 + (−8.9791 × gestational week).
      1.00 (0.56–1.78)0.59 (0.26–1.10)
      Indicated significant difference (P < .05) compared with combined “other + uncomplicated pregnancy” categories;
      0.27 (0.11–0.63)
      Indicated significant difference (P < .05) compared with combined “other + uncomplicated pregnancy” categories;
      0.18 (0.1–0.3)
      Indicated significant difference (P < .05) compared with combined “other + uncomplicated pregnancy” categories;
      0.74 (0.35–1.18)
      Other complications included spontaneous preterm delivery (3%; n = 38), chronic hypertension (2.2%; n = 28), gestational hypertension (6.8%; n = 86), gestational and pregestational diabetes mellitus (4.6%; n = 58), placental abruption (0.4%; n = 5), cholestasis of pregnancy (0.9%; n = 12), and placenta previa (0.2%; n = 3).
      Chaiworapongsa. Third-trimester screening by angiogenic/antiangiogenic factors for preeclampsia and stillbirth. Am J Obstet Gynecol 2013.
      a Data are given as mean ± SD;
      b Indicated significant difference (P < .05) compared with combined “other + uncomplicated pregnancy” categories;
      c Data are given as median (interquartile range) that were calculated among uncomplicated pregnancies (n = 886) by quantile regression:
      d Median = 1.8863+ (−0.0508 × gestational week);
      e Median = 354.3280 + (−8.9791 × gestational week).
      Table 2 displays the magnitude of association between abnormal biomarker profiles and late preeclampsia (overall and severe), delivery of SGA neonates (birthweight: <10%, <3%), and stillbirth. Patients with plasma PlGF/sEng or PlGF/sVEGFR-1 ratio concentrations of <0.3 MoM were significantly more likely to experience late preeclampsia (adjusted odds ratio [aOR], 7.1; 95% confidence interval [CI], 3.6–13.8; and 6.1; 95% CI, 3.1–11.8, respectively) and severe late preeclampsia (aOR, 16.1; 95% CI, 5.8–44.6 and 12.2; 95% CI, 4.6–32, respectively) than those with MoMs at or above the threshold (Table 2). The likelihood ratio of a positive test and sensitivity for either PlGF/sEng or PlGF/sVEGFR-1 ranged from 4.5–4.8 and 74–78%, respectively; both had a specificity of 84% for the identification of patients with severe late preeclampsia (Table 3).
      TABLE 2Likelihood of subsequent stillbirth, preeclampsia, and small-for-gestational-age neonate
      Dependent variableOthers (n/N)Outcome (n/N)UnadjustedAdjusted
      Odds ratio
      Data represent the likelihood of outcome in subjects with abnormal analyte ratio concentrations (above/below MoM cutoff) relative to patients with normal analyte ratio concentration multiples of the median cutoff;
      95% CIOdds ratio
      Data represent the likelihood of outcome in subjects with abnormal analyte ratio concentrations (above/below MoM cutoff) relative to patients with normal analyte ratio concentration multiples of the median cutoff;
      95% CI
      Preeclampsia (n = 40)
       PlGF/sVEGFR-1 <0.3 MoM199/1229 (16.2%)23/40 (57.5%)7.94.1–15.26.13.1–11.8
       PlGF/sEng <0.3 MoM196/1229 (15.9%)24/40 (60.0%)5.91.9–18.77.13.6–13.8
      Severe preeclampsia (n = 23)
       PlGF/sVEGFR-1 <0.3 MoM205/1246 (16.5%)17/23 (73.9%)11.92.2–66.012.20.6–32.0
       PlGF/sEng <0.3 MoM202/1246 (16.3%)18/23 (78.3%)11.72.1–64.616.15.8–44.6
      Small-for-gestational-age <10th percentile (n = 108)
       PlGF/sVEGFR-1 <0.3 MoM184/1161 (15.8%)38/108 (35.2%)4.22.4–7.33.02.0–4.7
       PlGF/sEng <0.3 MoM189/1161 (16.3%)31/108 (28.7%)3.62.0–6.42.01.3–3.1
      Small-for-gestational-age <3rd percentile (n = 23)
       PlGF/sVEGFR-1 <0.3 MoM210/1246 (16.9%)12/23 (52.2%)6.92.4–19.45.52.3–13.1
       PlGF/sEng <0.3 MoM209/1246 (16.8%)11/23 (47.8%)7.02.5–19.84.41.8–10.4
      Stillbirth (n = 5)
      Odds ratio was adjusted for gestational age at venipuncture (continuous).
       PlGF/sVEGFR-1 <0.12 MoM71/1264 (5.6%)4/5 (80%)20.14.8–84.323.15.6–95.4
       PlGF/sEng <0.2 MoM137/1264 (10.8%)3/5 (60%)8.42.0–35.19.12.2–37.2
      Medians were calculated among uncomplicated pregnancies (n = 886) by quantile regression (PlGF/sVEGFR-1: median = 1.8863 + (−0.0508 × gestational week); PlGF/sEng: median = 354.3280 + (−8.9791 × gestational week); cutoffs were selected based on inspection of receiver operating characteristic curves. The prediction of preeclampsia and small-for-gestational-age was adjusted for maternal age (continuous), combined parity and history of preeclampsia, prepregnancy body mass index (continuous), and tobacco use. PlGF/sVEGFR-1 MoM cutoff: <0.12 (or 5th -6th percentile of uncomplicated pregnancies) for stillbirth and <0.3 (or 17th percentile of uncomplicated pregnancies) for preeclampsia and small-for-gestational-age. PlGF/sEng MoM cutoff: <0.2 (or 11th percentile of uncomplicated pregnancies) for stillbirth and <0.3 (or 17th percentile of uncomplicated pregnancies) for preeclampsia and small-for-gestational-age.
      CI, confidence interval; MoM, multiples of the median; PlGF/sEng, placental growth factor/soluble endoglin; PlGF/sVEGFR-1, placental growth factor/soluble vascular endothelial growth factor receptor-1.
      Chaiworapongsa. Third-trimester screening by angiogenic/antiangiogenic factors for preeclampsia and stillbirth. Am J Obstet Gynecol 2013.
      a Data represent the likelihood of outcome in subjects with abnormal analyte ratio concentrations (above/below MoM cutoff) relative to patients with normal analyte ratio concentration multiples of the median cutoff;
      b Odds ratio was adjusted for gestational age at venipuncture (continuous).
      TABLE 3Predictive performance of maternal plasma concentrations of angiogenic and antiangiogenic factors for stillbirth and preeclampsia screening
      Diagnostic performance metricsPreeclampsia (n = 40)Severe preeclampsia (n = 23)Stillbirth (n = 5)
      Estimate95% CIEstimate95% CIEstimate95% CI
      PlGF/sVEGFR-1
       Sensitivity, %5840–737452–908028–100
       Specificity, %8382–868481–869493–96
        Positive predictive value, %106–1585–1251–13
        Negative predictive value, %9897–999999–10010099–100
        False positive rate, %1614–181614–1964–7
        False negative rate, %4327–592610–48200.5–72
        Positive likelihood ratio3.62.6–4.84.53.4–5.914.28.7–23.3
        Negative likelihood ratio0.50.4–0.70.30.2–0.60.20.04–1.22
      PlGF/sEng
       Sensitivity, %6043–757856–936015–95
       Specificity, %8482–868482–868987–91
        Positive predictive value, %117–1685–1320.4–6
        Negative predictive value, %9897–999999–1009999–100
        False positive rate, %1614–181614–18119–13
        False negative rate, %4025–57227–44405–85
        Positive likelihood ratio3.82.8–5.04.83.8–6.25.52.7–11.5
        Negative likelihood ratio0.50.3–0.70.30.1–0.60.40.2–1.3
      Medians were calculated among uncomplicated pregnancies (n = 886) by quantile regression (PlGF/sVEGFR-1: median = 1.8863 + (−0.0508 × gestational week); PlGF/sEng: median = 354.3280 + (−8.9791 × gestational week); cutoffs were selected based on inspection of receiver operating characteristic curves. PlGF/sVEGFR-1 multiples of the median cutoff: <0.12 (or 5th -6th percentile of uncomplicated pregnancies) for stillbirth and <0.3 (or 17th percentile of uncomplicated pregnancies) for preeclampsia; PlGF/sEng multiples of the median cutoff: <0.2 (or 11th percentile of uncomplicated pregnancies) for stillbirth and <0.3 (or 17th percentile of uncomplicated pregnancies) for preeclampsia.
      CI, confidence interval; MoM, multiples of the median; PlGF/sEng, placental growth factor/soluble endoglin; PlGF/sVEGFR-1, placental growth factor/soluble vascular endothelial growth factor receptor-1.
      Chaiworapongsa. Third-trimester screening by angiogenic/antiangiogenic factors for preeclampsia and stillbirth. Am J Obstet Gynecol 2013.
      The addition of the PlGF/sEng or PlGF/sVEGFR-1 ratio to the clinical risk factors increased the AUC from 0.76 to 0.88 and 0.86, respectively, for the prediction of severe late preeclampsia (P = .03 and .06). With a fixed false-positive rate of 15%, both the PlGF/sEng ratio and PlGF/sVEGFR-1 ratios achieved a sensitivity of 74% in predicting severe PE. These biomarkers in the third trimester outperformed those obtained previously at 6-15 and 20-25 weeks of gestation and UADV that was assessed at 20-25 weeks of gestation for the prediction of severe late preeclampsia (each P ≤ .02; Figure 2). Further, the addition of the PlGF/sVEGFR-1 or the PlGF/sEng ratio measured in the third trimester to clinical risk factors (age, body mass index, combined parity and history of preeclampsia, and tobacco use) yielded significantly greater sensitivity (74%) at a fixed false-positive rate of 15%, compared with a model that used the same biomarker ratios measured in the second trimester, clinical risk factors, and abnormal UADV values that were obtained at 20-25 weeks of gestation (P = .008 and .03, respectively). The direction, magnitude, and significance of these associations also persisted during sensitivity analyses performed excluding patients with a history of preeclampsia (n = 37) based on their elevated a priori risk in the current pregnancy.
      Figure thumbnail gr2
      FIGURE 2Comparison of ROC curves for the identification of severe late preeclampsia
      Areas under the ROC curves achieved by plasma concentrations of A, placental growth factor (PlGF)/soluble endoglin (sEng) ratio or B, PlGF/soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) ratio. Multiples of the median at 6-15, 20-25, and 30-34 weeks of gestation and uterine (Ut.) artery Doppler velocimetry at 20-25 weeks of gestation are shown.
      GA, gestational age; ROC, receiver-operating characteristic.
      Chaiworapongsa. Third-trimester screening by angiogenic/antiangiogenic factors for preeclampsia and stillbirth. Am J Obstet Gynecol 2013.
      Although patients with plasma PlGF/sVEGFR-1 or PlGF/sEng ratio concentrations of <0.3 MoM were more likely to experience SGA without preeclampsia (aOR, 2-3; Table 2), the addition of these biomarkers to demographic/perinatal data did not improve the AUC (0.64 vs 0.62; P = .2 and .6; respectively). Subgroup analysis that focused on patients with severe SGA (birthweight less than the third percentile; n = 23) indicated that the adjusted odds ratio of patients with PlGF/sVEGFR-1 or PlGF/sEng ratio <0.3 MoM to experience severe SGA ranged from 4.4–5.5 (Table 2). However, the addition of these biomarkers to clinical risk factors did not significantly improve the AUC (P > .05).
      Patients with a PlGF/sVEGFR-1 ratio of <0.12 MoM were significantly more likely to have a stillbirth than patients with a MoM ratio at or above the threshold (aOR, 23.1; 95% CI, 5.6–95.4). This cutoff had a sensitivity of 80%, a specificity of 94%, and a likelihood ratio of a positive test of 14.2 for the identification of a subsequent stillbirth at >34 weeks of gestation (Table 3). Compared with a model that included only clinical data (maternal age, combined parity and history of preeclampsia, body mass index, and tobacco use), the addition of the PlGF/sVEGFR-1 ratio or the PlGF/sEng ratio to clinical data increased the AUC from 0.7 to 0.91 (P = .03 and .06, respectively; Figure 3). The association between an abnormal ratio of angiogenic/antiangiogenic factors and stillbirth at or near term was also observed in the subsequent case-control study performed in a different population (Table 4). A maternal plasma concentration of PlGF/sVEGFR-1 ratio ≤0.046 or PlGF/sEng ratio ≤11.7 pg/ng at 30-34 weeks had a sensitivity of 80% and a specificity of 93% for the identification of subsequent stillbirth (Figure 4).
      Figure thumbnail gr3
      FIGURE 3Comparison of ROC curves for the identification of stillbirth
      Areas under the ROC curves achieved by plasma concentrations of placental growth factor (PIGF)/soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) ratio or PIGF/soluble endoglin (sEng) ratio at 30-34 weeks' gestation in combination with clinical data were compared with that achieved by clinical data alone.
      GA, gestational age; ROC, receiver-operating characteristic.
      Chaiworapongsa. Third-trimester screening by angiogenic/antiangiogenic factors for preeclampsia and stillbirth. Am J Obstet Gynecol 2013.
      TABLE 4Demographic and obstetrical characteristics of the population in the case-control study
      VariableControl (n = 30)Fetal death (n = 5)P value
      Maternal age, y
      Data are given as median (interquartile range).
      21.5 (19.8–23.2)26 (21.5–36.0).154
      Gestational age at venipuncture, wk
      Data are given as median (interquartile range).
      32.9 (32.1–33.6)33.4 (32–33.7).493
      Body mass index, kg/m2
      Data are given as median (interquartile range).
      26.1 (21.1–35.8)24.6 (17.9–44.5).873
      Nulliparity, n (%)16 (53.3)3 (60).585
      Tobacco use00
      African American80% (24)80% (4).90
      Gestational age at delivery, wk
      Data are given as median (interquartile range).
      39.5 (38.9–40.6)37.7 (34.7–38.9).047
      Birthweight, g
      Data are given as median (interquartile range).
      3273 (3165–3478)2305 (1635–3360).030
      Placental growth factor, pg/mL646 (279–1108)97 (63–640).016
      Soluble vascular endothelial growth factor receptor-1, pg/mL
      Data are given as median (interquartile range).
      2779 (1822–4349)6333 (3740–6908).016
      Soluble endoglin, ng/mL
      Data are given as median (interquartile range).
      7.5 (5.7–10.1)23.8 (14.4–33.4).002
      Placental growth factor/soluble vascular endothelial growth factor receptor-1 ratio
      Data are given as median (interquartile range).
      0.25 (0.09–0.5)0.02 (0.009–0.1).002
      Placental growth factor/soluble endoglin ratio, pg/ng
      Data are given as median (interquartile range).
      96 (28–167)6.9 (2.3–28).002
      Chaiworapongsa. Third-trimester screening by angiogenic/antiangiogenic factors for preeclampsia and stillbirth. Am J Obstet Gynecol 2013.
      a Data are given as median (interquartile range).
      Figure thumbnail gr4
      FIGURE 4ROC curves for the identification of subsequent stillbirth in the case-control study
      Maternal plasma concentrations of placental growth factor (PlGF)/soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) or PlGF/soluble endoglin (sEng) at 30-34 weeks of gestation were used to identify patients at risk of stillbirth. Area under the ROC curves = 0.91 for both biomarkers.
      GA, gestational age; ROC, receiver-operating characteristic.
      Chaiworapongsa. Third-trimester screening by angiogenic/antiangiogenic factors for preeclampsia and stillbirth. Am J Obstet Gynecol 2013.
      Table 5 displays the obstetrical events at delivery, gestational age at venipuncture, and placental pathologic conditions of each patient with a stillbirth in the cohort and case-control studies. Among patients with a stillbirth in the cohort study, the interval from venipuncture to the diagnosis of stillbirth ranged from 2.2–6.1 weeks (median, 4.5 weeks). One patient was diagnosed with gestational diabetes mellitus, and another had an abruptio placentae. Three patients had histologic placental lesions consistent with maternal underperfusion, according to the criteria of the Society for Pediatric Pathology.
      • Redline R.W.
      • Heller D.
      • Keating S.
      • Kingdom J.
      Placental diagnostic criteria and clinical correlation: a workshop report.
      Chronic chorioamnionitis and hyalinized avascular villi (consistent with fetal thrombotic vasculopathy), were observed in the other 2 cases. None of the cases included in the cohort study had a fetal autopsy. Of 5 cases included in the case-control study, 2 women had a diagnosis of diabetes mellitus and the remaining 3 had a diagnosis of severe preeclampsia, chronic hypertension, and Marfan's syndrome, respectively. The interval from venipuncture to the diagnosis of stillbirth ranged from 2.4–5.4 weeks (median, 4 weeks). All 4 cases of stillbirth who had plasma concentrations of angiogenic/antiangiogenic factor ratios below the aforementioned cutoff in the case-control study also had lesions in the placenta suggestive of maternal underperfusion. Two stillbirths had a karyotype performed, and the results were 46 XY. Among 4 cases with available fetal autopsy results, 1 had lesions in the fetal brain consistent with acute hypoxic/ischemic damage in the grey matter.
      TABLE 5Obstetrics events at delivery, gestational age at venipuncture, and placental lesions of patients with stillbirth
      CaseObstetrics events at deliveryPlGF/sVEGFR-1 ratio, MoMGestational age, wkBirthweight in grams (percentile)Placental lesions consistent with maternal underperfusionPlacental lesionsFetal autopsy
      At venous samplingAt delivery
      Cohort study
       1Normal blood pressure0.0732 1/734 3/72200 (50%)YesDiffuse chronic villitis; persistent muscularization of basal plate arteriesNot available
       2Gestational diabetes mellitus noncompliance with care0.1031 3/734 4/72280 (58%)YesIncreased syncytial knotNot available
       3Blood pressure 140/90 mm Hg, urine protein dipstick negative, placental abruption0.043135 4/73000 (92%)NoChronic chorioamnionitisNot available
       4Normal blood pressure, decreased fetal movement, thick meconium-stained amniotic fluid0.0833 3/739 3/73650 (74%)YesIncreased intervillous fibrin; prominent nucleated red blood cells, absence of physiologic change of the spiral arteriesNot available
       5Normal blood pressure0.7832 2/738 3/73350 (60.5%)NoHyalinized avascular villi; fetal thrombotic vasculopathyNot available
      Case-control studyPlGF/sVEGFR-1 ratio, pg/pg
       1Gestational diabetes mellitus class A2: poorly controlled glucose0.0233 5/737 5/73620 (81.5%)YesMicroscopic chorionic pseudo cysts in placental membranesNo congenital anomalies; no cause found
       2Pregestational diabetes mellitus class B: poorly controlled glucose0.0433 5/739 1/73100 (28.1%)YesRecent villous infarction; persistent muscularization of basal plate arteriesAcute hypoxic/ischemic gray matter damage and subarachnoid hemorrhage; no congenital anomalies
       3Severe preeclampsia0.0131 3/734 2/72040 (15%)YesRecent villous infarctionNo congenital anomalies; no cause found
       4Chronic hypertension0.00532 4/7351231 (1%)YesRemote villous infarction; increased syncytial knotsNot available
       5Marfan's syndrome0.1933 3/738 4/72305 (1%)NoNormalNo congenital anomalies; no cause found
      MoM, multiples of the median; PlGF/sVEGFR-1, placental growth factor/soluble vascular endothelial growth factor receptor-1.
      Chaiworapongsa. Third-trimester screening by angiogenic/antiangiogenic factors for preeclampsia and stillbirth. Am J Obstet Gynecol 2013.

      Comment

      Principal findings

      This is the first prospective cohort study to evaluate the predictive performance of angiogenic/antiangiogenic factors in the third trimester for the identification of patients with late preeclampsia, severe late preeclampsia, SGA without preeclampsia, and stillbirth. The principal findings were: (1) A maternal plasma concentration of PlGF/sEng <0.3 MoM at 30-34 weeks' gestation was associated with late preeclampsia (aOR, 7) and severe late preeclampsia (aOR, 16). With a fixed false-positive rate of 15%, both the PlGF/sEng and PlGF/sVEGFR-1 ratios achieved a sensitivity of 74% for the identification of severe late preeclampsia; (2) The ratio of PlGF/sEng or PlGF/sVEGFR-1 in the third trimester outperformed ratios obtained at 6-15 and 20-25 weeks of gestation and abnormal UADV obtained at 20-25 weeks of gestation for the identification of severe late preeclampsia (comparisons of AUC; each P ≤ .02); (3) A maternal plasma concentration of PlGF/sVEGFR-1 ratio <0.12 MoM at 30-34 weeks of gestation was significantly associated with a subsequent stillbirth (aOR, 23). This cutoff had a sensitivity of 80%, a specificity of 94%, and a likelihood ratio of a positive result of 14 for the identification of patients destined to have a stillbirth; and (4) Although low maternal plasma concentrations of PlGF/sVEGFR-1 and PlGF/sEng ratios were associated with a significant increase in the likelihood of the development of SGA, these biomarkers did not improve the identification of SGA from the models using clinical factors alone.

      Rationale for the examination of biomarkers in the third trimester

      The following advantages underlie the use of biomarkers in the third trimester, in addition to the first or second trimesters: (1) Testing performed closer to the event of interest or diagnosis usually yields better results than tests performed earlier in gestation. Several studies of screening tests in the first or second trimester for conditions related to placental dysfunction (preeclampsia, SGA, or fetal death) using either biochemical markers
      • Jauniaux E.
      • Gulbis B.
      • Tunkel S.
      • Ramsay B.
      • Campbell S.
      • Meuris S.
      Maternal serum testing for alpha-fetoprotein and human chorionic gonadotropin in high-risk pregnancies.
      • Smith G.C.
      • Crossley J.A.
      • Aitken D.A.
      • et al.
      First-trimester placentation and the risk of antepartum stillbirth.
      • Spencer K.
      • Yu C.K.
      • Cowans N.J.
      • Otigbah C.
      • Nicolaides K.H.
      Prediction of pregnancy complications by first-trimester maternal serum PAPP-A and free beta-hCG and with second-trimester uterine artery Doppler.
      • Dugoff L.
      • Hobbins J.C.
      • Malone F.D.
      • et al.
      Quad screen as a predictor of adverse pregnancy outcome.
      • Odibo A.O.
      • Sehdev H.M.
      • Stamilio D.M.
      • Macones G.A.
      Evaluating the thresholds of abnormal second trimester multiple marker screening tests associated with intra-uterine growth restriction.
      • Gagnon A.
      • Wilson R.D.
      • Audibert F.
      • et al.
      Obstetrical complications associated with abnormal maternal serum markers analytes.
      • Olsen R.N.
      • Woelkers D.
      • Dunsmoor-Su R.
      • Lacoursiere D.Y.
      Abnormal second-trimester serum analytes are more predictive of preterm preeclampsia.
      or UADV
      • Poon L.C.
      • Akolekar R.
      • Lachmann R.
      • Beta J.
      • Nicolaides K.H.
      Hypertensive disorders in pregnancy: screening by biophysical and biochemical markers at 11-13 weeks.
      • Papageorghiou A.T.
      • Yu C.K.
      • Bindra R.
      • Pandis G.
      • Nicolaides K.H.
      Multicenter screening for pre-eclampsia and fetal growth restriction by transvaginal uterine artery Doppler at 23 weeks of gestation.
      • Papageorghiou A.T.
      • Leslie K.
      Uterine artery Doppler in the prediction of adverse pregnancy outcome.
      • Smith G.C.
      • Yu C.K.
      • Papageorghiou A.T.
      • Cacho A.M.
      • Nicolaides K.H.
      Maternal uterine artery Doppler flow velocimetry and the risk of stillbirth.
      indicate that both are strongly associated with complications that develop earlier in pregnancy and therefore, are temporally close to the assessment of biomarkers.
      • Smith G.C.
      Researching new methods of screening for adverse pregnancy outcome: lessons from pre-eclampsia.
      • Smith G.C.
      • Shah I.
      • White I.R.
      • Pell J.P.
      • Crossley J.A.
      • Dobbie R.
      Maternal and biochemical predictors of antepartum stillbirth among nulliparous women in relation to gestational age of fetal death.
      Our findings that plasma concentrations of the ratio between angiogenic/antiangiogenic factors outperformed those that are obtained in the first 2 trimesters for the identification of patients with late preeclampsia strongly support this view; (2) The risk for a prospective stillbirth increases after 34 weeks of gestation
      • MacDorman M.F.
      • Kirmeyer S.
      Fetal and perinatal mortality, United States, 2005.
      and similarly, the prevalence of late-onset preeclampsia is much higher than that of early-onset disease;
      • Sibai B.
      • Dekker G.
      • Kupferminc M.
      Pre-eclampsia.
      • Kusanovic J.P.
      • Romero R.
      • Chaiworapongsa T.
      • et al.
      A prospective cohort study of the value of maternal plasma concentrations of angiogenic and anti-angiogenic factors in early pregnancy and midtrimester in the identification of patients destined to develop preeclampsia.
      and (3) The strategy of testing at the beginning of the third trimester to assess the risk of disease or pregnancy complications could be considered for patients who did not receive earlier prenatal care or undergo testing.
      A disadvantage of performing a screening test in the third trimester is that this may be too late to implement therapeutic interventions that can reverse a pathophysiologic process responsible for disease. However, the precise mechanisms of late-onset preeclampsia are unknown, and there is no effective intervention even if at-risk patients are identified in early gestation. Although a recent metaanalysis suggests that the administration of aspirin at before 16 weeks of gestation may prevent preeclampsia,
      • Bujold E.
      • Roberge S.
      • Lacasse Y.
      • et al.
      Prevention of preeclampsia and intrauterine growth restriction with aspirin started in early pregnancy: a meta-analysis.
      this strategy is not effective to prevent preeclampsia at term.
      • Roberge S.
      • Villa P.
      • Nicolaides K.
      • et al.
      Early administration of low-dose aspirin for the prevention of preterm and term preeclampsia: a systematic review and meta-analysis.
      Thus, a method to identify patients at risk for late preeclampsia is needed, given that it accounts for most severe maternal morbidity, including eclampsia,
      • Tuffnell D.J.
      • Jankowicz D.
      • Lindow S.W.
      • et al.
      Outcomes of severe pre-eclampsia/eclampsia in Yorkshire 1999/2003.
      • Liu S.
      • Joseph K.S.
      • Liston R.M.
      • et al.
      Incidence, risk factors, and associated complications of eclampsia.
      • Duley L.
      • Gulmezoglu A.M.
      • Henderson-Smart D.J.
      • Chou D.
      Magnesium sulphate and other anticonvulsants for women with pre-eclampsia.
      especially in developing countries.
      • Kenneth L.
      • Hall D.R.
      • Gebhardt S.
      • Grove D.
      Late onset preeclampsia is not an innocuous condition.
      Furthermore, this study, which examined the predictive performance of biomarkers in the third trimester in a low-risk population, is consistent with the recently proposed approach for the screening of adverse pregnancy outcomes that focuses on the prevention of pregnancy complications at term in low-risk, unselected populations.
      • Smith G.C.
      Researching new methods of screening for adverse pregnancy outcome: lessons from pre-eclampsia.
      However, although an appreciable magnitude of the association was observed between these biomarkers in the third trimester and late severe preeclampsia (which outperformed the screening implemented in earlier gestations), further studies are necessary to explain why some women with distinctly abnormal angiogenic/antiangiogenic profiles have uncomplicated pregnancies. Answers to this question would allow for even greater predictive performance than observed in this study.

      Risk assessment for stillbirth: a neglected area of prenatal care

      Our findings open new avenues for understanding the pathophysiologic process of the disease, risk assessment, and prevention of stillbirth, which is a neglected area of prenatal care.
      • Froen J.F.
      • Cacciatore J.
      • McClure E.M.
      • et al.
      Stillbirths: why they matter.
      • Lawn J.E.
      • Blencowe H.
      • Pattinson R.
      • et al.
      Stillbirths: Where? When? Why? How to make the data count?.
      In our cohort, there were no cases of intrapartum stillbirth. Among 8 stillbirths that were identified with these biomarkers from the cohort and case-control studies, 7 had placental lesions suggestive of maternal underperfusion.
      • Redline R.W.
      • Heller D.
      • Keating S.
      • Kingdom J.
      Placental diagnostic criteria and clinical correlation: a workshop report.
      These lesions, although observed in 15% of uncomplicated term pregnancies, are found more frequently in preeclampsia (relative risk 2-3).
      • Ogge G.
      • Chaiworapongsa T.
      • Kusanovic J.P.
      • et al.
      Evidence that placental lesions are more frequent in early-onset than in late-onset preeclampsia.
      Another patient with a stillbirth had chronic chorioamnionitis, a lesion associated with evidence of maternal antifetal rejection and fetal death.
      • Lee J.
      • Romero R.
      • Dong Z.
      • et al.
      Unexplained fetal death has a biological signature of maternal anti-fetal rejection: chronic chorioamnionitis and alloimmune anti-human leucocyte antigen antibodies.
      Although the precise mechanisms responsible for stillbirth are unknown, it appears that the biomarkers investigated in this study may be able to identify a large fraction of stillbirths that result from placenta-related, rather than nonplacenta-related, causes (such as cord accident, fetal thrombosis, or fetomaternal hemorrhage). This interpretation is consistent with the findings from a recent study that demonstrated an association between stillbirth at or near term and UADV in the second trimester, which indicated that an increase in impedance to blood flow to the placenta is one of the major risk factors for stillbirth at term.
      • Singh T.
      • Leslie K.
      • Bhide A.
      • D'Antonio F.
      • Thilaganathan B.
      Role of second-trimester uterine artery Doppler in assessing stillbirth risk.
      Moreover, because markers of placental dysfunction (such as high maternal serum alpha-fetoprotein or human chorionic gonadotropin), are associated with an increased risk of unexplained stillbirth and other pregnancy complications (such as preeclampsia or SGA),
      • Dugoff L.
      • Hobbins J.C.
      • Malone F.D.
      • et al.
      Quad screen as a predictor of adverse pregnancy outcome.
      • Odibo A.O.
      • Sehdev H.M.
      • Stamilio D.M.
      • Macones G.A.
      Evaluating the thresholds of abnormal second trimester multiple marker screening tests associated with intra-uterine growth restriction.
      • Gagnon A.
      • Wilson R.D.
      • Audibert F.
      • et al.
      Obstetrical complications associated with abnormal maternal serum markers analytes.
      • Smith G.C.
      • Shah I.
      • White I.R.
      • Pell J.P.
      • Crossley J.A.
      • Dobbie R.
      Maternal and biochemical predictors of antepartum stillbirth among nulliparous women in relation to gestational age of fetal death.
      • Spencer K.
      Second-trimester prenatal screening for Down syndrome and the relationship of maternal serum biochemical markers to pregnancy complications with adverse outcome.
      • Yaron Y.
      • Cherry M.
      • Kramer R.L.
      • et al.
      Second-trimester maternal serum marker screening: maternal serum alpha-fetoprotein, beta-human chorionic gonadotropin, estriol, and their various combinations as predictors of pregnancy outcome.
      • Lepage N.
      • Chitayat D.
      • Kingdom J.
      • Huang T.
      Association between second-trimester isolated high maternal serum maternal serum human chorionic gonadotropin levels and obstetric complications in singleton and twin pregnancies.
      it is possible that a subset of unexplained stillbirths, preeclampsia, and SGA represent different clinical manifestations of a placental response from insults at different gestational ages. Evidence in support of this hypothesis is that rats subjected to reduced uteroplacental perfusion by the application of clips to the abdominal aorta at different gestational ages had a different magnitude of change in angiogenic/antiangiogenic imbalance, fetal growth restriction, and the severity of placental ischemia–induced systemic hypertension.
      • Banek C.T.
      • Bauer A.J.
      • Gingery A.
      • Gilbert J.S.
      Timing of ischemic insult alters fetal growth trajectory, maternal angiogenic balance and markers of renal oxidative stress in the pregnant rat.
      Our findings of an association between abnormal angiogenic/antiangiogenic factors and stillbirth, preeclampsia, and SGA without preeclampsia strengthen this hypothesis.

      Limitations of the study

      To facilitate comparison of the predictive performance of angiogenic/antiangiogenic factors in the third trimester with that of these biomarkers in the first and second trimester and UADV in the second trimester, a number of patients without plasma samples in the first 2 trimesters and those patients without UADV information were excluded from the cohort. However, the distribution of baseline characteristics (maternal age, body mass index, nulliparity, combined parity, and previous preeclampsia) did not differ significantly from patients included in the current study compared with the overall cohort, and there were no significant differences in the risk of stillbirth or SGA between the full and subcohort. However, by design, participants in the subcohort were more likely to deliver at >34 weeks of gestation. Patients in the subcohort were also less likely to experience late preeclampsia (3.2% vs 4.8%; P = .03), which indicates that our results for preeclampsia may be biased toward the null hypothesis.
      Another limitation is that the determination of plasma concentrations of these biomarkers was performed in stored samples. However, serum PlGF and sVEGFR-1 concentrations are quite stable when stored at −80°C,
      • Law L.W.
      • Sahota D.S.
      • Chan L.W.
      • Chen M.
      • Lau T.K.
      • Leung T.Y.
      Effect of long-term storage on placental growth factor and fms-like tyrosine kinase 1 measurements in samples from pregnant women.
      and the duration of sample storage did not differ significantly by pregnancy outcome. Therefore, this is unlikely to have introduced bias into our study.
      A limited number of patients with stillbirth were included in this cohort of a low-risk population. This is a common problem of cohort studies with rare outcomes. We used statistical methods to address possible over-fitting of models and replicated our findings in a separate case-control study performed in a different population. Altogether, our results indicate that the ratio of maternal plasma concentrations of angiogenic/antiangiogenic factors in the third trimester have value in risk assessment for stillbirth, given the high likelihood ratio of a positive test (LR, 14.2), although further study is necessary both to validate these findings and to determine generalizability.
      A low positive predictive value, consistent with that observed in this study, is often invoked as a limitation of a test. However, it is noteworthy that positive predictive values are dependent on the prevalence of the disease and will always be low when the condition under study is rare. This is the case for stillbirth, which had a prevalence of 0.4% in our study. Even if we had a test with 99% sensitivity and 99% specificity, the positive predictive value, given the prevalence of disease, would be 28%. Thus, most patients who are screened positive would not have the disease (false-positive). Yet, this scenario occurs daily in the practice of obstetrics (eg, most patients who undergo mid trimester amniocentesis or chorionic villous sampling do not have aneuploidy).

      Conclusion

      Risk assessment for severe late preeclampsia and stillbirth in the third trimester may be possible with the determination of maternal plasma concentrations of angiogenic and antiangiogenic factors at 30-34 weeks of gestation. Of interest, the StAmP trial (http://www.birmingham.ac.uk/research/activity/index.aspx), a randomized controlled trial to determine the effect of pravastatin on the changes of maternal angiogenic/antiangiogenic factor concentrations in patients with a diagnosis of early-onset preeclampsia, is ongoing in the United Kingdom. Statins have the potential to reverse the abnormalities in angiogenic/antiangiogenic factors
      • Kumasawa K.
      • Ikawa M.
      • Kidoya H.
      • et al.
      Pravastatin induces placental growth factor (PGF) and ameliorates preeclampsia in a mouse model.
      demonstrated in unexplained stillbirth,
      • Romero R.
      • Chaiworapongsa T.
      • Erez O.
      • et al.
      An imbalance between angiogenic and anti-angiogenic factors precedes fetal death in a subset of patients: results of a longitudinal study.
      and may represent an intervention for patients identified with the approach herein. Other proposed therapeutic interventions to reverse an antiangiogenic state during pregnancy include the administration of VEGF 121
      • Li Z.
      • Zhang Y.
      • Ying M.J.
      • et al.
      Recombinant vascular endothelial growth factor 121 attenuates hypertension and improves kidney damage in a rat model of preeclampsia.
      or extracorporeal removal of sVEGFR-1.
      • Thadhani R.
      • Kisner T.
      • Hagmann H.
      • et al.
      Pilot study of extracorporeal removal of soluble fms-like tyrosine kinase 1 in preeclampsia.
      Accordingly, biomarkers investigated in this study in the third trimester may be useful as an additional tool for risk stratification in future interventional trials for the prevention of stillbirth and/or severe late preeclampsia at or near term. A specific clinical example is that patients at risk for stillbirth, after being identified by the markers proposed herein, can undergo intensive antepartum surveillance and deliver at or near term once the risks of prolonging pregnancy outweigh those of complications of prematurity.

      Acknowledgment

      The authors thank Professor Gordon C.S. Smith of Cambridge University, UK, for his advice and helpful discussions about the analyses performed in this study.

      References

        • Romero R.
        • Lockwood C.
        • Oyarzun E.
        • Hobbins J.C.
        Toxemia: new concepts in an old disease.
        Semin Perinatol. 1988; 12: 302-323
        • Redman C.W.
        • Sargent I.L.
        Latest advances in understanding preeclampsia.
        Science. 2005; 308: 1592-1594
        • Berg C.J.
        • Callaghan W.M.
        • Syverson C.
        • Henderson Z.
        Pregnancy-related mortality in the United States, 1998 to 2005.
        Obstet Gynecol. 2010; 116: 1302-1309
        • Zhang J.
        • Meikle S.
        • Trumble A.
        Severe maternal morbidity associated with hypertensive disorders in pregnancy in the United States.
        Hypertens Pregnancy. 2003; 22: 203-212
        • Brosens I.A.
        Morphological changes in the utero-placental bed in pregnancy hypertension.
        Clin Obstet Gynaecol. 1977; 4: 573-593
        • Brosens J.J.
        • Pijnenborg R.
        • Brosens I.A.
        The myometrial junctional zone spiral arteries in normal and abnormal pregnancies: a review of the literature.
        Am J Obstet Gynecol. 2002; 187: 1416-1423
        • Pijnenborg R.
        • Anthony J.
        • Davey D.A.
        • et al.
        Placental bed spiral arteries in the hypertensive disorders of pregnancy.
        BJOG. 1991; 98: 648-655
        • Robertson W.B.
        • Brosens I.
        • Dixon G.
        Maternal uterine vascular lesions in the hypertensive complications of pregnancy.
        Perspect Nephrol Hypertens. 1976; 5: 115-127
        • Brosens I.
        • Pijnenborg R.
        • Vercruysse L.
        • Romero R.
        The “Great Obstetrical Syndromes” are associated with disorders of deep placentation.
        Am J Obstet Gynecol. 2011; 204: 193-201
        • Romero R.
        • Kusanovic J.P.
        • Chaiworapongsa T.
        • Hassan S.S.
        Placental bed disorders in preterm labor, preterm PROM, spontaneous abortion and abruptio placentae.
        Best Pract Res Clin Obstet Gynaecol. 2011; 25: 313-327
        • Sacks G.P.
        • Studena K.
        • Sargent K.
        • Redman C.W.
        Normal pregnancy and preeclampsia both produce inflammatory changes in peripheral blood leukocytes akin to those of sepsis.
        Am J Obstet Gynecol. 1998; 179: 80-86
        • Redman C.W.
        • Sacks G.P.
        • Sargent I.L.
        Preeclampsia: an excessive maternal inflammatory response to pregnancy.
        Am J Obstet Gynecol. 1999; 180: 499-506
        • Gervasi M.T.
        • Chaiworapongsa T.
        • Pacora P.
        • et al.
        Phenotypic and metabolic characteristics of monocytes and granulocytes in preeclampsia.
        Am J Obstet Gynecol. 2001; 185: 792-797
        • Friedman S.A.
        • Schiff E.
        • Emeis J.J.
        • Dekker G.A.
        • Sibai B.M.
        Biochemical corroboration of endothelial involvement in severe preeclampsia.
        Am J Obstet Gynecol. 1995; 172: 202-203
        • Lyall F.
        • Greer I.A.
        The vascular endothelium in normal pregnancy and pre-eclampsia.
        Rev Reprod. 1996; 1: 107-116
        • Roberts J.M.
        • Taylor R.N.
        • Goldfien A.
        Clinical and biochemical evidence of endothelial cell dysfunction in the pregnancy syndrome preeclampsia.
        Am J Hypertens. 1991; 4: 700-708
        • Roberts J.M.
        Endothelial dysfunction in preeclampsia.
        Semin Reprod Endocrinol. 1998; 16: 5-15
        • Taylor R.N.
        • de Groot C.J.
        • Cho Y.K.
        • Lim K.H.
        Circulating factors as markers and mediators of endothelial cell dysfunction in preeclampsia.
        Semin Reprod Endocrinol. 1998; 16: 17-31
        • Chaiworapongsa T.
        • Romero R.
        • Yoshimatsu J.
        • et al.
        Soluble adhesion molecule profile in normal pregnancy and pre-eclampsia.
        J Matern Fetal Neonatal Med. 2002; 12: 19-27
        • Petrozella L.
        • Mahendroo M.
        • Timmons B.
        • Roberts S.
        • McIntire D.
        • Alexander J.M.
        Endothelial microparticles and the antiangiogenic state in preeclampsia and the postpartum period.
        Am J Obstet Gynecol. 2012; 207: 140-146
        • de Boer K.
        • ten Cate J.W.
        • Sturk A.
        • Borm J.J.
        • Treffers P.E.
        Enhanced thrombin generation in normal and hypertensive pregnancy.
        Am J Obstet Gynecol. 1989; 160: 95-100
        • Chaiworapongsa T.
        • Yoshimatsu J.
        • Espinoza J.
        • et al.
        Evidence of in vivo generation of thrombin in patients with small-for-gestational-age fetuses and pre-eclampsia.
        J Matern Fetal Neonatal Med. 2002; 11: 362-367
        • Cadroy Y.
        • Grandjean H.
        • Pichon J.
        • et al.
        Evaluation of six markers of haemostatic system in normal pregnancy and pregnancy complicated by hypertension or pre-eclampsia.
        BJOG. 1993; 100: 416-420
        • Higgins J.R.
        • Walshe J.J.
        • Darling M.R.
        • Norris L.
        • Bonnar J.
        Hemostasis in the uteroplacental and peripheral circulations in normotensive and pre-eclamptic pregnancies.
        Am J Obstet Gynecol. 1998; 179: 520-526
        • Hayashi M.
        • Inoue T.
        • Hoshimoto K.
        • Negishi H.
        • Ohkura T.
        • Inaba N.
        Characterization of five marker levels of the hemostatic system and endothelial status in normotensive pregnancy and pre-eclampsia.
        Eur J Haematol. 2002; 69: 297-302
        • Hubel C.A.
        Oxidative stress in the pathogenesis of preeclampsia.
        Proc Soc Exp Biol Med. 1999; 222: 222-235
        • Vaughan J.E.
        • Walsh S.W.
        Oxidative stress reproduces placental abnormalities of preeclampsia.
        Hypertens Pregnancy. 2002; 21: 205-223
        • Myatt L.
        • Kossenjans W.
        • Sahay R.
        • Eis A.
        • Brockman D.
        Oxidative stress causes vascular dysfunction in the placenta.
        J Matern Fetal Med. 2000; 9: 79-82
        • Zhou X.
        • Zhang G.Y.
        • Wang J.
        • Lu S.L.
        • Cao J.
        • Sun L.Z.
        A novel bridge between oxidative stress and immunity: the interaction between hydrogen peroxide and human leukocyte antigen G in placental trophoblasts during preeclampsia.
        Am J Obstet Gynecol. 2012; 206 (447-16)
        • Torry D.S.
        • Wang H.S.
        • Wang T.H.
        • Caudle M.R.
        • Torry R.J.
        Preeclampsia is associated with reduced serum levels of placenta growth factor.
        Am J Obstet Gynecol. 1998; 179: 1539-1544
        • Tidwell S.C.
        • Ho H.N.
        • Chiu W.H.
        • Torry R.J.
        • Torry D.S.
        Low maternal serum levels of placenta growth factor as an antecedent of clinical preeclampsia.
        Am J Obstet Gynecol. 2001; 184: 1267-1272
        • Maynard S.E.
        • Min J.Y.
        • Merchan J.
        • et al.
        Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia.
        J Clin Invest. 2003; 111: 649-658
        • Koga K.
        • Osuga Y.
        • Yoshino O.
        • et al.
        Elevated serum soluble vascular endothelial growth factor receptor 1 (sVEGFR-1) levels in women with preeclampsia.
        J Clin Endocrinol Metab. 2003; 88: 2348-2351
        • Taylor R.N.
        • Grimwood J.
        • Taylor R.S.
        • McMaster M.T.
        • Fisher S.J.
        • North R.A.
        Longitudinal serum concentrations of placental growth factor: evidence for abnormal placental angiogenesis in pathologic pregnancies.
        Am J Obstet Gynecol. 2003; 188: 177-182
        • Tsatsaris V.
        • Goffin F.
        • Munaut C.
        • et al.
        Overexpression of the soluble vascular endothelial growth factor receptor in preeclamptic patients: pathophysiological consequences.
        J Clin Endocrinol Metab. 2003; 88: 5555-5563
        • Chaiworapongsa T.
        • Romero R.
        • Espinoza J.
        • et al.
        Evidence supporting a role for blockade of the vascular endothelial growth factor system in the pathophysiology of preeclampsia: Young Investigator Award.
        Am J Obstet Gynecol. 2004; 190: 1541-1547
        • Venkatesha S.
        • Toporsian M.
        • Lam C.
        • et al.
        Soluble endoglin contributes to the pathogenesis of preeclampsia.
        Nat Med. 2006; 12: 642-649
        • Weissgerber T.L.
        • Roberts J.M.
        • Jeyabalan A.
        • et al.
        Haptoglobin phenotype, angiogenic factors, and preeclampsia risk.
        Am J Obstet Gynecol. 2012; 206: 358
        • von Dadelszen P.
        • Magee L.A.
        • Roberts J.M.
        Subclassification of preeclampsia.
        Hypertens Pregnancy. 2003; 22: 143-148
        • Romero R.
        • Vizoso J.
        • Emamian M.
        • et al.
        Clinical significance of liver dysfunction in pregnancy-induced hypertension.
        Am J Perinatol. 1988; 5: 146-151
        • Romero R.
        • Mazor M.
        • Lockwood C.J.
        • et al.
        Clinical significance, prevalence, and natural history of thrombocytopenia in pregnancy-induced hypertension.
        Am J Perinatol. 1989; 6: 32-38
        • Long P.A.
        • Abell D.A.
        • Beischer N.A.
        Fetal growth retardation and pre-eclampsia.
        BJOG. 1980; 87: 13-18
        • Ogge G.
        • Chaiworapongsa T.
        • Kusanovic J.P.
        • et al.
        Evidence that placental lesions are more frequent in early-onset than in late-onset preeclampsia.
        Reprod Sci. 2011; 18: 124A
        • Sibai B.M.
        Preeclampsia as a cause of preterm and late preterm (near-term) births.
        Semin Perinatol. 2006; 30: 16-19
        • Sibai B.
        • Dekker G.
        • Kupferminc M.
        Pre-eclampsia.
        Lancet. 2005; 365: 785-799
        • Ales K.L.
        • Charlson M.E.
        Epidemiology of preeclampsia and eclampsia.
        Am J Obstet Gynecol. 1991; 165: 238
        • Tuffnell D.J.
        • Jankowicz D.
        • Lindow S.W.
        • et al.
        Outcomes of severe pre-eclampsia/eclampsia in Yorkshire 1999/2003.
        BJOG. 2005; 112: 875-880
        • McIntire D.D.
        • Leveno K.J.
        Neonatal mortality and morbidity rates in late preterm births compared with births at term.
        Obstet Gynecol. 2008; 111: 35-41
        • Lubow J.M.
        • How H.Y.
        • Habli M.
        • Maxwell R.
        • Sibai B.M.
        Indications for delivery and short-term neonatal outcomes in late preterm as compared with term births.
        Am J Obstet Gynecol. 2009; 200: e30-e33
        • Ananth C.V.
        • Vintzileos A.M.
        Maternal-fetal conditions necessitating a medical intervention resulting in preterm birth.
        Am J Obstet Gynecol. 2006; 195: 1557-1563
        • Kenneth L.
        • Hall D.R.
        • Gebhardt S.
        • Grove D.
        Late onset preeclampsia is not an innocuous condition.
        Hypertens Pregnancy. 2010; 29: 262-270
        • Liu S.
        • Joseph K.S.
        • Liston R.M.
        • et al.
        Incidence, risk factors, and associated complications of eclampsia.
        Obstet Gynecol. 2011; 118: 987-994
        • Duley L.
        • Gulmezoglu A.M.
        • Henderson-Smart D.J.
        • Chou D.
        Magnesium sulphate and other anticonvulsants for women with pre-eclampsia.
        Cochrane Database Syst Rev. 2010; (CD000025)
        • Moodley J.
        Maternal deaths associated with hypertensive disorders of pregnancy: a population-based study.
        Hypertens Pregnancy. 2004; 23: 247-256
        • Romero R.
        Prenatal medicine: the child is the father of the man.
        J Matern Fetal Neonatal Med. 2009; 22: 636-639
        • Fretts R.C.
        Etiology and prevention of stillbirth.
        Am J Obstet Gynecol. 2005; 193: 1923-1935
        • Froen J.F.
        • Cacciatore J.
        • McClure E.M.
        • et al.
        Stillbirths: why they matter.
        Lancet. 2011; 377: 1353-1366
        • Lawn J.E.
        • Yakoob M.Y.
        • Haws R.A.
        • Soomro T.
        • Darmstadt G.L.
        • Bhutta Z.A.
        3.2 million stillbirths: epidemiology and overview of the evidence review.
        BMC Pregnancy Childbirth. 2009; 9: S2
        • Bhutta Z.A.
        • Yakoob M.Y.
        • Lawn J.E.
        • et al.
        Stillbirths: what difference can we make and at what cost?.
        Lancet. 2011; 377: 1523-1538
        • Flenady V.
        • Middleton P.
        • Smith G.C.
        • et al.
        Stillbirths: the way forward in high-income countries.
        Lancet. 2011; 377: 1703-1717
        • Lawn J.E.
        • Blencowe H.
        • Pattinson R.
        • et al.
        Stillbirths: Where?.
        Lancet. 2011; 377: 1448-1463
        • McClure E.M.
        • Saleem S.
        • Pasha O.
        • Goldenberg R.L.
        Stillbirth in developing countries: a review of causes, risk factors and prevention strategies.
        J Matern Fetal Neonatal Med. 2009; 22: 183-190
        • Menezes E.V.
        • Yakoob M.Y.
        • Soomro T.
        • Haws R.A.
        • Darmstadt G.L.
        • Bhutta Z.A.
        Reducing stillbirths: prevention and management of medical disorders and infections during pregnancy.
        BMC Pregnancy Childbirth. 2009; 9: S4
        • Yakoob M.Y.
        • Lawn J.E.
        • Darmstadt G.L.
        • Bhutta Z.A.
        Stillbirths: epidemiology, evidence, and priorities for action.
        Semin Perinatol. 2010; 34: 387-394
        • Smith G.C.
        Predicting antepartum stillbirth.
        Clin Obstet Gynecol. 2010; 53: 597-606
        • Hui D.
        • Okun N.
        • Murphy K.
        • Kingdom J.
        • Uleryk E.
        • Shah P.S.
        Combinations of maternal serum markers to predict preeclampsia, small for gestational age, and stillbirth: a systematic review.
        J Obstet Gynaecol Can. 2012; 34: 142-153
        • Reuvekamp A.
        • Velsing-Aarts F.V.
        • Poulina I.E.
        • Capello J.J.
        • Duits A.J.
        Selective deficit of angiogenic growth factors characterises pregnancies complicated by pre-eclampsia.
        BJOG. 1999; 106: 1019-1022
        • Ahmed A.
        New insights into the etiology of preeclampsia: identification of key elusive factors for the vascular complications.
        Thromb Res. 2011; 127: S72-S75
        • Luttun A.
        • Carmeliet P.
        Soluble VEGF receptor Flt1: the elusive preeclampsia factor discovered?.
        J Clin Invest. 2003; 111: 600-602
        • Bujold E.
        • Romero R.
        • Chaiworapongsa T.
        • et al.
        Evidence supporting that the excess of the sVEGFR-1 concentration in maternal plasma in preeclampsia has a uterine origin.
        J Matern Fetal Neonatal Med. 2005; 18: 9-16
        • Crispi F.
        • Dominguez C.
        • Llurba E.
        • Martin-Gallan P.
        • Cabero L.
        • Gratacos E.
        Placental angiogenic growth factors and uterine artery Doppler findings for characterization of different subsets in preeclampsia and in isolated intrauterine growth restriction.
        Am J Obstet Gynecol. 2006; 195: 201-207
        • Chaiworapongsa T.
        • Romero R.
        • Gotsch F.
        • et al.
        Low maternal concentrations of soluble vascular endothelial growth factor receptor-2 in preeclampsia and small for gestational age.
        J Matern Fetal Neonatal Med. 2008; 21: 41-52
        • Chaiworapongsa T.
        • Romero R.
        • Tarca A.L.
        • et al.
        A decrease in maternal plasma concentrations of sVEGFR-2 precedes the clinical diagnosis of preeclampsia.
        Am J Obstet Gynecol. 2010; 202 (550-10)
        • Benton S.J.
        • Hu Y.
        • Xie F.
        • et al.
        Angiogenic factors as diagnostic tests for preeclampsia: a performance comparison between two commercial immunoassays.
        Am J Obstet Gynecol. 2011; 205 (469-8)
        • Garovic V.D.
        The role of angiogenic factors in the prediction and diagnosis of preeclampsia superimposed on chronic hypertension.
        Hypertension. 2012; 59: 555-557
        • Ghosh S.K.
        • Raheja S.
        • Tuli A.
        • Raghunandan C.
        • Agarwal S.
        Serum PLGF as a potential biomarker for predicting the onset of preeclampsia.
        Arch Gynecol Obstet. 2012; 285: 417-422
        • Chaiworapongsa T.
        • Romero R.
        • Savasan Z.A.
        • et al.
        Maternal plasma concentrations of angiogenic/anti-angiogenic factors are of prognostic value in patients presenting to the obstetrical triage area with the suspicion of preeclampsia.
        J Matern Fetal Neonatal Med. 2011; 24: 1187-1207
        • Hagmann H.
        • Thadhani R.
        • Benzing T.
        • Karumanchi S.A.
        • Stepan H.
        The promise of angiogenic markers for the early diagnosis and prediction of preeclampsia.
        Clin Chem. 2012; 58: 837-845
        • Molvarec A.
        • Szarka A.
        • Walentin S.
        • Szucs E.
        • Nagy B.
        • Rigo Jr, J.
        Circulating angiogenic factors determined by electrochemiluminescence immunoassay in relation to the clinical features and laboratory parameters in women with pre-eclampsia.
        Hypertens Res. 2010; 33: 892-898
        • Rana S.
        • Powe C.E.
        • Salahuddin S.
        • et al.
        Angiogenic factors and the risk of adverse outcomes in women with suspected preeclampsia.
        Circulation. 2012; 125: 911-919
        • Verlohren S.
        • Stepan H.
        • Dechend R.
        Angiogenic growth factors in the diagnosis and prediction of pre-eclampsia.
        Clin Sci (Lond). 2012; 122: 43-52
        • Staff A.C.
        • Braekke K.
        • Johnsen G.M.
        • Karumanchi S.A.
        • Harsem N.K.
        Circulating concentrations of soluble endoglin (CD105) in fetal and maternal serum and in amniotic fluid in preeclampsia.
        Am J Obstet Gynecol. 2007; 197: 176
        • Smith G.C.
        • Wear H.
        The perinatal implications of angiogenic factors.
        Curr Opin Obstet Gynecol. 2009; 21: 111-116
        • Weed S.
        • Bastek J.A.
        • Anton L.
        • Elovitz M.A.
        • Parry S.
        • Srinivas S.K.
        Examining the correlation between placental and serum placenta growth factor in preeclampsia.
        Am J Obstet Gynecol. 2012; 207: 140-146
        • Maynard S.E.
        • Karumanchi S.A.
        Angiogenic factors and preeclampsia.
        Semin Nephrol. 2011; 31: 33-46
        • Lam C.
        • Lim K.H.
        • Karumanchi S.A.
        Circulating angiogenic factors in the pathogenesis and prediction of preeclampsia.
        Hypertension. 2005; 46: 1077-1085
        • Karumanchi S.A.
        • Maynard S.E.
        • Stillman I.E.
        • Epstein F.H.
        • Sukhatme V.P.
        Preeclampsia: a renal perspective.
        Kidney Int. 2005; 67: 2101-2113
        • Levine R.J.
        • Karumanchi S.A.
        Circulating angiogenic factors in preeclampsia.
        Clin Obstet Gynecol. 2005; 48: 372-386
        • Chaiworapongsa T.
        • Espinoza J.
        • Gotsch F.
        • et al.
        The maternal plasma soluble vascular endothelial growth factor receptor-1 concentration is elevated in SGA and the magnitude of the increase relates to Doppler abnormalities in the maternal and fetal circulation.
        J Matern Fetal Neonatal Med. 2008; 21: 25-40
        • Crispi F.
        • Llurba E.
        • Dominguez C.
        • Martin-Gallan P.
        • Cabero L.
        • Gratacos E.
        Predictive value of angiogenic factors and uterine artery Doppler for early- versus late-onset pre-eclampsia and intrauterine growth restriction.
        Ultrasound Obstet Gynecol. 2008; 31: 303-309
        • Asvold B.O.
        • Vatten L.J.
        • Romundstad P.R.
        • Jenum P.A.
        • Karumanchi S.A.
        • Eskild A.
        Angiogenic factors in maternal circulation and the risk of severe fetal growth restriction.
        Am J Epidemiol. 2011; 173: 630-639
        • Gourvas V.
        • Dalpa E.
        • Konstantinidou A.
        • Vrachnis N.
        • Spandidos D.A.
        • Sifakis S.
        Angiogenic factors in placentas from pregnancies complicated by fetal growth restriction [review].
        Mol Med Report. 2012; 6: 23-27
        • Espinoza J.
        • Chaiworapongsa T.
        • Romero R.
        • et al.
        Unexplained fetal death: another anti-angiogenic state.
        J Matern Fetal Neonatal Med. 2007; 20: 495-507
        • Chaiworapongsa T.
        • Romero R.
        • Kusanovic J.P.
        • et al.
        Unexplained fetal death is associated with increased concentrations of anti-angiogenic factors in amniotic fluid.
        J Matern Fetal Neonatal Med. 2010; 23: 794-805
        • Chaiworapongsa T.
        • Kusanovic J.P.
        • Savasan Z.A.
        • et al.
        Fetal death: a condition with a dissociation in the concentrations of soluble vascular endothelial growth factor receptor-2 between the maternal and fetal compartments.
        J Matern Fetal Neonatal Med. 2010; 23: 960-972
        • Bixel K.
        • Silasi M.
        • Zelop C.M.
        • et al.
        Placental origins of angiogenic dysfunction in mirror syndrome.
        Hypertens Pregnancy. 2012; 31: 211-217
        • Espinoza J.
        • Romero R.
        • Nien J.K.
        • et al.
        A role of the anti-angiogenic factor sVEGFR-1 in the ‘mirror syndrome’ (Ballantyne's syndrome).
        J Matern Fetal Neonatal Med. 2006; 19: 607-613
        • Kanter D.
        • Lindheimer M.D.
        • Wang E.
        • et al.
        Angiogenic dysfunction in molar pregnancy.
        Am J Obstet Gynecol. 2010; 202: 184-185
        • Koga K.
        • Osuga Y.
        • Tajima T.
        • et al.
        Elevated serum soluble fms-like tyrosine kinase 1 (sFlt1) level in women with hydatidiform mole.
        Fertil Steril. 2010; 94: 305-308
        • Kusanovic J.P.
        • Romero R.
        • Espinoza J.
        • et al.
        Twin-to-twin transfusion syndrome: an antiangiogenic state?.
        Am J Obstet Gynecol. 2008; 198: 382-388
        • Llurba E.
        • Marsal G.
        • Sanchez O.
        • et al.
        Angiogenic and antiangiogenic factors before and after resolution of maternal mirror syndrome.
        Ultrasound Obstet Gynecol. 2011; (Epub ahead of print)
        • Rana S.
        • Venkatesha S.
        • DePaepe M.
        • Chien E.K.
        • Paglia M.
        • Karumanchi S.A.
        Cytomegalovirus-induced mirror syndrome associated with elevated levels of circulating antiangiogenic factors.
        Obstet Gynecol. 2007; 109: 549-552
        • Chaiworapongsa T.
        • Romero R.
        • Kim Y.M.
        • et al.
        Plasma soluble vascular endothelial growth factor receptor-1 concentration is elevated prior to the clinical diagnosis of pre-eclampsia.
        J Matern Fetal Neonatal Med. 2005; 17: 3-18
        • Moore Simas T.A.
        • Crawford S.L.
        • Solitro M.J.
        • Frost S.C.
        • Meyer B.A.
        • Maynard S.E.
        Angiogenic factors for the prediction of preeclampsia in high-risk women.
        Am J Obstet Gynecol. 2007; 197: 244-248
        • Erez O.
        • Romero R.
        • Espinoza J.
        • et al.
        The change in concentrations of angiogenic and anti-angiogenic factors in maternal plasma between the first and second trimesters in risk assessment for the subsequent development of preeclampsia and small-for-gestational age.
        J Matern Fetal Neonatal Med. 2008; 21: 279-287
        • Romero R.
        • Nien J.K.
        • Espinoza J.
        • et al.
        A longitudinal study of angiogenic (placental growth factor) and anti-angiogenic (soluble endoglin and soluble vascular endothelial growth factor receptor-1) factors in normal pregnancy and patients destined to develop preeclampsia and deliver a small for gestational age neonate.
        J Matern Fetal Neonatal Med. 2008; 21: 9-23
        • Unal E.R.
        • Robinson C.J.
        • Johnson D.D.
        • Chang E.Y.
        Second-trimester angiogenic factors as biomarkers for future-onset preeclampsia.
        Am J Obstet Gynecol. 2007; 197: 211-214
        • Widmer M.
        • Villar J.
        • Benigni A.
        • Conde-Agudelo A.
        • Karumanchi S.A.
        • Lindheimer M.
        Mapping the theories of preeclampsia and the role of angiogenic factors: a systematic review.
        Obstet Gynecol. 2007; 109: 168-180
        • Park C.W.
        • Park J.S.
        • Shim S.S.
        • Jun J.K.
        • Yoon B.H.
        • Romero R.
        An elevated maternal plasma, but not amniotic fluid, soluble fms-like tyrosine kinase-1 (sFlt-1) at the time of mid-trimester genetic amniocentesis is a risk factor for preeclampsia.
        Am J Obstet Gynecol. 2005; 193: 984-989
        • Romero R.
        • Chaiworapongsa T.
        • Erez O.
        • et al.
        An imbalance between angiogenic and anti-angiogenic factors precedes fetal death in a subset of patients: results of a longitudinal study.
        J Matern Fetal Neonatal Med. 2010; 23: 1384-1399
        • Smith G.C.
        • Crossley J.A.
        • Aitken D.A.
        • et al.
        Circulating angiogenic factors in early pregnancy and the risk of preeclampsia, intrauterine growth restriction, spontaneous preterm birth, and stillbirth.
        Obstet Gynecol. 2007; 109: 1316-1324
        • Parra-Cordero M.
        • Rodrigo R.
        • Barja P.
        • et al.
        Prediction of early and late pre-eclampsia from maternal characteristics, uterine artery Doppler and markers of vasculogenesis during the first trimester of pregnancy.
        Ultrasound Obstet Gynecol. 2012; (Epub ahead of print)
        • Di L.G.
        • Ceccarello M.
        • Cecotti V.
        • et al.
        First trimester maternal serum PIGF, free beta-hCG, PAPP-A, PP-13, uterine artery Doppler and maternal history for the prediction of preeclampsia.
        Placenta. 2012; 33: 495-501
        • Poon L.C.
        • Akolekar R.
        • Lachmann R.
        • Beta J.
        • Nicolaides K.H.
        Hypertensive disorders in pregnancy: screening by biophysical and biochemical markers at 11-13 weeks.
        Ultrasound Obstet Gynecol. 2010; 35: 662-670
        • Pedrosa A.C.
        • Matias A.
        Screening for pre-eclampsia: a systematic review of tests combining uterine artery Doppler with other markers.
        J Perinat Med. 2011; 39: 619-635
        • Wikstrom A.K.
        • Larsson A.
        • Eriksson U.J.
        • Nash P.
        • Norden-Lindeberg S.
        • Olovsson M.
        Placental growth factor and soluble FMS-like tyrosine kinase-1 in early-onset and late-onset preeclampsia.
        Obstet Gynecol. 2007; 109: 1368-1374
        • Robinson C.J.
        • Johnson D.D.
        • Chang E.Y.
        • Armstrong D.M.
        • Wang W.
        Evaluation of placenta growth factor and soluble Fms-like tyrosine kinase 1 receptor levels in mild and severe preeclampsia.
        Am J Obstet Gynecol. 2006; 195: 255-259
        • Levine R.J.
        • Maynard S.E.
        • Qian C.
        • et al.
        Circulating angiogenic factors and the risk of preeclampsia.
        N Engl J Med. 2004; 350: 672-683
        • Kusanovic J.P.
        • Romero R.
        • Chaiworapongsa T.
        • et al.
        A prospective cohort study of the value of maternal plasma concentrations of angiogenic and anti-angiogenic factors in early pregnancy and midtrimester in the identification of patients destined to develop preeclampsia.
        J Matern Fetal Neonatal Med. 2009; 22: 1021-1038
        • Levine R.J.
        • Lam C.
        • Qian C.
        • et al.
        Soluble endoglin and other circulating antiangiogenic factors in preeclampsia.
        N Engl J Med. 2006; 355: 992-1005
        • Ohkuchi A.
        • Hirashima C.
        • Matsubara S.
        • et al.
        Alterations in placental growth factor levels before and after the onset of preeclampsia are more pronounced in women with early onset severe preeclampsia.
        Hypertens Res. 2007; 30: 151-159
        • Powers R.W.
        • Jeyabalan A.
        • Clifton R.G.
        • et al.
        Soluble fms-Like tyrosine kinase 1 (sFlt1), endoglin and placental growth factor (PlGF) in preeclampsia among high risk pregnancies.
        PLoS One. 2010; 5: e13263
        • Srinivas S.K.
        • Larkin J.
        • Sammel M.D.
        • et al.
        The use of angiogenic factors in discriminating preeclampsia: are they ready for prime time?.
        J Matern Fetal Neonatal Med. 2010; 23: 1294-1300
        • Kleinrouweler C.
        • Wiegerinck M.
        • Ris-Stalpers C.
        • et al.
        Accuracy of circulating placental growth factor, vascular endothelial growth factor, soluble fms-like tyrosine kinase 1 and soluble endoglin in the prediction of pre-eclampsia: a systematic review and meta-analysis.
        BJOG. 2012; 119: 778-787
        • Smith G.C.
        Researching new methods of screening for adverse pregnancy outcome: lessons from pre-eclampsia.
        PLoS Med. 2012; 9: e1001274
        • American College of Obstetricians and Gynecologists
        ACOG practice bulletin no. 33: diagnosis and management of preeclampsia and eclampsia.
        Obstet Gynecol. 2002; 99: 159-167
        • Gonzalez R.P.
        • Gomez R.M.
        • Castro R.S.
        • et al.
        [A national birth weight distribution curve according to gestational age in Chile from 1993 to 2000].
        Rev Med Chil. 2004; 132: 1155-1165
        • MacDorman M.F.
        • Kirmeyer S.
        The challenge of fetal mortality.
        NCHS Data Brief. 2009; 16: 1-8
        • Albaiges G.
        • Missfelder-Lobos H.
        • Lees C.
        • Parra M.
        • Nicolaides K.H.
        One-stage screening for pregnancy complications by color Doppler assessment of the uterine arteries at 23 weeks' gestation.
        Obstet Gynecol. 2000; 96: 559-564
        • Yu C.K.
        • Khouri O.
        • Onwudiwe N.
        • Spiliopoulos Y.
        • Nicolaides K.H.
        • Fetal Medicine Foundation Second-Trimester Screening Group
        Prediction of pre-eclampsia by uterine artery Doppler imaging: relationship to gestational age at delivery and small-for-gestational age.
        Ultrasound Obstet Gynecol. 2008; 31: 310-313
        • Koenker R.
        • Bassett G.
        Regression quantiles.
        Econometrica. 1978; 46: 36-50
        • Steyerberg E.W.
        • Eijkemans M.J.
        • Harrell Jr, F.E.
        • Habbema J.D.
        Prognostic modelling with logistic regression analysis: a comparison of selection and estimation methods in small data sets.
        Stat Med. 2000; 19: 1059-1079
        • Harrell Jr, F.E.
        • Lee K.L.
        • Mark D.B.
        Multivariable prognostic models: issues in developing models, evaluating assumptions and adequacy, and measuring and reducing errors.
        Stat Med. 1996; 15: 361-387
        • DeLong E.R.
        • DeLong D.M.
        • Clarke-Pearson D.L.
        Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach.
        Biometrics. 1988; 44: 837-845
        • Redline R.W.
        • Heller D.
        • Keating S.
        • Kingdom J.
        Placental diagnostic criteria and clinical correlation: a workshop report.
        Placenta. 2005; 26: S114-S117
        • Jauniaux E.
        • Gulbis B.
        • Tunkel S.
        • Ramsay B.
        • Campbell S.
        • Meuris S.
        Maternal serum testing for alpha-fetoprotein and human chorionic gonadotropin in high-risk pregnancies.
        Prenat Diagn. 1996; 16: 1129-1135
        • Smith G.C.
        • Crossley J.A.
        • Aitken D.A.
        • et al.
        First-trimester placentation and the risk of antepartum stillbirth.
        JAMA. 2004; 292: 2249-2254
        • Spencer K.
        • Yu C.K.
        • Cowans N.J.
        • Otigbah C.
        • Nicolaides K.H.
        Prediction of pregnancy complications by first-trimester maternal serum PAPP-A and free beta-hCG and with second-trimester uterine artery Doppler.
        Prenat Diagn. 2005; 25: 949-953
        • Dugoff L.
        • Hobbins J.C.
        • Malone F.D.
        • et al.
        Quad screen as a predictor of adverse pregnancy outcome.
        Obstet Gynecol. 2005; 106: 260-267
        • Odibo A.O.
        • Sehdev H.M.
        • Stamilio D.M.
        • Macones G.A.
        Evaluating the thresholds of abnormal second trimester multiple marker screening tests associated with intra-uterine growth restriction.
        Am J Perinatol. 2006; 23: 363-367
        • Gagnon A.
        • Wilson R.D.
        • Audibert F.
        • et al.
        Obstetrical complications associated with abnormal maternal serum markers analytes.
        J Obstet Gynaecol Can. 2008; 30: 918-949
        • Olsen R.N.
        • Woelkers D.
        • Dunsmoor-Su R.
        • Lacoursiere D.Y.
        Abnormal second-trimester serum analytes are more predictive of preterm preeclampsia.
        Am J Obstet Gynecol. 2012; 207: 228.e1-228.e7
        • Papageorghiou A.T.
        • Yu C.K.
        • Bindra R.
        • Pandis G.
        • Nicolaides K.H.
        Multicenter screening for pre-eclampsia and fetal growth restriction by transvaginal uterine artery Doppler at 23 weeks of gestation.
        Ultrasound Obstet Gynecol. 2001; 18: 441-449
        • Papageorghiou A.T.
        • Leslie K.
        Uterine artery Doppler in the prediction of adverse pregnancy outcome.
        Curr Opin Obstet Gynecol. 2007; 19: 103-109
        • Smith G.C.
        • Yu C.K.
        • Papageorghiou A.T.
        • Cacho A.M.
        • Nicolaides K.H.
        Maternal uterine artery Doppler flow velocimetry and the risk of stillbirth.
        Obstet Gynecol. 2007; 109: 144-151
        • Smith G.C.
        • Shah I.
        • White I.R.
        • Pell J.P.
        • Crossley J.A.
        • Dobbie R.
        Maternal and biochemical predictors of antepartum stillbirth among nulliparous women in relation to gestational age of fetal death.
        BJOG. 2007; 114: 705-714
        • MacDorman M.F.
        • Kirmeyer S.
        Fetal and perinatal mortality, United States, 2005.
        Natl Vital Stat Rep. 2009; 57: 1-19
        • Bujold E.
        • Roberge S.
        • Lacasse Y.
        • et al.
        Prevention of preeclampsia and intrauterine growth restriction with aspirin started in early pregnancy: a meta-analysis.
        Obstet Gynecol. 2010; 116: 402-414
        • Roberge S.
        • Villa P.
        • Nicolaides K.
        • et al.
        Early administration of low-dose aspirin for the prevention of preterm and term preeclampsia: a systematic review and meta-analysis.
        Fetal Diagn Ther. 2012; 31: 141-146
        • Lee J.
        • Romero R.
        • Dong Z.
        • et al.
        Unexplained fetal death has a biological signature of maternal anti-fetal rejection: chronic chorioamnionitis and alloimmune anti-human leucocyte antigen antibodies.
        Histopathology. 2011; 59: 928-938
        • Singh T.
        • Leslie K.
        • Bhide A.
        • D'Antonio F.
        • Thilaganathan B.
        Role of second-trimester uterine artery Doppler in assessing stillbirth risk.
        Obstet Gynecol. 2012; 119: 256-261
        • Spencer K.
        Second-trimester prenatal screening for Down syndrome and the relationship of maternal serum biochemical markers to pregnancy complications with adverse outcome.
        Prenat Diagn. 2000; 20: 652-656
        • Yaron Y.
        • Cherry M.
        • Kramer R.L.
        • et al.
        Second-trimester maternal serum marker screening: maternal serum alpha-fetoprotein, beta-human chorionic gonadotropin, estriol, and their various combinations as predictors of pregnancy outcome.
        Am J Obstet Gynecol. 1999; 181: 968-974
        • Lepage N.
        • Chitayat D.
        • Kingdom J.
        • Huang T.
        Association between second-trimester isolated high maternal serum maternal serum human chorionic gonadotropin levels and obstetric complications in singleton and twin pregnancies.
        Am J Obstet Gynecol. 2003; 188: 1354-1359
        • Banek C.T.
        • Bauer A.J.
        • Gingery A.
        • Gilbert J.S.
        Timing of ischemic insult alters fetal growth trajectory, maternal angiogenic balance and markers of renal oxidative stress in the pregnant rat.
        Am J Physiol Regul Integr Comp Physiol. 2012; 303: R658-R664
        • Law L.W.
        • Sahota D.S.
        • Chan L.W.
        • Chen M.
        • Lau T.K.
        • Leung T.Y.
        Effect of long-term storage on placental growth factor and fms-like tyrosine kinase 1 measurements in samples from pregnant women.
        J Matern Fetal Neonatal Med. 2010; 23: 1475-1480
        • Kumasawa K.
        • Ikawa M.
        • Kidoya H.
        • et al.
        Pravastatin induces placental growth factor (PGF) and ameliorates preeclampsia in a mouse model.
        Proc Natl Acad Sci USA. 2011; 108: 1451-1455
        • Li Z.
        • Zhang Y.
        • Ying M.J.
        • et al.
        Recombinant vascular endothelial growth factor 121 attenuates hypertension and improves kidney damage in a rat model of preeclampsia.
        Hypertension. 2007; 50: 686-692
        • Thadhani R.
        • Kisner T.
        • Hagmann H.
        • et al.
        Pilot study of extracorporeal removal of soluble fms-like tyrosine kinase 1 in preeclampsia.
        Circulation.