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The prediction of fetal death with a simple maternal blood test at 24-28 weeks: a role for angiogenic index-1 (PlGF/sVEGFR-1 ratio)

  • Tinnakorn Chaiworapongsa
    Affiliations
    Perinatology Research Branch, Eunice Kennedy Shriver 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 School of Medicine, Detroit, MI
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  • Roberto Romero
    Correspondence
    Corresponding author: Roberto Romero, MD, DMedSci.
    Affiliations
    Perinatology Research Branch, Eunice Kennedy Shriver 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, University of Michigan, Ann Arbor, MI

    Department of Epidemiology and Biostatistics, Michigan State University, East Lansing, MI

    Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI
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  • Offer Erez
    Affiliations
    Perinatology Research Branch, Eunice Kennedy Shriver 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 School of Medicine, Detroit, MI

    Department of Obstetrics and Gynecology, Soroka University Medical Center, School of Medicine, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beersheba, Israel
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  • Adi L. Tarca
    Affiliations
    Perinatology Research Branch, Eunice Kennedy Shriver 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 School of Medicine, Detroit, MI
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  • Agustin Conde-Agudelo
    Affiliations
    Perinatology Research Branch, Eunice Kennedy Shriver 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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  • Piya Chaemsaithong
    Affiliations
    Perinatology Research Branch, Eunice Kennedy Shriver 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 School of Medicine, Detroit, MI
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  • Chong Jai Kim
    Affiliations
    Perinatology Research Branch, Eunice Kennedy Shriver 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 Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea
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  • Yeon Mee Kim
    Affiliations
    Perinatology Research Branch, Eunice Kennedy Shriver 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 Pathology, Haeundae Paik Hospital, Inje University College of Medicine, Busan, Republic of Korea
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  • Jung-Sun Kim
    Affiliations
    Perinatology Research Branch, Eunice Kennedy Shriver 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 Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea
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  • Bo Hyun Yoon
    Affiliations
    Perinatology Research Branch, Eunice Kennedy Shriver 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, Seoul National University College of Medicine, Seoul, Republic of Korea
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  • Sonia S. Hassan
    Affiliations
    Perinatology Research Branch, Eunice Kennedy Shriver 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 School of Medicine, Detroit, MI
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  • Lami Yeo
    Affiliations
    Perinatology Research Branch, Eunice Kennedy Shriver 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 School of Medicine, Detroit, MI
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  • Steven J. Korzeniewski
    Affiliations
    Perinatology Research Branch, Eunice Kennedy Shriver 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 School of Medicine, Detroit, MI

    Department of Epidemiology and Biostatistics, Michigan State University, East Lansing, MI
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Published:October 13, 2017DOI:https://doi.org/10.1016/j.ajog.2017.10.001

      Background

      Fetal death is an obstetrical syndrome that annually affects 2.4 to 3 million pregnancies worldwide, including more than 20,000 in the United States each year. Currently, there is no test available to identify patients at risk for this pregnancy complication.

      Objective

      We sought to determine if maternal plasma concentrations of angiogenic and antiangiogenic factors measured at 24-28 weeks of gestation can predict subsequent fetal death.

      Study Design

      A case-cohort study was designed to include 1000 randomly selected subjects and all remaining fetal deaths (cases) from a cohort of 4006 women with a singleton pregnancy, enrolled at 6-22 weeks of gestation, in a pregnancy biomarker cohort study. The placentas of all fetal deaths were histologically examined by pathologists who used a standardized protocol and were blinded to patient outcomes. Placental growth factor, soluble endoglin, and soluble vascular endothelial growth factor receptor-1 concentrations were measured by enzyme-linked immunosorbent assays. Quantiles of the analyte concentrations (or concentration ratios) were estimated as a function of gestational age among women who delivered a live neonate but did not develop preeclampsia or deliver a small-for-gestational-age newborn. A positive test was defined as analyte concentrations (or ratios) <2.5th and 10th centiles (placental growth factor, placental growth factor/soluble vascular endothelial growth factor receptor-1 [angiogenic index-1] and placental growth factor/soluble endoglin) or >90th and 97.5th centiles (soluble vascular endothelial growth factor receptor-1 and soluble endoglin). Inverse probability weighting was used to reflect the parent cohort when estimating the relative risk.

      Results

      There were 11 fetal deaths and 829 controls with samples available for analysis between 24-28 weeks of gestation. Three fetal deaths occurred <28 weeks and 8 occurred ≥28 weeks of gestation. The rate of placental lesions consistent with maternal vascular underperfusion was 33.3% (1/3) among those who had a fetal death <28 weeks and 87.5% (7/8) of those who had this complication ≥28 weeks of gestation. The maternal plasma angiogenic index-1 value was <10th centile in 63.6% (7/11) of the fetal death group and in 11.1% (92/829) of the controls. The angiogenic index-1 value was <2.5th centile in 54.5% (6/11) of the fetal death group and in 3.7% (31/829) of the controls. An angiogenic index-1 value <2.5th centile had the largest positive likelihood ratio for predicting fetal death >24 weeks (14.6; 95% confidence interval, 7.7–27.7) and a relative risk of 29.1 (95% confidence interval, 8.8–97.1), followed by soluble endoglin >97.5th centile and placental growth factor/soluble endoglin <2.5th, both with a positive likelihood ratio of 13.7 (95% confidence interval, 7.3–25.8) and a relative risk of 27.4 (95% confidence interval, 8.2–91.2). Among women without a fetal death whose plasma angiogenic index-1 concentration ratio was <2.5th centile, 61% (19/31) developed preeclampsia or delivered a small-for-gestational-age neonate; when the 10th centile was used as the cut-off, 37% (34/92) of women had these adverse outcomes.

      Conclusion

      (1) A maternal plasma angiogenic index-1 value <2.5th centile (0.126) at 24-28 weeks of gestation carries a 29-fold increase in the risk of subsequent fetal death and identifies 55% of subsequent fetal deaths with a false-positive rate of 3.5%; and (2) 61% of women who have a false-positive test result will subsequently experience adverse pregnancy outcomes.

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      we sought to validate and extend these findings by determining whether plasma angiogenic index-1 at 24-28 weeks of gestation could be used as a biomarker to identify patients at risk for a subsequent fetal death.

      Materials and Methods

      Study design and participants

      This was a case-cohort study. We randomly selected 1000 patients from a cohort of 4006 pregnant women enrolled in a longitudinal study previously reported by our group.
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      The remaining women in the original cohort who had a fetal death, but were not selected in the random sample of 1000 women, were subsequently added to the case cohort. Women who had multiple gestations or any of the following conditions at the time of enrollment were excluded from this study: active vaginal bleeding; severe maternal morbidity (ie, renal insufficiency, congestive heart disease, and chronic respiratory insufficiency); chronic hypertension requiring medication; asthma requiring systemic steroids; requirement of antiplatelet or nonsteroidal antiinflammatory drugs; active hepatitis; or fetal chromosomal abnormalities and congenital anomalies. All study participants provided written informed consent and were followed up until delivery. The use of clinical data and biological specimens obtained from these women for research purposes was approved by the Institutional Review Boards of Wayne State University and the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, US Department of Health and Human Services.

      Clinical definitions

      The following definitions were used in this study:
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        Fetal death: diagnosed as the death of the fetus >20 weeks of gestation and confirmed by ultrasound examination.
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      • 2)
        Preeclampsia: diagnosed by the presence of systolic blood pressure ≥140 mm Hg or diastolic blood pressure ≥90 mm Hg on at least 2 occasions, 4 hours to 1 week apart, and of proteinuria ≥300 mg in a 24-hour urine collection or by 1 dipstick with ≥1+.
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        We have used this definition after analysis from the Collaborative Perinatal Project, which was reported in detail by Friedman and Neff,
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        and the reasons described by one of the authors.
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        Moreover, the definition outlined above was the one used in our center to collect outcome information at the time the patients were recruited and delivered.
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        Small-for-gestational-age (SGA) neonate: a birthweight <10th centile for gestational age at delivery according to a US reference population.
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      • 4)
        Preterm delivery: a delivery occurring <37th week of gestation.

      Sample collection and immunoassays

      Patients were scheduled to donate maternal plasma in EDTA tubes at enrollment, then every 4 weeks until the 24th week of gestation, and biweekly thereafter until delivery. Samples were centrifuged and stored at –70°C. Maternal plasma concentrations of sVEGFR-1, PlGF, and sEng were measured by immunoassays (R&D Systems, Minneapolis, MN) as previously described.
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      The interassay and intraassay coefficients of variation of the assays were 3.2% and 2.9% for sVEGFR-1, 4.5% and 5.0% for sEng, and 3.8% and 4.7% for PlGF, respectively. The sensitivity of each assay was 14.6 pg/mL for sVEGFR-1, 0.04 ng/mL for sEng, and 4.8 pg/mL for PlGF. Laboratory personnel performing the assays were blinded to the clinical information.

      Histologic placental examination

      Placentas were examined according to standardized protocols by perinatal pathologists blinded to clinical diagnoses and obstetrical outcomes. Placental lesions consistent with maternal vascular lesions of underperfusion (now known as maternal vascular malperfusion) were diagnosed using criteria established by the Perinatal Section of the Society for Pediatric Pathology
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      and were classified as the following: (1) villous changes, which are further subdivided into abrupt onset (remote villous infarcts, recent villous infarcts), gradual onset with intermediate duration (increased syncytial knots, villous agglutination, increased intervillous fibrin), or gradual onset with prolonged duration (distal villous hypoplasia); and (2) vascular lesions (persistent muscularization of the basal plate arteries, mural hypertrophy of the decidual arterioles, acute atherosis of the basal plate arteries and/or the decidual arterioles).
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      Classification of placental lesions.

      Statistical analysis

      Using quantile regression, which does not assume data normality, we estimated the percentiles of maternal plasma concentration of analytes and their ratios in a subset of controls (ie, women who delivered live babies at term and did not have preeclampsia or SGA neonate). Data were first log-transformed, a standard approach to improve normality of distribution
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      and to reduce the nonlinearity of the relation between the response and gestational age at sample collection. Linear quantile regression was then used iteratively to estimate the quantiles at discrete values of gestation, in narrow windows of gestational age, over which the linear assumption holds. The resulting estimated percentiles were not smooth because they were obtained from different models (fitted to the different narrow gestational-age windows); therefore, we fitted a 5th-degree polynomial function to smooth the estimated percentiles.
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      The quantile regression was performed using the quantreg package of the R open source statistical software (URL http://www.R-project.org/).
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      Positive tests were defined as analyte concentrations (or their ratios) <2.5th and 10th centiles (PlGF, PlGF/sVEGFR-1, and PlGF/sEng) or >90th and 97.5th centiles (sVEGFR-1 and sEng). Data from the 11 fetal deaths and 829 controls with available measurements were used to construct receiver operating characteristic (ROC) curves and to determine the sensitivity, specificity, and likelihood ratios (positive and negative). Likelihood ratios for a positive test result >10 and likelihood ratios for a negative test result <0.1 were taken as strong predictive evidence under most circumstances. Moderate prediction could be achieved with likelihood ratios of 5-10 and 0.1-0.2, whereas ratios <5 and >0.2 were only minimally predictive.
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      Using the whole cohort in the analysis of case-cohort data.
      For demographic data analysis, we used a χ2 or Fisher exact test and reported proportions for categorical variables, and medians and interquartile ranges for continuous variables.

      Results

      There were 24 fetal deaths (0.6%, 24/4006): 6 were initially selected with 994 controls in the random sample of 1000 women. Following this selection process, we added the remaining 18 patients with a fetal death from the parent cohort to the cases group, reaching a total of 24 patients. Of the 24 fetal deaths, 12 were diagnosed <28 weeks, and 12 diagnosed ≥28 weeks of gestation.
      Table 1 shows the characteristics of the cases and controls. Cases were delivered at a lower median gestational age (P < .0001) and had a lower median birthweight (P < .0001). There were no significant differences between cases and controls in median maternal age and the proportion of smokers, nulliparity, or African American ethnicity.
      Table 1Descriptive characteristics of the study participants
      CharacteristicNo fetal death, n = 994Fetal death, n = 24P
      Maternal age, y23 (20–27)22.5 (20–29.5).59
      Smoker206 (20.8)5 (20.8)1
      Nulliparity381 (38.7)8 (33.3).64
      African American921 (92.7)23 (95.8)1
      Prepregnancy BMI26.6 (22.5–32.5)26.8 (23.2–33.3).54
      GA at delivery, wk39.1 (37.9–40.1)28.3 (23–31.5)<.0001
      Birthweight, g3172.5 (2800–3485)924.5 (493.5–1400)<.0001
      Data presented as median (interquartile range) for continuous variables and n (%) for categorical variables.
      BMI, body mass index; GA, gestational age.
      Chaiworapongsa et al. Angiogenic index-1 (PlGF/sVEGFR-1 ratio), biomarker for subsequent fetal death. Am J Obstet Gynecol 2017.
      Patients with a fetal death had a higher rate of placental lesions consistent with maternal vascular underperfusion than the remainder of the study cohort (fetal death, 58.3% [14/24] vs the remainder of the cohort, 21.3% [206/968], and an odds ratio of 5.2 [95% CI, 2.1-13.2], P = .0001). Among women with a fetal death, the rate of placental lesions consistent with maternal vascular underperfusion was 42% (5/12) in those diagnosed <28 weeks of gestation and 75% (9/12) in those diagnosed ≥28 weeks of gestation (Fisher exact test, P = .2).

      The changes in angiogenic and antiangiogenic profiles at 24-28 weeks of gestation in women with a subsequent fetal death

      This analysis included only patients whose blood samples were collected between 24-28 weeks of gestation. Seven women had a fetal death <24 weeks of gestation and were excluded from further analysis. Additionally, 6 patients were excluded, as samples were not available at the desired gestational-age interval.
      Eleven women with a fetal death were available for further analysis; 8 had a fetal death ≥28 weeks and 3 of these women had a fetal death <28 weeks of gestation. From the remaining case cohort, blood samples collected at 24-28 weeks of gestation were available from 829 controls.
      Figure 1 shows the changes in the maternal plasma sVEGFR-1, sEng, and PlGF concentrations, and the PlGF/sEng ratio for fetal death cases superimposed on the estimated quantiles for the controls (after excluding patients with preeclampsia and those with an SGA neonate) (see Table 2 for quantile values of all analytes and ratios). Similar data for maternal plasma angiogenic index-1 are shown in Figure 2. The value of the 2.5th centile of angiogenic index-1 was constant (0.126) between 24-28 weeks of gestation and corresponds to 0.2 multiple of the median.
      Figure thumbnail gr1
      Figure 1Concentration of angiogenic and antiangiogenic factors in patients with fetal death
      For each fetal death case, data for 1 sample collected at 24-28 weeks are shown. Cases without placental lesions of maternal vascular underperfusion (MVU) (triangles) and cases with MVU (circles). Lines correspond with percentiles of analytes (or ratios) (2.5th, 10th, 50th, 90th, and 97.5th centiles). Distribution of raw longitudinal data used to derive percentiles is shown using heat map; color intensity denotes frequency of data points for corresponding gestational age: A, soluble vascular endothelial growth factor receptor (sVEGFR)-1; B, soluble endoglin (sEng); C, placental growth factor (PlGF); and D, PlGF/sEng ratio.
      Chaiworapongsa et al. Angiogenic index-1 (PlGF/sVEGFR-1 ratio), biomarker for subsequent fetal death. Am J Obstet Gynecol 2017.
      Table 2Percentiles (2.5th, 10th, 50th, 90th, and 97.5th) of concentrations of placental growth factor, soluble vascular endothelial growth factor receptor-1, soluble endoglin, ratios of placental growth factor/soluble vascular endothelial growth factor receptor-1 and placental growth factor/soluble endoglin, according to gestational age at sample collection
      GA, gestational age; PlGF, placental growth factor; sEng, soluble endoglin; sVEGFR, soluble vascular endothelial growth factor receptor.
      Chaiworapongsa et al. Angiogenic index-1 (PlGF/sVEGFR-1 ratio), biomarker for subsequent fetal death. Am J Obstet Gynecol 2017.
      Figure thumbnail gr2
      Figure 2Angiogenic index-1 at 24-28 weeks in patients with subsequent fetal death
      For each fetal death case, data for 1 sample collected at 24-28 weeks are shown. Cases without placental lesions of maternal vascular underperfusion (MVU) (triangles) and cases with MVU (circles). Lines correspond with percentiles of angiogenic index-1 as described by labels in graph (2.5th, 10th, 50th, 90th, and 97.5th centiles). Distribution of raw longitudinal data used to derive percentiles is shown using heat map; color intensity denotes frequency of data points for corresponding gestational age and angiogenic index-1 value.
      PlGF, placental growth factor; sVEGFR, soluble vascular endothelial growth factor receptor.
      Chaiworapongsa et al. Angiogenic index-1 (PlGF/sVEGFR-1 ratio), biomarker for subsequent fetal death. Am J Obstet Gynecol 2017.
      Table 3 shows the prediction performance for fetal death >24 weeks when defining a positive test based on abnormal angiogenic and antiangiogenic factor concentrations and ratios measured at 24-28 weeks of gestation. An angiogenic index-1 value <2.5th centile had the largest positive likelihood ratio (14.6; 95% CI, 7.7–27.7) and RR (29.1; 95% CI, 8.8–97.1), followed by sEng concentrations >97.5th centile and PlGF/sEng ratios <2.5th, both of which had a positive likelihood ratio of 13.7 (95% CI, 7.3–25.8) and a RR of 27.4 (95% CI, 8.2–91.2).
      Table 3Prediction performance of maternal plasma angiogenic and antiangiogenic factor concentrations and ratios (at 24-28 weeks of gestation) for subsequent fetal death (from 24-37.6 weeks of gestation)
      PredictorCut-offRelative riskAUCSensitivitySpecificityPositive likelihood ratioNegative likelihood ratio
      sVEGFR-1>909.4 (2.9–31.1)0.850.55 (0.23–0.83)0.89 (0.87–0.91)4.9 (2.8–8.7)0.51 (0.27–0.98)
      sVEGFR-1>97.510.8 (2.8–41.4)0.850.27 (0.06–0.61)0.97 (0.95–0.98)8.4 (3–23.5)0.75 (0.52–1.08)
      sEng>9014 (4.1–48.3)0.820.64 (0.31–0.89)0.89 (0.87–0.91)5.9 (3.6–9.5)0.41 (0.19–0.89)
      sEng>97.527.4 (8.2–91.2)0.820.55 (0.23–0.83)0.96 (0.94–0.97)13.7 (7.3–25.8)0.47 (0.25–0.9)
      PlGF<107.7 (2.3–25.5)0.730.55 (0.23–0.83)0.87 (0.84–0.89)4.1 (2.3–7.2)0.52 (0.27–1)
      PlGF<2.516.7 (5–55.4)0.730.45 (0.17–0.77)0.95 (0.94–0.97)9.9 (4.8–20.3)0.57 (0.33–0.98)
      PlGF/sVEGFR-1<1013.7 (4–47.1)0.790.64 (0.31–0.89)0.89 (0.87–0.91)5.7 (3.5–9.3)0.41 (0.19–0.89)
      PlGF/sVEGFR-1<2.529.1 (8.8–97.1)0.790.55 (0.23–0.83)0.96 (0.95–0.97)14.6 (7.7–27.7)0.47 (0.25–0.9)
      PlGF/sEng<1012 (3.5–41.1)0.770.64 (0.31–0.89)0.87 (0.85–0.9)5.1 (3.1–8.2)0.42 (0.19–0.91)
      PlGF/sEng<2.527.4 (8.2–91.2)0.770.55 (0.23–0.83)0.96 (0.94–0.97)13.7 (7.3–25.8)0.47 (0.25–0.9)
      Data presented as percentiles for cut-off values, and as estimates (95% CI) for relative risk, sensitivity, specificity, and likelihood ratios.
      AUC, area under receiver operating characteristic curve; PlGF, placental growth factor; sEng, soluble endoglin; sVEGFR, soluble vascular endothelial growth factor receptor.
      Chaiworapongsa et al. Angiogenic index-1 (PlGF/sVEGFR-1 ratio), biomarker for subsequent fetal death. Am J Obstet Gynecol 2017.
      Table 4 presents the number of cases and controls with maternal plasma angiogenic index-1 values <10th and 2.5th centiles. Using the 10th percentile cut-off, maternal plasma angiogenic index-1 had a sensitivity of 63.6% (7/11), a false-positive rate of 11.1% (92/829), and a positive likelihood ratio of 5.7. Using the 2.5th centile cut-off, maternal plasma angiogenic index-1 had a sensitivity of 54.5% (6/11), a false-positive rate of 3.7% (31/829), and a positive likelihood ratio of 14.6 (Table 3). The whole range of sensitivities and corresponding false-positive rates of angiogenic index-1 is shown by the ROC curve in Figure 3 (area under the curve, 0.79; 95% CI, 0.61–0.97, P = .0005).
      Table 4Contingency table for association between abnormal maternal plasma angiogenic index-1 and fetal death >24 weeks
      Angiogenic index-1Fetal death>24 wkControls
      <10th Centile792
      ≥10th Centile4737
      Total11829
       <2.5th Centile631
       ≥2.5th Centile5798
      Total11829
      Chaiworapongsa et al. Angiogenic index-1 (PlGF/sVEGFR-1 ratio), biomarker for subsequent fetal death. Am J Obstet Gynecol 2017.
      Figure thumbnail gr3
      Figure 3Prediction performance of angiogenic index-1 for detection of subsequent fetal death
      Area under curve (AUC) of ROC curve for detection of all patients with subsequent fetal death (bold line) is 0.79, 95% confidence interval (CI) 0.61–0.97, P = .0005. AUC of ROC curve for detection of subsequent fetal death of patients who also had placental lesions consistent with maternal vascular underperfusion (broken line) is 0.89, 95% CI 0.71-1.00, P = .00007.
      Chaiworapongsa et al. Angiogenic index-1 (PlGF/sVEGFR-1 ratio), biomarker for subsequent fetal death. Am J Obstet Gynecol 2017.
      Eight of the 11 cases had placental lesions consistent with maternal vascular underperfusion. The positive likelihood ratio of angiogenic index-1 in these 8 cases increased from 5.7 to 7.9 at the 10th centile cut-off, and from 14.6 to 20.0 at the 2.5th centile cut-off, as 3 of the 4 cases missed by a positive angiogenic index-1 at the 10th centile did not have placental lesions consistent with maternal vascular underperfusion. In other words, angiogenic index-1 identified 7 of 8 (87.58%) subsequent fetal deaths associated with placental lesions consistent with maternal vascular underperfusion (Figure 2). The performance of the test in the detection of fetal death in patients with maternal vascular lesions of underperfusion is shown by the ROC curve in Figure 3 (area under the curve, 0.89; 95% CI 0.71–1.00, P = .00007).

      The risk of fetal death in patients with abnormal angiogenic index-1

      In the tested cohort in which the prevalence of fetal death >24 weeks was 1.3% (11/840), 7% of women who had a positive angiogenic index-1 value at the 10th centile had a subsequent fetal death, and 16% of those who had a positive angiogenic index-1 value at the 2.5th centile had a subsequent fetal death (Table 4). When weighting of cases and controls in the tested cohort was performed so that the weight of cases was the same as the prevalence of fetal death in the full cohort (0.4%, 17/4006), the risk of fetal death >24 weeks was determined to be 2% (PPV) for a positive angiogenic index-1 value at the 10th centile, and 6% (PPV) for a positive angiogenic index-1 value at the 2.5th centile.

      Pregnancy outcomes of women with abnormal angiogenic index-1 values who did not have a fetal death

      Of the 31 women who had false-positive test results at the 2.5th centile cut-off, 48% (15/31) developed preeclampsia, 39% (12/31) delivered an SGA neonate, and 61% (19/31) had at least 1 of these outcomes. Of the 92 women who had false-positive test results at the 10th centile cut-off, 23% (21/92) developed preeclampsia, 24% (22/92) delivered an SGA neonate, and 37% (34/92) had at least 1 of these outcomes.

      Comment

      Principal findings of the study

      First, the maternal plasma angiogenic index-1 value (PlGF/sVEGFR-1 maternal plasma concentration ratio) at 24-28 weeks can identify women who subsequently have a fetal death. Second, an angiogenic index-1 ratio <2.5th centile had a positive likelihood ratio of 14.6 for the identification of subsequent fetal death and can detect half of all fetal deaths (sensitivity, 54.5%) in the late second and third trimesters of pregnancy. Third, in the subset of fetal deaths associated with placental lesions consistent with maternal vascular underperfusion, the positive likelihood ratio of an angiogenic index-1 value <2.5th centile increased to 20.0. Finally, 61% of women whose angiogenic index-1 value at 24-28 weeks was <2.5th centile, who did not have a subsequent fetal death, developed preeclampsia or delivered an SGA neonate.

      Placental lesions and fetal death

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      and infection
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      and maternal antifetal rejection
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      become the most common causes >26 weeks until term,
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      after which the etiology in most cases, especially >40 weeks, is unknown.
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      In this study, the frequency of placental lesions consistent with maternal vascular underperfusion was significantly higher in cases than controls, supporting the view that the most common cause of fetal death in the late second and third trimesters of pregnancy is placental in origin.

      What is the association between fetal death and maternal plasma angiogenic index-1?

      The placenta is a network of blood vessels surrounded by the trophoblast,
      • Burton G.J.
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      What is the placenta?.
      and the processes of vasculogenesis and angiogenesis are crucial for the success of pregnancy. Indeed, deletion of a single vascular endothelial growth factor allele in an embryo (haploinsufficiency) is lethal.
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      An imbalance in the maternal plasma angiogenic and antiangiogenic factors has been reported in women with preeclampsia
      • Chaiworapongsa T.
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      • et al.
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      • Kusanovic J.P.
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      • 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.
      • Chaiworapongsa T.
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      • 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.
      • Chaiworapongsa T.
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      • et al.
      Plasma concentrations of angiogenic/anti-angiogenic factors have prognostic value in women presenting with suspected preeclampsia to the obstetrical triage area: a prospective study.
      • Soto E.
      • Romero R.
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      • et al.
      Late-onset preeclampsia is associated with an imbalance of angiogenic and anti-angiogenic factors in patients with and without placental lesions consistent with maternal underperfusion.
      • Vaisbuch E.
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      Circulating angiogenic and antiangiogenic factors in women with eclampsia.
      • Levine R.J.
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      The use of angiogenic biomarkers in maternal blood to identify which SGA fetuses will require a preterm delivery and mothers who will develop pre-eclampsia.
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      • Romero R.
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      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.
      • Erez O.
      • Romero R.
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      • 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.
      • Chaiworapongsa T.
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      Evidence supporting a role for blockade of the vascular endothelial growth factor system in the pathophysiology of preeclampsia. Young Investigator Award.
      • Chaiworapongsa T.
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      Plasma soluble vascular endothelial growth factor receptor-1 concentration is elevated prior to the clinical diagnosis of pre-eclampsia.
      an SGA neonate,
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      The use of angiogenic biomarkers in maternal blood to identify which SGA fetuses will require a preterm delivery and mothers who will develop pre-eclampsia.
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      • Erez O.
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      • 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.
      • Chaiworapongsa T.
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      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.
      and a subset of those who had a spontaneous preterm parturition,
      • Chaiworapongsa T.
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      A subset of patients destined to develop spontaneous preterm labor has an abnormal angiogenic/anti-angiogenic profile in maternal plasma: evidence in support of pathophysiologic heterogeneity of preterm labor derived from a longitudinal study.
      mirror syndrome,
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      A role of the anti-angiogenic factor sVEGFR-1 in the 'mirror syndrome' (Ballantyne's syndrome).
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      twin-to-twin transfusion syndrome,
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      Twin-to-twin transfusion syndrome: an antiangiogenic state?.
      and fetal death.
      • Romero R.
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      • et al.
      An imbalance between angiogenic and anti-angiogenic factors precedes fetal death in a subset of patients: results of a longitudinal study.
      • Espinoza J.
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      • et al.
      Unexplained fetal death: another anti-angiogenic state.
      These alterations in the maternal plasma concentrations of PlGF and sVEGFR-1 can be detected at the time of disease and at several weeks prior to the onset of the different obstetrical syndromes.

      What are the clinical implications of a low maternal plasma angiogenic index-1?

      In a previous study,
      • Korzeniewski S.J.
      • Romero R.
      • Chaiworapongsa T.
      • et al.
      Maternal plasma angiogenic index-1 (placental growth factor/soluble vascular endothelial growth factor receptor-1) is a biomarker for the burden of placental lesions consistent with uteroplacental underperfusion: a longitudinal case-cohort study.
      we reported that maternal plasma angiogenic index-1 measured at 30-34 weeks of gestation identified 80% of subsequent late fetal deaths. Among those who had a fetal death, identified by the maternal plasma angiogenic index-1 value, 80% had maternal vascular underperfusion.
      • Korzeniewski S.J.
      • Romero R.
      • Chaiworapongsa T.
      • et al.
      Maternal plasma angiogenic index-1 (placental growth factor/soluble vascular endothelial growth factor receptor-1) is a biomarker for the burden of placental lesions consistent with uteroplacental underperfusion: a longitudinal case-cohort study.
      This result, coupled with our findings that women who subsequently had a fetal death have higher maternal plasma sVEGFR-1 and lower PlGF concentrations, as early as 20 weeks of gestation, compared to those with a normal pregnancy, led us to explore whether the ratio between these 2 analytes was predictive of fetal death as early as 24 weeks of gestation. This study found that a maternal plasma angiogenic index-1 value <2.5th centile at 24-28 weeks of gestation (ie, <0.126) could identify 54.5% of subsequent fetal deaths. A patient whose plasma angiogenic index-1 at 24-28 weeks of gestation is <0.126 has a 6% chance of a fetal death, while a patient whose plasma angiogenic index-1 is >0.126 has only a 0.2% chance of a subsequent fetal death.
      Two factors support choosing the 2.5th centile over the 10th centile as the cut-off to define a positive angiogenic index-1. First, angiogenic index-1 is constant <2.5th centile but not <10th centile, so a single value can be used to determine whether a test is positive or negative using the 2.5th centile as the cut-off. Second, the proportion of false positives associated with preeclampsia or an SGA neonate was higher for false positives <2.5th centile (61%) than for the 10th centile (37%), making a positive result <2.5th centile more clinically significant than a positive result <10th centile, even if the latter cut-off is slightly more sensitive to predict a subsequent fetal death.
      A preliminary report indicating that statins may improve pregnancy outcome by reducing the antiangiogenic state in pregnancy
      • Chaiworapongsa T.
      • Romero R.
      • Korzeniewski S.J.
      • et al.
      Pravastatin to prevent recurrent fetal death in massive perivillous fibrin deposition of the placenta (MPFD).
      has increased the potential value of screening pregnancies for the PlGF/sVEGFR-1 concentration. We previously reported a case
      • Chaiworapongsa T.
      • Romero R.
      • Korzeniewski S.J.
      • et al.
      Pravastatin to prevent recurrent fetal death in massive perivillous fibrin deposition of the placenta (MPFD).
      in which a patient with recurrent fetal losses due to maternal perivillous fibrin deposition was successfully treated with pravastatin, and the treatment improved her PlGF/sVEGFR-1 concentration ratio, leading to a successful pregnancy. Maternal perivillous fibrin deposition has a stereotypic pattern for angiogenic index-1,
      • Whitten A.E.
      • Romero R.
      • Korzeniewski S.J.
      • et al.
      Evidence of an imbalance of angiogenic/antiangiogenic factors in massive perivillous fibrin deposition (maternal floor infarction): a placental lesion associated with recurrent miscarriage and fetal death.
      and we provided evidence that this may reflect (at least, in some cases) maternal antifetal rejection.
      • Romero R.
      • Whitten A.
      • Korzeniewski S.J.
      • et al.
      Maternal floor infarction/massive perivillous fibrin deposition: a manifestation of maternal antifetal rejection?.
      Since treatment with pravastatin can reverse a low plasma PlGF/sVEGFR-1 concentration ratio, therapeutic trials of pravastatin in women who have a low plasma PlGF/sVEGFR-1 concentration ratio are warranted, especially given the negligible risks resulting from a false-positive screening test result. Other interventions that could be effective include metformin
      • Brownfoot F.C.
      • Hastie R.
      • Hannan N.J.
      • et al.
      Metformin as a prevention and treatment for preeclampsia: effects on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion and endothelial dysfunction.
      and proton pump inhibitors.
      • Onda K.
      • Tong S.
      • Beard S.
      • et al.
      Proton pump inhibitors decrease soluble fms-like tyrosine kinase-1 and soluble endoglin secretion, decrease hypertension, and rescue endothelial dysfunction.
      • Cluver C.A.
      • Walker S.P.
      • Mol B.W.
      • et al.
      Double blind, randomized, placebo-controlled trial to evaluate the efficacy of esomeprazole to treat early onset pre-eclampsia (PIE trial): a study protocol.
      Thus, the maternal plasma angiogenic index-1 value measured at 24-28 weeks of gestation can serve as a biomarker to identify women at risk of having a subsequent fetal death or developing other obstetrical complications. The availability of potentially effective treatments makes the formulation of practical screening programs using plasma angiogenic index-1 a clinical priority.

      Strengths and limitations of this study

      The strengths of this study are as follows. First, the use of a longitudinal case-cohort study design included a random sample of about one quarter of the full cohort of 4006 women and all remaining women with a fetal death in the parent cohort. Unlike a nested case-control study, this case-cohort design allowed us to estimate RR and to account for other complications that may involve a common pathway with fetal death. Second, we demonstrated that an angiogenic index-1 value <2.5th centile has a higher positive likelihood ratio for patients whose placenta had histologic lesions of maternal vascular underperfusion, emphasizing that this risk-assessment method is also related to the underlying mechanism of disease leading to fetal death. Finally, the proportion of placental-associated fetal deaths in this study is comparable to that reported in another study.
      • Helgadottir L.B.
      • Turowski G.
      • Skjeldestad F.E.
      • et al.
      Classification of stillbirths and risk factors by cause of death–a case-control study.
      Limitations of this study are as follows. First, in the study design, the subcohort of 1000 random individuals was not selected completely at random from the full cohort but rather from all patients who had at least 3 blood samples available for analysis. Such longitudinal data are best suited for establishing reference intervals in the control group. Second, not all of the patients selected for inclusion in the case cohort provided a sample during the 24- to 28-week interval for analysis. Third, the small number of late fetal deaths included in the analysis is a limitation; however, this reflects the prevalence of the disease in the population, and, in spite of this, we demonstrated a highly positive result. Finally, although an abnormal angiogenic index-1 value had the highest RR and positive likelihood ratio for fetal death, a cost-effectiveness analysis is still warranted to determine the optimal screening method, since sEng (Figure 1, B) alone had a similar prediction performance for this outcome.

      Conclusions

      First, a maternal plasma angiogenic index-1 value <2.5th centile at 24-28 weeks of gestation carries a 29-fold increase for the risk of subsequent fetal death and identifies 55% of these patients at a very low (3.7%) false-positive rate. Second, 61% of the women who had a false-positive result subsequently had adverse pregnancy outcomes (preeclampsia or an SGA neonate).

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