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Altered angiogenesis as a common mechanism underlying preterm birth, small for gestational age, and stillbirth in women living with HIV

Open AccessPublished:October 11, 2017DOI:https://doi.org/10.1016/j.ajog.2017.10.003

      Background

      Angiogenic processes in the placenta are critical regulators of fetal growth and impact birth outcomes, but there are limited data documenting these processes in HIV-infected women or women from low-resource settings.

      Objective

      We sought to determine whether angiogenic factors are associated with adverse birth outcomes in HIV-infected pregnant women started on antiretroviral therapy.

      Study Design

      This is a secondary analysis of samples collected as part of a clinical trial randomizing pregnant women and adolescents infected with HIV to lopinavir/ritonavir-based (n = 166) or efavirenz-based (n = 160) antiretroviral therapy in Tororo, Uganda. Pregnant women living with HIV were enrolled between 12-28 weeks of gestation. Plasma samples were evaluated for angiogenic biomarkers (angiopoietin-1, angiopoietin-2, vascular endothelial growth factor, soluble fms-like tyrosine kinase-1, placental growth factor, and soluble endoglin) by enzyme-linked immunosorbent assay between: 16-<20, 20-<24, 24-<28, 28-<32, 32-<36, 36-<37 weeks of gestation. The primary outcome was preterm birth.

      Results

      In all, 1115 plasma samples from 326 pregnant women and adolescents were evaluated. There were no differences in angiogenic factors according to antiretroviral therapy group (P > .05 for all). The incidence of adverse birth outcomes was 16.9% for spontaneous preterm births, 25.6% for small-for-gestational-age births, and 2.8% for stillbirth. We used linear mixed effect modelling to evaluate longitudinal changes in angiogenic factor concentrations between birth outcome groups adjusting for gestational age at venipuncture, maternal age, body mass index, gravidity, and the interaction between treatment arm and gestational age. Two angiogenic factors–soluble endoglin and placental growth factor–were associated with adverse birth outcomes. Significantly higher concentrations of soluble endoglin throughout gestation were found in study participants destined to deliver preterm [likelihood ratio test, χ2(1) = 12.28, P < .0005] and in those destined to have stillbirths [χ2(1) = 5.67, P < .02]. By contrast, significantly lower concentrations of placental growth factor throughout gestation were found in those destined to have small-for-gestational-age births [χ2(1) = 7.89, P < .005] and in those destined to have stillbirths [χ2(1) = 21.59, P < .0001].

      Conclusion

      An antiangiogenic state in the second or third trimester is associated with adverse birth outcomes, including stillbirth in women and adolescents living with HIV and receiving antiretroviral therapy.

      Key words

      Click Supplemental Materials and Video under article title in Contents at ajog.org

      Introduction

      It is estimated that 17.8 million of 36.7 million people living with HIV in 2015 were women and girls of reproductive age (>15 years).
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      Angiogenic processes in the placenta affect pregnancy, but there are limited data documenting these processes in the context of HIV infection and low-resource settings where rates of adverse birth outcomes are the highest. We hypothesize that alterations in angiogenic factors are associated with adverse birth outcomes in pregnant WLHIV. To test this hypothesis we longitudinally characterized circulating plasma levels of angiogenic proteins in a cohort of WLHIV initiated on ART.
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      • Luwedde F.
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      In this report we describe the kinetics of angiogenic factors over pregnancy, compare angiogenic factors between different ART regimens (efavirenz- vs lopinavir/ritonavir-based ART), and evaluate whether angiogenic factors predict adverse birth outcomes (spontaneous PTB, SGA, and stillbirth).

      Materials and Methods

      Ethics statement

      Written informed consent was obtained from all participants. Ethical approval was received from Makerere University School of Medicine (Sept. 20, 2009; reference 2009-141); the Uganda National Council for Science and Technology; the University of California–San Francisco (Aug. 9, 2009; reference 10-02958); and the University Health Network (March 13, 2014; reference 14-7313-AE). This trial was registered: ClinicalTrials.gov (identifier: NCT00993031).

      Study population

      Plasma samples were collected from pregnant WLHIV participating in a randomized controlled trial of protease inhibitor vs nonnucleoside reverse transcriptase inhibitor–based ART in Tororo, Uganda, from 2009 through 2013.
      • Natureeba P.
      • Ades V.
      • Luwedde F.
      • et al.
      Lopinavir/ritonavir-based antiretroviral treatment (ART) versus efavirenz-based ART for the prevention of malaria among HIV-infected pregnant women.
      Participants were HIV-infected, ≥16 years of age, and pregnant (12-28 weeks of gestation by last menstrual period with confirmation by ultrasound). Eligibility for enrollment was not dependent on CD4 cell count. Patients were ineligible to participate if they had received ART (including any abbreviated monotherapy) or dual therapy with nevirapine in the last 24 months. Subjects received standard antenatal care according to Ugandan Ministry of Health Guidelines (http://www.health.go.ug/docs/ucg_2010.pdf). Blood pressure and urine protein was assessed at enrollment and routine antenatal visits. Participants on protease inhibitor–based ART received lopinavir/ritonavir (n = 166) and those on nonnucleoside reverse transcriptase inhibitor–based ART received efavirenz (n = 160). Socioeconomic status was assessed as described.
      • Young S.
      • Murray K.
      • Mwesigwa J.
      • et al.
      Maternal nutritional status predicts adverse birth outcomes among HIV-infected rural Ugandan women receiving combination antiretroviral therapy.

      Laboratory assays

      Blood collection and laboratory work was performed at baseline and at all subsequent antenatal visits. Clinical tests included complete blood cell count and determination of CD4+/CD8+ T-lymphocyte subsets. Standardized assessments were completed at delivery including gestational age and birthweight (using an electronic scale).

      Study outcomes

      Women were eligible for inclusion in this secondary analysis if they had a singleton pregnancy with known birth outcome, and samples collected within 6 prespecified gestational age bins: 16-<20, 20-<24, 24-<28, 28-<32, 32-<36, 36-<37 weeks of gestation. The primary exposure was biomarker levels and the primary outcome was PTB (so samples were not tested >36 weeks of completed gestation). PTB was defined as delivery <37 weeks of gestation (ultrasound dated), SGA was defined using INTERGROWTH standards,
      • Villar J.
      • Cheikh Ismail L.
      • Victora C.G.
      • et al.
      International standards for newborn weight, length, and head circumference by gestational age and sex: the Newborn Cross-Sectional Study of the Intergrowth-21st Project.
      and stillbirth defined as intrauterine fetal demise ≥20 weeks of gestation.

      Enzyme-linked immunosorbent assays

      EDTA plasma samples were collected and stored at −80°C prior to testing. Samples were tested in Uganda using commercially available enzyme-linked immunosorbent assays (Duosets, R&D Systems, Minneapolis, MN) with the following ranges, dilution factors, and intraassay coefficients of variation: Ang-1 (313-20,000 pg/mL, 1:10, 5.6%); Ang-2 (93.8-6000 pg/mL, 1:20, 6.1%); sEng (250-16,000 pg/mL, 1:20, 7.4%); sFlt-1 (250-16,000 pg/mL, 1:5, 11.9%); PlGF (63.0-4000 pg/mL, 1:5, 8.1%), and VEGF (31-2000 pg/mL, 1:2). All testing was performed blinded to group and outcome.

      Statistical analysis

      Statistical analysis was performed using STATA v14 (StataCorp, College Station, TX), R v3.2.1

      R Core Team. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. 2017. http://www.R-project.org/.

      (R Foundation for Statistical Computing), and GraphPad Prism v6 (GraphPad Software Inc, La Jolla, CA) software. Descriptive statistics were calculated as n (%) and median (interquartile range). The χ2 and Fisher exact tests were used to compare categorical variables. Linear regression was used to assess whether biomarker levels changed over pregnancy. To assess the effect of gestational age on angiogenic factor concentrations between birth outcome groups, we used the lme4
      • Bates D.
      Fitting linear mixed-effects models using lme4.
      package in R

      R Core Team. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. 2017. http://www.R-project.org/.

      to construct linear mixed effects (LME) models with random intercept and random slope, adapting the approach employed by Romero et al.
      • 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.
      For each biomarker and outcome, we constructed a null model with 4 fixed effects: the linear effect of gestational age, maternal age, body mass index (BMI), and gravidity. To make the intercept meaningful, the gestational age variable was shifted such that the lowest gestational age in our data set (the baseline samples) would be the intercept’s x-value. We also included the interaction between gestational age and treatment arm, to control for the possibility that the treatment affected the biomarker’s rate of change. Treatment arm was omitted as a main effect to constrain the groups to have the same intercept, as baseline differences between the (randomly allocated) groups would have been due to chance. In 2 additional models, we added the birth outcome as a fixed effect: an additive, no-interaction model and an interaction model. Both models were the same as the null but also included outcome as a main effect; and the interaction model further included the interaction between outcome and gestational age. For none of the biomarkers or birth outcomes did the interaction term significantly improve the model fit (P > .05 for all). Therefore, the data favor the more parsimonious additive models. For models in which the groups had different intercepts, the fitted lines did not significantly converge or diverge over time. For random effects, all models included a by-participant intercept and by-participant slope for the effect of gestational age. The biomarker levels were transformed using the natural logarithm to stabilize their variance. Residual plots did not show any apparent deviation from homoscedasticity or normality. Statistical significance was assessed using likelihood ratio (LR) tests, which compared in a stepwise fashion the null model, the additive model, and the interaction model. As the PlGF values vary quadratically over time, we added a quadratic interaction term to the model, which significantly improved the model fit [LR test, χ2(1) = 157.15, P < .0001].

      Results

      Description of the study population

      A total of 1115 plasma samples were evaluated from 326 women and adolescents (Figure 1). The demographic characteristics of the study population are presented in Table 1. The median age of women was 30 years with a median BMI at enrollment of 21.4 kg/m2. The majority of participants (n = 271, 83%) were multigravida. None of the participants were diagnosed with chronic hypertension, preeclampsia, or eclampsia during the study period. Adverse birth outcomes in the cohort were common with 18.1% (n = 59) of participants having a low-birthweight infant (compared to a national estimate of 12%

      United Nations Children's Fund and World Health Organization. Low birthweight: country, regional and global estimates. UNICEF New York: 2004.

      ), 16.9% (n = 55) spontaneous PTB, 25.6% (n = 81) SGA, and 2.8% (n = 9) stillborn. The median gestational age of stillbirth deliveries was 31 weeks of gestation. Cause of fetal demise was not ascertained. Malaria infection status and birth outcomes did not differ between participants receiving lopinavir/ritonavir compared to those receiving efavirenz (Supplemental Table 1).
      Figure thumbnail gr1
      Figure 1Flow chart of maternal plasma samples processed by gestational age
      ART, antiretroviral therapy; EFV, efavirenz; LPV/r, lopinavir/ritonavir.
      Conroy et al. Angiogenic factors across pregnancy in women living with HIV. Am J Obstet Gynecol 2017.
      Table 1Descriptive characteristics of study population
      Cohort, n = 326
      Demographics
      Age, y30 (26–33)
      BMI, kg/m221.4 (19.9–23.0)
      Socioeconomic status, tertile
       1111 (35.9)
       2135 (43.7)
       363 (20.4)
      Gestational age at enrollment, wk23.6 (19.6–27.9)
      Previous pregnancies
       020 (6.1)
       135 (10.7)
       2271 (83.1)
      Laboratory characteristics
      Hemoglobin level, g/dL11.0 (10.2–11.8)
      White blood cell count, cells/mm35050 (4200–6200)
      Platelet count, ×109/L210 (173–252)
      CD4+ T-cell count, cells/mm3369 (271–504)
      HIV RNA load, log10 copies/mL4.2 (3.9–4.8)
      Delivery characteristics
      Gestational age delivery, wk38 (37–40)
      Birthweight, kg2890 (2670–3230)
      Preterm birth55 (16.9)
      Small for gestational age81 (25.6)
      Stillbirth9 (2.8)
      Placental malaria
      Defined by positive finding of placental blood smear or polymerase chain reaction.
      24 (8.7)
      Continuous variables expressed as median (interquartile range), categorical variables expressed as n (%).
      BMI, body mass index.
      Conroy et al. Angiogenic factors across pregnancy in women living with HIV. Am J Obstet Gynecol 2017.
      a Defined by positive finding of placental blood smear or polymerase chain reaction.

      Longitudinal changes in angiogenic factors over pregnancy

      There were no differences in angiogenic proteins by trial arm (Supplemental Figure 1), so subsequent analysis was conducted using the combined cohort. We plotted the longitudinal kinetics of angiogenic factors over pregnancy and observed declining levels of Ang-2 across gestation, and increasing levels PlGF, sFlt-1, and sEng (P < .0001 for all) (Figure 2). There were no differences in circulating Ang-1 levels across gestation. Median plasma Ang-2 decreased from 6.6 ng/mL at 16-20 weeks to 2.5 ng/mL by 37 weeks of gestation. Median levels of sEng increased from 9.1-14.7 ng/mL and sFlt-1 increased from 1.9-6.4 ng/mL from 16-20 and 37 weeks of gestation (Figure 2). PlGF levels were 0.24 ng/mL at 16-20 weeks, peaked at 0.84 ng/mL at 28-32 weeks, and declined to 0.40 ng/mL by 37 weeks of gestation (Figure 2). VEGF-A levels were largely undetectable with 87% of samples having concentrations below the bottom standard of 31.3 pg/mL.
      Figure thumbnail gr2
      Figure 2Angiogenic factors in HIV infected study participants receiving antiretroviral therapy
      Scatter plot of plasma levels of angiogenic factors plotted according to gestational age of sample collection: A, placental growth factor (PlGF); B, soluble fms-like tyrosine kinase (sFlt)-1; C, soluble endoglin (sEng); D, angiopoietin (Ang)-2; and E, Ang-1. Line indicates best fit line with 95% confidence intervals.
      Conroy et al. Angiogenic factors across pregnancy in women living with HIV. Am J Obstet Gynecol 2017.

      Relationship between angiogenic factors and immune status

      To determine whether angiogenic factors were associated with maternal health or immune status, we conducted nonparametric bivariate correlations comparing angiogenic proteins and enrollment laboratory tests. There were no differences between angiogenic proteins measured in the first gestational age bin (16-<20 weeks of gestation) and enrollment hemoglobin, platelet count, viral load, or CD4 count (P > .05 for all).

      Relationship between angiogenic factors and birth outcomes

      LME modeling evaluated the longitudinal changes in angiogenic factor concentrations between birth outcome groups. The models adjusted for gestational age at venipuncture, maternal age, BMI, gravidity, and the interaction between treatment arm and gestational age (Tables 2 and 3, and Table S2, Table S3, Table S4). Two angiogenic factors–sEng and PlGF–were associated with adverse birth outcomes. Significantly higher concentrations of sEng throughout gestation were found in study participants destined to deliver preterm [LR test, χ2(1) = 12.28, P < .0005] (Figure 3, A ) and in those destined to have stillbirths [χ2(1) = 5.67, P < .02] (Figure 3, B, and Table 2). By contrast, significantly lower concentrations of PlGF throughout gestation were found in those destined to have SGA births [χ2(1) = 7.89, P < .005] (Figure 3, C) and in those destined to have stillbirths [χ2(1) = 21.59, P < .0001] (Figure 3, D, and Table 3). To show the natural variability of biomarkers over gestation among individual participants, we generated trellis plots for a random subset of participants with the fitted regression line from the LME model conditional on fixed effects only (Supplemental Figure 2, Supplemental Figure 3, Supplemental Figure 4, Supplemental Figure 5).
      Table 2Linear mixed effect modelling of longitudinal changes in soluble endoglin and adverse birth outcomes
      Preterm birthStillbirth
      BetaSEBetaSE
      Fixed terms
      (Intercept)2.137330.17602.127620.1788
      Birth outcome0.167450.04750.264590.1110
      Gestational age (shifted)0.040930.00300.040640.0030
      Maternal age–0.001020.0047–0.001540.0048
      Enrollment BMI–0.004550.0063–0.002970.0064
      Gravidity–0.012700.0109–0.010470.0111
      Gestational age: treatment arm interaction–0.005070.0025–0.004710.0026
      No. of subjects320320
      Observations10851085
      LR test against null modelχ2(1) = 12.282, P < .0005χ2(1) = 5.6717, P < .02
      Linear mixed effect modelling evaluated longitudinal changes in angiogenic factor concentrations between birth outcome groups. Models adjusted for gestational age at venipuncture, maternal age, BMI, gravidity, and interaction between treatment arm and gestational age.
      BMI, body mass index; LR, likelihood ratio.
      Conroy et al. Angiogenic factors across pregnancy in women living with HIV. Am J Obstet Gynecol 2017.
      Table 3Linear mixed effect modelling longitudinal changes in placental growth factor and adverse birth outcomes
      Small for gestational ageStillbirth
      BetaSEBetaSE
      Fixed terms
      (Intercept)–3.038170.6134–3.143400.5889
      Birth outcome–0.380940.1357–1.663800.3520
      Gestational age (shifted)0.362860.02640.368280.0259
      Gestational age (shifted) squared–0.013790.0010–0.014050.0010
      Maternal age0.010620.01630.008810.0156
      Enrollment BMI–0.009810.0218–0.008760.0208
      Gravidity0.061390.03790.075300.0359
      Gestational age: treatment arm interaction–0.003840.0087–0.004210.0085
      No. of subjects311320
      Observations10801104
      LR test against null modelχ2(1) = 7.892, P < .005χ2(1) = 21.595, P < .0001
      Linear mixed effect modelling evaluated longitudinal changes in angiogenic factor concentrations between birth outcome groups. Models adjusted for gestational age at venipuncture, maternal age, BMI, gravidity, and interaction between treatment arm and gestational age.
      BMI, body mass index; LR, likelihood ratio.
      Conroy et al. Angiogenic factors across pregnancy in women living with HIV. Am J Obstet Gynecol 2017.
      Figure thumbnail gr3
      Figure 3Antiangiogenic shift is associated with adverse birth outcomes in women living with HIV receiving antiretroviral therapy
      Individual data points colored by birth outcome. Overlaid regression lines are from linear mixed effects models, fitted for subject with average values (conditional on fixed effects only).
      AGA, appropriate for gestational age; PlGF, placental growth factor; PTB, preterm birth; sEng, soluble endoglin; SGA, small for gestational age.
      Conroy et al. Angiogenic factors across pregnancy in women living with HIV. Am J Obstet Gynecol 2017.

      Comment

      Principal findings of the study

      Angiogenic processes in the placenta are critical regulators of fetal growth, but there are limited data documenting these processes in low-resource settings where maternal infections are common. In this study we assessed longitudinal concentrations in angiogenic proteins in HIV-infected pregnant women and adolescents started on ART. There were no differences in angiogenic factors by treatment arm. All proteins except Ang-1 and VEGF-A were dynamically regulated over gestation. Ang-2 levels declined, while sFlt-1 and sEng increased over gestation, and PlGF increased up to 32 weeks of gestation and then declined. Altered expression of angiogenic factors was associated with spontaneous PTB, SGA, and stillbirth. These data suggest an early shift toward an antiangiogenic state is a common pathway associated with adverse birth outcomes in WLHIV, consistent with data from HIV-uninfected women.
      • 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.
      • Mijal R.S.
      • Holzman C.B.
      • Rana S.
      • Karumanchi S.A.
      • Wang J.
      • Sikorskii A.
      Mid-pregnancy levels of angiogenic markers as indicators of pathways to preterm delivery.
      • 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.
      • 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.
      • Chaiworapongsa T.
      • Romero R.
      • Tarca A.
      • et al.
      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.
      • McDonald C.R.
      • Darling A.M.
      • Conroy A.L.
      • et al.
      Inflammatory and angiogenic factors at mid-pregnancy are associated with spontaneous preterm birth in a cohort of Tanzanian women.
      • Darling A.M.
      • McDonald C.R.
      • Conroy A.L.
      • et al.
      Angiogenic and inflammatory biomarkers in midpregnancy and small-for-gestational-age outcomes in Tanzania.
      Placental malaria was uncommon in this study due to the distribution of insecticide-treated bed nets and daily prophylaxis with trimethoprim-sulfamethoxazole.

      Comparison with previous studies

      We evaluated 1115 plasma samples collected from 326 women and adolescents between 16-<37 weeks of gestation. We compared our findings directly to results from women enrolled in a clinical trial receiving calcium supplementation in pregnancy with longitudinal assessment of sFlt-1, PlGF and VEGF,
      • Levine R.J.
      • Maynard S.E.
      • Qian C.
      • et al.
      Circulating angiogenic factors and the risk of preeclampsia.
      and sEng
      • Levine R.J.
      • Lam C.
      • Qian C.
      • et al.
      Soluble endoglin and other circulating antiangiogenic factors in preeclampsia.
      between 8-42 weeks of gestation.
      • Joffe G.M.
      • Esterlitz J.R.
      • Levine R.J.
      • et al.
      The relationship between abnormal glucose tolerance and hypertensive disorders of pregnancy in healthy nulliparous women. Calcium for Preeclampsia Prevention (CPEP) study group.
      Both sFlt-1 and sEng levels increased over pregnancy starting at 24-28 weeks of gestation. While levels of sFlt-1 increased 3-fold over the third trimester, levels of sEng reached a plateau by 32-26 weeks of gestation. PlGF levels increased in early pregnancy, peaked between 28-32 weeks of gestation, and declined. Our results were consistent with those from the calcium trial suggesting temporal regulation of angiogenic factors is tightly controlled across pregnancy. In both studies, VEGF was measured but was undetectable in the majority of samples.
      Despite their important regulatory role in placental vascular development, data on Ang kinetics in pregnancy are limited. Our observations are consistent with a report of decreased placental Ang-2 messenger RNA over pregnancy.
      • Geva E.
      • Ginzinger D.G.
      • Zaloudek C.J.
      • Moore D.H.
      • Byrne A.
      • Jaffe R.B.
      Human placental vascular development: vasculogenic and angiogenic (branching and nonbranching) transformation is regulated by vascular endothelial growth factor-a, angiopoietin-1, and angiopoietin-2.
      Placental expression of Ang-2 messenger RNA is strongly correlated with circulating Ang-2 protein levels.
      • Geva E.
      • Ginzinger D.G.
      • Zaloudek C.J.
      • Moore D.H.
      • Byrne A.
      • Jaffe R.B.
      Human placental vascular development: vasculogenic and angiogenic (branching and nonbranching) transformation is regulated by vascular endothelial growth factor-a, angiopoietin-1, and angiopoietin-2.
      Our results are further supported by a study examining plasma Ang levels between 10-37 weeks of gestation where Ang-2 levels decreased and Ang-1 remained the same across gestation.
      • Bolin M.
      • Wiberg-Itzel E.
      • Wikström A.-K.
      • et al.
      Angiopoietin-1/angiopoietin-2 ratio for prediction of preeclampsia.
      Collectively these data demonstrate the Ang-Tie-2 axis is dynamically regulated over pregnancy.
      There are limited data on the impact of maternal HIV infection on expression of angiogenic factors. Lower PlGF levels have been reported in pregnant WLHIV in South Africa
      • Govender N.
      • Naicker T.
      • Moodley J.
      Maternal imbalance between pro-angiogenic and anti-angiogenic factors in HIV-infected women with pre-eclampsia.
      ; however, the sample size was small (n = 27 HIV-uninfected, n = 31 WLHIV), and samples were collected a week later in WLHIV. Other studies examining angiogenic factors in nonpregnant individuals infected with HIV have shown increases in the Ang-2:Ang-1 ratio associated with acute HIV infection, and decreased Ang-1 in chronic disease.
      • Graham S.M.
      • Rajwans N.
      • Jaoko W.
      • et al.
      Endothelial activation biomarkers increase after HIV-1 acquisition: plasma vascular cell adhesion molecule-1 predicts disease progression.
      In HIV-infected Kenyan women with advanced infection, Ang-2 levels decreased and Ang-1 levels increased following the initiation of ART.
      • Graham S.M.
      • Rajwans N.
      • Tapia K.A.
      • et al.
      A prospective study of endothelial activation biomarkers, including plasma angiopoietin-1 and angiopoietin-2, in Kenyan women initiating antiretroviral therapy.
      On a cellular level, HIV-infected cells release transactivator of transcription, which accumulates on and is taken up by endothelial cells where it acts in synergy with VEGF-A to modify the cytoskeletal structure of endothelium.
      • Urbinati C.
      • Nicoli S.
      • Giacca M.
      • et al.
      HIV-1 Tat and heparan sulfate proteoglycan interaction: a novel mechanism of lymphocyte adhesion and migration across the endothelium.
      • Das J.R.
      • Gutkind J.S.
      • Ray P.E.
      Circulating fibroblast growth factor-2, HIV-Tat, and vascular endothelial cell growth factor-A in HIV-infected children with renal disease activate Rho-A and Src in cultured renal endothelial cells.
      There have been a number of studies investigating the relationship between placental angiogenesis, vascular remodeling, and stillbirth. In a study of 22 unexplained stillbirths and 44 age-matched live-born controls, stillbirth was associated with increased placental microvascular density, vasculopathy, and increased vascular permeability,
      • Plunkett B.A.
      • Fitchev P.
      • Doll J.A.
      • et al.
      Decreased expression of pigment epithelium derived factor (PEDF), an inhibitor of angiogenesis, in placentas of unexplained stillbirths.
      suggesting increased vascular remodeling in terminal stillbirth placentae. In contrast, in 1269 singleton women with samples collected between 30-34 weeks of gestation, a low PlGF/sFlt-1 ratio was associated with stillbirth.
      • Chaiworapongsa T.
      • Romero R.
      • Korzeniewski S.J.
      • et al.
      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.
      Levels of sFlt-1 and sEng in the highest quartile of amniotic fluid were associated with increased odds of stillbirth in women with unexplained fetal death.
      • Chaiworapongsa T.
      • Romero R.
      • Kusanovic J.P.
      • et al.
      Plasma soluble endoglin concentration in pre-eclampsia is associated with an increased impedance to flow in the maternal and fetal circulations.
      In a longitudinal nested case-control study of women with a fetal death compared to those with an appropriate-for-gestational-age term delivery, the first trimester (weeks 7-11) was characterized by a proangiogenic phenotype (low sFlt-1, low sEng, high PlGF) and this shifted in favor of an antiangiogenic phenotype over the second and third trimester (between 23-41 weeks of gestation).
      • 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.
      Another cohort showed low sFlt-1 and PlGF levels in the first trimester were associated with spontaneous abortion.
      • Andersen L.B.
      • Dechend R.
      • Karumanchi S.A.
      • et al.
      Early pregnancy angiogenic markers and spontaneous abortion: an Odense Child Cohort study.
      Our data support and extend the hypothesis that an antiangiogenic state in the second and third trimester is associated with subsequent stillbirth with lower PlGF and higher sEng levels. As the placental vascular network undergoes continual growth and remodeling, it requires the coordinated regulation of angiogenic factors in a spatial, temporal, and quantitative manner. Additional studies are needed to assess how temporal changes in angiogenic factors during pregnancy relate to the vascular phenotype observed in the placenta at delivery.
      Beginning in the second trimester the placenta undergoes a continuous process of sprouting angiogenesis, intercalated growth, and intussusception. This remodeling allows for increased placental volume and surface area for nutrient exchange to support the rapidly growing fetus. Dysregulation of the pathways that mediate these processes midpregnancy may result in preterm delivery or fetal growth restriction if the placenta cannot support this rapid growth. Relative increases in sEng levels across gestation were associated with spontaneous PTB in this cohort, supporting the hypothesis that an antiangiogenic environment can contribute to premature birth.
      • Chaiworapongsa T.
      • Romero R.
      • Tarca A.
      • et al.
      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.
      • McDonald C.R.
      • Darling A.M.
      • Conroy A.L.
      • et al.
      Inflammatory and angiogenic factors at mid-pregnancy are associated with spontaneous preterm birth in a cohort of Tanzanian women.
      Likewise, reduced PlGF across gestation was associated with SGA, consistent with previous literature linking PlGF to placental and fetal growth and development.
      • 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.
      • Darling A.M.
      • McDonald C.R.
      • Conroy A.L.
      • et al.
      Angiogenic and inflammatory biomarkers in midpregnancy and small-for-gestational-age outcomes in Tanzania.

      Strengths and weaknesses

      This study has several strengths including ultrasound-confirmed gestational dating and frequent blood sampling. Despite routine clinical monitoring over pregnancy including monthly blood pressure and proteinuria assessments, no women in this cohort developed preeclampsia. This study is the first to present detailed kinetics data from a low-resource setting where the burden of disease is greatest, but for which we have the least amount of data. The lack of an HIV-uninfected comparison group in this study is a limitation that prevents us from discussing the generalizability of the findings or the relative impact of maternal HIV infection. While we did not observe any changes in angiogenic factor expression by treatment arm, CD4 count, or HIV-1 RNA viral load at enrollment, additional studies are needed to delineate the role of HIV-1 infection on the expression of angiogenic factors to determine whether infection itself modifies also the risk of adverse birth outcomes through dysregulated angiogenesis.

      Research and clinical implications

      This study was conducted in a rural area of southeastern Uganda where the low incidence of preeclampsia is consistent with clinical reports from the region. While the reason for this is unknown, we speculate that a relative absence of risk factors for preeclampsia, including nulliparity, coupled with lower weight gain over gestation contributed to a lower risk of preeclampsia.
      • Gavard J.A.
      Gestational weight gain and maternal and neonatal outcomes in underweight pregnant women: a population-based historical cohort study.
      The median weekly weight gain in the study was 0.2 kg with nearly 20% of women gaining no weight over the study period.
      • Koss C.A.
      • Natureeba P.
      • Plenty A.
      • et al.
      Risk factors for preterm birth among HIV-infected pregnant Ugandan women randomized to lopinavir/ritonavir- or efavirenz-based antiretroviral therapy.
      Additional studies are needed to validate these findings in HIV-infected women and in populations where preeclampsia is more common to ascertain the generalizability of these findings, and whether early assessment of angiogenic markers may have utility in identifying high-risk pregnancies.

      Conclusions

      Early changes in angiogenic proteins may have predictive utility in identifying women at risk of adverse birth outcomes in WLHIV receiving ART. While these findings need to be prospectively validated, early identification of women at increased risk of adverse birth outcomes may facilitate enhanced monitoring and referral to health care facilities equipped to manage high-risk pregnancies (Video).

      Acknowledgment

      This study would not have been possible without the participation, input, and work of the mothers, field teams, nursing and medical staff, and members of the Infectious Diseases Research Collaboration involved in this trial. We would like to thank Mr Ssemanda Cephus for assistance with plate mapping and aliquoting samples.

      Appendix

      Table S1Descriptive characteristics of the study population by ART treatment arm
      Baseline CharacteristicTreatment ArmP Value
      Efavirenz-Based ART, n=160Lopinavir/ritonavir -Based ART, n=166
      Age (years)30 [26, 33]29.2 ± 5.30.623
      BMI (kg/m2)21.2 [19.6, 22.9]21.6 [20.2, 23.2]0.074
      Socioeconomic status (tertile)0.611
       154 (36.2)57 (35.6)
       268 (45.6)67 (41.9)
       327 (18.1)36 (22.)
      Gestational age enrolment (weeks)23.6 [19.7, 27.6]23.5 [19.4, 27.0]0.711
      Previous pregnancies
       013 (8.3)7 (4.2)0.275
       115 (9.6)20 (12.1)
       ≥2132 (82.2)139 (83.6)
      Hemoglobin level (g/dL)11.0 [10.2, 11.8]11.1 [10.2, 11.7]0.855
      White blood cell count (cells/mm3)4900 [4100, 6100]5200 [4300, 6400]0.134
      Platelet count (x109/L)218.0 [177.5, 255.5]201.0 [167.0, 239.5]0.080
      CD4+T-cell count (cells/mm3)373.0 (269.3-497.8)368.0 (280.5-507.5)0.575
      HIV RNA load (log10copies/mL)4.286 [3.427, 4.837]4.097 [3.328, 4.755]0.468
      Outcome Characteristics
      Gestational age delivery (weeks)39 (37-40)38 (37-39)0.061
      Birth weight (kg)2910 (2680-3240)2880 (2650-3210)0.503
      Preterm birth24 (15.0)31 (18.7)0.376
      Small-for-gestational age44 (27.2)37 (23.9)0.502
      Stillbirth4 (2.5)5 (3.0)1.000
      Placental malaria
      Placental malaria defined by a positive finding of a placental blood smear or PCR.
      14 (10.2)10 (7.1)0.363
      Continuous variables expressed as median (interquartile range), categorical variables expressed as n (%).
      Conroy et al. Angiogenic factors across pregnancy in women living with HIV. Am J Obstet Gynecol 2017.
      1 Placental malaria defined by a positive finding of a placental blood smear or PCR.
      Table S2Linear mixed effects models of angiogenic markers and preterm birth
      Ang-1Ang-2sFlt-1PlGFsEng
      BetaSEBetaSEBetaSEBetaSEBetaSE
      Fixed Terms
      (Intercept)2.134790.44452.175190.3960−0.435200.5145−3.269560.61192.137330.1760
      Preterm0.133870.1206−0.158450.10680.179020.1499−0.161960.16170.167450.0475
      Gestational age (shifted)−0.008440.0064−0.063470.00530.154260.00940.369990.02600.040930.0030
      Gestational age (shifted) squared−0.014070.0010
      Maternal age−0.003040.0120−0.014710.0109−0.003750.01320.006460.0162−0.001020.0047
      Enrolment BMI0.002970.01610.006550.0144−0.020360.0181−0.002980.0216−0.004550.0063
      Gravidity−0.024310.02770.019200.0253−0.024790.03080.081500.0375−0.012700.0109
      Gestational age: treatment arm interaction0.001320.0062−0.002900.00630.004370.0058−0.004020.0086−0.005070.0025
      Number of subjects320312320320320
      Observations11041049110411041085
      LR Test against Null Modelχ2(1) = 1259, p > 0.05χ2(1) = 2.233, p > 0.05χ2(1) = 1.444, p > 0.05χ2(1) = 1.020, p > 0.05χ2(1) = 12.282, p < 0.0005*
      Conroy et al. Angiogenic factors across pregnancy in women living with HIV. Am J Obstet Gynecol 2017.
      Table S3Linear mixed effects models of angiogenic markers and small-for-gestational age
      Ang-1Ang-2sFlt-1PlGFsEng
      BetaSEBetaSEBetaSEBetaSEBetaSE
      Fixed Terms
      (Intercept)2.141910.45592.132460.4024−0.394290.5229−3.038170.61342.126740.1820
      Stillbirth−0.007120.10280.133170.0920−0.033390.1140−0.380940.1357−0.001890.0408
      Gestational age (shifted)−0.007600.0064−0.062470.00530.151380.00950.362860.02640.040840.0030
      Gestational age (shifted) squared−0.013790.0010
      Maternal age−0.003740.0124−0.015080.0111−0.003390.01360.010620.0163−0.000600.0049
      Enrolment BMI0.003010.01650.004890.0147−0.019530.0185−0.009810.0218−0.004000.0066
      Gravidity−0.019630.02870.025060.0259−0.024510.03190.061390.0379−0.011340.0114
      Gestational age: treatment arm interaction0.001130.0063−0.002960.00640.005190.0059−0.003840.0087−0.004450.0026
      Number of subjects311303311311311
      Observations10801025108010801061
      LR Test against Null Modelχ2(1) = 0.0, p > 0.05χ2(1) = 2.128, p > 0.05χ2(1) = 0.0878, p > 0.05χ2(1) = 7.892, p < 0.005*χ2(1) = 0.0025, p > 0.05
      Conroy et al. Angiogenic factors across pregnancy in women living with HIV. Am J Obstet Gynecol 2017.
      Table S4Linear mixed effects models of angiogenic markers and stillbirth
      Ang-1Ang-2sFlt-1PlGFsEng
      BetaSEBetaSEBetaSEBetaSEBetaSE
      Fixed Terms
      (Intercept)2.140070.44682.200690.3979−0.408890.5146−3.143400.58892.127620.1788
      Stillbirth0.016160.2756−0.247060.2320−0.340660.3784−1.663800.35200.264590.1110
      Gestational age (shifted)−0.008870.0064−0.063290.00530.152780.00930.368280.02590.040640.0030
      Gestational age (shifted) squared−0.014050.0010
      Maternal age−0.003090.0120−0.014860.0109−0.003620.01320.008810.0156−0.001540.0048
      Enrolment BMI0.003690.01610.004800.0145−0.019590.0181−0.008760.0208−0.002970.0064
      Gravidity−0.023310.02780.018850.0253−0.023590.03080.075300.0359−0.010470.0111
      Gestational age: treatment arm interaction0.001490.0062−0.003160.00630.004270.0058−0.004210.0085−0.004710.0026
      Number of subjects320312320320320
      Observations11041049110411041085
      LR Test against Null Modelχ2(1) = 0.0045, p > 0.05χ2(1) = 1.129, p > 0.05χ2(1) = 0.819, p > 0.05χ2(1) = 21.595, p < 0.0001*χ2(1) = 5.6717, p < 0.02*
      Conroy et al. Angiogenic factors across pregnancy in women living with HIV. Am J Obstet Gynecol 2017.
      Figure thumbnail fx1
      Supplemental Figure 1Longitudinal changes in angiogenic factors by treatment arm
      Scatter plot of plasma levels of angiogenic factors plotted by treatment arm with nonnucleoside reverse transcriptase inhibitor (NNRTI)-based antiretroviral therapy (ART) in red and protease inhibitor (PI)-based ART in blue. Biomarkers plotted according to gestational age of sample collection: A, placental growth factor (PlGF); B, soluble fms-like tyrosine kinase (sFlt)-1; C, soluble endoglin (sEng); D, angiopoietin (Ang)-2; and E, Ang-1. Line indicates best fit line with 95% confidence intervals.
      Conroy et al. Angiogenic factors across pregnancy in women living with HIV. Am J Obstet Gynecol 2017.
      Figure thumbnail fx2
      Supplemental Figure 2Representative plots of soluble endoglin (sEng) levels over gestation in participants destined to have preterm or term delivery
      Trellis plots of 60 randomly selected subjects (n = 30 term, n = 30 preterm). Solid line depicts fitted regression line from linear mixed effect model (conditional on fixed effects only).
      Conroy et al. Angiogenic factors across pregnancy in women living with HIV. Am J Obstet Gynecol 2017.
      Figure thumbnail fx3
      Supplemental Figure 3Representative plots of soluble endoglin (sEng) levels over gestation in participants destined to have livebirth or stillbirth infant
      Trellis plots of 18 randomly selected subjects (n = 9 livebirth, n = 9 stillbirth). Solid line depicts fitted regression line from linear mixed effect model (conditional on fixed effects only).
      Conroy et al. Angiogenic factors across pregnancy in women living with HIV. Am J Obstet Gynecol 2017.
      Figure thumbnail fx4
      Supplemental Figure 4Representative plots of placental growth factor (PlGF) levels over gestation in participants destined to have small-for-gestational-age (SGA) or appropriate-for-gestational-age (AGA) infant
      Trellis plots of 60 randomly selected subjects (n = 30 AGA, n = 30 SGA). Solid line depicts fitted regression line from linear mixed effect model (conditional on fixed effects only).
      Conroy et al. Angiogenic factors across pregnancy in women living with HIV. Am J Obstet Gynecol 2017.
      Figure thumbnail fx5
      Supplemental Figure 5Representative plots of placental growth factor (PlGF) levels over gestation in women destined to have livebirth or stillbirth infant
      Trellis plots of 18 randomly selected subjects (n = 9 livebirth, n = 9 stillbirth). Solid line depicts fitted regression line from linear mixed effect model (conditional on fixed effects only).
      Conroy et al. Angiogenic factors across pregnancy in women living with HIV. Am J Obstet Gynecol 2017.

      Supplementary Data

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