ZAPPS cohort description
This study used clinical data and biorepository specimens collected from pregnant women enrolled in the Zambian Preterm Birth Prevention Study (ZAPPS; ClinicalTrials.gov Identifier: NCT02738892). ZAPPS is an ongoing observational antenatal cohort established at the Women and Newborn Hospital of the University Teaching Hospital in Lusaka to characterize factors associated with adverse birth outcomes. The data and specimens used in the current analysis are all derived from participants in the first phase of ZAPPS, which completed enrollment in September 2017 and follow-up in June 2018 [15, 16].
ZAPPS participants are enrolled before 24 gestational weeks, as determined by ultrasound at screening. All participants are tested for HIV with the Alere Determine HIV-1/2 test kit (Abbot Diagnostics, Lake Forest, Illinois, USA). Positive results are confirmed with the SD Bioline 3.0 (SD Biostandard Diagnostics, Gurgaon, Haryana, India). Women newly diagnosed with HIV are counseled and referred to the appropriate HIV care and treatment services. In Zambia, immediate ART initiation is recommended for pregnant and breastfeeding women, with most pregnant women receiving the first-line combination of tenofovir disoproxil fumarate, emtricitabine, and efavirenz. For participants who test positive for HIV, viral load is measured at enrollment using a GeneXpert (Cepheid, Sunnyvale, California, USA).
Participants who enroll in ZAPPS are provided routine antenatal care at enrollment and monthly thereafter in line with Zambian guidelines. At delivery, research nurses collect details regarding each participant’s labor and delivery course, as well as maternal and neonatal outcomes by either direct observation/record review at the time of delivery, or at first contact with the participant postpartum [15, 16].
We performed a nested case–control study comparing serum concentrations of VEGF-A, sEng, PlGF, and sFLT-1 between cases and controls. Cases were defined as participants with HIV at enrollment for whom a serum sample collected before 16 gestational weeks was available. Because concentrations of these protein biomarkers are known to vary over the course of pregnancy , controls were chosen from participants without HIV at enrollment and matched on gestational age at sample collection.
All ZAPPS participants provide individual written informed consent before undergoing study procedures. The University of Zambia Biomedical Research Ethics Committee, the National Health Research Authority of the Zambian Ministry of Health, and the University of North Carolina at Chapel Hill Institutional Review Board each approved the use of these clinical data and biological specimens for research purposes.
As part of the ZAPPS protocol, maternal serum samples are collected at the enrollment visit. Specimens are transferred from the study clinic to the UNC GPZ on-site research laboratory within two hours of specimen collection. There, specimens are processed into 1 mL aliquots and then transferred to -80 °C freezers. All serum samples used in this analysis were subjected to no more than two freeze–thaw cycles.
Concentrations of VEGF-A, sEng, PlGF, and sFLT-1 were measured in thawed serum using the Luminex MAGPIX analyzer (Merck, Darmstadt, Germany) with kits purchased from R&D Biosystems (Minneapolis, MN; catalogue # LXSAHM; Lot # L127927) at our laboratory in Lusaka. VEGF-A was run separately from sEng, PlGF, and sFLT-1 to avoid antibody cross-reactivity. Samples were processed according to manufacturer instructions. Serum was diluted in calibrator diluent at a 1:3 ratio. A minimum of 50 microparticles were captured for each analyte.
Descriptive statistics were collected as part of the ZAPPS protocol at the first antenatal visit. We reported these statistics for cases and controls as n (%) and median (interquartile range). We used Wilcoxon rank sum test to compare continuous variables and the χ2 test to compare categorical variables between cases and gestational age-matched controls.
Biomarker concentrations were reported as medians and interquartile ranges (IQR) for cases and controls. In unadjusted analysis, we used the non-parametric Wilcoxon rank-sum test to assess differences in biomarker concentration between cases and controls. In adjusted analysis, we constructed a linear regression model for each biomarker using log-transformed biomarker concentration as the dependent variable and HIV status as the independent variable, adjusting for potential confounders based on our demographic data and variables that have the potential to be confounding based on reports in the literature. The PlGF/sFLT-1 ratio has been cited as a useful predictor of placental dysfunction . To see if this ratio differed among cases and controls we build a linear regression model with log-transformed PlGF/sFLT-1 as the dependent variable and HIV status as the independent variable. We also conducted an adjusted analysis, adjusting for potential confounders as described above.
Among participants with HIV, we conducted unadjusted analyses to quantify the association of angiogenic biomarker concentration by viral load (i.e., detectable vs. undetectable) at enrollment and by timing of ART initiation (preconceptional vs. not preconceptional). For these analyses we used the Wilcoxon rank-sum non-parametric test of association.
We built unadjusted and adjusted logistic regression models to investigate the relationship between biomarker concentration and adverse outcomes in our cohort. Preeclampsia was defined as both elevated blood pressure (≥ 140 mmHg systolic or ≥ 90 mmHg diastolic) and proteinuria (≥ 2 protein on urine dipstick). PTB was defined as delivery between 16 and 36 6/7 gestational weeks. The lower limit of 16 weeks was chosen based on evidence that the mechanisms underlying preterm birth at 16 weeks do not substantially differ from the mechanisms underlying preterm birth after 20 weeks [19, 20]. Among participants who experienced a PTB in this nested study, the median gestational age at delivery was 31.9 weeks (IQR 20.6 – 35.3 weeks). As angiogenic imbalance could contribute to both spontaneous (e.g., spontaneous labor onset in the setting of placental abruption) and provider-initiated preterm birth phenotypes (e.g., induction of labor for preeclampsia), we did not differentiate between spontaneous PTB and provider-initiated PTB in case selection. We used small-for-gestational age infant (SGA) as a proxy for FGR and defined SGA as birth weight below the 10th percentile for gestational age based on INTERGROWTH-21st standards . Stillbirth was defined as infants born beyond 16 weeks gestational age without signs of life. Finally, we also evaluated a composite adverse birth outcome of PTB and/or stillbirth at any gestational age. In adjusted analysis, we adjusted for potential confounders based on our demographic data and variables that have the potential to be confounding based on prior reports. In all cases, biomarkers were converted to deciles before inclusion as the independent variable in the logistic regression model so that the odds ratio represents the odds of the adverse event of interest associated with a one decile increase in biomarker concentration.
The sample size for this study was driven by the availability of eligible samples in the Lusaka biorepository. All samples that met the eligibility criteria for cases (collected from women with HIV at the appropriate gestational age) were included in the study. These cases were matched based on gestational age with controls at a ratio of 1 case to 1 control. All statistical analyses were performed using STATA (version 15.1; StataCorp LP, College Station, TX). Statistical significance was set at a threshold of p < 0.05.