Association of mid‐trimester maternal angiogenic biomarkers with small‐for‐gestational‐age infants in an urban Zambian cohort: a nested case‐control study

Abstract Objective To investigate whether angiogenic biomarker concentrations differ between women who deliver small‐for‐gestational‐age (SGA) infants (<10th centile birth weight for gestational age) compared with controls, because identifying SGA risk early could improve outcomes. Methods This case‐control study compared serum concentrations of angiogenic biomarkers before 24 weeks of pregnancy from 62 women who delivered SGA infants (cases) and 62 control women from an urban Zambian cohort. Odds of delivering an SGA infant were calculated using conditional logistic regression. Results Placental growth factor (PlGF), soluble fms‐like tyrosine kinase (sFLT‐1) and soluble endoglin (sEng) in controls were 37.74 pg/mL (interquartile range [IQR] 23.12–63.15), 2525.18 pg/mL (IQR 1502.21–4265.54) and 2408.18 pg/mL (IQR 1854.87–3017.94), respectively. SGA cases had higher PlGF (40.50 pg/mL, IQR 22.81–67.94) and sFLT‐1 (2613.06 pg/mL, IQR 1720.58–3722.50), and lower sEng (2038.06 pg/mL, IQR 1445.25–3372.26). Participants with sEng concentration below and concomitant sFLT‐1 concentration above their respective thresholds (n = 40) had five‐fold higher odds of SGA (adjusted odds ratio 4.77, 95% confidence interval 1.61–14.1; P = 0.005). Conclusion Biomarker concentrations were similar between cases and controls. Participants with concomitant low sEng and high sFLT‐1 had the highest odds of SGA, suggesting that a combination of biomarkers may better for predicting SGA than single biomarkers.


| INTRODUC TI ON
Low birth weight, which results from the overlapping conditions of prematurity and small for gestational age (SGA), contributes to over half of newborn deaths in sub-Saharan Africa. 1 Compared with neonates with a birth weight appropriate for gestational age, those born with a birth weight below the 10th centile for gestational age are at higher risk of neonatal mortality, 2 poor neurocognitive outcomes, 3 and chronic illness. 4,5 Although correct classification of SGA requires accurate gestational age estimation with ultrasound, largely unavailable to the world's most at-risk women, globally, SGA affects 10% of neonates by definition, but is as high as 20% in low-and middle-income countries. 6 Resource scarcity further compounds the burden of SGA in settings with limited neonatal supportive technologies and long-term care options.
Clinical detection of fetal growth restriction, a commonly used proxy for SGA outcome, is linked to decreased perinatal mortality. 7 However, many infants at risk for growth restriction are missed by available methods of fetal surveillance, particularly in underresourced settings. 8 The capacity to accurately identify women at risk of delivering SGA babies is therefore a matter of considerable clinical and public health importance worldwide.
Normal placental vascularization, angiogenesis, and growth rely on a tightly regulated signaling pathway involving the pro-angiogenic factors vascular endothelial growth factor A (VEGF-A) and placental growth factor (PlGF), along with binding proteins conventionally thought to have anti-angiogenic activity, the soluble forms of endoglin (sEng) and fms-like tyrosine kinase (sFLT-1). 9,10 Dysregulation of these proteins is implicated in adverse birth outcomes, including hypertensive disorders, fetal growth restriction, and stillbirth. 11,12 Abnormal concentrations of these factors can be detected in maternal blood weeks before the clinical manifestation of obstetric disease, 12,13 and are potential targets for predictive tests of placentally-derived pregnancy complications.
The current study aimed to assess whether mid-trimester angiogenic biomarker concentrations were predictive of SGA in an urban Zambian cohort. We hypothesized that angiogenic biomarker concentrations would differ between women who delivered SGA neonates compared with those who delivered neonates that were either appropriate or large for gestational age.

| RE SULTS
A total of 124 participants were included in this analysis: 62 cases who delivered SGA infants and 62 controls (  Figure 2). Ratios of PlGF/ sEng, PlGF/sFLT-1, and sEng/sFLT-1 were also similar between cases and controls ( Table 3). VEGF-A levels were below minimum detectable levels and were excluded from analysis.   Table 4).
a Conditional logistic models adjusted for parity, marital status, maternal weight at enrollment, and hypertension at enrolment.
The ALK-1 pathway induces angiogenesis through the proliferation and migration of endothelial cells, whereas the ALK-5 pathway has the reverse effect. 19 Outside the optimal range, sEng concentration may cause TGFβ to shift towards either a pro-angiogenic pathway or an anti-angiogenic pathway in a dose-dependent manner. Given that these biomarkers have complicated pathways that may act in opposing manners based on the physiological circumstance, further studies are warranted to fully elucidate their mechanisms of action and downstream effects. 20 The similarities in the overall concentrations of angiogenic biomarkers between cases and controls may suggest that other mechanisms of poor fetal growth that are prevalent in low-and middle-income countries may contribute to birthweight below the tenth centile in our cohort. SGA includes both constitutionally small but healthy neonates and those whose growth was restricted in utero due to placental dysfunction, maternal conditions, or fetal genetic abnormalities. case-control analysis, may have limited the proportion of cases resulting from angiogenic dysregulation compared with constitutional smallness and pathological SGA driven by non-placental factors.
Some SGA biomarker studies that reported a significant betweengroup difference employed birth weights below the 5th 18,32 or 2.5th centile, 33 which likely concentrated any discernible effect.
Additionally, although our sample size for this exploratory analysis was within the range of many studies of biomarker prediction of SGA, 25 our wide confidence intervals indicate that future studies with larger sample sizes are needed.
Our results suggest that single angiogenic biomarker concentrations before 24 weeks of gestation may be of limited utility, but risk stratification using interactions of multiple biomarkers may perform better. Ethnogeographic variations in the relationship between angiogenic biomarkers and fetal growth restriction merit further study.
Discovery of assays that can accurately identify those fetuses at highest risk for SGA early in gestation could improve management and reduce adverse neonatal outcomes.

ACK N OWLED G M ENTS
The study team acknowledges the contribution of our study participants and their families, as well as the staff of UNC Global Projects Zambia. We also thank Dr. Edmund Njeru, Professor Robin Bailey, Dr. Bethany Atkins, Shadi Farazahdi, Dr. Ioannis Baltas, and Jennifer Winston for their assistance with the study design and analysis plan.

CO N FLI C T S O F I NTE R E S T
The authors have no conflicts of interest.