Prenatal sFlt-1/PlGF value for prediction of adverse neonatal outcome of isolated ultrasonic small-for-gestational-age fetus in normotensive pregnant women: A prospective cohort study

Not only preeclampsia but also small-for-gestational-age (SGA) neonates in the absence of preeclampsia are at increased risk of morbidity and mortality. Early recognition of fetuses at increased risk of being growth-restricted enables more appropriate surveillance and optimization of management for reduced risk of adverse neonatal outcomes. We investigated potential value of soluble fms-like tyrosine kinase-1 (sFlt-1) to placental growth factor (PlGF) ratio, estimated in late-second and early-third trimester respectively, for prediction of SGA neonates with poor neonatal outcome.

no adverse outcome. A cutoff value of 28.15 for the sFlt-1/PlGF ratio at 29 to 36+6weeks significantly predicted SGA neonates who had adverse outcome, with sensitivity and specificity of 76.9% and 88%, respectively.

Conclusion
In this study, sFlt-1/PlGF ratio of SGA with adverse neonatal outcome group was significantly higher than control group. This study suggests the feasibility of the sFlt-1/PlGF ratio as helpful objective measurement for predicting the adverse SGA neonatal outcome by providing sFlt-1/PlGF cutoff value, besides ultrasound biometry measurement.

Background
Generally, small-for-gestational-age (SGA) fetuses are defined as those having gestational weights below the tenth percentile. During the prenatal period, ultrasound SGA fetuses are detected at a rate of around 5~8% of all late-phase pregnancies (1). When SGA fetuses with combined clinically suspected pre-eclampsia (PE) status are detected on prenatal ultrasonography, they are carefully evaluated for Intrauterine Growth Restriction (IUGR) status, which can lead to fetal death due to pathologic intrauterine fetal asphyxia. With no clinical PE clues or any other abnormal findings of the Doppler index, which is the tool of diagnosis of fetal asphyxia, incidentally detected isolated ultrasonic SGA fetuses have been given normal antenatal care (2)(3)(4). Hence these fetuses have long been considered to be constitutionally small babies with a good perinatal outcome. However, as clinical study results have accumulated over the recent decades, it has become clear that SGA fetuses have poorer perinatal outcomes, such as suboptimal neurodevelopment and higher postnatal cardiovascular risk, than do appropriate for gestational age (AGA) newborns (2)(3)(4)(5)(6)(7)(8)(9). One of those studies reported that impaired fetal growth is a serious complication in pregnancy, being a major determinant of perinatal morbidity and mortality (9).
Although the exact pathophysiologies of poor fetal growth remain unknown, hypoperfusion 4 from incomplete trophoblastic invasion is known to be the most crucial causative factor for SGA. Impaired trophoblast invasion results in decreased feto-placental perfusion and placental dysfunction, which, finally, leads to poor pregnancy outcomes. The placenta of SGA infants shows reduced intervillous space volume and parenchyme, with few chorionic villies. A well-known diagnostic tool of pathologic fetal growth restriction is the UA Doppler index. Increased UA systolic-diastolic ratio is considered to be indicative of poorly vascularized placental villi status, though such status is seen only in extremely progressed cases. With earlier detection of pathologic fetal growth restriction, other Doppler ultrasound indices, such as reduced values of middle cerebral artery (MCA) Doppler or cerebroplacental ratio (CPR), have been clinically applied. However, ultrasound surveillance requires trained personnel and equipment, which may not be readily available for all diagnosed SGA pregnancies(1).
Consequently, a clinical need has emerged to develop additional biochemical markers to improve current diagnostic accuracy and reduce the need for ultrasound-based examinations for detection of SGA fetuses. The most widely studied angiogenic markers are placental growth factor (PlGF) and soluble fms-like tyrosine kinase-1 (sFlt-1).
Uteroplacental insufficiency is associated with unbalanced angiogenesis, which results in up-regulation of sFlt-1 and down-regulation of PlGF. So, the sFlt-1/PlGF ratio is significantly higher in cases of uteroplacental insufficiency such as PE originating from placental insufficiency, and has been commercialized and employed in cases of suspected PE to predict poor maternal and perinatal outcomes and help clinicians to determine the most appropriate delivery times.
However, clinicians have to be concerned not only about fetal growth restriction combined with PE but also isolated ultrasonic SGA with no other abnormalities and the optimal management of such cases. Despite the fact that isolated SGA fetuses are also known to 5 be associated with placenta insufficiency, there has been a tendency to focus on mostly SGA with PE and to study only the relevant biomarkers. There has been little research on biomarkers for poor perinatal outcomes in cases of isolated SGA in the absence of PE. As not only PE neonates but also SGA neonates in the absence of PE are at increased risk of morbidity and mortality after birth, a new approach and a different perspective are needed for evaluation of isolated SGA. Early recognition of fetuses at increased risk of being only growth-restricted might enable more appropriate surveillance and (thereby) optimized management, which have been shown to reduce the risk of adverse fetal outcome.
In the present study, we investigated the potential value of maternal serum levels of the sFlt-1/PlGF ratio for prediction of adverse neonatal outcome for isolated SGA fetuses in the absence of PE, and also investigated whether other factors can predict poor perinatal outcomes. Additionally, we endeavored to determine the clinically significant cut-off value of sFlt-1/PlGF for prediction of poor neonatal outcome in cases of isolated ultrasonic SGA fetuses.

Methods
Ours was a prospective cohort study that included 530 singleton pregnant women who had weeks and 20 weeks, 6 days, 4 markers (alpha-fetoprotein (AFP), unconjugated estriol (uE3), inhibin-A, and human chorionic gonadotropin (hCG)) were measured as a Down syndrome screening procedure. All of the markers were measured using a UniCel DxI 800 analyzer (Beckman Coulter Inc., Fullerton, CA, USA), and the values were transformed to multiples of the median (MoM) after adjusting for gestational age and maternal BMI.
We also measured, as additional angiogenic biomarkers, the maternal plasma levels of the sFlt-1 and PlGF values at both early-phase gestation and late-phase gestation. After clotting, the samples were centrifuged, and plasma was stored at −80°C. The sFlt-1 and PlGF levels of each of the samples were measured simultaneously using the fully automated Roche Diagnostics Elecsys assay (Roche Diagnostics, Penzberg, Germany), and the sFlt-1/PlGF ratio was calculated.
At each trimester, an ultrasound scan also was performed. Fetal biparietal diameter, femur length, and abdominal and head circumference were measured using the ATL-5000 In our study, the control group was defined as AGA with no concerns regarding PE before or after delivery. The diagnosis of PE was made according to the National High Blood 7 Pressure Education Program Working Group on High Blood Pressure in Pregnancy criteria.
Hypertension was defined as repeated systolic blood pressure measurements of ≥ 140 mmHg (Korotkoff phase 1) and diastolic blood pressure measurements of ≥ 90 mmHg (Korotkoff phase 5). Proteinuria was defined as repeated ≥ 1+ proteinuria on dipstick urinalysis or ≥ 300 mg of protein in a 24-hour urine collection sample. Our SGA group was defined as infant birth weight less than the tenth percentile of the corresponding curves after adjustment for gestational age.
Adverse neonatal outcomes regarded as requiring NICU admission were attributed to subcauses including jaundice, meconium aspiration syndrome, transient tachypnea of newborn, respiratory distress syndrome, necrostizing enterocolitis, sepsis, and use of ventilation.
Information on the presence of obstetric complications, pregnancy outcome and fetal outcome, including fetal sex, gestational age at delivery and birth weight, were obtained after delivery.
All of the statistical analyses were performed using SPSS for Windows version 21.0 (SPSS Inc., Chicago, IL, USA) software. All of the values were recorded in means (± SD), and the analysis method entailed use of the Mann-Whitney U test. Due to the fact that the sFlt-1/PlGF repeated-measure analysis of the second and third trimesters in each group did not satisfy the requirements for sphericity of data analysis, a mixed version of the general liner model, an analysis technique similar to repeated-measure ANOVA, was employed.
Values of p < 0.05 were considered statistically significant. Receiver operating characteristic (ROC) curves were constructed using logistic regression analysis. The resulting areas under the ROC curves (AUC) were compared by pairwise analysis.

Results
Among the total 530 study recruits, 22 (4.2%) pregnant women were diagnosed as PE.
After excluding these 22 PE singletons, 508 sets of pregnancy data were analyzed. Among these, 47 SGA singletons and 461 control-group subjects were enrolled and compared to identify the relationship between each of the markers and the customized birth weight.
The basic characteristics of the 2 subjects are summarized in Table 1 Among the serum levels measured at 24-28+6 weeks (early-phase gestation), the sFlt-1 level and sFlt-1/PlGF ratio (ratio I) were higher and the PlGF level was lower in the SGA group, but the differences were not statistically significant. The sFlt-1/PlGF ratio as determined at 29-36+6 weeks (late-phase gestation; ratio II), however, was significantly higher in the SGA group (14.42 vs. 28.62, p = 0.037). As is known, it has been statistically confirmed that neonatal birth weight and sFlt-1/PlGF ratio I, II were statistically were inversely proportional (data not shown). There were also significant differences in the uterine-artery Doppler ultrasonography measurements between the 2 groups. All of the 3 Doppler impedance indices were higher in the SGA group than in the control group (RI:  The neonatal outcome data is also presented in Table 2. Fifty-six (56) neonates were admitted to the NICU, and the admission rate was significantly higher in the SGA group To determine the clinically applicable cut-off value for prediction of SGA neonates, especially those who showed adverse neonatal outcome, Table 3  What should be the next step for clinicians in cases of incidentally detected ultrasonic SGA? Differential diagnosis between fetuses that are small due to 'placental intrauterine growth restriction' and those that are 'constitutionally small fetuses' has been considered a major area of interest in clinical obstetrics (10)(11)(12)(13)(14). The important preliminary step would be to determine PE coexistence. Not only maternal blood pressure and proteinurea, but Gestation-age-specific sFlt/PlGF ratio cut-offs of >85 (20+0 to 33+6 weeks) and >110 (34+0 weeks to delivery) have been shown to be highly suggestive of PE. In the PROGNOSIS study, a single sFlt-1/PlGF ratio cut-off (<38) was validated as accurately and reliably ruling out PE within 1 week (negative predictive value > 96%) and confirming PE (> = 38) within 4 weeks (positive predictive value >25%). As such, it can be considered to be of great value to both clinicians and patients. The rate of incidence of PE in Asian women, however, is relatively lower than in other ethnic groups (e.g., African-Americans), which fact has resulted in a paucity of PE studies on Asian populations. The present study's population, uniquely, was Asian (i.e., Korean) women. Especially for such a country as Korea, in which the incidence of PE is low, our present results suggest an optimal and novel approach that utilizes the sFlt-1/PlGF ratio as a highly effective prognostic indicator of adverse outcome in cases of isolated SGA status.
It must be noted, though, that at the third trimester, unlike the first and second, SGA and placental insufficiency might not be noticeable, because SGA determination is made only by ultrasound biometry routinely. Thus, besides ultrasound biometry measurement for prediction of SGA, objective measurements are needed.
This study is the first (as ascertained by a PubMed search of English language papers from 1996 to December 2018 using the search terms 'PlGF' and 'sFlt-1') to examine, among a cohort of SGA fetuses, the utility of angiogenic factors for prediction of adverse outcome in cases where PE is absent. Over the last few years, several reports have provided evidence that angiogenic markers also differ with respect to late-pregnancy complications.
Accordingly, PlGF levels are lower in pregnancies with SGA. However, no studies have evaluated angiogenic factors to determine whether they predict poor outcome among SGA fetuses or how they compare with Doppler ultrasound surveillance. In several preceding studies, maternal serum angiogenic factors in SGA-pregnancy women with PE have been scrutinized. One study reported that among patients suspected of having SGA fetuses, abnormal maternal plasma concentrations of angiogenic/anti-angiogenic factors were 5-9 and 8-9 times more likely to lead to PE or preterm delivery (21). Ignacio et al. reported the following median values of sFlt-1/PlGF: control group 11.0, fetal growth restriction group 116.8, PE 66.5, and PE combined with fetal growth restriction group 165.4 (22). However, they could not determine the cut-off value. In our study, we demonstrated that the early-sFlt-1/PlGF ratio was not remarkable in predicting obstetric outcome but that increased sFlt-1/PlGF ratio checked at 29 to 36+6 weeks (late-phase gestation) was strongly predictive of adverse neonatal outcome with a cut-off value of 28.15 (76.9% sensitivity and 88% specificity, AUC area: 0.907). Moreover, increased sFlt-1/PlGF ratio checked at the same time with a cut-off value of 11.25 was shown to be a clinically predictive marker of real SGA neonates in cases of ultrasonic SGA (60.0% sensitivity and 61.9% specificity; AUC area: 0.663; 95% CI, 0.564-0.762) beyond the clinical and ultrasonic values.
The PAPP-A, which is produced by developing trophoblast cells, in the maternal serum is known to decrease in PE populations (23)(24)(25). In a multicenter study of 8839 women, a significant relationship between low PAPP-A levels and IUGR, preterm delivery, PE and stillbirth were reported (26). Low levels of PlGF and PAPP-A in the maternal serum may reflect impaired placentation, which results in the development of fetal growth restriction (27). In our study, HCG and E3 levels in SGA group were lower than control group. There is no established association with the HCG and E3 levels in SGA group, but it is thought to be a good indicator for predicting poor fetal growth if more results are built up in the future.
This study has important clinical implications. Effective screening for detection of adverse perinatal outcome in cases of isolated SGA is an area of unmet clinical need. In the study, we serially evaluated not only UtA Doppler but also sFlt-1/PlGF ratio data for cases of 13 suspected SGA, determining that sFlt-/PlGF can be an independent marker of poor neonatal outcome. Such biomarkers can be identified from a simple blood test; thus, this approach can be especially useful in areas where access to Doppler examination expertise is limited. If these results are corroborated by others, the strong potential of angiogenic biomarkers for risk stratification in cases of ultrasonic isolated SGA and subsequent reduction of both morbidity and healthcare costs in the management of SGA fetuses will be confirmed.

Conclusion
At the third trimester, unlike the first and second, SGA and placental insufficiency might not be noticeable, because SGA determination is made only by ultrasound biometry. Thus, besides ultrasound biometry measurement for prediction of SGA, this study explored the feasibility of the early-third-trimester sFlt/PlGF ratio as an objective measurement and a possibly more useful predictor of SGA and adverse neonatal outcome than maternal