Placental growth factor as a predictive marker of preeclampsia in twin pregnancy

Study design and participants

This was a single-center, prospective observational study with data collected retrospectively in twin pregnancies. Women aged 18 years and older who presented with suspected preeclampsia with a live twin pregnancy, booking for antenatal care between 24 weeks 0 days–36 weeks 6 days gestation at Hospital of Maternal and Health of Tangshan between July 2020 and February 2022 were invited to participate. Suspected preeclampsia was defined if one or more of the following signs or symptoms are met: headache with visual disturbances, epigastric or right upper-quadrant pain, dipstick proteinuria, new-onset or worsening of existing hypertension, a headache with visual disturbances, abnormal maternal blood tests that were suggestive of disease (such as hepatic or thrombocytopenia or renal dysfunction), and fetal growth restriction. This study was approved by the Ethics Committee of the Hospital of Maternal and Health of Tangshan, and all participants signed informed consent.

Definitions and outcome measures

The outcome was the diagnosis of preeclampsia , as defined by the international Society for the Study of Hypertension in Pregnancy 2018 recommendations [12].

Materials

Maternal blood sample was collected from each participant at recruitment. A volume of 5 mL of maternal blood was drawn using EDTA tubes for plasma samples. Samples were inverted at least five times after blood drawing and left to clot at room temperature for 30 min before centrifugation. Plasma was collected after centrifugation and stored at 2° C–8° C until analysis. All samples were analyzed within 24 h.

Plasma levels of PlGF were measured in batch using a commercial PlGF assay at clinical laboratory, on an automated immunoassay platform (iRaTe 1,600/300; Aucheer, China). Sample analysis was performed according to the manufacturer’s instruction.

The assay uses fluorescent labeled recombinant murine monoclonal antibodies and detects PlGF specifically and quantitatively, in the range of 12–3,000 pg/mL, in≈12 min. The assay coefficients of variation for PlGF was ＜15 %.

Data collection and follow up

Baseline characteristics and confirmation eligibility were obtained during participants interview. Gestational age was determined based on the last menstrual period. Maternal characteristics and medical, obstetric histories were recorded; maternal weight and height were measured. Mean arterial pressure (MAP) was measured by validated automated devices (BP3AQ1 Microlife, Taipei, Taiwan) according to the manufacture’s protocol. Pregnancy outcomes were obtained from the medical file of patient. Patients’ follow-up continued until hospital discharge.

Statistical analysis

The formulae for calculating multiple of the median (MoM) values of PlGF were determined by multivariate regression analysis of factors including gestational age and maternal characteristics(maternal age, maternal weight, parity, conception, diabetes mellitus) of singleton pregnancies using the clinical data from Hospital of Maternal and Health of Tangshan. Measured values of PlGF were converted into MoMs.

Statistical Package of SPSS version 19.0 was used for analysis. A receiver operating characteristic (ROC) curve analysis was performed to determine the optimal cutoff predictive value of PlGF and PlGF MoM for preeclampsia. Chi-square test, T-test, Kruskal Wallis test and ANOVA were used for data analysis All statistical tests were bilateral. A p-value of <0.05 was considered statistically significant.

Demographics and characteristics

Between July 2020 and February 2022, a total of 298 twin pregnancies were enrolled, of which 141 were suspected with preeclampsia and eligible for analysis. Baseline characteristics and laboratory findings based on the clinical diagnosis at time of study entry are shown in Table 1. A total of 36 patients developed preeclampsia were observed, and the incidence of preeclampsia was 25.53 %.

Placental growth factor in twin pregnancies with and without the development of preeclampsia

The distribution of PlGF and PlGF MoMs, between 24 and 36 weeks gestation, of twin pregnancies suspected with preeclampsia was shown in Figure 1. MoM calculations were not adjusted for chorionicity because there was no difference in PlGF levels based on chorionicity (Figure 2).

Figure 1:

The distribution of placental growth factor (PlGF) and PlGF multiple of the median (MoM) in twin pregnancies with suspected preeclampsia. (A) Shows the distribution of PlGF value and (B) shows the distribution of PlGF MoM between 24 weeks 0 days–36 weeks 6 days gestation in twin pregnancies clinically suspected of having preeclampsia. The blue dots means PlGF concentration in (A) and PlGF MoM in. (B) The red line means the median of PlGF concentration in (A) and the median of PlGF MoM in (B).

Figure 2:

Placental growth factor levels based on chorionicity. (A) Shows the PlGF value in monochorionic and dichorionic pregnancies (p=0.707). (B) Shows the PlGF MoM in monochorionic and dichorionic pregnancies (p=0.234). The bottom edge and the top edge of each box represents the minimum value and the 90 % percentile, respectively. The band within the box represents the median value.

Patients who developed preeclampsia had significantly lower PlGF levels (p＜0.001), both PlGF values and PlGF MoMs, compared with unaffected pregnancies (Figure 3A and B). The results showed that, between 24 and 36 weeks gestation, PlGF showed well separation between the two groups, indicating that PlGF could be used to predict the risk of preeclampsia in twin pregnant women suspected with preeclampsia.

Figure 3:

Placental growth factor (PlGF) value and PlGF multiple of the median (MoM) in women with preeclampsia and without preeclampsia. (A) Shows the PlGF level and (B) shows the PlGF MoM. The bottom edge and the top edge of each box represents the minimum value and the 90 % percentile, respectively. The band within the box represents the median value.

The performance of placental growth factor in twin pregnancies to predict preeclampsia

We further preformed the ROC analysis for PlGF and PlGF MoM. The PlGF and PlGF MoM showed good performance in diagnosis of preeclampsia. No significant difference in the effect of PlGF (AUC, 0.863 [0.796–0.931]; sensitivity, 86.1 % [74.8–97.4 %]; specificity, 80 % [72.3–87.7 %]) and PlGF MOM (AUC, 0.865 [0.794–0.936]; sensitivity, 83.3 % [74.1–97.3 %]; specificity, 81.0 % [73.4–88.5 %]) was observed in the prediction of preeclampsia (Table 2). The optimal cutoff value for preeclampsia predicting was 215 pg/mL for PlGF or 0.564 for PlGF MoM (Figure 4).

Figure 4:

Predictive performance of placental growth factor (PlGF) value and PlGF multiple of the median (MoM) in predicting preeclampsia receiver-operating characteristic curve(ROC) analysis of PlGF and PlGF MoM as a predictor of preeclampsia. Area under receiver operating curve (AUC) denotes area under the curve.

We also compared the prediction performance for preeclampsia using a cutoff value of 215 pg/mL with using PlGF percentiles at different gestation. As shown in Table 3, the cutoff of the 15th percentile had a good predictive performance (66.7 % sensitivity, 90.5 % specificity, 84.4 % accuracy) but not superior to the performance of the cutoff value of 215 pg/mL.

We further try to evaluate the predictive performance of PlGF and PlGF MoM for the short-term occurring of preeclampsia after recruitment. Results showed that PlGF cutoff of 215 pg/mL and 0.564 MoM had a similar high accuracy in determine preeclampsia. The sensitivity of PlGF for predicting preeclampsia within 1 week, 2 weeks, and 4 weeks was 100 % [51.7 %, 100.0 %], 100 % [62.9 %, 100.0 %], and 93.8 % [67.6 %, 99.7 %], respectively, and the negative predictive values were 100 % [94.8 %, 100 %], 100 % [95.0 %, 100 %], and 98.9 % [93.0 %, 99.9 %], respectively (Figure 5 and Table 4). The sensitivity of PlGF MoM for predicting preeclampsia within 1 week, 2 weeks, and 4 weeks was 100 % [51.7 %, 100.0 %], 100 % [62.9 %, 100.0 %], and 87.5 % [60.4 %, 97.8 %], respectively, and the negative predictive values were 100 % [95.0 %, 100 %], 100 % [95.0 %, 100 %], and 97.8 % [91.5 %, 99.6 %], respectively (Figure 5 and Table 4). No significantly difference between the performance of PlGF and PlGF MoM.

Figure 5:

Predictive performance of placental growth factor (PlGF) value and PlGF multiple of the median(MoM) in predicting the short-term occurring of preeclampsia. Receiver-operating characteristic curve (ROC) analysis of PlGF and PlGF MoM as a predictor of preeclampsia within 1 week (A), 2 weeks (B) and 4weeks (C). Area under receiver operating curve (AUC) denotes area under the curve.

Given the very wide range of gestational age at recruitment, we divided the enrolled women into two groups, women enrolled at less than 32 weeks of gestation and ≥32 weeks of gestation, and evaluated the predictive performance of PLGF cut-off value of 215 pg/mL for preeclampsia in the two groups, respectively. Due to there were no preeclampsia occured within 1 week of PlGF testing in women enrolled at less than 32 weeks of gestation, we only evaluated the PlGF performance for predicting preeclampsia within 2 weeks, and 4 weeks in this group. In women enrolled at less than 32 weeks of gestation, the sensitivity of PlGF for predicting preeclampsia in within 2 weeks, and 4 weeks was 100 % [5.5 %, 100 %] and 100 % [31 %, 100 %], respectively; the specificity was 70.8 % [60.5 %, 79.4 %] and 72.3 % [62.0 %, 80.8 %], respectively; the positive predictive value was 0 % [0 %, 14.6 %] and 10.3 % [2.7 %, 28.5 %], respectively; the negative predictive value was 100 % [85.4 %, 100 %] and 100 % [93.3 %, 100 %], respectively (Figure 6 and Table 5). In women enrolled at≥32 weeks of gestation, the sensitivity of PlGF for predicting preeclampsia in within 1week, 2 weeks, and 4 weeks was 100 % [51.7 %, 100.0 %], 100 % [59.8 %, 100 %] and 92.3 % [62.1 %, 99.6 %], respectively; the specificity was 55.3 % [38.5 %, 71.0 %], 58.3 % [40.9 %, 70.4 %] and 64.5 % [45.4 %, 80.2 %], respectively; the positive predictive value was 26.1 % [11.1 %, 48.7 %], 34.8 % [17.2 %, 57.2 %] and 52.2 % [17.2 %, 57.2 %], respectively; the negative predictive value was 100 % [80.8 %, 100 %], 100 % [80.8 %, 100 %] and 95.2 % [74.1 %, 99.8 %], respectively (Figure 7 and Table 6).

Figure 6:

Predictive performance of placental growth factor (PlGF) value in predicting the short-term occurring of preeclampsia between 24 weeks 0 days and 31 weeks 6 days. Receiver-operating characteristic curve (ROC) analysis of PlGF as a predictor of preeclampsia within 2 and 4 weeks between 24 weeks 0 days and 31 weeks 6days. Area under receiver operating curve (AUC) denotes area under the curve.

Figure 7:

Predictive performance of placental growth factor (PlGF) value in predicting the short-term occurring of preeclampsia between 32 weeks 0 days and 36 weeks 6 days gestation. Receiver-operating characteristic curve (ROC) analysis of PlGF as a predictor of preeclampsia within 1 week, 2 and 4weeks between 32 weeks 0 days and 36 weeks 6 days gestation. Area under receiver operating curve (AUC) denotes area under the curve.

Mean arterial pressure slightly increase the predictive performance for preeclampsia

Given that blood pressure was measured by all participants, and the MAP was considered as a critical marker for predicting preeclampsia [13], whether adding MAP would improve the performance remained unclear. The result showed that MAP slightly increase the predictive performance for preeclampsia by PlGF (AUC 0.891 vs. AUC 0.863, Figure 8). The optimal calculation model of PlGF plus MAP was presented as follows: YPlGF+MAP=exp(−7.91318 + 0.07924*MAP −0.00789*PlGF).

Figure 8:

Predictive performance of placental growth factor (PlGF) value and PlGF plus mean arterial pressure (MAP) in predicting preeclampsia green line indicate the performance of PlGF plus MAP (AUC=0.891), which is slightly better than the performance of PlGF only (red line) (AUC=0.863). MAP only performed worse in predicting preeclampsia (AUC=0.735).