Although large amount of research has been focused on preeclampsia prediction during pregnancy, very few serum prediction markers have been successfully implemented in clinical practice. With the low prevalence of preeclampsia in the general pregnant population, the application of specific laboratory test(s) would be costly to apply universally during pregnancy. In the publication for evaluating the preeclampsia predictive value of sFlt-1/PlGF by Zeisler et al., the authors narrowed down the targeting patients who presented with preeclampsia-related clinical and/or laboratory presentations (7). A similar patient recruiting strategy was adopted in our study. With a narrow focus on the subgroup of patients more likely to develop preeclampsia, medical resources may be better directed at high-risk patients; however, unlike universal screening, stratifying pregnant women based on their clinical symptoms and/or usual laboratory findings certainly requires extra effort. Whether an economic benefit exists in the overall management of preeclampsia remains a question.
According to a meta-analysis on the sFlt-1/PlGF ratio, which was recently considered one of the most promising serum markers in preeclampsia prediction, the authors found an overall sensitivity of 80%, a specificity of 92%, a positive likelihood ratio of 10.5 and a negative likelihood ratio of 0.22 after pooling 15 studies involving 534 cases and 19587 controls (20). A 4-week observation window along with the cut-off value of 38 was applied in the paper by Zeisler et al., which showed that the sFlt-1/PlGF ratio could accurately exclude preeclampsia occurrence in suspicious patients, with an AUC of 0.90 in the ROC analysis compared to an AUC of 0.67 in our study with follow-up until delivery (7); however, for the remaining markers included in present study, the observation window was yet to be well-defined; delivery remained the mainstream endpoint in most of the preeclampsia prediction studies (5, 21). The average interval between blood sampling and preeclampsia diagnosis was 7 weeks with our prospective cohort, which provided important clinical evidence for future validation studies. Interestingly, with the previously reported cut-off value of 38 for the sFlt-1/PlGF ratio and 4-week observation window, only 15 recruited subjects developed preeclampsia in our study, which was 30.5% (15/49) of the total preeclampsia-positive patients (Supplementary Table 5). Moreover, the NPV (94.4%) was close to that previously reported (7); the sensitivity (40.0%), specificity (83.4%) and PPV (16.7%) were much lower with our cohort (Supplementary Table 5), suggesting that ethnicity may be a confounding factor for the application of the sFlt-1/PlGF ratio and the cut-off value needs to be further optimized for Chinese populations before clinical implementation.
The hemostatic factors such as TM and tPAI-C were found to be related with the incidence and severity of PE decades ago (22). In preeclampsia patients, significant endothelial disturbance and procoagulant potential, along with aberrant expression of these hemostatic factors, were reported in previous studies (14,15); however, whether they could be useful in preeclampsia prediction has yet to be investigated. With our cohort, no difference was observed between the preeclampsia positive and negative groups, indicating their limited values in preeclampsia prediction (Table 2).
The excessive activation and poor regulation of the complement system at the maternal-fetal interface contributes to the development of preeclampsia (23). More importantly, a recent study by Jia et al. showed that the complement factors C1q, B and H were able to diagnose early-onset severe preeclampsia with AUCs of 0.81, 0.74 and 0.68, respectively. To further evaluate their potential utility in preeclampsia prediction, the circulating levels were measured in the present study. No significant difference was found between the preeclampsia-positive and preeclampsia-negative groups (Table 2).
The two glycoproteins that were included in our testing panel, GlyFn and PAPP-A2, have been widely studied in preeclampsia. As an abundant protein with a wide spectrum of functions, serum GlyFn was found to be highly elevated during both the early and late pregnancies of the preeclampsia patients (13, 24). More interestingly, in a 2020 study by Huhn et al. (8), the GlyFn level in a prospective cohort identified with preeclampsia-specific high-risk factors was reported to show satisfactory preeclampsia prediction with an AUC of 0.94 in the ROC analysis. In our study, GlyFn was also increased, although not significantly, in the patient group that developed preeclampsia. This apparent discrepancy may be introduced by differences in the sample size and patient recruiting criteria of the two studies. In addition, a point-of-care testing system that employed fluorescently labeled fibronectin polyclonal antibodies in testing strips was used in Huhn’s study, compared with the traditional ELISA assay applied with our serum samples. This distinction may further lead to the differential performance of the GlyFn level in preeclampsia prediction. The glycoprotein PAPP-A2, involved in cleaving insulin-like growth factor binding protein in the placenta, was found to be helpful in diagnosing (12) and predicting preeclampsia (8). In our study, the PAPP-A2/PlGF ratio (p=0.003) was found to be a better marker than PAPP-A2 (p=0.032) alone (Table 2), with an adjusted AUC of 0.72 (Table 3). Interestingly, PAPP-A protein, which has similar biological functions as PAPP-A2 and was a more extensively studied marker for aneuploidies and preeclampsia prediction, was found to be decreased in most of the previous preeclampsia studies (25).
As one of the essential criteria for the diagnosis of preeclampsia (6), proteinuria itself was not a sufficient predictor for the occurrence or the adverse outcomes of preeclampsia (26). Conversely, the common renal function tests such as BUN, Cre, UA and Cysc were shown to be potentially valuable in preeclampsia diagnosis and prediction. For example, the BUN (27) and BUN/Cre ratio (28) were both found increased in the preeclampsia patients compared with normal controls. Cysc, the alternative test of Cre used in glomerular filtration rate estimation, was found to be elevated in preeclampsia patients (29) and was able to predict preeclampsia in combination with neutrophil gelatinase-associated lipocalin (AUC=0.88) (30). Moreover, Cysc was reported as a predictor of preterm labor in severe preeclampsia patients, although the physiological increase of Cysc during pregnancy may pose an additional confounding factor in its clinical evaluation (31). In a prospective study with a relatively large cohort (n=9522) by Rezk et al., the serum UA level during the second trimester was found to be a useful preeclampsia predictor for women at moderate or low risk (32). More interestingly, an elevated UA level was later reported to be a risk factor for women with gestational hypertension to develop preeclampsia and deliver small-for-gestational-age infants (33). We observed similar findings in which all the renal markers included (BUN, Cre, UA and Cysc) were significantly increased in the patients who developed preeclampsia before delivery. Of them, UA was the most promising predictor with the greatest AUCs (0.73 and 0.77, before and after adjustment, respectively) in the ROC analyses (Figure 2 and Table 3).
There are a few limitations in this study. First, in the prospective studies of preeclampsia after 20 GWs with patients suspected for preeclampsia development, there were no widely accepted or universal inclusion/exclusion criteria. For instance, the BP cut-off values used by Huhn for enrollment were 140/90 mmHg (systolic/diastolic). However, the BP elevation standards were not clearly specified in Zeisler’s study (7); our clinician team decided to take a more cautionary move and had used 120/80 mmHg as the cut-offs. Moreover, the patients on anti-hypertensive treatment were excluded in both Zeisler’s (7) and our studies, but were allowed in Huhn’s cohort (8). The use of anti-hypertensive medicine could have interfered the pathological development of preeclampsia and potentially compromise the predictive efficacy of the serum markers. Second, a longer period of time from blood sampling to preeclampsia diagnosis (average 7 weeks in present study) was used compared to other studies with prediction of imminent preeclampsia that took place in 4 weeks or less (7, 8) after sampling. Although using delivery as the endpoint in our study seemed reasonable since the appropriate observation window was not well-defined, the elongated study period may decrease the sensitivities or detection rates of the serum markers. More specifically, in this scenario, we would anticipate an increase of false negative samples that would have been determined as true negatives if shorter observation window (such as 4 weeks) was used. With these two limitations combined, the sensitivities for sFlt-1/PlGF ratio and other selected markers (BUN, Cre, UA, Cysc, GlyFn/PlGF and PAPP-A2/PlGF) were only between 25-36% with a specificity of 90% (Supplementary Table 4), compared with the overall sensitivities of >80% in the studies with similar prospective cohort (7, 8, 20).
In conclusion, in a prospective cohort suspected of preeclampsia development, the angiogenic modulators sFlt-1 and PlGF; the renal function markers BUN, Cre, UA, and Cysc; and the glycoprotein PAPP-A2 were significantly altered between the two groups. However, with not-well-defined patient recruitment/excluding criteria and observation window, the clinical utilities of the serum biomarkers in preeclampsia prediction are limited based on the data summarized in present study. Future studies with independent cohorts of larger sizes are warranted to further reveal the values of these serum markers in preeclampsia prediction.