The incidence of preeclampsia (PE) is about 2–8%, making it one of the leading causes of perinatal morbidity, and responsible for 10–15% of the maternal deaths in the world (1, 2). Women with PE present a higher risk of cardiovascular morbidity, multisystemic complications, and a long-term increased risk for cardiovascular diseases (3) and Mortality (4). Early-onset PE (diagnosed before 32 weeks of gestation) is also frequently associated with intrauterine growth restriction (IUGR), and therefore affecting perinatal outcomes and long-term offspring development (5, 6). Late-onset PE, perhaps clinically less severe but much more common, is also an important cause of maternal and neonatal morbidity and mortality with long-term consequences (7).

Although a complete understanding of the pathogenesis of PE remains unclear, the current theory suggests a two-stage process. The first stage is caused by shallow invasion of the trophoblast, resulting in inadequate remodeling of the spiral arteries. This is presumed to lead to the second stage, which involves the maternal response to endothelial dysfunction and imbalance between angiogenic and antiangiogenic factors (8). In PE and IUGR, this vascular remodeling does not exist or it is incomplete, leading to placental hypoperfusion associated with endothelial cell dysfunction (9). For a correct placental development, a balance between the production of angiogenic (Placental Growth Factor, PlGF) and antiangiogenic factors (soluble Fms-like tyrosine kinase-1, sFlt-1) is required. PIGF and sFlt-1 are partially produced by the syncytiotrophoblast. Angiogenic biomarkers (PlGF and sFlt-1) have shown their ability to predict PE and its complications (10).

We have effective preventive strategies to reduce the incidence of PE. The nightly low dose (150 mg) administration of acetyl salicylic acid (ASA) before 16 weeks of gestational age until term is associated with a 90% reduction in the incidence of preterm-PE (11, 12) and IUGR (13, 14), as well as with a 70% reduction of the neonatal mean stay in intensive care units (ICUs) (15). Therefore, it is essential to develop universal PE screening strategies during the first trimester of pregnancy to determine the risk of each patient, selecting those high-risk candidates to start as soon as possible for the prophylactic treatment with low-dose ASA (16, 17).

Universal PE screening has been proven to be cost-effective (18). PE screening models based on maternal demographic characteristics and risk factors have been proposed (19), with a very low detection rate and an excess in false-positive rate (20). Hence, different multivariate models have been developed. A competitive risk approach at 11–13 weeks by means of a combination of maternal characteristics (age, parity, medical history as thrombophilia, nephrological diseases, and chronic hypertension), maternal biophysical variables [mean arterial pressure (MAP) and Doppler of Uterine Arteries (Uterine Artery Pulsatility Index, UTPI)], and biochemical variables (PlGF) has shown a prediction rate of 90% for early-PE and 75% for late-onset PE, with a 10% false-positive rate (21, 22). Additionally, observational retrospective studies have reported that including antiangiogenic biomarkers such as sFlt-1 could improve the first-trimester screening performance (23).

Unfortunately, universal implementation of any first-trimester screening system, such as angiogenic and antiangiogenic markers (PlGF and/or sFlt-1), entails a high financial cost, unaffordable for many healthcare systems. For that reason, more economical screening alternatives have been explored. In this line, taking advantage of the resources used on a regular basis for the screening of chromosomal abnormalities, an alternative multivariate model based on maternal characteristics, MAP, and Uterine Arteries Pulsatility Index combined with Pregnancy-Associated Plasma Protein A levels (PAPP-A), has shown a detection rate of early-onset PE of 80.8% and for late-onset PE of 39.6%, with a 10% false-positive rate (24).

Recent retrospective studies with large sample sizes (25, 26) suggest that a two-step screening protocol, performing only angiogenic markers in 30% of the population classified as moderate or high risk of PE after an economical first step (24), would correctly select high-risk patients for developing PE, but lower-economic cost. However, these results come from retrospective cohorts after universal screening models with PlGF, and therefore with potential intervention biases.

The aim of this study is to prospectively determine, in a real clinical setting, the predictive and preventive capacity of a universal PE first trimester two-step sequential screening model, determining PlGF only in those patients previously classified as intermediate risk by means of a multivariate model based on medical history, MAP, PAPP-A, and UTPI. We hypothesize that this screening model will achieve similar diagnostic performance as the universal determination of PlGF but at a lower economic cost.

As a secondary objective, in every intermediate and high-risk patient, sFlT-1 values will be determined to assess their potential performance in the screening for PE. Even though this data will be blinded to the researchers and will not be considered for the clinical management. We also evaluate the predictive and preventive capacity of this first-trimester sequential screening model for the development of IUGR.

The most important aim of this project is to develop an efficient first-trimester screening of PE using resources already used during pregnancy control. All the clinical data, sonography, and blood test results will be collected during the regular pregnancy control without the need for additional appointments, explorations, or extraordinary blood extractions.

Maternal characteristics will be prospectively recorded at the time of recruitment. Pregnancy outcomes will be registered during pregnancy and confirmed after delivery. Fetal crown-rump length (CRL) (27), and transabdominally uterine artery Doppler will be determined in all patients at the routine first-trimester ultrasound (11⁰-13⁶ weeks) by experienced fetal medicine specialists. Blood pressure (BP) will be measured once, after 5-min of rest with women seated at the time of inclusion. The MAP will be calculated as: diastolic BP + (systolic–diastolic BP)/3.

First-trimester routine blood samples will be performed between 10⁰ and 13⁶ weeks. PAPP-A (taken routinely for trisomy screening) will be initially determined in every patient. Serum remaining samples will be stored at −80°C, to be able to analyze PlGF and sFlT-1 after the first-trimester ultrasound in those patients with an estimated risk of PE risk ≥ 1/500 after the first step of screening. The serum will be separated by centrifugation at 1,500 g for 10 minutes at 4°C, and concentrations of PAPP-A, PlGF, and sFlT-1 will be determined by electrochemiluminescence immunoassays, fully automated on the Cobas e 601 analyzer, Roche Diagnostics. Multiples of the median (MoM) values for PAPP-A, PlGF, and sFlT-1, calculated from locally derived normal medians using the above-mentioned multivariate Gaussian distribution model, will be considered for analysis. To calculate the risk of PE, we will use the SsdwLab6 version 6.1 package (SBP Soft 2007 S.L.), previously developed in the pilot study.

The main outcome is the diagnosis of PE during pregnancy, according to the definition of the International Society for the Study of Hypertension in Pregnancy (ISSHP). Thus, the diagnosis will be based on systolic BP ≥ 140 mmHg or diastolic BP ≥ 90 mmHg on repeated occasions after 20 weeks' gestation, and proteinuria [dipstick urinalysis ≥ 1+ or protein/creatinine ratio ≥ 30 mg/mmol (0.3 mg/mg)] or another maternal organ dysfunction. PE will be classified according to gestational age at delivery into early-onset (<34 weeks), preterm (<37 weeks), and term (>37 weeks). Table 3 describes the secondary outcomes that will be assessed during the project.

Since we have an effective preventive treatment with early administration of low dose ASA, universal screening for PE is cost effective (18), and a clinical and moral obligation. The objective of this project is to validate a first-trimester screening of PE protocol that guarantees access to all pregnant women, without sacrificing quality. For this reason, in the first step of the screening, we will calculate the risk of each patient according to variables already included in the standard pregnancy control. As there is no economic cost involved, we ensure universal accessibility for all pregnant women to screening for PE. However, the estimation of angiogenic markers is the strategy with the best performance when it comes to screening the risk of PE (21, 22). By adding PlGF determination (and blinded sFlT-1) to only a reduced proportion of patients, we believe that we can offer an early PE screening with maximum quality, while marginally increasing the economic cost. This design has been previously proposed after retrospective analysis of randomized clinical trials based on the universal PE screening with PlGF (25, 26). Our objective is to validate these results in a multicenter, real-world clinical setting study. This sample size assures enough statistical power for the main outcomes. We consider that the design and methodology of the project guarantees the external validity of the results.

Some limitations of our study need to be mentioned. First, our design is not a randomized control trial, therefore it does not include a control group without intervention. Several randomized clinical trials have already shown the efficacy of the first-trimester PE screening for the early implementation of low-dose ASA prophylaxis. Our objective is to validate a two-step screening system previously proposed by retrospectively analyzing high methodological quality clinical trials in a real clinical scenario. We have not considered the creation of a non-intervention group in the design, as our intention is to compare our results with all the screening strategies published to date. However, we have quality information on historical patient cohorts prior to the implementation of PE screening in all centers. As a contingency plan, if we consider it necessary at the end of the study, we could also compare the results with the group of patients in whom we have not been able to screen for PE in the first trimester for other reasons. We assume that the study design allows us less control over the results than a clinical trial, as it is subjected to the potential biases of routine clinical practice. However, we consider that this is, on the other hand, one of the differentiating points of this project, since it offers us results with high external validity and transferability to clinical reality. Second, for clinical purposes to be able to publish our results as soon as possible, follow-up of the offspring is limited to the neonatal period. However, this is not an important limitation, since it is not the objective of the project, and since all the study patients are registered, a long-term cohort study with these neonates could be considered.

The studies involving human participants were reviewed and approved by Aragon Research Ethics Committee (CEICA) on 3 July 2020 (15/2020). The patients/participants provided their written informed consent to participate in this study.

EL, CT, MF, CL, and DO: conception and design of the research. DO: principal investigator. CT: co-investigator. SD, LR, AR-R, JM, TB, MF, AM, and SR-M: collaborators. CT, CL, and DO: wrote the first version of the manuscript, drafting, and revision of the manuscript. All authors revised it and contributed significantly in writing the final version that was accepted.

This study was supported by the government of Spain, Plan Estatal de I+D+I and Instituto de Salud Carlos III- Subdirección General de Evaluación y Fomento de la Investigación Sanitaria, project PI19/00692, and the European Regional Development Fund (FEDER).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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