Preeclampsia (PE) is a severe pregnancy complication characterized by the development of high blood pressure and proteinuria after 20 weeks of gestation. Characterized by persistent high systolic/diastolic blood pressure (≥140/90 mm Hg) and proteinuria (≥300 mg/24 h) after 20 weeks of gestation in women with previously normal blood pressure [1]. Currently, there are many etiological theories regarding PE, including theories of nutritional deficiency, endothelial injury, and excessive inflammatory immune response. However, the underlying molecular mechanisms are still unclear. It is generally believed that the main pathogenic factor is insufficient maternal vascular remodeling, which leads to placental dysfunction [2]. It often leads to maternal organ dysfunction and fetal complications. Globally, PE affects 2% to 5% of pregnant women, making it a leading cause of maternal mortality [3]. Recognized risk factors for PE include advanced maternal age, obesity, primiparity, multiple pregnancies, and a family or personal medical history of liver and kidney diseases, among others [4]. The pathogenesis of PE and its early prediction remain prominent topics in obstetrics [5], [6]. The clinical diagnosis of PE primarily relies on monitoring blood pressure and testing for proteinuria. However, these methods have limited predictive value for early detection of PE in pregnancy. For example, some women with early-onset PE may not show signs of high blood pressure or proteinuria until later stages, complicating effective early prediction and diagnosis [7]. Thus, early and accurate prediction and diagnosis of PE are crucial for improving maternal and infant health Fig. 1.

Due to the multifactorial nature of PE, employing a combination of multiple indicators is crucial to enhance predictive accuracy. Identifying early diagnostic biomarkers significant for PE can lay the groundwork for early prediction in clinical practice, enabling timely interventions through preventive medications and other strategies. Studies have shown that placental growth factor (PLGF), a critical placental secretion for angiogenesis, reflects placental function through its potent pro-angiogenic effects on placental blood vessels [4]. In normal pregnancies, serum PLGF levels exhibit a peak-shaped pattern, increasing with gestational age in early to mid-pregnancy, reaching a peak around 30 weeks, and then gradually decreasing [8]. The reduction in serum PLGF levels is significantly more pronounced in women with severe PE than those with mild PE and normal pregnancies. Consequently, assessing the ratio of soluble fms-like tyrosine kinase-1 (sFlt-1) to PLGF in serum offers valuable insights into the likelihood of PE [9]. However, detecting serum PLGF and sFlt-1 requires invasive tests, demanding high proficiency in instrument operation and skilled personnel.

Misfolded proteins detected in the urine, blood, and placental tissues of pregnant women with PE present another avenue for diagnosis [10], [11]. Several proteins, such as amyloid beta-peptide, alpha-1 antitrypsin, albumin, IgG k-free light chains, and ceruloplasmin, are altered in PE, accumulating amyloid-like aggregates in the placenta and body fluids. These findings present an opportunity to create new diagnostic methods, as amyloids possess distinct characteristics [12]. The levels of these proteins are notably higher in the urine of PE patients than in normal pregnancies [13], [14]. These misfolded proteins have the distinctive property of binding to Congo Red dye (mainly due to misfolded proteins rich in abnormal beta-folded structures), detectable through a simple, rapid, and non-invasive colorimetric technique known as the Congo Red Dot Paper (CRD) Test. This method, requiring minimal technical expertise, has been adopted in several countries, though the effectiveness of its quality control varies [15], [16], [17]. Therefore, evaluating the utility of CRD indicators in specific national contexts is essential. Moreover, integrating PLGF and CRD measurements with conventional markers like urinary protein, hypertension, and BMI may significantly improve the precision and sensitivity of PE prediction Fig. 2.

Building on the aforementioned research, we utilize a prospective cohort design to monitor levels of misfolded proteins in urine and placental growth factor (PLGF) in serum across various gestational stages. The findings will be correlated with clinical diagnoses derived from comprehensive examinations. The primary objective is to assess the predictive efficacy of combining urine CRD test with serum PLGF measurements for detecting PE in pregnant women.