Urinary Misfolded Proteins based detection for Imminent Prediction of Pre-eclampsia in pregnant women with suspected pre-eclampsia: A protocol of a prospective non-interventional study

Background: Pre-eclampsia (PE) is the most common hypertensive diseases, affecting 2% - 8% of all pregnancies. High maternal and fetal mortality of PE is due to a lack of early identification of affected pregnant women for more close care. Recent data suggested that the misfolded protein might be a promising biomarker for PE prediction. Objective: The main purpose of this trial is to evaluate the value of a urine Congophilia based detection of misfolded proteins for imminent prediction of PE in women presenting with suspected PE. Methods: In this prospective cohort, at least 300 pregnant women with clinical suspicion of PE will be enrolled. Consecutive urine samples will be collected, blinded and tested for misfolded proteins and other PE-related biomarkers at enrollment and 4 weeks thereafter. Clinical assessments of PE status and related complications for all subjects will be performed at regular intervals using strict diagnostic criteria. Investigators and participants will remain blinded to the results. Follow-up


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Introduction Background
Pre-eclampsia (PE) is the most common hypertensive diseases in pregnancy, characterized by the elevated blood pressure and proteinuria after 20 weeks of gestation [1].The incidence of PE is approximately 2% -8% [2,3] worldwide, and each year, an estimated 76,000 women and 500,000 fetuses die from PE and related complications [4].Symptoms of PE are not specific, including headache, visual impairment, upper abdominal pain, and shortness of breath, among others.In severe cases, it may develop into eclampsia, which involves seizures or coma [5][6][7].PE may also lead to placental abruption, intrauterine growth restriction, HELLP syndrome (a life-threatening liver and Tang et al blood clotting disorder), and other serious health consequences for both mothers and fetus [8,9].To date the treatment of PE involving anti-hypertensive medications such as methyldopa, labetalol, and hydralazine are commonly used to lower blood pressure and prevent further maternal complications [10][11][12], and magnesium sulfate is also administered to prevent seizures in severe cases [13].However, the only curative treatment of PE is to deliver the placenta.Current clinical strategy is focused on the early screening and close monitoring of high risk pregnancies, since lowdose aspirin therapy has proved to be effective in reducing the risk of the disease by 62% [14].
Although biomarkers and ultrasonic techniques have been evaluated in last decades, none has adequate positive or negative predictive values for the prediction of PE [15][16][17].
PE appears to progress in two stages [6]: The first, preclinical stage, is characterized by insufficient endovascular invasion of fetal extravillous cytotrophoblasts into the maternal spiral arterioles, resulting in decreased placental perfusion and local hypoxia.In the second stage, oxidative stress reaction is induced.Inflammatory factors released by the placenta enter into maternal blood circulation, causing damage to the maternal vascular endothelium and excessive inflammatory response, leading to vasoconstriction and diversified organ damage.Recently, the increased level of misfolded proteins has been identified in the urine of PE patients [18], which indicated that PE might share the same pathophysiologic features with recognized protein misfolding disorders like Alzheimer's disease.In PE patients, misfolded proteins such as amyloid beta and transthyretin can accumulate in the circulation and exert neurotoxic effects, activate inflammatory cascades leading to endothelial dysfunction and oxidative stress.Congo red is a kind of synthetic diazo dye with specific affinity to misfolded proteins.Buhimschi IA [18] first designed a Congo red dot test using this feature (congophilia) to detect the misfolded protein in PE patients' urine, and demonstrated that congophilia measurement could serve as a promising diagnostic tool for PE.Li XM [19] has later developed a simple and rapid point of care testing tool to detect the presence of misfolded proteins in the urine based on the visual observation, and achieved the overall sensitivity at 73.6%, and up to 83% in severe PE cases.
The traditional approach for PE screening is to identify risk factors from maternal demographic characteristics and medical history, which was only able to identify 35% of total PE and 40% of preterm PE at false-positive rate of about 10% [20,21].Other non-invasive laboratory method by measurement of urine soluble fms-like tyrosine kinase 1 (sFLT-1) and placental growth factor (PLGF) ratio was also proved to be effective in PE diagnosis, achieving 70%-72.5% sensitivity at Tang et al 5%-14% false positive [22,23].It is possible that combining urine PLGF and sFLT-1 testing with other biomarkers may yield the best predictive performance.In this trial, we used a new congophilia based detection tool alone or combined with clinical variables and other urine biomarkers to predict the onset of PE before clinical manifestation.Our research aimed to help physicians to identify PE patients earlier and proactively manage pregnant women with signs or symptoms of PE but not achieving the diagnostic criteria.

Objective
The primary objective of the study is to assess the short-term predictive potential of the newly

Study design
This is a prospective non-interventional clinical trial planned in accordance with the principles of evidence-based medicine using the PICO criteria.The trial is being conducted in accordance with ethical principles derived from the Declaration of Helsinki, and is consistent with Good Clinical Practice (GCP) and applicable regulatory requirements.The clinical research ethics committee of Women's hospital, school of medicine, Zhejiang University has reviewed the trial protocol, and the full ethical approval has been granted.Each eligible woman identified is required to give written informed consent prior to inclusion in the trial.
Apart from sample collecting and required assessment during follow-up, the study does not involve any further impact on patient's clinical procedures.The patients' PE status will be kept confidential from laboratory technicians, and neither the investigator nor the patient will know the detection results, which ensures that the clinical therapeutic scheme and the pregnancy outcomes measurement will not be affected.The study procedure has been outlined in fig. 1.
Tang et al (1) New onset of elevated blood pressure (systolic ≥130 mmHg and/or diastolic ≥80 mmHg); (2) Aggravation of preexisting hypertension (non-hypertensive patients increased more than 30 mmHg (systolic) /15 mmHg (diastolic) from the basic blood pressure, hypertensive patients showed a significant increase trend or beyond controlled based on the original treatment); (3) New onset of proteinuria (no urinary tract infection);

Sample size
The target negative predictive value was 99% and the target positive predictive value was 54.0%.
The test positive rate of 15% was assumed.Type I error is 0.05.The sample size of 280 should be enough to detect NPV>90% and PPV>25% with the power of 80%.So at least 300 eligible subjects will be included in this study considering a drop-out rate of 6%.To avoid an over-representation of late-onset PE, no more than 50% of pregnancies enrolled after 32 gestational weeks.Besides, subjects with gestational week＜28+0 at inclusion shall not less than 20%.

Study groups: classification of cases and controls
Standard diagnostics [24]

Outcome Measures
The Primary outcome is the onset of PE after inclusion.The secondary outcomes are maternal and fetal adverse outcomes as follows: maternal death, pulmonary edema, acute renal failure, cerebral hemorrhage, cerebral thrombus, disseminated intravascular coagulation (DIC), eclampsia, HELLP syndrome, rupture of placenta, placental abruption, perinatal death, stillbirth, intrauterine growth restriction (IUGR), small for gestational age (SGA), respiratory distress, necrotizing enteritis, intraventricular hemorrhage or subdural and cerebral hemorrhage, neonatal hypoxic encephalopathy or periventricular leukomalacia.In addition, date and mode of delivery, the fetal weight, apgar score are also the important outcome measures.

Urine sampling and storage
All enrolled subjects will be provided with a sterile cup to collect no less than 5mL of midstream urine at each visit.The sampled urine tubes shall be frozen and stored at -20 not later than 4 hours ℃ after collection.Samples will be shipped frozen to local laboratory for further analysis.

Urine coding
At enrollment, each subject was assigned a specific study number to protect their anonymity.The leading principle investigators will ensure that the participants' anonymity is maintained in the trial database and during the whole research process.Samples collected from one subject at one time will be labeled with the same number to allow tracking and identification of any aliquot prepared from the original urine and for sample programming in laboratory analyzers.Subjects' test results will be held securely and separately from the CRFs, ensuring that clinical decisions will not be interfered by test results.

Laboratory detection
Urine samples will be thawed at room temperature, and centrifuged at the rate of 3000 rpm for 10 minutes.Supernatant will be used for detection.
Urine misfolded proteins will be detected by Pre-Eclampsia Detection kit (Shuwen, China) since misfolded proteins can selectively bound to the Congo red.When a mixture of the Congo red and Tang et al urine from a pregnant woman is loaded onto a chromatographic column mounted in a detection cuvette, the presence (positive) or absence (negative) of misfolded proteins can be determined based on the color of eluent in the cuvette.The eluent will also be applied for quantitative measurement using HACH DR1900 instrument by Abs 520 nm -Abs 600 nm.

Selection of predictor variables
According to the objective of this study, the misfolded protein level in urine will be considered as a predictor candidate for PE onset or adverse outcomes.Other pre-set variables include: (1) demographic factors including high risk factors of PE; (2) urine biomarkers reported in publications; (3) medication; (4) routine clinical variables or laboratory tests in daily practice (Table 2).The determination of valid variables is based on the statistical analysis results with a significance level <0.05.

Statistical Analysis
All variables will be compared between case and control groups.Either parametric or non-parametric tests will be applied for continuous variables and Chi-square tests will be used for categorical variables.The ROC analysis will be used when applicable.Univariable or multivariable logistic regression will be applied to develop the prediction models.All p-values will be two-tailed.The statistical analysis will be run on SPSS (IBM, Armonk, NY) statistical software.

Results
The study is in the patients recruitment phase.Study recruitment started in July 2023.As of October 2023, a total of 85 pregnant women with clinical suspicion of PE have been included in, and will be continuously followed up and monitored with their PE status until 42 days postpartum.Statistical analysis is scheduled to start after all subjects reached the follow-up endpoint and full clinical information data collected.

Study Implications
In previous study, many potential PE biomarkers have been identified using samples from diagnosed patients comparing with normal pregnancies, which is more convenient and pragmatic.Theses on blood samples with precise instrument [26][27][28], which limits the home self-inspection and timely identification of the disease.Congophilia represent a promising new approach for detection of PE by targeting the misfolded proteins in urine, providing a simple, rapid, and cost-effective method for early diagnosis of this complex condition.The tool can be performed using small volumes of urine, and no extra instrument is needed, making it suitable for the resource-limited settings and even for home self-test.Therefore, the affordable, non-invasive and foolproof-to-use of this POCT detection tool will enable its widely accessibility at bed site of the central hospital, low-resource setting regions and even at home, which would significantly reduce the serious socioeconomic burden attributed to PE.

Limitations
Utilising prospective cohort in undiagnosed populations provides the best avenue for discovering predictive biomarkers, but these markers or combinations must be rigorously validated in internal/ external cohorts to ensure their potential for PE prediction.The single-centre nature of this study and prospective recruitment may imply the selection bias.Besides, the enrollment mainly conducted at prenatal clinics, and this might introduce selection bias regarding severity of disease where women with sudden onset of organ complications are more often seen at emergency clinic.Therefore, future investigation with larger populations and assessment of long-term outcomes of infants will be necessary to ensure the accuracy of the prediction model obtained in this trial and to establish the clinical utility of this tool for routine use in pregnancy care.

Figure 1
Figure 1 Flow chart of participants in the trial.

( 4 ) 1 .
One or more clinical symptoms showed suspected PE (other possible causes have been ruled out): Upper abdominal pain  Headache with impaired vision  Fetal growth restriction  Thrombocytopenia (platelets count < 100×10 9 /L); Liver function impairment (elevated blood concentrations of liver transaminases to twice normal concentration)  Renal impairment (serum creatinine >1.1 mg/dL or a doubling of the serum creatinine concentration in the absence of other renal disease); Exclusion criteria Confirmed diagnosis of PE/ chronic hypertension with PE (superimposed PE) 2. Confirmed diagnosis of eclampsia 3. Confirmed diagnosis of HELLP syndrome 4. Fetal chromosomal abnormalities 5. Concomitant participation in another clinical study 6.Received investigational intervention drugs in past 3 months 7. Visible hematuria 8. Preexisting renal disease studies have generated excellent hypotheses for potential PE biomarkers, but with less focus on prediction.It is possible that biomarkers deferentially expressed in patients with PE versus those without will fail in predictive verification study.Thus, prospective cohort with uncertain subsequent PE status would provide the best avenue for exploring PE prediction models.To our best knowledge, this is the first prospective study to explore the predictive value of a Congophilia based detection tool in clinical suspicious PE women in China.It will be conducted in a high-quality tertiary hospital focused on women health.Maternal age 35 years or older, nulliparity, preexisting hypertension, diabetes, autoimmune diseases (SLE, APS) are the high-risk factors for PE[25], and will be included as predictive variables in our study.High risk pregnant women are recommended to use low-dose aspirin to prevent PE, so it is appropriate to consider the medication of aspirin as a variable candidate.The crucial biomarkers in PE[22,23] such as PLGF, sFLT-1, and routine clinical variables or laboratory tests are also under evaluation to build a prediction model.Another important feature of this study is using blinding, which ensures that the participants' laboratory data has no influence on the diagnosis of PE as well as the clinical decisions of treatment.PE remains one of the most severe pregnancy complications associated with substantial maternal and perinatal morbidity, and with the current pregnancy trend of advancing age and obesity, the morbidity of PE might increase inevitably in the future.The only definitive treatment for PE is to terminate the pregnancy, so early screening and continuous monitoring during pregnancy are particularly important for pregnant women with high risk of PE.At present, effective screening tests for PE have not yet been identified.The commonly used clinical screening methods are mostly based

Table 1 . Summary of the data collection procedure Visit 1 Visit 2 Visit 3 Visit 4 Visit 5 Delivery Postpartum Unscheduled visit
Inclusion / exclusion criteria ★