Multiple sFlt-1 measurements for prognosis of premature preeclampsia

Abstract

The present invention relates to a method for prognosis, prediction, risk assessment and / or risk stratification of preeclampsia in a pregnant subject comprising determining the level of soluble fms-like tyrosine kinase-1 (sFlt-1) or fragments thereof in a sample that has been isolated from the pregnant subject. The invention also relates to a method for prognosis, prediction, risk assessment and / or risk stratification of premature preeclampsia in a pregnant subject. The invention also relates to sFlt-1 and PIGF, optionally combined measurements in combination with consideration of one or more additional factors selected from pregnant and lying-in woman age, body mass index, uterine artery Doppler measurements and / or mean arterial pressure (MAP). The invention also relates to a kit for carrying out the method of the invention comprising a detection reagent for determining the level of sFlt-1 or a fragment thereof in a sample from a subject, and optionally for determining the level of at least one additional biomarker as described herein in a sample from a subject.

Description

Technical Field

[0001] The present invention belongs to the field of clinical and molecular diagnosis and prognosis of medical conditions, particularly preeclampsia (PE).

[0002] Accordingly, the present invention relates to a method for prognosticating, predicting, risk assessing, and / or risk stratifying preeclampsia in a pregnant subject, comprising determining the level of sFlt-1 or a fragment thereof in a sample isolated from the pregnant subject; wherein said level of sFlt-1 or a fragment thereof indicates the likelihood of preeclampsia.

[0003] The present invention also relates to a method for prognosticating, predicting, risk assessing, and / or risk stratifying early-onset preeclampsia (EO-PE) in a pregnant subject, comprising (a) determining a first level of soluble fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof in a first sample isolated from the subject, wherein the first sample is isolated before the end of the 14th week of pregnancy, (b) determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, wherein the second sample is isolated after the first sample, and (c) wherein a higher second level of sFlt-1 or a fragment thereof compared to the first level indicates the occurrence of early-onset preeclampsia before the end of the 33rd week of pregnancy (before 231 days).

[0004] The present invention also relates to a method for prognosticating, predicting, risk assessing, and / or risk stratifying early-onset preeclampsia in a pregnant subject, comprising determining the level of soluble fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof and the level of placental growth factor (PIGF) or a fragment thereof in a first sample and / or a second sample. The present invention also relates to measuring sFlt-1 and PIGF, optionally in combination with considering one or more additional factors selected from maternal age, body mass index, uterine artery Doppler measurements, and / or mean arterial pressure (MAP).

[0005] The present invention also relates to a kit for implementing the method of the present invention, the kit comprising detection reagents for determining the level of sFlt-1 or a fragment thereof in a sample from a subject, and optionally for determining the level of at least one additional biomarker (such as PIGF) as described herein in a sample from a subject. Background Art

[0006] Preeclampsia (PE) is a pregnancy-specific hypertensive disorder and a leading cause of maternal and perinatal morbidity and mortality globally. The World Health Organization (WHO) estimates that 16% of global maternal deaths (approximately 63,000 maternal deaths per year) are caused by PE alone. Infants are also at risk. Preeclampsia complicates approximately 2% to 8% of all pregnancies and is a major cause of maternal and fetal death globally (Duley 2009, Semin Perinatal: 33:130-37). Preeclampsia is generally defined as pregnancy-related or induced hypertension and proteinuria that develops after 20 weeks of gestation (140 days). Early-onset preeclampsia (EO-PE) is a low-prevalence subgroup of preeclampsia cases, occurring in 0.2% to 0.4% of all pregnancies, and is associated with various adverse perinatal outcomes such as intrauterine fetal death (IUFD) and high perinatal mortality.

[0007] In resource-limited settings, the risk of maternal death is much higher. The most commonly recognized factor contributing to maternal and fetal morbidity in most cases is the failure to timely identify preeclampsia. As a result, pregnant women are not able to receive effective monitoring or treatment until long after the development of complications associated with the disease, including elevated blood pressure and proteinuria. Additionally, pregnant women at little or no risk of developing such a disease must undergo unnecessary symptomatic testing throughout their pregnancy because no caregiver has an effective means by which to rule out their risk in the early stages of pregnancy.

[0008] WO 2008 / 103202 A2 discloses a method for diagnosing pregnancy-related hypertensive disorders by measuring COMT, HIF-l[α], EPO, LDH-A, ET-I, transferrin, transferrin receptor, and flk-I, free VEGF, total VEGF, sFlt-1, PIGF. Alterations in the expression levels of these polypeptides compared to reference levels are indicators of pregnancy-related hypertensive disorders.

[0009] WO 2006 / 069373 A2 discloses a method for diagnosing a pregnant woman as having or being at risk of developing a hypertensive disorder. The levels of sFlt-1 and placental growth factor (PIGF) are measured in a urine sample. The ratio of sFlt-1 expression to PIGF expression is used as an indicator as to whether a pregnant woman is at risk of developing a hypertensive disorder.

[0010] WO 2004 / 008946 A2 discloses a method for treating or preventing preeclampsia or eclampsia in a subject, comprising the step of administering a compound capable of binding to soluble fms-like tyrosine kinase 1 (sFlt-1). It is also disclosed that elevated sFlt-1 concentrations in a patient prior to the onset of preeclampsia are due to an acute elevation of sFlt-1 within 5 weeks prior to the onset of the clinical disease.

[0011] Myatt et al. (BJOG: International Journal of Obstetrics and Gynaecology, Vol. 120, No. 10, 2013) disclosed the measurement of PIGF, sFlt-1, and sEng during the first and second trimesters of pregnancy in low-risk patients. Samples were taken at 9-12 weeks, 15-18 weeks, and 23-26 weeks of pregnancy. The changes in biomarker levels from the first trimester to the second trimester and their association with the occurrence of early-onset preeclampsia were studied.

[0012] Palm et al. (Acta Obstetricia and Gynecologica Scandinavica, Vol. 90, No. 11, 2011) disclosed the measurement of sFlt-1, PIGF, and VEGF-A during pregnancy and postpartum in healthy patients without pregnancy complications. Preferably, at least six samples were taken at 12 weeks, 20 weeks, 32 weeks, 36 weeks, 40 weeks, and postpartum. An increase in sFlt-1 levels was observed in these subjects during pregnancy. However, the prediction of the occurrence of preeclampsia based on these biomarker measurements was not disclosed.

[0013] De Kat et al. (Cardiovascular Health, Vol. 16, 2019) disclosed an overview of models known in the prior art for predicting preeclampsia. PIGF and sFlt-1 were disclosed as biomarkers for predicting preeclampsia.

[0014] Staff et al. (Cardiovascular Health, Vol. 1, No. 1, 2010) disclosed various biomarkers for predicting preeclampsia, including sFlt-1 and PIGF. In this document, conflicting studies on the prediction of preeclampsia based on sFlt-1 measurement were cited. The document included several studies that disclosed the prediction of early-onset preeclampsia by measuring sFIt-1, but also presented studies that disclosed that sFlt-1 measurements at several time points during pregnancy were not suitable for predicting preeclampsia.

[0015] To date, the most invasive treatment for preeclampsia is to terminate the pregnancy by vaginal preterm birth or cesarean preterm birth. As mentioned above, in cases of preeclampsia occurring before 34 weeks of gestation, maternal risks are significantly increased and fetal viability is significantly impaired. Therefore, attempts should be made to delay delivery in order to improve neonatal survival rates. Tsakiridis et al. (Obstetrical and Gynecological Survey, Vol. 76, No. 10) emphasized the content of pregnancy-related guidelines, stating that high-risk patients should take low-dose aspirin as early as possible, ideally from the first trimester of pregnancy until delivery or until 36 to 37 weeks of pregnancy (until 246 to 259 days).

[0016] Improving the prognosis of preeclampsia in women who are clinically asymptomatic or suspected of having or developing preeclampsia during the first trimester of pregnancy has important clinical significance.

[0017] In its most severe forms (such as early-onset PE), it is necessary to start treatment as early as possible, ideally as early as 11 weeks of pregnancy (71 to 77 days of gestation). Currently, the FMF algorithm detects approximately 60% to 66% of preterm PE and approximately 70% of early-onset PE. The Fetal Medicine Foundation (FMF) screening algorithm takes into account a variety of maternal characteristics and medical histories, including factors such as blood pressure, pregnancy-associated plasma protein A and placental growth factor, crown-rump length, and uterine artery pulsatility index. However, the FMF algorithm is complex and relies on uterine artery Doppler measurements, so this technique is generally not applicable to all pregnant subjects.

[0018] In addition, sFlt-1 levels vary significantly between different gestational ages and show a correlation with preeclampsia, but both low levels (e.g., below the healthy average) or high levels (e.g., above the healthy average) have been observed, and currently the kinetics of sFlt-1 levels and its relationship with PE remain unclear. The results regarding sFlt-1 concentrations during pregnancy relative to the risk of PE are conflicting, with studies reporting decreased, increased, or no change in sFlt-1 concentrations during pregnancy in which PE develops (Pihl et al., Fetal Diagn Ther 2020; 47:277-283; Akolekar et al., Prenat Diagn. March 2010; 30(3):191-7).

[0019] There is an urgent need in the art for improved and simplified means for determining the risk of preeclampsia (especially early-onset preeclampsia) in the early stages of pregnancy. SUMMARY OF THE INVENTION

[0020] According to the prior art, the technical problem to be solved by the present invention is to provide improved or alternative means for prognosticating, predicting, risk assessing and / or risk stratifying preeclampsia in pregnant subjects. Another object of the present invention is to provide means for early prognosing or risk assessing preeclampsia. Another object of the present invention is to provide a prognostic method for risk assessing preeclampsia within the first trimester of pregnancy. Other objects of the present invention relate to providing means for improving and / or simplifying the screening or prognosis of early-onset preeclampsia in early pregnancy, which means increase sensitivity and preferably do not require uterine artery Doppler measurements.

[0021] This problem is solved by the features of the independent claims. The dependent claims provide preferred embodiments of the present invention.

[0022] Accordingly, the present invention relates to a method for prognosticating, predicting, risk assessing and / or risk stratifying early-onset preeclampsia in a pregnant subject, comprising

[0023] a. determining a first level of soluble fms-like tyrosine kinase-1

[0024] (sFlt-1) or a fragment thereof in a first sample isolated from the subject, wherein the first sample is isolated before gestational age (GA) reaches 90 days,

[0025] b. determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, wherein the second sample is isolated after the first sample,

[0026] c. wherein a higher second level of sFlt-1 or a fragment thereof compared to the first level indicates early-onset preeclampsia occurring before the end of week 33 of pregnancy (before GA reaches 231 days).

[0027] Accordingly, the method described herein enables early pregnancy EO-PE risk assessment, thereby providing the possibility for clinical practitioners to initiate appropriate prophylactic treatment early in pregnancy. In the most severe forms of PE, such as EO-PE, treatment is recommended to be initiated as early as possible, preferably as early as week 16 of pregnancy (GA from 106 days to 112 days), more preferably as early as week 11 of pregnancy (GA from 71 days to 77 days). Until the present invention was proposed, diagnostic assays were available for PE prognosis, but early prognosis of EO-PE mainly relied on the FMF algorithm using uterine artery Doppler measurements. Accordingly, the present invention enables a simple and reliable molecular diagnostic and / or prognostic method for identifying subjects at risk of EO-PE at very early gestational age (GA).

[0028] In addition, sFlt-1 levels appear to vary significantly between different gestational ages. Although sFlt-1 appears to be associated with preeclampsia, both low levels (e.g., below the healthy average) and high levels (e.g., above the healthy average) have been observed, and the kinetics of sFlt-1 levels and their relationship to PE are still unclear. Thus, another difficulty faced by diagnostic experts or medical practitioners in EO-PE risk assessment is that sFlt-1 also appears to be a promising biomarker that has some relationship with PE, and the lack of knowledge of sFlt-1 kinetics throughout pregnancy makes it difficult to interpret sFlt-1 levels and their significance regarding PE risk, especially EO-PE risk. For example, if the exact date of conception is unknown or inaccurately or falsely recorded, sFlt-1 levels obtained in routine tests may be misleading and provide false negative or false positive results regarding PE or EO-PE risk. Specifically, sFlt-1 levels obtained from a single sample within weeks 12 to 13 of pregnancy (GA days 78 to 91) may be misleading and provide the most likely false negative results because the levels of subjects who do not develop EO-PE and subjects who develop EO-PE do not differ substantially within weeks 12 to 13 of pregnancy (GA days 78 to 91) (see Figure 15 B).

[0029] In an embodiment, the gestational age of a pregnant subject is determined by abdominal ultrasound and / or vaginal ultrasound before obtaining the first sample from the subject.

[0030] For example, the results of Pihl et al. (Fetal Diagn Ther 2020;47:277-283) outlining sFlt-1 concentrations during pregnancy relative to PE risk are conflicting, with studies reporting decreased, increased, or no change in sFlt-1 concentrations during pregnancy in which PE develops. However, the present invention is now able to implement a novel method of using sFlt-1 measurements in PE and EO-PE risk assessment.

[0031] The present inventors have determined that sFlt-1 levels are significantly but negatively correlated with the risk of EO-PE within 90 days before GA full term (low sFlt-1 levels are associated with an increased risk of EO-PE). However, sFlt-1 assessments at 90 - 100 days, even in subjects who went on to develop EO-PE, showed no significant difference from the healthy population mean. Additionally, after 100 days, e.g., between 140 and 154 days after GA full term, sFlt-1 levels are significantly positively correlated with the risk of EO-PE (high sFlt-1 levels are associated with an increased risk of EO-PE). Accordingly, the present inventors have developed a prognostic analysis protocol in which two samples are obtained from a pregnant subject, and an increase in sFlt-1 levels at the required time points indicates an elevated risk of EO-PE relative to the healthy population mean. By obtaining a second sample after the first sample according to the present invention, false negative results can be advantageously reduced, especially in early pregnancy, e.g., within the first trimester and at the start of the second trimester of pregnancy.

[0032] As can be seen from Figure 1 and the following examples, women (N = 10) who developed EO-PE had sFlt-1 levels below the median before 90 days of pregnancy (p < 0.01). Between 90 and 100 days of pregnancy, on average, women (N = 24) who developed EO-PE had sFlt-1 levels equal to the mean. Between 140 and 154 days of pregnancy, women (N = 4) who developed EO-PE had sFlt-1 levels above the median. As can be seen from Figure 14 and Figure 15 B, similar results were observed. These results allow us to conclude that in women who will develop early preeclampsia (before 34 weeks, before 232 days of GA full term), sFlt-1 levels are abnormally decreased in early pregnancy (before 90 days of pregnancy) and will gradually increase to normal levels by the end of the first trimester (between 90 and 100 days), and then increase abnormally thereafter (after 140 days of pregnancy).

[0033] Aspects of the present invention are unified by, benefit from, are based on, and / or are associated with the following common finding: that the level of sFlt-1 or a fragment thereof in a sample from a pregnant subject is significantly lower than the healthy mean within 90 days before GA full term and increases to the healthy mean or above after 100 days of GA full term, such that an increase in sFlt-1 levels indicates the likelihood of early-onset preeclampsia. Accordingly, advantageously, compared to an analysis protocol that makes prognostic statements based on a single sample obtained from a subject, the prognostic analysis protocol of the present invention, in which two samples are obtained from a pregnant subject, results in a reduced false negative rate.

[0034] The present invention provides an effective and reliable test for healthcare practitioners (such as doctors, nurses, emergency department personnel, etc.) to rapidly and accurately assess the likelihood of a pregnant subject developing PE (e.g., preterm PE, spontaneous abortion, EO-PE, and severe PE, as well as related complications such as preterm birth and small for gestational age), particularly EO-PE. Typically, pregnant women with little or no risk of developing such conditions must undergo unnecessary symptomatic testing throughout their pregnancy because no caregiver has an effective means to rule out their risk early in pregnancy.

[0035] High levels of placental soluble fms-like tyrosine kinase (sFlt-1) are closely associated with PE in the second trimester of pregnancy (weeks 14 to 27 of gestation, GA 92 days to 189 days) and the third trimester of pregnancy (weeks 28 to birth, GA 190 days to birth). Surprisingly, according to the present invention, the level of sFlt-1 or its fragment in the first 90 days of pregnancy is also associated with early-onset PE, whereby a low sFlt-1 level indicates EO-PE.

[0036] Based on this surprising finding, the present invention provides a means for identifying pregnant subjects at increased or high risk of developing EO-PE, as well as for identifying patients who are less likely to develop such complications or in whom the development of such complications can actually be excluded, which is achieved by measuring the level of sFlt-1 or its fragment in a sample isolated from the patient.

[0037] Another advantage resulting from this surprising finding is that, in addition to the conventional combination of biomarkers, clinical parameters, and imaging procedures used for prognosticating PE, the prognostic biomarker sFlt-1 can also be used in any healthcare setting, regardless of whether a device for measuring uterine artery pulsatility index (UAPI) is available, for which a special ultrasound device is typically required, as well as an expert to operate the device and perform the measurement. Thus, a direct minimally invasive test can be performed.

[0038] It is also noteworthy that the Aspirin for Evidence-Based Preeclampsia Prevention (ASPRE) trial, a multicenter trial that included women identified as being at high risk of preterm PE according to the FMF algorithm, who were randomly assigned to receive aspirin or placebo from 11 to 14 weeks of gestation (GA 71 days to 98 days) to 37 weeks of gestation (GA 259 days), showed that low-dose aspirin daily reduced preterm PE by 62% (relative risk, 0.38; 95% confidence interval [CI], 0.20 - 0.74) compared with the placebo group.

[0039] This effective treatment can now be applied to pregnant subjects in the early stages of pregnancy if they can be accurately predicted to be at high risk of developing EO-PE within the first 12 or 14 weeks of pregnancy (GA from 78 days to 98 days). Based on this surprising finding, another advantage is brought: the risk of developing EO-PE can be predicted within the first trimester of pregnancy, so that subjects predicted to be at high risk of developing PE can be given preventive mild treatment, medical treatment, or even bed rest and frequent monitoring, rather than any severe measures such as early termination of pregnancy after developing PE in the third trimester of pregnancy.

[0040] In one embodiment, the present invention relates to a method for prognosis, prediction, risk assessment, and / or risk stratification of early-onset preeclampsia in a pregnant subject, comprising

[0041] a. determining a first level of soluble fms-like tyrosine kinase-1

[0042] (sFlt-1) or a fragment thereof in a first sample isolated from the subject, wherein the first sample is isolated before the end of the 13th week of pregnancy (before GA of 90 days),

[0043] b. determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, wherein the second sample is isolated after the first sample,

[0044] c. wherein a higher second level of sFlt-1 or a fragment thereof compared to the first level indicates early-onset preeclampsia before the end of the 33rd week of pregnancy (before GA of 231 days).

[0045] In one embodiment, the present invention relates to a method for prognosis, prediction, risk assessment, and / or risk stratification of early-onset preeclampsia in a pregnant subject, comprising

[0046] a. determining a first level of soluble fms-like tyrosine kinase-1

[0047] (sFlt-1) or a fragment thereof in a first sample isolated from the subject, wherein the first sample is isolated before the end of the 13th week of pregnancy (before GA of 90 days),

[0048] b. determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, wherein the second sample is isolated after the first sample, and the second sample is isolated after the end of the 20th week of pregnancy (after GA of 140 days),

[0049] c. A higher second level of sFlt-1 or a fragment thereof compared to the first level indicates early-onset preeclampsia occurring before the end of 33 weeks of gestation (before 231 days of GA).

[0050] In one embodiment, the present invention relates to a method for prognosticating, predicting, risk-assessing, and / or risk-stratifying early-onset preeclampsia in a pregnant subject, comprising

[0051] a. Determining a first level of soluble fms-like tyrosine kinase-1

[0052] (sFlt-1) or a fragment thereof in a first sample isolated from the subject, wherein the first sample is isolated before 90 days of GA,

[0053] b. Determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, wherein the second sample is isolated after the first sample, and wherein the second sample is isolated after 140 days of GA,

[0054] c. A higher second level of sFlt-1 or a fragment thereof compared to the first level indicates early-onset preeclampsia occurring before 231 days of GA.

[0055] In an embodiment, the first sample is isolated from the subject before the end of 12 weeks or 14 weeks of gestation (before 100 days of GA).

[0056] In an embodiment, the first sample is isolated from the subject before the end of 7 weeks or 9 weeks of gestation (before 63 days of GA).

[0057] In an embodiment, the first sample is isolated from the subject before the end of 8 weeks or 9 weeks of gestation (before 63 days of GA).

[0058] Considering the data presented herein, sFlt-1 levels appear to increase over time during pregnancy, and the increase is evident from before 90 days of GA to between 90 and 100 days of GA, from between 90 and 100 days of GA to later time points, and from before 90 days of GA to later time points, such as between 140 and 154 days or later ( Figure 1 、 Figure 14 and Figure 15, and the following examples). Thus, the first sample is obtained and evaluated before GA reaches 100 days, then subsequent samples are measured, and an increase in the sFlt-1 level in the second sample is detected, indicating the risk of EO-PE in the subject. In addition, the first sample is obtained and evaluated before GA reaches 90 days, then subsequent samples are measured, and an increase in the sFlt-1 level in the second sample is detected, indicating the risk of EO-PE in the subject. In addition, the first sample is obtained and evaluated before GA reaches 63 days, then subsequent samples are measured, and an increase in the sFlt-1 level in the second sample is detected, indicating the risk of EO-PE in the subject.

[0059] In an embodiment, the first sample is isolated from the subject before the end of the 13th week of pregnancy (before GA reaches 90 days). In an embodiment, the first sample is isolated from the subject before the end of the 9th week of pregnancy (before GA reaches 63 days). In an embodiment, the first sample is isolated from the subject before the end of the 8th week of pregnancy (before GA reaches 56 days). In an embodiment, the first sample is isolated from the subject before the end of the 7th week of pregnancy (before GA reaches 49 days).

[0060] As outlined in detail below, the level of sFlt-1 in samples from pregnant subjects is significantly lower than the healthy average in the 90 days before GA reaches full term and increases to the healthy average or above 100 days after GA reaches full term. Therefore, an increase in the sFlt-1 level indicates the likelihood of early-onset preeclampsia. Specifically, the level before GA reaches 90 days is significantly lower than the healthy average. Therefore, obtaining the first sample within 90 days after GA reaches full term is a preferred embodiment of the present invention.

[0061] In an embodiment, the sFlt-1 level in a sample obtained from a pregnant subject with early-onset preeclampsia that develops before the end of the 33rd week of pregnancy (before 231 days after GA reaches full term) in the 90 days before GA reaches full term is lower than the 10th percentile of the sFlt-1 level of the healthy population in the 90 days before GA reaches full term. In an embodiment, the sFlt-1 level in a sample obtained from a pregnant subject with early-onset preeclampsia that develops before the end of the 33rd week of pregnancy (before 231 days after GA reaches full term) in the 90 days before GA reaches full term is lower than the 15th percentile of the sFlt-1 level of the healthy population in the 90 days before GA reaches full term, such as lower than the 15th, 14th, 13th, 12th, 11th, or 10th percentile.

[0062] In an embodiment, the sFlt-1 level in a second sample obtained from a pregnant subject with early-onset preeclampsia that develops before the end of 33 weeks of gestation (before GA reaches 231 days) after a first sample is higher than the 90th percentile of the sFlt-1 level in a healthy population. In an embodiment, the sFlt-1 level in a sample obtained from a pregnant subject with early-onset preeclampsia that develops before the end of 33 weeks of gestation (before GA reaches 231 days) after a first sample is higher than the 95th percentile of the sFlt-1 level in a healthy population, such as higher than the 95th, 96th, 97th, 98th, or 99th percentile.

[0063] In an embodiment, the sFlt-1 level in a second sample obtained from a pregnant subject with early-onset preeclampsia that develops before the end of 33 weeks of gestation (before GA reaches 231 days) after the end of 13 weeks of gestation (after GA reaches 90 days) is higher than the 90th percentile of the sFlt-1 level in a healthy population. In an embodiment, the sFlt-1 level in a sample obtained from a pregnant subject with early-onset preeclampsia that develops before the end of 33 weeks of gestation (before GA reaches 231 days) after the end of 13 weeks of gestation (after GA reaches 90 days) is higher than the 95th percentile of the sFlt-1 level in a healthy population, such as higher than the 95th, 96th, 97th, 98th, or 99th percentile.

[0064] In an embodiment, the sFlt-1 level in a second sample obtained from a pregnant subject with early-onset preeclampsia that develops before the end of 33 weeks of gestation (before GA reaches 231 days) between the end of 20 weeks of gestation and the end of 22 weeks of gestation (between GA reaching 140 days and 154 days) is higher than the 90th percentile of the sFlt-1 level in a healthy population. In an embodiment, the sFlt-1 level in a sample obtained from a pregnant subject with early-onset preeclampsia that develops before the end of 33 weeks of gestation (before GA reaches 231 days) between the end of 20 weeks of gestation and the end of 22 weeks of gestation (between GA reaching 140 days and 154 days) is higher than the 95th percentile of the sFlt-1 level in a healthy population, such as higher than the 95th, 96th, 97th, 98th, or 99th percentile.

[0065] In one embodiment, the healthy population refers to a group of pregnant subjects who do not develop early-onset preeclampsia before the end of 33 weeks of gestation (before GA reaches 231 days). In one embodiment, samples are obtained from a healthy population of the same gestational age as the pregnant subject from whom samples are obtained according to the method of the present invention.

[0066] In one embodiment, the MoM of the sFlt-1 level in a first sample obtained from a pregnant subject with early-onset preeclampsia that develops before the end of week 33 of gestation (before GA reaches 231 days) is less than 1.0, preferably less than 0.9, more preferably less than 0.8, such as 0.99, 0.98, 0.97, 0.96, 0.95, 0.94, 0.93, 0.92, 0.91, 0.90, 0.89, 0.88, 0.87, 0.86, 0.85, 0.84, 0.83, 0.82, 0.81, 0.80, 0.79, 0.78, 0.77, 0.76, 0.75, 0.74, 0.73, 0.72, 0.71, 0.70, 0.68, 0.65, 0.60, 0.55, 0.50, 0.45, 0,40, 0.35, 0.30, 0.25, 0.20, 0.15, and 0.10 before the end of week 13 of gestation (before GA reaches 90 days).

[0067] In one embodiment, the MoM of the sFlt-1 level in a first sample obtained from a pregnant subject with early-onset preeclampsia that develops before the end of week 33 of gestation (before GA reaches 231 days) is less than 1.0, preferably less than 0.9, more preferably less than 0.8, such as 0.99, 0.98, 0.97, 0.96, 0.95, 0.94, 0.93, 0.92, 0.91, 0.90, 0.89, 0.88, 0.87, 0.86, 0.85, 0.84, 0.83, 0.82, 0.81, 0.80, 0.79, 0.78, 0.77, 0.76, 0.75, 0.74, 0.73, 0.72, 0.71, 0.70, 0.68, 0.65, 0.60, 0.55. 0.50, 0.45, 0,40, 0.35, 0.30, 0.25, 0.20, 0.15, and 0.10 before the end of week 9 of gestation (before GA reaches 63 days).

[0068] In one embodiment, the MoM of the sFlt-1 level in a second sample obtained from a pregnant subject with early-onset preeclampsia that develops before the end of week 33 of gestation (before GA reaches 231 days) is greater than 1.0, preferably greater than 2.0, more preferably greater than 3.0, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4,.2.5, 2.6, 2.7, 2.9, 3.0, 3.2, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.5, and 5.0 after the end of week 13 of gestation (after GA reaches 90 days).

[0069] In one embodiment, the MoM of the sFlt-1 level in a second sample obtained from a pregnant subject with early-onset preeclampsia that develops before the end of 33 weeks of gestation (before GA reaches 231 days) after the end of 13 weeks of gestation (after GA reaches 90 days) is greater than 1.0, preferably greater than 2.0, more preferably greater than 3.0, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.9, 3.0, 3.2, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.5, and 5.0.

[0070] In one embodiment, the MoM of the sFlt-1 level in a second sample obtained from a pregnant subject with early-onset preeclampsia that develops before the end of 33 weeks of gestation (before GA reaches 231 days) between the end of 20 weeks of gestation and the end of 22 weeks of gestation (between GA reaching 140 days and 154 days) is greater than 1.0, preferably greater than 2.0, more preferably greater than 3.0, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.9, 3.0, 3.2, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.5, and 5.0.

[0071] In one embodiment, the MoM for a healthy population that does not develop early-onset preeclampsia before the end of 33 weeks of gestation (before GA reaches 231 days) is 1.0.

[0072] In one embodiment, the median multiple (MoM) of the first level is less than 1.0, and the MoM of the second level is greater than 1.0, preferably greater than 2.0, more preferably greater than 3.0, indicating early-onset preeclampsia before GA reaches 231 days.

[0073] In an embodiment, the first sample is isolated from the subject after the end of the 13th week of gestation (after GA is 90 days). In an embodiment, the second sample is isolated from the subject after the end of the 14th week of gestation (after GA is 100 days). In an embodiment, the second sample is isolated from the subject between the 11th and 13th weeks of gestation (between GA of 71 days and 91 days). In an embodiment, the second sample is isolated from the subject after the end of the 10th week of gestation (after GA is 70 days). In an embodiment, the first sample is isolated from the subject after the end of the 13th week of gestation (after GA is 91 days). In an embodiment, the second sample is isolated from the subject during the second trimester of pregnancy. Considering the sFlt-1 kinetics disclosed herein, isolating the second sample after the first sample and detecting an increase in sFlt-1 in the second sample indicates the risk of EO-PE. However, by obtaining and testing the second sample, such as after 90 days or after 100 days, an even greater and more significant difference between the first and second samples can be determined, thus indicating the risk of EO-PE. Additionally, by obtaining and testing the first sample before 90 days of gestation, or even before 63 days or even before 49 days, an early indication of the EO-PE risk is obtained, allowing for early initiation of treatment or increased monitoring rates to improve maternal and fetal health.

[0074] According to the present invention, the term "indicates", for example, in the context of "indicates early-onset preeclampsia", is intended as a term of risk and / or likelihood. Preferably, an "indication" of the presence or absence or subsequent occurrence of early-onset preeclampsia (or other condition) is intended as a risk assessment and generally should not be construed in a restrictive manner as deterministically indicating the absolute presence or absence of the condition.

[0075] In other embodiments, non-limitingly, the second sample can be obtained 1 day after the first sample, or 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days after the first sample.

[0076] In one embodiment, the second sample is obtained at least 1 week after the first sample.

[0077] In one embodiment, the second sample is obtained at least 2 weeks after the first sample.

[0078] In one embodiment, the second sample is obtained at least 3 weeks after the first sample.

[0079] In an embodiment, the second sample is isolated after the end of the 20th week of pregnancy (after 140 days GA). In an embodiment, the second sample is isolated between the end of the 20th week and the end of the 22nd week of pregnancy (140 days to 154 days GA).

[0080] As outlined in the examples herein, the sFlt-1 levels observed at these time points during pregnancy allow for the determination of an even greater and / or more significant difference between the first and second samples, thus indicating the risk of EO-PE.

[0081] In an embodiment, the sFlt-1 level in the second sample is at least 1% higher than that in the first sample. In other embodiments, the sFlt-1 level in the second sample is at least 5%, 10%, 15%, 20%, 25% or 30% higher than that in the first sample, even if the individual measurement points are lower or at the same mean compared to a healthy population (reference level).

[0082] In an embodiment, the first level is below the reference level, which is preferably the population mean and / or median of a healthy population.

[0083] In an embodiment, the second level is equal to or higher than the reference level, which is preferably the population mean and / or median of a healthy population.

[0084] As Figure 1 and shown in the examples below, subjects who do not develop EO-PE exhibit significantly different sFlt-1 levels from those who go on to develop EO-PE. To determine the sFlt-1 levels associated with risk, a comparison between the first and second samples and / or a comparison with a reference value can be made.

[0085] In an embodiment of the method, after obtaining the first sample, the subject is at 9 to 11 weeks of pregnancy (57 days to 77 days GA). In an embodiment of the method, after obtaining the first sample, the subject is at 11 weeks of pregnancy (71 days to 77 days GA). In an embodiment of the method, the gestational age (GA) of the subject is 50 to 90 days, preferably 60 to 90 days, or 70 to 90 days, such as GA being 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 or 90 days.

[0086] In an embodiment of the method, after obtaining the first sample, the subject is at 7 to 9 weeks of gestation (GA 43 to 63 days). In an embodiment of the method, after obtaining the first sample, the subject is at 8 to 9 weeks of gestation (GA 50 to 63 days). In an embodiment of the method, the gestational age (GA) of the subject is 43 to 63 days, preferably 50 to 63 days, such as GA being 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 or 63 days.

[0087] In an embodiment of the method, after obtaining the first sample, the subject is at 7 to 11 weeks of gestation (GA 43 to 77 days). In an embodiment of the method, after obtaining the first sample, the subject is at 8 to 11 weeks of gestation (GA 50 to 77 days). In an embodiment of the method, the gestational age (GA) of the subject is 43 to 77 days, preferably 50 to 77 days, such as GA being 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90 days.

[0088] As mentioned above, these early time points for sampling and testing enable early treatment to be initiated, which may be crucial for effectively addressing and / or preventing the occurrence of severe forms of PE.

[0089] In one embodiment of the method:

[0090] a. The first level is below the reference level, and the second level is equal to or higher than the reference level, where the reference level is preferably the population mean and / or median of a healthy population, or

[0091] b. The first level is equal to or below the reference level, and the second level is higher than the reference level, where the reference level is preferably the population mean and / or median of a healthy population, or

[0092] c. The first level is below the reference level, and the second level is higher than the reference level, where the reference level is preferably the population mean and / or median of a healthy population.

[0093] In an embodiment, the present invention relates to a method for prognosticating, predicting, risk assessing, and / or risk stratifying early-onset preeclampsia in a pregnant subject, comprising

[0094] a. determining a first level of soluble fms-like tyrosine kinase-1

[0095] (sFlt-1) or a fragment thereof in a first sample isolated from the subject, wherein the first sample is isolated before the end of week 13 of gestation (before 90 days GA),

[0096] b. determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, wherein the second sample is isolated after the first sample,

[0097] c. wherein the first level is below a reference level, and the second level is equal to or above the reference level, and a higher second level of sFlt-1 or a fragment thereof compared to the first level indicates early-onset preeclampsia before the end of week 33 of gestation (before 231 days GA).

[0098] In an embodiment, the present invention relates to a method for prognosticating, predicting, risk assessing, and / or risk stratifying early-onset preeclampsia in a pregnant subject, comprising

[0099] a. determining a first level of soluble fms-like tyrosine kinase-1

[0100] (sFlt-1) or a fragment thereof in a first sample isolated from the subject, wherein the first sample is isolated before the end of week 13 of gestation (before 90 days GA),

[0101] b. determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, wherein the second sample is isolated after the first sample,

[0102] c. wherein the first level is equal to or below a reference level, and the second level is above the reference level, and a higher second level of sFlt-1 or a fragment thereof compared to the first level indicates early-onset preeclampsia before the end of week 33 of gestation (before 231 days GA).

[0103] In an embodiment, the present invention relates to a method for prognosticating, predicting, risk assessing, and / or risk stratifying early-onset preeclampsia in a pregnant subject, comprising

[0104] a. determining a first level of soluble fms-like tyrosine kinase-1

[0105] a first level of (sFlt-1) or a fragment thereof, wherein the first sample is isolated before the end of week 13 of gestation (before 90 days GA),

[0106] b. determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, wherein the second sample is isolated after the first sample,

[0107] c. wherein the first level is below a reference level, and the second level is above the reference level, and a higher second level of sFlt-1 or a fragment thereof compared to the first level indicates early-onset preeclampsia before the end of week 33 of gestation (before 231 days GA).

[0108] Additional embodiments and aspects of the invention relate to methods described herein for indicating intrauterine fetal death (IUFD).

[0109] In one embodiment of the method, a higher second level of sFlt-1 or a fragment thereof compared to the first level additionally indicates a subsequent occurrence of intrauterine fetal death (IUFD). In embodiments, the indication of a subsequent occurrence of intrauterine fetal death is related to an increased risk for the patient compared to the average risk of a subsequent IUFD in the healthy population.

[0110] Embodiments of the invention relate to prognosis, prediction, risk assessment, and / or risk stratification for IUFD. The IUFD prognosis can be independent of the EO-PE prognosis or can be combined with the EO-PE prognosis. In other words, EO-PE can occur in combination with IUFD, or IUFD can occur independently of EO-PE. Features of the methods and kits described herein regarding the EO-PE prognosis apply equally to the IUFD prognosis and vice versa.

[0111] As shown in more detail below, when samples are obtained between 90 and 100 days GA, the combined use of sFlt-1 and PIGF enables prognosis of IUFD ( Figure 11 ) while when samples are obtained before 90 days GA, a statistically improved IUFD prognosis is shown ( Figure 12 ).

[0112] Other embodiments regarding combinations with other biomarkers :

[0113] In one embodiment, the method further comprises:

[0114] a. determining the level of placental growth factor (PIGF) or a fragment thereof in the first sample and / or the second sample,

[0115] b. wherein a combination of the level of sFlt-1 or a fragment thereof and the level of said PIGF or a fragment thereof indicates a likelihood of early-onset preeclampsia occurring before the end of week 33 of gestation (before GA reaches 231 days).

[0116] In an embodiment, the method of the invention further comprises determining the level of placental growth factor (PIGF) or a fragment thereof in a first sample and / or a second sample from said patient, wherein a combination of the level of sFlt-1 or a fragment thereof and the level of PIGF or a fragment thereof in the first sample and / or the second sample indicates early-onset preeclampsia occurring before the end of week 33 of gestation (before GA reaches 231 days).

[0117] As shown in more detail below, when samples are obtained in early pregnancy, e.g., before the end of week 13 of gestation (before GA reaches 90 days), the combined use of sFlt-1 and PIGF results in a statistically improved prognosis for EO-PE. Notably, PIGF is generally effective in prognosticating EO-PE when measured after GA reaches 90 days, while sFlt-1 does not appear to provide a reliable prognostic indication in a single measurement after GA reaches 90 days ( Figure 7 ). Surprisingly, when measured before GA reaches 13 weeks (within 90 days), both sFlt-1 and PIGF are able to achieve a prognosis for EO-PE, but sFlt-1 appears to provide greater sensitivity at comparable specificity values, preferably higher than 0.6 ( Figure 8 ). Also surprisingly, the combined analysis of sFlt-1 and PIGF shows that an unexpected synergistic enhancement of the EO-PE prognosis occurs when measured before GA reaches 90 days ( Figure 9 ).

[0118] In an embodiment, the method further comprises:

[0119] a. determining or providing the maternal age, body mass index, and / or uterine artery Doppler measurements of said subject,

[0120] b. wherein a combination of the level of sFlt-1 or a fragment thereof, preferably in combination with the level of PIGF or a fragment thereof, and the maternal age, body mass index, and / or uterine artery Doppler measurements of the subject indicates early-onset preeclampsia occurring before the end of week 33 of gestation (before GA reaches 231 days).

[0121] In an embodiment, the method further comprises:

[0122] a. determining or providing the mean arterial pressure (MAP) level of said subject,

[0123] b. A combination of the level of sFlt-1 or a fragment thereof, preferably in combination with the level of PIGF or a fragment thereof, and further in combination with the MAP level of the subject indicates early-onset preeclampsia occurring before the end of 33 weeks of gestation (before 231 days of GA).

[0124] As follows Figure 10 As shown, the combination of sFlt-1 and PIGF measurements shows further improved diagnostic ability when combined with additional uterine artery Doppler measurements.

[0125] In an embodiment, the method comprises:

[0126] a. Determining the level of sFlt-1 or a fragment thereof and the level of PIGF or a fragment thereof in a first sample and / or a second sample isolated from the subject, and

[0127] b. Determining or providing the maternal age, body mass index (BMI), and uterine artery Doppler measurements of the subject, and optionally the mean arterial pressure (MAP),

[0128] c. A combination of the level of sFlt-1 or a fragment thereof, the level of PIGF or a fragment thereof, with the maternal age, body mass index (BMI), and uterine artery Doppler measurements of the subject, and optionally the mean arterial pressure (MAP) indicates the likelihood of early-onset preeclampsia occurring before the end of 33 weeks of gestation (before 231 days of GA).

[0129] In an embodiment, the method comprises:

[0130] a. Determining the level of sFlt-1 or a fragment thereof and the level of PIGF or a fragment thereof in a first sample and / or a second sample isolated from the subject, and

[0131] b. Determining or providing the maternal age, body mass index (BMI), and mean arterial pressure (MAP) of the subject, and optionally the uterine artery Doppler measurements,

[0132] c. A combination of the level of sFlt-1 or a fragment thereof, the level of PIGF or a fragment thereof, with the maternal age, body mass index (BMI), and mean arterial pressure (MAP) of the subject, and optionally the uterine artery Doppler measurements indicates the likelihood of early-onset preeclampsia occurring before the end of 33 weeks of gestation (before 231 days of GA).

[0133] In an embodiment, the method further comprises determining or providing the maternal age, body mass index, and / or uterine artery Doppler measurements of a subject, wherein the level of sFlt-1 or a fragment thereof in the first sample and / or the second sample, in combination with the maternal age, body mass index, and / or uterine artery Doppler measurements of the subject, preferably in combination with the level of PIGF or a fragment thereof in the first sample and / or the second sample and / or preferably in combination with the mean arterial pressure (MAP) level of the subject, indicates early-onset preeclampsia before the end of 33 weeks of gestation (before 231 days of GA).

[0134] In an embodiment of the invention, the level of sFlt-1 or a fragment thereof, preferably the level of the sFlt-1 or a fragment thereof, the level of the PIGF or a fragment thereof, in combination with the maternal age, body mass index (BMI), and uterine artery Doppler measurements of the subject, and optionally the mean arterial pressure (MAP), indicates an early onset of preeclampsia starting from 20 weeks of gestation until the end of 33 weeks of gestation (before 231 days of GA).

[0135] In an embodiment of the invention, the level of sFlt-1 or a fragment thereof, preferably the level of the sFlt-1 or a fragment thereof, the level of the PIGF or a fragment thereof, in combination with the maternal age, body mass index (BMI), and uterine artery Doppler measurements of the subject, and optionally the mean arterial pressure (MAP), further indicates the subsequent incidence of intrauterine fetal death (IUFD).

[0136] In an embodiment of the invention, the first sample and / or the second sample is a body fluid sample, such as a blood sample, such as a venous blood sample, a capillary blood sample, a serum sample, a plasma sample, a vaginal secretion sample, a saliva sample, or an amniotic fluid sample, preferably a blood sample, a serum sample, or a plasma sample.

[0137] In an embodiment of the invention, a higher second level of sFlt-1 or a fragment thereof compared to the first level indicates initiation or modification of treatment of the subject to reduce the risk of developing preeclampsia, delay the onset time point of preeclampsia, and / or reduce the severity of preeclampsia, for example, by balancing the angiogenic / anti-angiogenic processes in placental development, reducing blood pressure, and / or protecting organ functions, such as the functions of the kidney and / or liver.

[0138] In embodiments of the present invention, the treatment is selected from the group consisting of: one or more diuretics, β-blockers, ACE inhibitors, angiotensin II receptor blockers, calcium channel blockers, α-blockers, methyldopa, central agonists and vasodilators, VEGF, PLGF, statins, arginine vasopressin receptor antagonists, L-arginine, citrulline, arginase inhibitors (nor-NOHA), iron chelators (deferoxamine), heparin, magnesium sulfate, diazepam, phenytoin, vitamin D, calcium, selenium inhibitory molecules, extracorporeal extraction such as apheresis, lifestyle advice, ambulation monitoring, increasing the frequency of maternal and fetal monitoring, preferably low-dose acetylsalicylic acid or metformin.

[0139] In embodiments of the present invention, the treatment comprises administering acetylsalicylic acid.

[0140] In embodiments of the present invention, the treatment comprises administering metformin.

[0141] Accordingly, the present invention relates to a method for treating a pregnant subject to reduce the risk of early-onset preeclampsia, comprising

[0142] a. prognosticating, predicting, risk-assessing, and / or risk-stratifying early-onset preeclampsia in a pregnant subject, comprising

[0143] - determining a first level of soluble fms-like tyrosine kinase-1

[0144] (sFlt-1) or a fragment thereof in a first sample isolated from the subject, wherein the first sample is isolated before the end of week 14 of gestation (before 100 days GA),

[0145] - determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, wherein the second sample is isolated after the first sample,

[0146] - wherein a higher second level of sFlt-1 or a fragment thereof compared to the first level indicates early-onset preeclampsia occurring before the end of week 33 of gestation (before 231 days GA), and

[0147] b. administering a treatment to the subject to reduce the risk of development, delay the time point of onset, and / or reduce the severity of preeclampsia.

[0148] Accordingly, the present invention relates to a method for treating a pregnant subject to reduce the risk of early-onset preeclampsia, comprising:

[0149] a. prognosticating, predicting, risk-assessing, and / or risk-stratifying early-onset preeclampsia in a pregnant subject, comprising

[0150] - Determine a first level of soluble fms-like tyrosine kinase-1

[0151] (sFlt-1) or a fragment thereof in a first sample isolated from the subject, wherein the first sample is isolated before the end of the 13th week of pregnancy (before 90 days of GA),

[0152] - Determine a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, wherein the second sample is isolated after the first sample,

[0153] - wherein a higher second level of sFlt-1 or a fragment thereof compared to the first level indicates early-onset preeclampsia occurring before the end of the 33rd week of pregnancy (before 231 days of GA), and

[0154] b. Administer a treatment to the subject to reduce the risk of development, delay the time point of onset, and / or reduce the severity of preeclampsia.

[0155] Accordingly, the present invention relates to a method for treating a pregnant subject to reduce the risk of early-onset preeclampsia, comprising:

[0156] a. Prognosing, predicting, risk-assessing, and / or risk-stratifying early-onset preeclampsia in a pregnant subject, comprising

[0157] - Determine a first level of soluble fms-like tyrosine kinase-1

[0158] (sFlt-1) or a fragment thereof in a first sample isolated from the subject, wherein the first sample is isolated before the end of the 9th week of pregnancy (before 63 days of GA),

[0159] - Determine a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, wherein the second sample is isolated after the first sample,

[0160] - wherein a higher second level of sFlt-1 or a fragment thereof compared to the first level indicates early-onset preeclampsia occurring before the end of the 33rd week of pregnancy (before 231 days of GA), and

[0161] b. Administer a treatment to the subject to reduce the risk of development, delay the time point of onset, and / or reduce the severity of preeclampsia.

[0162] In an embodiment, the treatment administered involves or comprises balancing the angiogenic / anti-angiogenic processes in placental development, reducing blood pressure, and / or protecting organ functions such as the functions of the kidney and / or liver.

[0163] In embodiments of the treatment method, the treatment is selected from the group consisting of: one or more diuretics, β-blockers, ACE inhibitors, angiotensin II receptor blockers, calcium channel blockers, α-blockers, statins (such as pravastatin), methyldopa, central agonists and vasodilators, VEGF, PLGF, statins, arginine vasopressin receptor antagonists, L-arginine, citrulline, arginase inhibitors (nor-NOHA), iron chelators (deferoxamine), heparin, anticoagulants such as acetylsalicylic acid (also known as C9H8O4 or aspirin) and heparin, magnesium sulfate, diazepam, phenytoin, vitamin D, calcium, selenium inhibitory molecules, extracorporeal extraction such as apheresis, lifestyle advice, ambulation monitoring, increasing the frequency of maternal and fetal monitoring, preferably low-dose acetylsalicylic acid or metformin.

[0164] In an embodiment of the treatment method, the treatment includes administering an anticoagulant.

[0165] In an embodiment of the treatment method, the treatment includes administering acetylsalicylic acid.

[0166] In an embodiment of the treatment method, the treatment includes administering a low dose of acetylsalicylic acid, preferably administered at 75 mg to 150 mg per day, such as 75 mg, 80 mg, 81 mg, 85 mg, 90 mg, 95 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg.

[0167] In one embodiment of the treatment method, the treatment includes administering 81 mg of acetylsalicylic acid (aspirin) per day. In one embodiment, the treatment includes administering acetylsalicylic acid (aspirin). In one embodiment, the treatment including administering 81 mg of acetylsalicylic acid (aspirin) starts before 16 weeks of gestational age (before 106 days of GA).

[0168] In an embodiment of the treatment method, the treatment includes administering heparin.

[0169] In an embodiment of the treatment method, the treatment includes administering metformin.

[0170] In an embodiment, a level of sFlt-1 or a fragment thereof that is at least 6% lower than a reference sample indicates starting or modifying treatment of a subject to reduce the risk of developing PE, delaying the onset time point of PE, or at least reducing the severity of PE, such as balancing the angiogenic / anti-angiogenic process in placental development, reducing blood pressure, and protecting organ function, such as the function of the kidney or liver.

[0171] A gynecologist and / or an internist can determine the appropriate treatment for a subject based on the current condition with or without risk factors.

[0172] In an embodiment, aspirin treatment can be initiated before 16 weeks of gestation (before 106 days of GA), which is associated with a significant reduction in preterm PE. The Aspirin for Evidence-Based Preeclampsia Prevention (ASPRE) trial was a multicenter trial that enrolled women identified as being at high risk for preterm PE according to the FMF algorithm and randomly assigned them to receive aspirin or placebo from 11 to 14 weeks of gestation (71 to 98 days of GA) to 37 weeks of gestation (259 days of GA). The results showed that compared with the placebo group, daily low-dose aspirin reduced preterm PE by 62% (relative risk, 0.38; 95% confidence interval [CI], 0.20 - 0.74).

[0173] In an embodiment of the present invention, the subject has one or more risk factors selected from the group consisting of: hypothyroidism, hyperthyroidism, BMI over 24, first pregnancy, history of preeclampsia, race at risk of impairment, multiple pregnancy, migraine, lupus, coagulation disorders (such as increased platelet aggregation), inflammatory diseases, incipient heart disease, diabetes, chronic kidney disease, and chronic hypertension.

[0174] In an embodiment of the present invention, the method further comprises determining the level of at least one additional biomarker or a fragment thereof in a first sample and / or a second sample from the patient, wherein the at least one additional biomarker is selected from the group consisting of: βhCG, copeptin, vasopressin, troponin, BNP, ANP, CRP, eosinophils / leukocytes, IL6, IL11, MR-proADM, VEGF, PAPP-A, PIGF, endoglin, pro-Epil, PP-13, ADAM-12, vitamin D, inhibin-a, activin-a, pentraxin-3, p-selectin, free fetal hemoglobin, alpha-1-microglobulin, unconjugated estriol, alpha-fetoprotein, GDF15, neurophysin 2, LNPEP, ESM1, HGF, pikachurin, haptoglobin, pp13, uE3, CT-proET1, ADAM12, sTNFαR1, RBP4, ICAM, cell-free fetal DNA, FSTL3, visfatin, AFP, MMP9, TIMP1, Flt1, PCT, SHGB, creatinine, GBP1, IGFALS, urinary protein, PAI1 / PAI2, catechol-o-methyltransferase (COMT), heme breakdown products (bilirubin, biliverdin, carbon monoxide, ferritin), arginine breakdown products (urea, ornithine, citrulline, apolipoprotein H, argininosuccinic acid, ammonia), uterine artery Doppler measurements (UtA-Pi), diastolic notch, MAP, blood pressure, smoking, leptin, genetic information, arginine, and cervical length, wherein the level of the at least one additional biomarker and the level of sFlt-1 or a fragment thereof indicate early-onset preeclampsia before the end of week 33 of gestation.

[0175] In an embodiment, the additional markers PAPP-A and / or PIGF are used in combination with sFlt-1 in the first sample and / or the second sample.

[0176] In an embodiment, the additional markers PAPP-A, PIGF, and / or βhCG are used in combination with sFlt-1 in the first sample and / or the second sample.

[0177] In an embodiment, the additional markers MAP, PAPP-A, and / or PIGF are used in combination with sFlt-1 in the first sample and / or the second sample.

[0178] In an embodiment, the additional markers MAP, PAPP-A, βhCG, and / or PIGF are used in combination with sFlt-1 in the first sample and / or the second sample.

[0179] In an embodiment of the present invention, the subject is a nulliparous woman.

[0180] In embodiments of the present invention, the subject has had one or more previous pregnancies.

[0181] In embodiments of the present invention, the subject is a multiple pregnancy.

[0182] In embodiments of the present invention, the subject is suspected of carrying a fetus with a chromosomal abnormality.

[0183] Another aspect of the present invention relates to a kit for carrying out the methods as described herein.

[0184] In an embodiment, the kit comprises:

[0185] - a detection reagent for determining the level of sFlt-1 or a fragment thereof in a sample from a subject, and

[0186] - computer software in the form of a computer-readable medium and / or computer-executable code, the computer-readable medium and / or the computer software being configured to compare two determined levels of sFlt-1 or a fragment thereof.

[0187] In an embodiment, the computer-readable medium and / or the computer software optionally comprises one or more reference levels of sFlt-1 or a fragment thereof, the reference level preferably corresponding to the population mean and / or median of a healthy population, and the computer-readable medium and / or computer software being configured to compare the two determined levels of sFlt-1 or a fragment thereof with the reference level.

[0188] In an embodiment, the computer-readable medium and / or computer software is optionally configured to compare the maternal age, body mass index and / or uterine artery Doppler measurements of the subject with one or more reference levels, the reference level preferably corresponding to the population mean and / or median of a healthy population. In embodiments of the present invention, the software in the kit or software connected to the kit is configured to enable comparison of the molecular markers determined, such as those described herein, and to provide a prognostic statement regarding the risk of EO-PE based on samples obtained at or after 90 days GA, such as a prognostic statement regarding the risk of EO-PE based on a first sample obtained at 90 days GA and a second sample obtained after 90 days GA.

[0189] In embodiments of the present invention, the kit comprises a physical disk or computer-readable medium having the software, alternatively, the kit provides a link or other code, such as a QR code, which is adapted to induce and / or provide a connection to a server via the internet, where the appropriate software is maintained and / or can be executed.

[0190] Additional aspects and embodiments of the present invention :

[0191] The present invention relates to a method for prognosticating, predicting, risk assessing and / or risk stratifying preeclampsia in a pregnant subject, comprising

[0192] a. determining the level of sFlt-1 or a fragment thereof in a sample isolated from the pregnant subject,

[0193] b. wherein the level of the sFlt-1 or a fragment thereof indicates the likelihood of preeclampsia.

[0194] The present invention also relates to a kit for implementing the method of the present invention, which comprises detection reagents for determining the level of sFlt-1 or a fragment thereof in a sample from a subject, and optionally for determining the level of at least one additional biomarker as described herein in a sample from a subject, wherein the kit further comprises reference levels, such as one or more cut-off levels, which correspond to reference levels indicating a high risk or a low risk of preeclampsia.

[0195] Accordingly, the present invention also relates to a method for prognosticating, predicting, risk assessing and / or risk stratifying preeclampsia in a pregnant subject, comprising

[0196] a. determining the level of sFlt-1 or a fragment thereof in a sample isolated from the pregnant subject,

[0197] b. wherein the level of the sFlt-1 or a fragment thereof indicates the likelihood of preeclampsia,

[0198] c. wherein the sample is isolated from the subject no later than the end of the 12th week of pregnancy (no later than 84 days GA).

[0199] In one embodiment, the subject is at 9 to 11 weeks of pregnancy (57 to 77 days GA), preferably at 11 weeks of pregnancy (71 to 77 days GA). In one embodiment, the subject is at 7 to 9 weeks of pregnancy (43 to 63 days GA), preferably at 8 to 9 weeks of pregnancy (50 to 63 days GA).

[0200] Surprisingly, significant differences in the sFlt-1 predictive values (AUC values) for early-onset PE were observed among women recruited at gestational week 11 (GA days 71 to 77), gestational week 12 (GA days 78 to 84), and gestational week 13 (GA days 85 to 91). The AUC value for the sFlt-1 level for predicting early-onset PE in the samples of subjects at gestational week 11 (GA days 71 to 77) was equal to 0.82. The AUC value for the sFlt-1 level for predicting early-onset PE in the samples of subjects at gestational week 12 (GA days 78 to 84) was equal to 0.62. The AUC value for the sFlt-1 level for predicting early-onset PE in the samples of subjects at gestational week 13 (GA days 78 to 91) was equal to 0.50. Surprisingly, a similar trend was also observed when predicting late-onset PE.

[0201] Based on this surprising finding, the advantage is that the likelihood of developing PE can be accurately predicted by measuring the level of sFlt-1 or its fragment in the early stage of pregnancy, especially at gestational week 11 (GA days 71 to 77).

[0202] In an embodiment, the maternal age is from 18 to 34 years old.

[0203] In an embodiment, the maternal age is over 34 years old.

[0204] Surprisingly, a difference in the sFlt-1 predictive values (AUC values) was observed between maternal ages of 18 to 34 years old and maternal ages over 34 years old.

[0205] Pregnant subjects with a maternal age ≥ 34 years old may have a higher probability of having premature birth, hypertension, superimposed PE, severe PE, and a decreased risk of chorioamnionitis.

[0206] Pregnant subjects with a maternal age ≥ 40 years old may have an increased probability of having mild PE, fetal distress, and fetal growth restriction.

[0207] Very advantageously, subjects in the designated risk groups (including maternal ages below 18 years old, over or equal to 34 years old, and over or equal to 40 years old) can receive an accurate prognosis for preeclampsia before the end of gestational week 12 or at a time point until the end of gestational week 12 (until GA day 84).

[0208] In an embodiment, the method as described herein includes:

[0209] a. Comparing the level of sFlt-1 or its fragment measured in the sample with a reference level derived from a reference sample.

[0210] b. A level of sFlt-1 or a fragment thereof that is lower than or equal to a reference level indicates a high risk of preeclampsia, or

[0211] c. A level of sFlt-1 or a fragment thereof that is higher than the reference level indicates a low risk of preeclampsia.

[0212] In an embodiment, the reference level is derived from a reference sample isolated from a pregnant subject who has never had or developed any pregnancy-related hypertensive disorder such as PE or eclampsia.

[0213] In an embodiment, an additional risk parameter is the sex of the fetus. Surprisingly, significant differences in the predicted values (AUC values) of sFlt-1 levels and a significant decrease in sFlt-1 levels compared to the reference level can be observed in samples from subjects carrying at least one female fetus or at least one male fetus. In an embodiment, the level of sFlt-1 in the subject sample is at least 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25% lower than the reference level. Based on this surprising finding, subjects carrying such a risk parameter can be predicted to be at risk of developing PE.

[0214] The present invention also relates to a method for identifying and treating a subject at risk of early-onset preeclampsia (occurring before the end of 33 weeks of gestation, before 231 days of GA), the method comprising:

[0215] (a) diagnosing, prognosticating, predicting, risk-assessing and / or risk-stratifying early-onset preeclampsia in a pregnant subject, including:

[0216] - determining a first level of soluble fms-like tyrosine kinase-1

[0217] (sFlt-1) or a fragment thereof in a first sample isolated from the subject, wherein the first sample is isolated before the end of 14 weeks of gestation (before 100 days of GA),

[0218] - determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, wherein the second sample is isolated after the first sample,

[0219] - wherein a higher second level of sFlt-1 or a fragment thereof compared to the first level indicates early-onset preeclampsia occurring before the end of 33 weeks of gestation (before 231 days of GA).

[0220] (b) Administer a treatment for early-onset preeclampsia to the subject, or administer a treatment to the subject to reduce the risk of early-onset preeclampsia.

[0221] The present invention also relates to a method for identifying a subject at risk of early-onset preeclampsia (occurring before the end of 33 weeks of gestation, before 231 days of GA) and treating the subject, the method comprising:

[0222] (a) Diagnosing, prognosticating, predicting, risk-assessing, and / or risk-stratifying early-onset preeclampsia in a pregnant subject, comprising:

[0223] - Determining a first level of soluble fms-like tyrosine kinase-1

[0224] (sFlt-1) or a fragment thereof in a first sample isolated from the subject, wherein the first sample is isolated before the end of 13 weeks of gestation (before 90 days of GA),

[0225] - Determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, wherein the second sample is isolated after the first sample,

[0226] - wherein a higher second level of sFlt-1 or a fragment thereof compared to the first level indicates early-onset preeclampsia occurring before the end of 33 weeks of gestation (before 231 days of GA).

[0227] (b) Administer a treatment for early-onset preeclampsia to the subject, or administer a treatment to the subject to reduce the risk of early-onset preeclampsia.

[0228] The present invention also relates to a method for identifying a subject at risk of early-onset preeclampsia (occurring before the end of 33 weeks of gestation, before 231 days of GA) and treating the subject, the method comprising:

[0229] (a) Diagnosing, prognosticating, predicting, risk-assessing, and / or risk-stratifying early-onset preeclampsia in a pregnant subject, comprising:

[0230] - Determining a first level of soluble fms-like tyrosine kinase-1

[0231] (sFlt-1) or a fragment thereof in a first sample isolated from the subject, wherein the first sample is isolated before the end of 9 weeks of gestation (before 63 days of GA),

[0232] - Determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, wherein the second sample is isolated after the first sample,

[0233] - A second level of sFlt-1 or a fragment thereof that is higher compared to the first level indicates early-onset preeclampsia occurring before the end of week 33 of gestation (before 231 days GA).

[0234] (b) Administering a treatment for early-onset preeclampsia to the subject, or administering a treatment to the subject to reduce the risk of early-onset preeclampsia.

[0235] The present invention also relates to a method for detecting soluble fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof in a sample from a subject, the method comprising:

[0236] - Providing a first sample from the subject, preferably a blood sample or a sample derived from a blood sample, which has a complex comprising sFlt-1 or a fragment thereof and at least one binding agent that binds thereto; and preferably providing another sample from the subject, preferably a blood sample or a sample derived from a blood sample, which has a complex comprising PIGF or a fragment thereof and at least one binding agent that binds thereto;

[0237] - Providing a second sample from the subject, preferably a blood sample or a sample derived from a blood sample, which has a complex comprising sFlt-1 or a fragment thereof and at least one binding agent that binds thereto; and preferably providing another sample from the subject, preferably a blood sample or a sample derived from a blood sample, which has a complex comprising PIGF or a fragment thereof and at least one binding agent that binds thereto;

[0238] - Wherein the sFlt-1 level in the second sample is higher than that in the first sample, and preferably wherein the PIGF level is higher in the second sample than in the first sample, or wherein the level is lower than or higher than a threshold (such as any of the thresholds disclosed herein), preferably the population mean and / or population median of the sFlt-1 level of normal pregnancy at any given time point from the corresponding patient population.

[0239] The present invention also relates to a method for treating early-onset preeclampsia and / or reducing the risk of early-onset preeclampsia, or for administering a treatment for early-onset preeclampsia to a subject, the method comprising:

[0240] - Administering a treatment for early-onset preeclampsia to the subject,

[0241] - Wherein it has been determined that the sFlt-1 level in a second body fluid sample (preferably a blood sample or a sample derived from a blood sample) of the subject is higher than the level in the first sample, or the level is lower than or higher than a threshold (such as any of the thresholds disclosed herein), preferably the population mean and / or population median of the sFlt-1 level of normal pregnancy at any given time point from the corresponding patient population.

[0242] Embodiments describing the method of the present invention can be used to describe the kits of the present invention, and vice versa. The features in any given embodiment of the method are applicable to other embodiments of the method, and the features of any given method are also applicable to other methods of the present invention. The present invention is unified in the novel and beneficial application of the prognostic marker sFlt-1, which is used to indicate the risk of developing EO-PE based on two samples. Therefore, the relevant features described for one aspect herein can be used to describe any given aspect of the present invention in a manner understandable to those skilled in the art.

[0243] Alternative embodiments :

[0244] In one embodiment, the present invention relates to a method for prognosticating, predicting, risk assessing, and / or risk stratifying early-onset preeclampsia in a pregnant subject, the method comprising

[0245] a. determining a first level of soluble fms-like tyrosine kinase-1

[0246] (sFlt-1) or a fragment thereof in a first sample isolated from the subject, wherein the first sample is isolated before the end of the 14th week of pregnancy,

[0247] b. determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, wherein the second sample is isolated after the first sample,

[0248] c. wherein a higher second level of sFlt-1 or a fragment thereof compared to the first level indicates the occurrence of early-onset preeclampsia before the end of the 33rd week of pregnancy.

[0249] In one embodiment, the first sample is isolated from the subject before the end of the 12th week of pregnancy.

[0250] In one embodiment, the second sample is isolated from the subject after the end of the 12th week of pregnancy.

[0251] In one embodiment, the second sample is isolated from the subject after the end of the 14th week of pregnancy.

[0252] In one embodiment, the second sample is isolated after the end of the 20th week of pregnancy, preferably between the end of the 20th week and the end of the 22nd week of pregnancy.

[0253] In one embodiment, the method further comprises determining the level of placental growth factor (PIGF) or a fragment thereof in a first sample and / or a second sample from the patient, wherein a combination of the level of sFlt-1 or a fragment thereof and the level of PIGF or a fragment thereof in the first sample and / or the second sample indicates early-onset preeclampsia before the end of week 33 of gestation.

[0254] In one embodiment, the method further comprises determining or providing the maternal age, body mass index, mean arterial pressure (MAP), and / or uterine artery Doppler measurements of a subject, wherein a combination of the level of sFlt-1 or a fragment thereof in the first sample and / or the second sample and the maternal age, body mass index, mean arterial pressure (MAP), and / or uterine artery Doppler measurements of the subject, preferably in combination with the level of PIGF or a fragment thereof in the first sample and / or the second sample, indicates early-onset preeclampsia before the end of week 33 of gestation. Detailed Description

[0255] All cited documents of this patent and non-patent literature are hereby incorporated by reference in their entirety.

[0256] The present invention relates to a method for prognosticating, predicting, risk-assessing, and / or risk-stratifying preeclampsia in a pregnant subject, comprising: a) determining the level of sFlt-1 or a fragment thereof in a sample isolated from the pregnant subject, b) wherein the level of the sFlt-1 or a fragment thereof indicates the likelihood of preeclampsia.

[0257] As used herein, the term "subject" shall mean a mammal, including but not limited to a human or non-human mammal, such as a cow, horse, dog, sheep, or cat. This definition includes pregnant, postpartum, and non-pregnant mammals.

[0258] In the present invention, the terms "risk assessment" and "risk stratification" relate to classifying a subject into different risk groups based on the further prognosis of the subject. Risk assessment also relates to stratifying the application of preventive measures and / or treatment measures. The term "therapy stratification" particularly relates to grouping or classifying patients into different groups, such as risk groups or treatment groups that receive certain different treatment measures according to their classification.

[0259] As used herein, "prognosis" relates to predicting the outcome or specific risks of a subject developing into PE. This may also include an estimate of the recovery opportunity or the opportunity of adverse outcomes of the subject. The assessment of the severity of PE may also be covered by the terms "prognosis" or "risk assessment" or "risk stratification".

[0260] As used herein, "preeclampsia" (PE) is used in its ordinary meaning. PE can be defined according to widely recognized criteria, such as a blood pressure of at least 140 / 90 mmHg and a urinary excretion of at least 0.3 grams of protein in 24-hour urine protein excretion (or at least +1 or more in a dipstick test), with both of the above parameters measured twice, at an interval of 4 to 6 hours.

[0261] Preeclampsia is considered a multisystem disorder characterized by hypertension with proteinuria or edema, or hypertension with proteinuria and edema, glomerular dysfunction, cerebral edema, hepatic edema, or coagulation abnormalities caused by pregnancy or recent pregnancy. Preeclampsia usually occurs after the 20th week of pregnancy. Preeclampsia is generally defined as some combination of the following symptoms: (1) systolic blood pressure (BP) > 140 mmHg and diastolic blood pressure > 90 mmHg after 20 weeks of pregnancy (GA after 140 days), usually measured twice at an interval of 4 to 168 hours, (2) new-onset proteinuria (dipstick method 1+, protein > 300 mg in a 24-hour urine collection, or protein / creatinine ratio > 0.3 in a single random urine sample), and (3) resolution of hypertension and proteinuria 12 weeks postpartum. Severe preeclampsia is generally defined as (1) diastolic blood pressure > 110 mmHg (usually measured twice at an interval of 4 to 168 hours) or (2) proteinuria characterized by a protein measurement of more than 3.5 g in a 24-hour urine collection, or protein at least 3+ measured by dipstick method in two random urine samples.

[0262] In preeclampsia, hypertension and proteinuria generally occur successively within 7 days. In severe preeclampsia, severe hypertension, severe proteinuria, and HELLP syndrome (hemolysis, elevated liver enzymes, low platelets) or eclampsia can occur simultaneously, or only one symptom may appear at a time. Severe preeclampsia may occasionally lead to the development of seizures. This severe form of the syndrome is called eclampsia. "Eclampsia" can also include dysfunction or damage to several organs or tissues, such as the liver (e.g., hepatocyte injury, periportal necrosis) and the central nervous system (e.g., cerebral edema and intracerebral hemorrhage). The cause of seizures is thought to be secondary to the development of cerebral edema and focal spasm of small renal blood vessels.

[0263] "Severe preeclampsia" or "preeclampsia of high severity" is also defined according to established criteria as a blood pressure of at least 160 / 110 mmHg at least twice, at an interval of 6 hours, and a protein greater than 5 grams in 24-hour urine protein excretion, or persistent proteinuria 3+ in a dipstick test.

[0264] Severe preeclampsia can include HELLP syndrome (hemolysis, elevated liver enzymes, low platelet count). Other factors of severe preeclampsia can include: intrauterine growth restriction (IUGR) in less than 10% percentile population according to US demographics; persistent neurological symptoms (headache, visual disturbances), epigastric pain, oliguria (less than 500 mL / 24 h), serum creatinine greater than 1.0 mg / dL, elevated liver enzymes (twice the normal value), thrombocytopenia (less than 100,000 cells / μL).

[0265] As used herein, "preterm birth" is defined as delivery before 37 completed weeks of gestation (before GA of 253 days).

[0266] As used herein, "preterm PE" is defined as PE with delivery before 37 completed weeks of gestation (before GA of 253 days).

[0267] As used herein, "early-onset preeclampsia" shall refer to the occurrence of symptoms of preeclampsia starting from the 20th week of gestation to the end of the 33rd week of gestation (GA of 148 days to 231 days). In an embodiment, early-onset preeclampsia refers to cases with delivery before 34 completed weeks of gestation (before GA of 232 days).

[0268] As used herein, "mid-onset preeclampsia" shall refer to the occurrence of symptoms of preeclampsia starting from the 34th week of gestation to the end of the 36th week of gestation (GA of 232 days to 252 days).

[0269] As used herein, "late-onset preeclampsia" shall refer to the occurrence of symptoms of preeclampsia starting from the 37th week of gestation (starting from GA of 253 days).

[0270] For example, "symptoms of preeclampsia" can refer to the following symptoms: (1) systolic blood pressure (BP) > 140 mmHg and diastolic blood pressure > 90 mmHg after 20 weeks of gestation, (2) new-onset proteinuria (urine dipstick method 1+, protein > 300 mg in 24-hour urine collection, or random urine protein / creatinine ratio > 0.3), and (3) hypertension and proteinuria resolve by 12 weeks postpartum. Symptoms of preeclampsia can also include renal dysfunction and glomerular endothelial hyperplasia or hypertrophy.

[0271] As used herein, "symptoms of eclampsia" refer to any of the following symptoms caused by pregnancy or recent pregnancy effects: seizure, coma, thrombocytopenia, hepatic edema, pulmonary edema, and cerebral edema.

[0272] "At risk of developing" a pregnancy-related hypertensive disorder such as preeclampsia or eclampsia means that the subject does not currently have a pregnancy-related hypertensive disorder, but has a higher than average likelihood of developing such a disorder. Such subjects at risk include pregnant subjects in whom the sFlt-1 level in the blood is reduced by at least 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25% compared to a reference level, without limitation. In some embodiments, the patient may not exhibit other signs of a pregnancy-related hypertensive disorder (such as preeclampsia).

[0273] As used herein, the term "likelihood of preeclampsia" means that the subject does not currently have preeclampsia, but has a higher than average likelihood of developing preeclampsia. This prognosis or risk assessment is based on the measured level of sFlt-1 or a fragment thereof. The level of sFlt-1 or a fragment thereof indicates the likelihood of developing preeclampsia. In one embodiment, the level of sFlt-1 or a fragment thereof measured in a sample is compared to a reference level, wherein a level of sFlt-1 or a fragment thereof that is less than or equal to the reference level indicates a high risk of preeclampsia, and a level of sFlt-1 or a fragment thereof that is greater than the reference level indicates a low risk of preeclampsia.

[0274] As used herein, "high risk of preeclampsia" shall mean a high risk of developing preeclampsia, but the subject does not currently have preeclampsia.

[0275] As used herein, "low risk of preeclampsia" shall mean a low risk of developing preeclampsia, but the subject does not currently have preeclampsia.

[0276] "Pregnancy-related hypertensive disorder" shall mean any condition or disease associated with or characterized by elevated blood pressure during pregnancy. These conditions and diseases include preeclampsia (including preterm preeclampsia, severe preeclampsia), eclampsia, gestational hypertension, HELLP syndrome (hemolysis, elevated liver enzymes, low platelets), placental abruption, chronic hypertension in pregnancy, intrauterine growth restriction pregnancy, and small for gestational age (SGA) pregnancy.

[0277] As used herein, the term "soluble Flt-1 (sFlt-1)" (soluble fms-like tyrosine kinase 1, also known as sVEGF-R1) refers to a soluble form of the Flt-1 receptor that is homologous to the protein defined by GenBank accession number U01134 or UniProt P17948 or the entry name VGFR1_HUMAN and has sFlt-1 biological activity. The biological activity of an sFlt-1 polypeptide can be determined using any standard method, for example, by measuring the binding of sFlt-1 to VEGF. sFlt-1 lacks the transmembrane domain and cytoplasmic tyrosine kinase domain of the Flt-1 receptor. sFlt-1 can bind to VEGF and PIGF with high affinity, but it cannot induce proliferation or angiogenesis and is thus functionally distinct from the Flt-1 and KDR receptors. sFlt-1 was initially purified from human umbilical vein endothelial cells and was later shown to be produced by trophoblast cells in vivo. As used herein, sFlt-1 includes any sFlt-1 family member or isotype.

[0278] As used herein, the term "specifically binds" means that a compound or antibody or any detection reagent recognizes and binds to a polypeptide (i.e., sFlt-1 or any fragment thereof), but does not substantially recognize and bind to other molecules in a sample (e.g., a biological sample that naturally contains the sFLt-1 polypeptide or any fragment thereof). In one embodiment, an antibody that specifically binds sFlt-1 does not bind Flt-1.

[0279] As used herein, "detection reagent" and the like refer to a reagent suitable for assaying a biomarker as described herein (e.g., sFlt-1, PAPP-A, PIGF). Such exemplary detection reagents are, for example, ligands such as antibodies or fragments thereof that specifically bind to a peptide or epitope of a biomarker as described herein. Such ligands can be used in immunoassays as described above. Other reagents used in immunoassays to measure the level of a biomarker can also be included in a kit and are also considered detection reagents herein. A detection reagent can also relate to a reagent for detecting a biomarker or a fragment thereof by a mass spectrometry-based method. Thus, such a detection reagent can also be a reagent for preparing a sample for MS analysis, such as an enzyme, a chemical, a buffer, etc. A mass spectrometer can also be considered a detection reagent.

[0280] A detection reagent according to the present invention can also be, for example, a calibration solution that can be used to assay and compare the levels of a biomarker.

[0281] According to the present invention, an antibody can be a monoclonal antibody as well as a polyclonal antibody. In particular, an antibody that specifically binds at least to sFlt-1 or a fragment thereof is used.

[0282] An antibody is considered specific if its affinity for the molecule of interest (e.g., sFlt-1 or a fragment thereof) is at least 50-fold, preferably 100-fold, and most preferably at least 1000-fold the affinity for other molecules contained in a sample containing the molecule of interest. How to develop and select antibodies with a given specificity is well known in the art. In the context of the present invention, monoclonal antibodies are preferably used as detection reagents. The antibody or antibody-binding fragment specifically binds to the marker or a fragment thereof as defined herein. In particular, the antibody or antibody-binding fragment binds to the sFlt-1 peptide as defined herein. Thus, the peptide as defined herein can also be an epitope to which the antibody specifically binds. Additionally, antibodies or antibody-binding fragments that specifically bind to sFlt-1 or a fragment thereof are used in the methods and kits of the present invention.

[0283] Further, antibodies or antibody-binding fragments that specifically bind to sFlt-1 or a fragment thereof and optionally to other markers of the present invention (such as PAPP-A or PIGF) are used in the methods and kits of the present invention.

[0284] Exemplary immunoassays can be luminescence immunoassay (LIA), radioimmunoassay (RIA), chemiluminescence immunoassay, fluorescence immunoassay, enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), bead arrays based on luminescence, bead arrays based on magnetic beads, protein microarray assays, rapid test formats, rare earth complex assays. Further, assays suitable for point-of-care testing and rapid test formats (such as immunochromatographic strip tests) can also be employed. Automated immunoassays, such as the B·R·A·H·M·S KRYPTOR assay, are also contemplated.

[0285] Alternatively, the scope of the present invention may cover other capture molecules or molecular scaffolds that specifically and / or selectively recognize sFlt-1, rather than antibodies. As used herein, the term "capture molecule" or "molecular scaffold" includes molecules that can be used to bind a target molecule or molecule of interest, i.e., an analyte (such as sFlt-1), from a sample. Thus, the capture molecule must be sufficiently shaped both spatially and in terms of surface characteristics such as surface charge, hydrophobicity, hydrophilicity, presence or absence of Lewis donors and / or acceptors, to specifically bind the target molecule or molecule of interest. Thus, binding can be mediated, for example, by ionic, van der Waals, π-π, σ-π, hydrophobic or hydrogen bond interactions, or a combination of two or more of the foregoing interactions or covalent interactions between the capture molecule or molecular scaffold and the target molecule or molecule of interest. In the context of the present invention, the capture molecule or molecular scaffold can, for example, be selected from the group consisting of nucleic acid molecules, carbohydrate molecules, PNA molecules, proteins, peptides and glycoproteins. Capture molecules or molecular scaffolds include, for example, nucleic acid aptamers, DARPins (designed ankyrin repeat proteins). Affimers, etc. are included.

[0286] The method according to the invention can further be embodied as a homogeneous method, in which the sandwich complex formed by one or more antibodies and the marker to be detected (sFlt-1 or a fragment thereof) remains suspended in the liquid phase. In this case, preferably, when two antibodies are used, the two antibodies are labeled with the respective parts of the detection system, which causes signal generation or signal triggering when the two antibodies are incorporated into a single sandwich structure. Such techniques will be embodied specifically as fluorescence enhancement or fluorescence quenching detection methods. A particularly preferred aspect relates to the use of pairs of detection reagents, such as those described in US4882733, EP0180492 or EP0539477 and the prior art cited therein. In this way, it becomes possible to measure only directly in the reaction mixture the reaction product of the two labeled components comprising a single immune complex. For example, such techniques are available under the trade name TRACE TM (Time Resolved Amplification Cryptate Emission) or KRYPTOR TM is provided, thus implementing the teachings of the applications cited above. Thus, in a particularly preferred aspect, a diagnostic device is used to carry out the methods provided herein. For example, the level of sFlt-1 or a fragment thereof, and / or the level of any additional markers (such as PAPP-A, PIGF) of the methods provided herein is determined. In a particularly preferred aspect, the diagnostic device is the B·R·A·H·M·S KRYPTOR.

[0287] In an embodiment, the quantitative determination of sFlt-1 can be performed by an automated immunofluorescence assay, namely the B·R·A·H·M·S sFlt-1 KRYPTOR assay, preferably in conjunction with the B·R·A·H·M·S PIGF plus KRYPTOR assay. The lower limit of detection and the upper limit of detection are 22 pg / mL and 90,000 pg / mL, and the B·R·A·H·M·S sFlt-1 KRYPTOR provides the measurement range required for reliable detection of clinical sFlt-1 values throughout pregnancy. This assay requires only 8 μL of serum sample to be isolated from the subject.

[0288] Those skilled in the art can obtain or develop means for identifying, measuring, assaying, and / or quantifying the above-mentioned sFlt-1 molecule or its fragments or variants, as well as other markers of the present invention, according to standard molecular biology practices.

[0289] The levels of the markers of the present invention (e.g., sFlt-1 or its fragments, PAPP-A or its fragments, or other markers) can also be determined by mass spectrometry (MS)-based methods. Such methods can include detecting the presence, amount, or concentration of one or more modified or unmodified fragment peptides (e.g., sFlt-1 or PAPP-A, PIGF) in the biological sample or in the protein digest (e.g., tryptic digest) from the sample, and optionally separating the sample by chromatography, followed by MS analysis of the prepared and optionally separated sample. For example, selected reaction monitoring (SRM), multiple reaction monitoring (MRM), or parallel reaction monitoring (PRM) mass spectrometry can be used for MS analysis, especially for determining the amount of sFlt-1 or its fragments.

[0290] As used herein, the term "mass spectrometry" or "MS" refers to an analytical technique for identifying compounds by their mass. To enhance the mass resolution and mass determination capabilities of mass spectrometry, the sample can be processed prior to MS analysis.

[0291] Accordingly, the present invention relates to MS detection methods that can be associated with immunocapture techniques, and sample preparation and / or chromatography methods, preferably in combination with liquid chromatography (LC), more preferably with high performance liquid chromatography (HPLC) or ultra-high performance liquid chromatography (UHPLC).

[0292] Sample preparation methods include techniques for lysing, separating, and digesting a sample into peptides, as well as techniques for consuming, enriching, dialyzing, desalting, alkylating, and / or reducing the peptides. However, these steps are optional. Analyte ions can be selectively detected by tandem mass spectrometry (MS / MS). Tandem mass spectrometry is characterized by a mass selection step (as used herein, the term "mass selection" means separating ions having a specific m / z or narrow m / z / s range), followed by fragmentation of the selected ions and mass analysis of the resulting product (fragment) ions.

[0293] As used herein, the term "detection reagent that specifically binds to sFlt-1 and its fragments" shall mean a detection reagent that recognizes and binds to sFlt-1 polypeptides and their fragments, but substantially does not recognize and bind to other molecules in a sample (e.g., a biological sample that naturally contains sFlt-1 polypeptides).

[0294] Detection reagents for determining the level of sFlt-1 or its fragments and optionally for determining the level of PAPP-A, PIGF, and / or their fragments are preferably selected from those necessary for performing the method, such as antibodies against sFlt-1; suitable labels (such as fluorescent labels), preferably two separate fluorescent labels suitable for use in a KRYPTOR assay; sample collection tubes.

[0295] As used herein, the term "determining the level of sFlt-1 or its fragments in a sample" means any means of determining sFlt-1 or its fragments.

[0296] As used herein, "fragment" shall mean a portion of a polypeptide or nucleic acid molecule. This portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of the reference nucleic acid molecule or polypeptide. Fragments can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 813, or more nucleotides, or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 186, 200, 250, 271, or more amino acids. Preferred fragments have sFlt-1 biological activity.

[0297] The sensitivity and specificity of a diagnostic and / or prognostic test depend not only on the analytical "quality" of the test, but also on the definition of an abnormal result. In implementation, the receiver operating characteristic curve (ROC curve) is typically calculated by plotting the variable value against its relative frequency in the "normal" population (i.e., apparently healthy individuals without infection) and the "disease" population (e.g., subjects with an infection). For any particular biomarker (such as sFlt-1), the distributions of biomarker levels in subjects with and without the disease / condition may overlap. In such cases, the test does not absolutely distinguish normal from disease with 100% accuracy, and the overlapping region can indicate where the test fails to distinguish normal from disease. A threshold is selected below which the test is considered abnormal, above which the test is considered normal, or below or above which the test indicates a particular condition, such as an infection. The area under the ROC curve is a measure of the probability that the measured value will allow correct discrimination of the condition. Even if the test result does not necessarily give an exact number, an ROC curve can be used. As long as the results can be ranked, an ROC curve can be created. For example, the test results for "disease" samples can be ranked according to degree (e.g., 1 = low, 2 = normal, 3 = high). This ranking can be correlated with the results in the "normal" population, and then an ROC curve can be created. These methods are well known in the art; see, for example, Hanley et al., 1982. Radiology 143:29-36. Preferably, the threshold is selected to provide the following area under the ROC curve: greater than about 0.5, more preferably greater than about 0.7, still more preferably greater than about 0.8, even more preferably greater than about 0.85, and most preferably greater than about 0.9. In this context, the term "about" means + / - 5% of the given measurement value.

[0298] The horizontal axis of the ROC curve represents (1 - specificity), which increases as the false positive rate increases. The vertical axis of the curve represents sensitivity, which increases as the true positive rate increases. Thus, for a particular cutoff value selected, the (1 - specificity) value can be determined, and the corresponding sensitivity can be obtained. The area under the ROC curve is a measure of the probability that the measured biomarker level will allow correct discrimination of the disease or condition. Thus, the area under the ROC curve can be used to determine the effectiveness of the test. The AUC (area under the curve) makes it easy to compare one ROC curve with another. An ROC curve with a larger AUC represents a better logistic regression.

[0299] As used herein, the multiple of the median (MoM) is a measure of the degree to which an individual test result, such as the sFlt-1 level of a subject, deviates from the median of a reference population (such as the median sFlt-1 level of a healthy population, such as the median sFlt-1 level of a population of pregnant subjects who have not developed EO-PE). The MoM is calculated by dividing the individual test result by the median of the reference population. Thus, the MoM value describes how much higher or lower the measured value is relative to the median of the reference population. In one embodiment, the MoM is adjusted for one or more additional factors, such as gestational age and / or BMI.

[0300] As used herein, terms such as "marker", "surrogate", "prognostic marker", "factor", or "biomarker" are used interchangeably and refer to a measurable and quantifiable biomarker (e.g., a specific protein or enzyme concentration or a fragment thereof, a specific hormone concentration or a fragment thereof, or the presence of a biological substance or a fragment thereof) that serves as an indicator for health and physiologically relevant assessments (such as the risk of a disease / condition / clinical situation, preferably an adverse event). A marker or biomarker is defined as a characteristic that can be objectively measured and evaluated as an indicator of a normal biological process, a pathogenic process, or a pharmacological response to a therapeutic intervention. Biomarkers can be measured in a sample (such as blood, plasma, urine, or a tissue assay).

[0301] The gestational stage at which the methods described herein can be implemented depends on various clinical factors, including the overall health of the subject and the severity of preeclampsia symptoms.

[0302] In certain embodiments, the method is performed on a subject no later than the end of the 12th week of gestation (before GA reaches 84 days). The end of the 12th week of gestation shall refer to the last day of the 12th week of gestation (GA reaches 84 days), or the second-to-last day (GA reaches 83 days), the third-to-last day (GA reaches 82 days), or the fourth-to-last day (GA reaches 81 days) of the 12th week of gestation.

[0303] In certain embodiments, the method is performed on a subject no later than the end of the 13th week of gestation (before GA reaches 90 days).

[0304] In some embodiments, the method is performed on a subject at 9 weeks of gestation (GA of 57 to 63 days). In one embodiment, the method is performed at 10 weeks of gestation (GA of 64 to 70 days). In one embodiment, the method is performed at 11 weeks of gestation (GA of 71 to 77 days). In an embodiment, the method is performed after 12 weeks of gestation (after GA of 84 days). In an embodiment, the method is performed at 13 weeks of gestation (GA of 85 to 91 days). In an embodiment, the method is performed at 14 weeks of gestation (GA of 92 to 98 days). In an embodiment, the method is performed between 15 and 20 weeks of gestation and including 15 and 20 weeks of gestation (GA between 99 and 140 days).

[0305] As used herein, "sample" shall refer to a body fluid sample, such as a blood sample, such as a venous blood sample, a capillary blood sample, a serum sample, a plasma sample, a vaginal secretion sample, a saliva sample, or an amniotic fluid sample, cerebrospinal fluid, preferably a blood sample, a serum sample, or a plasma sample.

[0306] In the context of the present invention, "plasma" is the almost cell-free supernatant obtained after centrifugation of anticoagulated blood. Exemplary anticoagulants include calcium ion-binding compounds, such as EDTA or citrate, and thrombin inhibitors (such as heparin salts or hirudin). Cell-free plasma can be obtained by centrifuging anticoagulated blood (e.g., citrated, EDTA-treated, or heparinized blood) at 2000g to 3000g for at least 15 minutes.

[0307] In the context of the present invention, "serum" is the liquid portion collected from whole blood after allowing it to clot. When centrifuging the clotted blood (coagulated blood), the supernatant can be obtained, and its composition is serum.

[0308] "Sample" also refers to a tissue biopsy (e.g., placental tissue), chorionic villus sample, cell, or other sample obtained from a subject. Ideally, the biological sample includes sFlt-1 nucleic acid molecules or polypeptides, or both.

[0309] As used herein, the term "reference sample" means any sample, standard, or level used for comparison purposes. A "normal reference sample" can be a previous sample taken from the same subject, i.e., a sample from a subject who is pregnant without any pregnancy-related hypertensive disorders (such as preeclampsia or eclampsia), a subject who is pregnant but the sample is taken in the first trimester of pregnancy (e.g., in the first or second trimester of pregnancy, or before the detection of a pregnancy-related hypertensive disorder (such as preeclampsia or eclampsia)), a subject who is pregnant but without a history of pregnancy-related hypertensive disorders (such as preeclampsia or eclampsia), a non-pregnant subject; a purified reference polypeptide sample with a known normal concentration (i.e., not indicating a pregnancy-related hypertensive disorder, such as preeclampsia or eclampsia).

[0310] As used herein, the term "reference level" refers to a value or number derived from a reference sample. A normal reference standard or level can be a value or number derived from a normal subject. Ideally, all reference samples, standards, and levels are matched to the subject sample by at least one of the following criteria: fetal gestational age, maternal age, pre-pregnancy maternal blood pressure, maternal blood pressure during pregnancy, mother's BMI, fetal weight, previous diagnosis of pregnancy-related hypertensive disorders, and family history of pregnancy-related hypertensive disorders.

[0311] In one embodiment, the reference level refers to a value derived from a pregnant subject who has not developed preeclampsia or a pregnancy-related hypertensive disorder (e.g., in the first trimester or second trimester of pregnancy, or before the detection of a pregnancy-related hypertensive disorder (such as preeclampsia or eclampsia)).

[0312] In an embodiment, the reference level refers to a value derived from a pregnant subject without a history of pregnancy-related hypertensive disorders (such as preeclampsia or eclampsia).

[0313] In one embodiment, the reference level and the level from the subject to be assayed, as used herein, preferably refer to the measured value of the protein level of sFlt-1 or a fragment thereof in a blood sample obtained by means of a Thermo Scientific B·R·A·H·M·S KRYPTOR assay, and the blood sample is preferably a whole blood sample, or a plasma or serum sample, obtained from a pregnant subject who has not developed preeclampsia. Thus, depending on the assay / measurement method employed, the values disclosed herein may vary to some extent, and the specific values disclosed herein are also intended to be understood in terms of the corresponding values determined by other methods. In an embodiment of the invention, a decrease in the level of sFlt-1 or a fragment thereof compared to the reference level can be any decrease in the range of 6% to 20% compared to the reference level, where the reference level can define the transition from low to high risk of developing PE. Any value within this range can be considered an appropriate reference level for high-risk and low-risk sFlt-1 levels. Additionally, a value that is less than or equal to such a reference level can indicate a high risk of preeclampsia, while a value that is greater than such a reference level can indicate a low risk of preeclampsia. Appropriate cut-off levels that can be used in the context of the present invention include, without limitation, at least 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25% change compared to the reference level.

[0314] As used herein, the term "positive reference" sample, standard or value is a sample or value or number derived from a subject known to have or have had a pregnancy-related hypertensive disorder such as preeclampsia or eclampsia. Depending on the context, the reference standard or level may also reflect the mean or average value of the nucleic acid, polypeptide or small molecule level from normal reference subjects or positive reference subjects. The reference may also be a chart, graph or standard curve representing the normal reference level of a polypeptide, nucleic acid or small molecule at any and / or all stages of pregnancy (e.g., weekly). Ideally, all reference samples, standards and levels match the subject sample by at least one of the following criteria: fetal gestational age, maternal age, pre-pregnancy maternal blood pressure, maternal blood pressure during pregnancy, maternal BMI, fetal weight, previous diagnosis of pregnancy-related hypertensive disorder, and family history of pregnancy-related hypertensive disorder.

[0315] As used herein, the term "history of pregnancy-related hypertensive disorder" shall refer to a previous diagnosis of a pregnancy-related hypertensive disorder (e.g., preeclampsia or eclampsia, or gestational hypertension) in the subject or a related family member.

[0316] As used herein, "gestational age" shall mean the age of the fetus calculated from the first day of the mother's last menstrual period. It also refers to the corresponding gestational age estimated by more precise methods in the art. In the case of in vitro fertilization, 14 days are added based on the known duration since fertilization. Gestational age can be determined by obstetric ultrasound examination.

[0317] As used herein, "maternal age" shall mean the age of the pregnant subject at the time of delivery.

[0318] As used herein, the term "pregnancy-induced hypertension" shall mean the development of hypertension without proteinuria after 20 weeks of pregnancy (after 140 days of GA).

[0319] As used herein, the term "polypeptide" refers to a polymer of amino acids and does not refer to a specific length. Thus, peptides, oligopeptides, and proteins are also included within the definition of polypeptides.

[0320] The term "comprising" is used herein to mean the phrase "comprising but not limited to" and may be used interchangeably therewith.

[0321] The term "such as" is used herein to mean the phrase "such as but not limited to" and may be used interchangeably therewith.

[0322] As used herein, the term "risk parameter" or "risk factor" is a health condition that predisposes a pregnant subject to developing preeclampsia. One of the risk parameters is the blood type of the parents, and the blood type of the biological father or biological mother is preferably AB. A further preferred blood type is the Rh factor of the parents, especially if the pregnant subject is Rh negative and the biological father of the fetus is Rh positive. Risk parameters include but are not limited to: hypothyroidism, hyperthyroidism, BMI over 24, first pregnancy, history of preeclampsia, race at risk of impairment, multiple pregnancy, migraine, lupus, coagulation disorders (such as increased platelet aggregation), inflammatory diseases, initial heart diseases, diabetes, chronic kidney disease, and chronic hypertension.

[0323] According to the method as described herein, an sFlt-1 level lower than or equal to the reference level indicates starting or modifying the treatment of the subject to reduce the risk of developing preeclampsia, delay the onset time point of preeclampsia, or at least reduce the severity of preeclampsia, such as balancing the angiogenic / anti-angiogenic process in placental development, reducing blood pressure, and protecting organ functions, such as the functions of the kidney or liver.

[0324] In an embodiment, an sFlt-1 level that is less than or equal to a reference level indicates the initiation or modification of treatment of a subject to reduce the risk of developing preeclampsia, delay the onset time point of preeclampsia, or at least reduce the severity of preeclampsia, such as balancing the angiogenic / anti-angiogenic processes in placental development, reducing blood pressure, protecting organ function, such as that of the kidney or liver. Such treatment also involves prenatal monitoring, lifestyle modification, nutritional supplementation, bed rest, restricted activity or regular exercise, nutritional measures such as reducing salt intake, and antioxidants such as vitamins C and E, garlic, and marine oils.

[0325] As used herein, the term "nulliparous" refers to a subject who has never given birth. "Singleton multiparous" shall refer to a subject who has given birth once, and "biparous multiparous" shall refer to a subject who has given birth twice. "Multiparous" shall refer to a subject who has given birth more than twice.

[0326] As used herein, the term "chromosomal abnormality" shall refer to chromosomal differences that may occur during fetal development. These abnormalities may be unique to the fetus or may be inherited from the parents. The abnormalities are divided into two categories: numerical refers to a different number of chromosomes than expected, such as monosomy or trisomy, and structural refers to translocations, deletions, duplications, ring formation due to a tear in a part of the chromosome, and chromosomal inversions. Chromosomal abnormalities include, but are not limited to, Down syndrome, Turner syndrome, Klinefelter syndrome, trisomy 13, trisomy 14, triple X syndrome, XYY syndrome, fragile X chromosome syndrome, and Cri-Du-Chat syndrome.

[0327] The invention also relates to kits, uses of the kits, and methods of using such kits. The invention relates to kits for carrying out the methods provided in the context herein. The definitions provided herein (e.g., those provided with respect to the methods) also apply to the kits of the invention. The kit may be part of a medical device that also houses calibrators, controls, buffer reagents, and may be used in combination with a diagnostic instrument and / or software. In particular, the invention relates to a kit for treatment monitoring, which treatment monitoring includes prognosticating, risk assessing, or risk stratifying subsequent adverse events to a patient's health, wherein the kit includes a detection reagent for determining the level of sFlt-1 or a fragment thereof, and optionally, additional reagents for determining the levels of other biomarkers in a sample from a subject, and reference data, such as a reference level, that corresponds to an sFlt-1 risk level and optionally corresponds to the levels of other biomarkers, wherein the reference data is preferably stored on a computer-readable medium and / or used in the form of computer-executable code that is configured to compare the determined level of sFlt-1 or a fragment thereof, and optionally, additionally the determined level of other biomarkers or fragments thereof with the reference data.

[0328] In one embodiment of the methods described herein, the method further comprises comparing a measured level of sFlt-1 or a fragment thereof to a reference level, threshold, and / or population mean corresponding to sFlt-1 or a fragment thereof in a patient at risk of developing PE, wherein the comparison is implemented in a computer processor using computer-executable code.

[0329] The methods of the invention can be implemented in part by a computer. For example, the step of comparing the level of a detected biomarker (e.g., sFlt-1 or a fragment thereof) to a reference level can be performed in a computer system. In the computer system, the measured level of the biomarker can be combined with other biomarker levels and / or clinical parameters of the subject in order to calculate a score that indicates prognosis, risk assessment, and / or risk stratification. For example, the measured values can be input into the computer system (manually by a healthcare professional or automatically from a device that has measured the corresponding biomarker levels). The computer system can be located directly at the point of care (e.g., primary care unit, hospital, or home environment), or can be located at a remote location connected via a computer network (e.g., via the Internet or a professional medical cloud system, optionally in combination with other IT systems or platforms such as a hospital information system (HIS)). Generally, the computer system stores these values (e.g., biomarker levels or clinical parameters such as age, blood pressure, weight, gender, etc., or pregnancy parameters such as UAPI, FMF algorithm, scores such as VOCAL score, BMI, etc.) on a computer-readable medium and then calculates a score based on predefined and / or pre-stored reference levels or reference values. The resulting score will be displayed and / or printed for the user (usually a healthcare professional such as a doctor or a patient). Alternatively or in addition, the associated prognosis, assessment, treatment guidance, patient management guidance, or stratification will be displayed and / or printed for the user (usually a healthcare professional such as a doctor or a patient).

[0330] In one embodiment of the present invention, a software system can be employed, in which machine learning algorithms are evident. Preferably, data from electronic health records (EHRs) are used to identify patients at risk of PE. Machine learning methods can be trained on EHR data from patients (such as laboratory, biomarker expression, vital signs, and demographics) using a random forest classifier. Machine learning is a type of artificial intelligence that enables a computer to learn complex patterns in data without explicit programming, as opposed to systems based on simpler rules. Early studies used electronic health record data to trigger alerts for detecting overall clinical deterioration. In one embodiment of the present invention, the processing of sFlt-1 levels can be incorporated into appropriate software for comparison with existing datasets. For example, sFlt-1 levels can also be processed in machine learning software to assist in predicting the incidence of PE.

[0331] "PAPP-A" is pregnancy-associated plasma protein A, pappalysin-1, specifically referring to the plasma protein used as a screening test between 8 and 14 weeks of pregnancy. A decrease in the level of this protein indicates an increased risk of Down syndrome, intrauterine growth retardation, preeclampsia, and stillbirth.

[0332] "PIGF" is placental growth factor (UniprotKB-Q6IB04), one of the members of the vascular endothelial growth factor (VEGF) family. PIGF is involved in the glycosylphosphatidylinositol-anchor biosynthesis pathway, which is part of glycolipid biosynthesis. The level of PIGF decreases in pregnant subjects destined to develop preeclampsia.

[0333] As used herein, reference data (such as reference levels) correspond to patient groups of maternal age up to 18 years, between 18 and 34 years, over 34 years, and optionally additional markers as described herein, preferably PAPP-A and / or PIGF levels, where the reference data are preferably stored on a computer-readable medium and / or used in the form of computer-executable code configured to compare the measured level of sFlt-1 or a fragment thereof, and optionally, in addition, the measured level of PAPP-A and / or PIGF or a fragment thereof with the reference data. "Reference data" includes additional reference levels corresponding to the following patient groups: AB blood type, Rh-negative blood type, the biological father of the fetus is Rh-positive and the subject itself is Rh-negative, carrying at least one female fetus, carrying at least one male fetus, nulliparous, having had one or more previous pregnancies, and / or suspected of carrying a fetus with chromosomal abnormalities. The reference data can also include an instruction manual on how to use the kit of the present invention.

[0334] The kit may additionally include items useful for obtaining a sample (such as a blood sample). For example, the kit may include a container, wherein the container includes means for attaching the container to a cannula or syringe, a syringe adapted to separate blood, having an internal pressure less than atmospheric pressure, such as adapted to draw a predetermined volume of sample into the container, and / or additionally includes a detergent, a chaotropic salt, a ribonuclease inhibitor, a chelating agent (such as guanidine isothiocyanate, guanidine hydrochloride, sodium dodecyl sulfate, polysorbate 20), an RNase inhibitor protein, and mixtures thereof, and / or a filtration system that houses nitrocellulose, silica matrix, ferromagnetic spheres, cupretrieve spill over, trehalose, fructose, lactose, mannose, polyethylene glycol, glycerol, EDTA, TRIS, limonene, xylene, benzoyl, phenol, mineral oil, aniline, pyrrole, citrate, and mixtures thereof. Description of the Drawings

[0335] The present invention is further described by the accompanying drawings. These drawings are not intended to limit the scope of the present invention.

[0336] Figure 1 : Box plots represent sFlt-1 levels before 90 days of GA, between 90 and 100 days of GA, or between 140 and 154 days of GA, comparing levels in subjects with or without EO-PE. The study involved 11,952 women recruited in the first trimester of pregnancy, of whom 11,918 did not develop PE and 34 developed early-onset PE. Before 90 days of pregnancy, all women with EO-PE cases (N = 10) had sFlt-1 levels below the median (p < 0.01). Between 90 and 100 days of pregnancy, on average, women with EO-PE (N = 24) had sFlt-1 levels equal to the mean. Between 140 and 154 days of pregnancy, all women with EO-PE (N = 4) had sFlt-1 levels above the median.

[0337] Figure 2 : Statistical evaluation of sFlt-1 levels in subjects, where samples were obtained in the first trimester of pregnancy. The study involved 11,952 women recruited in the first trimester of pregnancy, of whom 11,918 did not develop PE and 34 developed early-onset PE. It was observed that SFlt-1 in the first trimester was not associated with EO-PE (EO-PE: 0.98 MoM, compared to control: 1.00 MoM, p = 0.09).

[0338] Figure 3: Statistical evaluation of sFlt-1 levels in subjects, where samples were obtained after 12 6 / 7 weeks of GA (after 90 days of GA). This study involved 7,409 women recruited after 90 days of gestation, of which 7,385 did not develop PE and 24 developed early-onset PE. It was observed that sFlt-1 after 12 6 / 7 weeks (after 90 days of GA) was not associated with EO-PE (EO-PE: 1.00 MoM, compared to control: 1.00 MoM, p = 0.40).

[0339] Figure 4 : Figure 3 ROC curve of the data presented therein, which shows that sFlt-1 after 12 6 / 7 weeks (after 90 days of GA) is not associated with EO-PE.

[0340] Figure 5 : Statistical evaluation of sFlt-1 levels in subjects, where samples were obtained before 12 6 / 7 weeks of GA (before 90 days of GA). This study involved 4,543 women recruited before 90 days of gestation, of which 4,533 did not develop PE and 10 developed early-onset PE. It was observed that sFlt-1 before 12 6 / 7 weeks (before 90 days of GA) was negatively associated with EO-PE (EO-PE: 0.94 MoM, compared to control: 1.00 MoM, p = 0.003).

[0341] Figure 6 : Figure 5 ROC curve of the data presented therein, which shows that sFlt-1 before 12 6 / 7 weeks (before 90 days of GA) is associated with EO-PE. Measured values of sFlt-1 (adjusted for gestational age - MoM) and sFlt-1 (not adjusted for gestational age - raw value) can both predict early-onset PE and can be used as markers for early-onset PE (AUC: 0.74 (95% CI: 0.64 - 0.84), p < 0.01).

[0342] Figure 7 : ROC curve of sFlt-1 and PIGF level data in subjects, where samples were obtained after 12 6 / 7 weeks of GA (after 90 days of GA). After 90 days of gestation, PIGF is a strong marker for EO-PE, while sFlt-1 is not.

[0343] Figure 8 : ROC curve of sFlt-1 and PIGF level data in subjects, where samples were obtained before 12 6 / 7 weeks of GA (before 90 days of GA). Before 90 days of gestation, sFlt-1 is a strong marker for EO-PE, while PIGF is not as effective as after 90 days.

[0344] Figure 9 : ROC curves of sFlt-1 and PIGF level data in subjects, where samples were obtained before 12 6 / 7 weeks of GA (before 90 days of GA). In addition, combined data of both sFlt-1 and PIGF levels were used to show the ROC curve. The ROC curve shows the AUC values for the following: PIGF: 0.70 (95% CI: 0.56 - 0.85), sFlt-1: 0.74 (95% CI: 0.64 - 0.84), combination of the two markers: 0.85 (95% CI: 0.78 - 0.92).

[0345] Figure 10 : ROC curves of sFlt-1 and PIGF level data in subjects, where samples were obtained before 12 6 / 7 weeks of GA (before 90 days of GA). In addition, data of uterine artery Doppler measurements obtained in the study were combined in this analysis. Combined data of both sFlt-1 and PlGF levels were used to show the ROC curve. In addition, these combined levels were further combined with Doppler data. The ROC curve shows the AUC value for this Doppler combination as 0.87 (95% CI: 0.80 - 0.94).

[0346] Figure 11 : ROC curves of sFlt-1 and PIGF level data in subjects, where samples were obtained between 90 and 100 days of GA. The combination of sFlt-1 and PIGF predicted some cases of IUFD between 90 and 100 days of GA.

[0347] Figure 12 : ROC curves of sFlt-1 and PIGF level data in subjects, where samples were obtained before 90 days of GA. The combination of sFlt-1 and PlGF predicted IUFD, and thus, this association was stronger before 90 days of pregnancy compared to between 90 and 100 days of pregnancy (AUC: 0.72, 95% CI: 0.60 - 0.84).

[0348] Figure 13 : Box plots and ROC curves show the correlation between sFlt-1 levels (measured before 90 days of GA) in subjects and the estimated high or low risk determined using the FMF algorithm. It can be seen from the figure that high-risk patients determined using the FMF algorithm had significantly lower sFlt-1 levels before 90 days of GA.

[0349] Figure 14: sFlt-1 levels in subjects, where samples were obtained from 9 completed weeks of gestation to 12 6 / 7 weeks (57 days to 90 days of completed gestation). Each point represents the sFlt-1 level of an individual subject at the corresponding time point. Larger points represent the sFlt-1 levels of subjects who developed early-onset preeclampsia (EO-PE), and smaller points represent the sFlt-1 levels of subjects who did not develop EO-PE (no EO-PE). (B) sFlt-1 MoM in subjects, where samples were obtained from 9 completed weeks of gestation to 12 6 / 7 weeks (57 days to 90 days of completed gestation). Each point represents the sFlt-1 MoM of an individual subject at the corresponding time point. Larger points represent the sFlt-1 MoM of subjects who developed early-onset preeclampsia (EO-PE), and smaller points represent the sFlt-1 MoM of subjects who did not develop EO-PE (no EO-PE). The median sFlt-1 (MoM) for subjects who did not develop EO-PE was 1.00 (IQR: 0.76 - 1.34). The median sFlt-1 (MoM) for subjects who did develop EO-PE was 0.78 (IQR: 0.60 - 0.87). (C) ROC curve of data on sFlt-1 levels in subjects, where samples were obtained from 9 to 12 6 / 7 weeks of pregnancy (57 days to 90 days of completed gestation). N = 1108; 4 cases of EO-PE. The ROC curve shows an AUC value of 0.75 (95% CI: 0.63 - 0.87) for sFlt-1.

[0350] Figure 15: sFlt-1 levels in subjects, where samples were obtained from 21 to 40 weeks of gestation (GA of 141 to 280 days). Each point represents the sFlt-1 level of an individual subject at the corresponding time point. Larger points represent the sFlt-1 levels of subjects who developed early-onset preeclampsia (EO-PE), and smaller points represent the SFlt-1 levels of subjects who did not develop EO-PE (no EO-PE). The dashed line represents the overall trend of sFlt-1 levels for each group (no EO-PE and with EO-PE) from 21 to 40 weeks of gestation (GA of 141 to 280 days). Top: with EO-PE, bottom: no EO-PE. Subjects who developed EO-PE showed increased sFlt-1 levels between 21 weeks and 40 weeks of gestation (GA of 141 to 280 days) when compared to subjects who did not develop EO-PE (no EO-PE). (B) sFlt-1 levels in subjects, where samples were obtained from GA of 9 to 40 weeks (GA of 57 to 280 days). Each point represents the sFlt-1 level of an individual subject at the corresponding time point. Larger points represent the sFlt-1 levels of subjects who developed early-onset preeclampsia (EO-PE), and smaller points represent the SFlt-1 levels of subjects who did not develop EO-PE (no EO-PE). The dashed line represents the overall trend of sFlt-1 levels for each group (no EO-PE and with EO-PE) from 9 to 40 weeks of gestation (GA of 57 to 280 days). Top: with EO-PE, bottom: no EO-PE. Subjects who developed EO-PE showed decreased sFlt-1 levels before the end of 13 weeks of gestation (before GA of 90 days) and increased sFlt-1 levels after the end of 13 weeks of gestation (after GA of 90 days) when compared to healthy subjects who did not develop EO-PE. The sFlt-1 levels of subjects who developed EO-PE showed a significantly greater increase during pregnancy (9 to 40 weeks of gestation, GA of 57 to 280 days) when compared to patients who did not develop EO-PE.

[0351] Examples

[0352] Example 1 :

[0353] Study design :

[0354] Pregnant women were recruited between 11 and 14 weeks of gestation (GA 71 to 98 days) and followed up until delivery. sFlt-1 was measured using Thermo Scientific B·R·A·H·M·S KRYPTOR at recruitment and reported as multiples of the median (MoM) adjusted for gestational age. Median sFlt-1 levels were compared between the following groups of women: those who developed early-onset PE (<34 weeks, <GA 231 days); those who developed mid-onset PE (34 to 36 weeks, GA 232 to 252 days); those who developed late-onset PE (37 weeks or later, GA 253 days or later); and those who did not develop PE (controls). The area under the ROC curve (AUC) was used to estimate the potential predictive value of sFlt-1 for PE.

[0355] Results :

[0356] We included 12,383 participants who delivered after 16 weeks of gestation (GA 112 days), of whom 33 (0.3%) developed early-onset PE; 65 (0.5%) and 400 (4.0%) developed late-onset PE. We observed that sFlt-1 was lower in the first trimester in participants who developed early-onset or mid-onset PE (p = 0.02), but more importantly, this difference was mainly observed in participants recruited in the early pregnancy.

[0357] Significant differences in the predictive value for early-onset PE were observed between women recruited at the following times: week 11 of gestation (GA 71 to 77 days) (AUC: 0.82; 95% CI: 0.72 - 0.92; p < 0.001); week 12 of gestation (AUC: 0.62; 95% CI: 0.49 - 0.74; p = 0.06); and week 13 of gestation (AUC: 0.50; 95% CI: 0.32 - 0.67; p = 0.97). A similar trend was also observed in predicting mid-onset PE.

[0358] Conclusions :

[0359] Maternal sFlt-1 in the first trimester was reduced in women who would develop PE before term (<37 weeks; ≤GA 253 days). Its predictive value was significantly increased when sFlt-1 was collected at 12 weeks of gestation or earlier (before GA 78 days), and this particularity could explain the contradictions between the results of previous studies.

[0360] Example 2 :

[0361] Study design :

[0362] We performed a secondary analysis of a prospective cohort study of nulliparous women recruited between 11 and 14 weeks of gestation (GA 71 to 98 days). Maternal characteristics, mean arterial blood pressure, maternal serum biomarkers (pregnancy-associated plasma protein-A, placental growth factor, two risk cut-offs from the FMF algorithm (1 / 70 and 1 / 100), sFlt-1, and mean uterine artery pulsatility index) levels were obtained to calculate the risks of preterm PE and term PE compared with a control group of women who did not develop preterm PE. The detection rates, false-positive rates, and positive and negative predictive values of term PE and preterm PE as placental-mediated complications were estimated and calculated. Women reporting daily aspirin use were excluded. Concentrations of PAPP-A, PIGF, and sFlt-1 were measured using the Thermo Scientific B.R.A.H.M.S KRYPTOR automated assay. Preterm PE was defined as PE delivered before 37 weeks of gestation (before GA 253 days), and early-onset PE was defined as cases delivered before 34 weeks of gestation (before GA 231 days). Analyses were performed using the SAS statistical software package (version 9.3; SAS Institute Inc, Cary, NC). All analyses accounted for a 5% type I error.

[0363] Results :

[0364] We included 4,575 participants with complete observations. Twenty-nine patients developed preterm PE, while 194 women developed term PE, and 3,705 women did not develop placental-mediated complications (reference group). The median sFlt-1 value in the reference group was 1,023 pg / mL, with Q1–Q3 values of 771 pg / mL to 1,373 pg / mL. The median sFlt-1 value and Q1–Q3 for women who developed term PE were 933 pg / mL (726 pg / ml to 1,221 pg / ml). Thus, pregnant women who developed term PE showed an 8.8% decrease in median sFlt-1 levels and a median fold change (FC) of 0.91.

[0365] The median sFlt-1 value and Q1–Q3 for women who developed preterm PE were 852 pg / mL (658 pg / ml to 1,095 pg / ml). Thus, pregnant women who developed preterm PE showed a greater decrease in sFlt-1 than the term PE group, with a 16.7% decrease in median sFlt-1 at 11 to 14 weeks of gestation (GA 71 to 98 days) compared with the reference group, and a median fold change (FC) of 0.83.

[0366] The Q1 of the sFlt-1 level for preterm PE showed an FC of 0.85, a 14.7% decrease, compared with the reference group.

[0367] Compared with the reference group, Q1 of the sFlt-1 level in term PE showed an FC of 0.94, a decrease of 5.8%.

[0368] Compared with the reference group, Q3 of the sFlt-1 level in preterm PE showed an FC of 0.8, a decrease of 20%.

[0369] Compared with the reference group, Q3 of the sFlt-1 level in term PE showed an FC of 0.89, a decrease of 11%.

[0370] Conclusions :

[0371] Nulliparous women with a median decrease in sFlt-1 of at least 8.8% detected between 11 and 14 weeks of gestation (GA from 71 to 98 days) had an increased risk of developing term PE, and nulliparous women with a median decrease in sFlt-1 of at least 16.7% had an increased risk of developing preterm PE.

[0372] Example 3 :

[0373] Predicting early preeclampsia and preterm birth in the first trimester of pregnancy (prediction study) :

[0374] Pre-eclampsia is a pregnancy complication that affects 2% to 5% of pregnant women. This complication is one of the leading causes of maternal and neonatal death and morbidity worldwide. Early-onset pre-eclampsia requires delivery before 34 weeks of gestation (before GA of 232 days) and is associated with very prominent perinatal morbidity.

[0375] Online software (Fetal Medicine Foundation) combines biophysical factors (age, BMI, BP, medical history), ultrasound factors (uterine artery Doppler), and biochemical factors (PIGF and PAPP-A) measured in the first trimester of pregnancy. This software is currently available and suggests that it can predict more than 90% of early pre-eclampsia with a false-positive rate of less than 10%.

[0376] In the present invention study, additional patient groups were further included and investigated, such as patients under 18 years old, with certain rare blood types such as AB, Rh negative, multiple pregnancies, presence, experience of one or more previous pregnancies, presence of malformations or multiple malformation syndromes, and / or suspected or confirmed pregnancies with chromosomally abnormal (extremely high nuchal translucency) fetuses, to explore the correlation between biomarkers such as sFlt-1, PAPP-A, or PIGF and pre-eclampsia.

[0377] Objectives :

[0378] 1) Validate the FMF screening tool for early pre-eclampsia and other placenta-related pregnancy diseases (preterm pre-eclampsia, IUGR) between 11 and 13 weeks of gestation (GA from 71 to 91 days)

[0379] The screening effectiveness for the 3rd percentile, perinatal death

[0380] 2) Compare the screening effectiveness of the screening tool with and without uterine artery Doppler measurements

[0381] 3) Explore the efficacy of potential markers (serum sFlt-1, serum ADAM-12, serum PP-13, placental volume and subplacental volume, placental vascular distribution) in predicting preeclampsia

[0382] Methods :

[0383] Prospective observational study. Recruit nulliparous pregnant women with singleton pregnancies and no lethal fetal anomalies between 11 3 / 7 and 13 6 / 7 weeks of gestation (GA from 73 to 90 days). Complete a lifestyle questionnaire. Measure BMI and BP, and collect a blood sample (20 ml). Transvaginal uterine artery Doppler ultrasound examination and three-dimensional placental evaluation have been completed at this visit. Follow up the medical records approximately 1 month after the expected date of delivery

[0384] Analyze PAPP-A, PIGF, sFIt1, fbHCG, and AFP in serum within 10 days of recruitment using Thermo Scientific B·R·A·H·M·S KRYPTOR automated assays. At the end of the project, residual serum will be stored at -80 °C for additional analyses (PP-13, ADAM-12, vitamin D) to evaluate the possibility of improving the prediction model using promising markers (including placental volume and vascular distribution evaluated by three-dimensional ultrasound). For each participant, the risks of early preeclampsia and overall preeclampsia are calculated with the help of the FMF software, but they have been disclosed to them. The best sensitivity and specificity of the tool will be evaluated using the ROC curve

[0385] We estimated that the incidence of early-onset preeclampsia (<34 weeks, <GA of 231 days) in the nulliparous population (Quebec & Montreal) was 0.7%. We recruited 7554 women, demonstrating that the FMF screening tool had at least 80% sensitivity and 90% specificity, while the tool was expected to have 95% sensitivity and 92% specificity respectively

[0386] Objectives

[0387] Primary objectives

[0388] Validate the FMF early preeclampsia screening tool between 11 and 13 weeks of gestation (GA from 71 to 91 days)

[0389] Secondary objectives

[0390] 1) Evaluate the performance of the FMF test in predicting all cases of preeclampsia and predicting other placenta-related pregnancy diseases (early and severe preeclampsia, IUGR < 3rd percentile, perinatal death).

[0391] 2) Compare the screening effectiveness of the screening tool with and without uterine artery Doppler.

[0392] 3) If the FMF screening tool is not positively validated in our population; then evaluate the predictive value of each biomarker individually and assess whether they can be used in different prediction models.

[0393] 4) Evaluate the predictive value of potential biomarkers for early preeclampsia (sFlt-1, PP13, ADAM-12,

[0394] 25-OH-vitamin D, placental volume and placental / subplacental vascular distribution, sFlt-1).

[0395] 5) Evaluate the predictive value of cervical measurements in the first trimester for preterm birth.

[0396] I. Study citations

[0397] This is a prospective observational study of nulliparous women recruited in the first trimester, collecting and analyzing a large number of biomarkers in the first trimester, and following up until delivery to verify the presence of our primary and secondary outcomes.

[0398] II. Population and selection criteria

[0399] Inclusion criteria for the study group :

[0400] ■ Women with a singleton live pregnancy between 11 3 / 7 weeks and 13 6 / 7 weeks (GA from 80 days to 97 days).

[0401] ■ Nulliparous women (for any reason, not previously pregnant for up to 20 / 7 weeks (up to GA of 147 days))

[0402] Exclusion criteria for the study group :

[0403] ■ Women < 18 years old

[0404] ■ Women who cannot give informed consent (e.g., do not understand English and French)

[0405] ■ Women planning to deliver outside the participating center (at - Women giving birth to the God of Lévis are excluded, but they are eligible to participate in the project because they have consented in writing to the consultation of their files at the institution)

[0406] ■ Women who are positive for HIV or hepatitis C or chronic hepatitis B (not cured)

[0407] Further inclusion criteria for the study group :

[0408] ■ Multiple pregnancies (women carrying two fetuses with one having stopped growing are ineligible)

[0409] ■ Presence of fetal malformations or multiple malformation syndromes

[0410] ■ A nuchal translucency measurement of ≥ 3.5 mm is associated with a very high risk of chromosomal abnormalities and / or heart malformations and may affect the serum PAPP-A value.

[0411] ■ Fetal heart negative on the day of the recruitment visit

[0412] III. Conducting the study

[0413] 11 3 / 7 weeks - 13 6 / 7 weeks (GA from 80 days to 97 days)

[0414] Visiting nurse (11 3 / 7 weeks - 13 6 / 7 weeks) (GA from 80 days to 97 days)

[0415] First, the research nurse collects blood samples by venipuncture (2 × 5 ml tubes, BD Vacutainer SST). The tubes are gently inverted 5 times to mix the reagent with the blood thoroughly. The tubes are placed upright in a dark box and not taken out until processing (minimum 30 minutes, maximum 2 hours). Second, the research nurse measures the patient's blood pressure. Before the measurement, the patient must sit quietly for 5 minutes with legs uncrossed and not talking. The blood pressure is measured simultaneously on both arms (without a sleeve vest) three times using a pre-programmed Microlife electronic sphygmomanometer (model 33603).

[0416] Complete the patient questionnaire to understand their medical and obstetric family history and their socioeconomic background, including their date of birth, anthropometric data, tagabism, etc. (see the attached questionnaire). The total meeting time with the nurse is at most 30 minutes.

[0417] Visiting technician (11 3 / 7 weeks - 13 6 / 7 weeks, GA from 80 days to 97 days)

[0418] After the meeting with the nurse, all patients were interviewed by a radiology technician from the research team and had their nuchal translucency thickness measurements certified for ultrasound acquisition using a Voluson E8 Expert (GE Medical Systems, Milwaukee, WI, USA) equipped with a 4 to 8 MHz transducer. The instrument settings were the same for all patients, i.e., "angiography mode" = 100; "smoothness" = 4 / 5; FRQ = low; quality = normal; density = 6; "enhancement" = 16; balance = 175; WMF = low 1; "actual power" = 2 dB; "pulse repetition frequency" = 0.6 kHz; gain color = -7.2 dB

[0419] Ultrasound examinations were performed to confirm the eligibility criteria for the project:

[0420] 1) If the CCL ≥ 77 mm, crown - heel length (CCL) and biparietal diameter were used to confirm gestational age.

[0421] 2) Nuchal translucency thickness measurements were performed according to the standards of the Fetal Medicine Foundation. If the participant had a prescription for nuchal translucency thickness measurement, a result report was provided.

[0422] 3) In cases of malformation syndromes, multiple pregnancies, nuchal translucency thickness ≥ 3.5 mm and / or negative fetal heart rate, the patient was informed and referred to a doctor in charge of the project or their substitute. The ultrasound examination report of the results was sent to the patient's referring doctor.

[0423] The technician filled in the eligibility form accordingly. Participants who met at least one exclusion criterion were treated the same as any other participant, i.e., their blood samples were analyzed and all data that had been collected or would be collected until delivery were retained. All data of these participants were excluded from the main analysis.

[0424] The visit window between 11 3 / 7 weeks and 13 6 / 7 weeks of gestation (GA from 80 to 97 days) is very important to ensure the validity of certain data (biochemical and ultrasound data). Therefore, if the ultrasound dating test confirmed gestational age:

[0425] a) < 11 3 / 7 weeks (< GA of 80 days) (LCC < 45 mm): The visit was rescheduled to a day between 11

[0426] 3 / 7 weeks and 13 6 / 7 weeks of gestation (GA from 80 to 97 days). Blood sampling and ultrasound examination were repeated at this time. Two samples were retained, but only the second sample was used for the main analysis. In the case - control study, if a minimum number (n ≥ 5) of these patients developed early pre - eclampsia, the first sample was analyzed and compared with the second sample.

[0427] The willingness of the participants to revisit was respected. If a participant declined the second visit, the data collected were retained but excluded from the main analysis.

[0428] b) > 14 weeks (GA ≥ 98 days) (CCL > 84 mm): Blood samples, ultrasound data, and questionnaire data were retained as they were. No further visits were scheduled. These patients were excluded from the analysis of the primary objective.

[0429] Then, for research purposes, the following ultrasound measurements were performed:

[0430] 4) Using Doppler ultrasound, the left and right uterine arteries were observed, and the pulsatility index was measured according to the FMF criteria: The uterine artery was examined at the level of the internal carotid bone, and the pulsatility index was automatically calculated by the machine using the pulsatile flow curves of three subsequent and similar cardiac cycles. The measurements were performed in the sagittal and transverse planes, and the differences between the two were evaluated on samples from approximately 1000 cases to assess the reproducibility, duration, and efficiency of the two techniques. The presence or absence of notches on both sides was recorded (a notch was considered present if an early diastolic notch appeared in each cycle).

[0431] 5) Three-dimensional ultrasound examinations of the placenta and the subplacental area were performed with and without Doppler ultrasound.

[0432] This examination took approximately 30 seconds.

[0433] At the end of recruitment, for case-control analysis, a technician who was unaware of the clinical data performed the following volume measurements and calculations: Using VOCAL (Virtual Organ Computer-Aided Analysis) and a series of 6 slices of the placenta (each slice rotated 30 degrees horizontally from the previous slice in planes A and B), the placenta contour was manually drawn, taking care to exclude the uterine wall. Similarly, the volume of the subplacental myometrium was evaluated from the boundary between the placenta and the decidua-myometrium to the full thickness of the myometrium (up to 1 cm). These volume acquisitions were measured by the following variables:

[0434] a. Placental volume

[0435] b. Placental quotient (PQ = 1 / 4 placental volume / LCC).

[0436] c. The vascularization index (VI), flow index (Fl), and vascular flow index (VFI) of the placenta and the decidua-myometrium region will be evaluated using VOCAL software. VFI represents the number of voxels in the volume studied (expressed as a percentage). Fl is the average color value of all voxels representing the mean intensity of blood flow (expressed as an absolute value between 0 and 100). VIF is the average of the colors of all voxels in the region studied (gray and colored, expressed as an absolute value between 0 and 100).

[0437] 6) Cervical length: Measured transvaginally (intravaginal probe). This examination was only performed at the CHU in Quebec City and validated the preterm birth prediction that also exists in the FMF algorithm. This examination was not systematically performed on all participants but was carried out on the prescription of the attending physician.

[0438] 7) Ultrasonic acquisitions were also performed in the abdominal region to measure the posterior thickness of the visceral adipose tissue located between the medial margin of the rectus abdominis muscle and the anterior wall of the abdominal aorta. This ultrasonic measurement may have a stronger predictive power than BMI in predicting preeclampsia. The acquisition process only takes a few seconds.

[0439] The total time required for all ultrasonic acquisitions is between 15 minutes and 35 minutes. The research technicians are certified and licensed by the Fetal Medicine Foundation (FMF) and PQDT21 for nuchal translucency thickness and cervical measurement. They received a DVD or USB drive with fetal images as a token of appreciation for their participation.

[0440] Monitoring during pregnancy (34 0 / 7 weeks - 35 6 / 7 weeks, GA from 232 days to 251 days)

[0441] A survey (in electronic form, sent by email to the participants) was conducted at 34 weeks of gestation. This verified whether the patient's medications had changed during pregnancy and confirmed whether the pregnancy was proceeding normally, whether there had been a move, or whether there had been any complications to date. If the survey was not completed, an email reminder was automatically sent one week later. Telephone contact was made with those who did not respond to the survey later.

[0442] Postpartum follow-up (6 weeks after DPA)

[0443] The second electronic survey (sent by email to participants) was completed approximately 6 weeks after the participants' expected date of delivery. This enabled us to verify whether the participants or their babies had experienced any difficulties after childbirth. Among other things, we wanted to know about cases of postpartum preeclampsia, rare cases of perinatal or maternal death (regardless of which hospital these events occurred in). If the survey was not completed, an email reminder was automatically sent one week later. Those who did not respond to the survey later were contacted by phone. A procedure was implemented to ensure that participants who had a terminated pregnancy, an intrauterine death, or other adverse complications (mentioned in the 34-week survey) were not contacted again at 46 weeks if not necessary, to avoid inconvenience.

[0444] Follow-up at the end of pregnancy (1 month after DPA)

[0445] Data on pregnancy (pregnancy hypertension, preeclampsia, perinatal death, etc.), data on delivery (gestational age, etc.), and data on the newborn (gender, birth weight, etc.) were collected by research nurses from the patients' medical records after childbirth using CristalNet software. In rare cases where the patient gave birth at a non-participating center (not CHUL, HSFA, or Lévis), the patient was contacted to request permission to obtain this information at the place of delivery. A second data review was conducted directly from the medical records by a doctor (EB, KG, FA, or their substitute) for all suspected cases of preeclampsia and cases of delivery before 37 weeks (<10%, i.e., fewer than 532 records retrieved from the CHU-Q file and fewer than 228 records retrieved from the CHUSJ file).

[0446] Blood samples

[0447] Blood samples were placed in boxes at room temperature and transported to the laboratory, where they were centrifuged more than 30 minutes but less than 2 hours after puncture.

[0448] The serum collected after centrifugation (1200 x g, centrifuged for 10 minutes at room temperature) was transferred to 1 ml aliquots, and then PAPP-A, PIGF, sFlt-1, free bHCG, and maternal serum AFP were measured using a commercially available kit validated by FMF. For the CHU de Québec (CHUL and HSFA), the samples were stored at 4°C until assayed within 24 hours. For CHU Ste-Justine, the aliquots were kept at -20°C and sent to the CHU de Québec twice a month by registered mail with built-in dry ice for analysis and preservation. After the main analysis (serum PAPP-A, PIGF, and sFlt-1 assays), the remaining serum was frozen at -80°C for measurement of PP13, ADAM12, and vitamin D in the case-cohort substudy conducted at the end of the main study.

[0449] Case - cohort study

[0450] A case-cohort study nested within the main cohort evaluated the predictive performance of potential biomarkers for the prediction of early preeclampsia. At the end of the study, maternal serum sFlt-1, ADAM-12, PP-13, and vitamin D were measured using commercial kits in a randomly selected subgroup of women (about 236 women) and all early preeclampsia cases (about 45). The following variables of the same case-cohort were analyzed: placental volume (PV), placental and subplacental vascular distribution index (IV), blood flow index (FI), and vascular flow index (VFI).

[0451] IV. Judgment criteria

[0452] Primary endpoint: Preeclampsia requiring delivery before 34 weeks of gestation (before GA of 231 days) based on the gestational age determined by the date of the last menstrual period (DMD) or ultrasound examination at 11 to 13 weeks (GA of 71 to 91 days), if the latter differs from the DMD method by ≥5 days. Preeclampsia was diagnosed according to the following criteria: 1) gestational hypertension with systolic blood pressure ≥140 mmHg and / or diastolic blood pressure ≥90 mmHg (measured twice within 4 hours) in combination with any one of the following conditions: growth retardation <10th percentile, thrombocytopenia <100, AST and / or ALT twice the normal value, diastolic blood pressure ≥110 mmHg, and / or proteinuria (≥2+ on the test strip or >300 mg / 24 h).

[0453] Secondary endpoints:

[0454] Born <37 weeks (<GA of 253 days) (spontaneous delivery or PPROM before 37 weeks of gestation)

[0455] Born <34 weeks (<GA of 231 days) (spontaneous delivery or PPROM before 34 weeks of gestation)

[0456] Preeclampsia:

[0457] Preterm preeclampsia <37 weeks (<GA of 253 days)

[0458] Severe preeclampsia (any of its conditions: 1) systolic blood pressure ≥160 mmHg and diastolic blood pressure ≥110 mmHg after 4 hours of rest; 2) proteinuria ≥5 g / 24 h or ≥3+ on the stick; 3) oliguria, ≤400 ml / 24 h; visual disturbances or cerebral dysfunction; upper abdominal pain; pulmonary edema or cyanosis; thrombocytopenia <100,000 mm3.

[0459] Perinatal death (antenatal, up to 7 days after birth)

[0460] Growth retardation (<10th percentile), based on Canadian reference values

[0461] Severe stunting (<3rd percentile), based on Canadian reference values

[0462] Birth weight <2500 g

[0463] Birth weight <1500 g

[0464] Mean birth weight

[0465] V. Data analysis plan

[0466] Primary analysis: At the end of the study, the calculation of the early pre-eclampsia and pre-eclampsia risks was performed automatically by the FMF software using all the necessary collected data (including uterine Doppler measurements). Excluding the uterine Doppler measurements, the risk calculation was repeated automatically for all eligible participants.

[0467] The area under the curve, sensitivity, specificity, positive predictive value, and negative predictive value of the two screening methods were calculated using the ROC curve and different cut-off values and reported.

[0468] Results :

[0469] From Figures 1 to 12 It can be seen that the data obtained from the predictive study support the prognostic ability of sFlt-1 to identify patients at risk of EO-PE and / or IUFD when analyzing samples obtained before 90 days of GA.

[0470] From Figure 1 it can be seen that, before 90 days of gestation, women with EO-PE (N = 10) had sFlt-1 levels below the median (p < 0.01). Between 90 and 100 days of gestation, on average, women with EO-PE (N = 24) had sFlt-1 levels equal to the mean. Between 140 and 154 days of gestation, women with EO-PE (N = 4) had sFlt-1 levels above the median. This study allows us to conclude that, in women who will develop early pre-eclampsia (before 34 weeks of gestation, before 231 days of GA), sFlt-1 levels are abnormally decreased in the early pregnancy (before 90 days of gestation) and will gradually increase to normal levels by the end of the first trimester (between 90 and 100 days), and then increase abnormally thereafter (after 140 days of gestation).

[0471] Therefore, the increase in sFlt-1 levels between the first sample and the second sample can determine the risk pattern in the subject and potentially initiate appropriate treatment.

[0472] However, when sFlt-1 was measured from samples obtained throughout the first trimester, the measurements did not provide a statistical correlation with EO-PE ( Figure 2 ). It is also noteworthy that when measured from multiple samples obtained after 12 6 / 7 weeks (90 days) of GA, the sFlt-1 measurements also did not provide a statistical correlation with EO-PE ( Figure 3 , Figure 4 ).

[0473] Surprisingly, sFlt-1 measurements within 12 weeks of gestation or within 90 days showed a significant correlation with EO-PE, especially a negative correlation with EO-PE ( Figure 5 , Figure 6 ). Thus, measuring sFlt-1 within 90 days of GA enables reliable prediction of EA-PE at an early time point.

[0474] When samples were obtained early in pregnancy, such as before the end of 12 weeks of gestation (before 90 days of GA), the combined use of sFlt-1 and PIGF statistically improved the prognosis of EO-PE. It is noteworthy that PIGF is generally effective in prognosticating EO-PE when measured after 90 days of GA, while sFlt-1 does not seem to provide a reliable prognostic indication in a single measurement after 90 days of GA ( Figure 7 ). Surprisingly, when measured before 12 weeks of GA (within 90 days), both sFlt-1 and PIGF were able to achieve the prognosis of EO-PE, but sFlt-1 seems to provide greater sensitivity at comparable specificity values, preferably higher than 0.6 ( Figure 8 ). Equally surprisingly, the combined analysis of sFlt-1 and PIGF showed an unexpected synergistic enhancement of the EO-PE prognosis when measured before 90 days of GA ( Figure 9 ).

[0475] It was also found through this study that the combined use of uterine artery Doppler measurements (preferably in combination with sFlt-1, PIGF, maternal age, and BMI) showed an improved predictive ability for EO-PE in subjects compared to using sFlt-1 or PIGF alone ( Figure 10 ).

[0476] When samples were obtained between 90 days and 11 days of GA, the combined use of sFlt-1 and PIGF also enabled prognosticating IUFD ( Figure 11 ), while when samples were obtained before 90 days of GA, a statistically improved IUFD prognosis was shown ( Figure 12 ).

[0477] In addition, comparisons in the datasets obtained using the FMF algorithm and sFlt-1 analysis as described herein revealed a strong correlation between these two prognostic procedures. As can be seen from Figure 13 patients at high risk determined using the FMF algorithm had significantly lower sFlt-1 levels before 90 days GA. Considering that the FMF screening algorithm includes consideration of multiple maternal characteristics and medical histories, including factors such as blood pressure, pregnancy-associated plasma protein A and placental growth factor, crown-rump length, and uterine artery pulsatility index, the findings of the present invention represent a significant simplification of the prognostic method, which can achieve comparable risk assessment and thus potentially avoid the use of the more complex FMF method. Analyzing sFlt-1 before 90 days of pregnancy can actually predict how a pregnant woman will fare on a positive FMF test without the need for uterine artery Doppler assessment.

[0478] In summary, this study enabled us to conclude that in most pregnant women who will develop early-onset preeclampsia, sFLt-1 declines before 90 days of pregnancy, and in the same pregnant women, sFLt-1 subsequently rises and becomes abnormally high during the second trimester of pregnancy. Thus, sFlt-1 before 90 days of pregnancy can predict early-onset PE, especially when combined with PIGF and / or uterine artery Doppler (detection rate of 40% at a FPR of 10%). In addition, we also observed that the combination of these two markers can also predict approximately 35% of UFDI with a false positive rate of 10%.

[0479] The importance of this information is obvious, because the earlier aspirin therapy or similar therapies are initiated to attempt to address and potentially avoid EO-PE, the better the treatment outcome. Thus, the present invention provides an alternative and improved diagnostic method for identifying patients at risk of EO-PE by using biomarker analysis of samples obtained early in pregnancy, as early as before 90 days GA, and subsequently initiating treatment and subsequent treatment guidance.

[0480] Example 4 :

[0481] Pregnant women were recruited at 7 to 9 weeks of gestation (GA 43 to 63 days) and followed up until delivery. Serum samples obtained at recruitment and between 11 to 13 weeks and 20 to 22 weeks of gestation (GA 71 to 91 days and 140 to 154 days) were measured for sFlt-1 and optionally PAPP-a, PIGF, and / or βhCG using Thermo Scientific B·R·A·H·M·S KRYPTOR, and were preferably reported as multiples of the median (MoM) adjusted for gestational age and / or BMI. Median sFlt-1 levels were compared between the following women: those who developed early-onset PE (<34 weeks, <GA 232 days); those who developed mid-onset PE (34 to 36 weeks, GA 232 to 252 days); those who developed late-onset PE (37 weeks or later); and those who did not develop PE (controls). The area under the ROC curve (AUC) was used to estimate the potential predictive value of sFlt-1 and PAPP-a, PIGF, and βhCG for PE. Analyses such as univariate and multivariate regression analyses were performed using the SAS statistical software package (version 9.3; SAS Institute Inc, Cary, NC). All analyses accounted for a 5% type I error.

[0482] In addition, maternal and pregnancy characteristics (such as height, smoking, BMI, parity, fetal anomalies such as chromosomal or anatomical anomalies, e.g., identified by nuchal translucency thickness measurement, method of conception to blood test performance, adverse FMF risk score), mean arterial blood pressure, levels of maternal serum biomarkers (pregnancy-associated plasma protein-A, placental growth factor), 2 risk cut-offs (1 / 70 and 1 / 100 from the FMF algorithm), sFlt-1, and mean uterine artery pulsatility index were obtained to calculate the risks of preterm PE (including early-onset and mid-onset PE) and term PE compared to a reference group of women who did not develop preterm or term PE. The detection rates, false positive rates, and positive and negative predictive values of term and preterm PE as placental-mediated complications were estimated and calculated.

[0483] It can be determined that when analyzing samples obtained before 90 days of GA and even before 63 days of GA (before the end of the 9th week of pregnancy, before 63 days of GA), the prognostic ability of sFlt-1 in identifying patients at risk of EO-PE. In women who will develop early preeclampsia (before 34 weeks, before 232 days of GA), sFlt-1 levels decrease in the early pregnancy (before 90 days of pregnancy and even before 63 days of pregnancy), and gradually increase to normal levels at the end of the first trimester (between 90 and 100 days), and then increase abnormally (after 140 days of pregnancy). The increase in sFlt-1 levels between the first sample and the second sample can determine the risk pattern in the subject and potentially initiate appropriate treatment.

[0484] When the first sample is obtained in the early pregnancy, for example, before the end of the 12th week of pregnancy (before 90 days of GA), and even before the end of the 9th week of pregnancy (before 63 days of GA), the combined use of sFlt-1 and PIGF, PAPP-A, and / or βhGC statistically improves the EO-PE prognosis.

[0485] When measured before the end of 12 weeks of GA (within 90 days) and even before the end of 9 weeks of GA (within 63 days), sFlt-1 and additional PIGF, PAPP-A, and / or βhCG can perform EO-PE prognosis. When measured before 90 days of GA and even before 63 days, the combined analysis of sFlt-1 and PIGF, PAPP-a, and / or βHCG can synergistically enhance the EO-PE prognosis.

[0486] The importance of early prognosis is crucial because the earlier aspirin therapy or similar therapy is initiated to attempt to resolve and potentially avoid EO-PE, the better the treatment effect. Therefore, the present invention achieves an alternative and improved diagnostic method for identifying patients at risk of EO-PE through biomarker analysis of samples obtained in the early pregnancy, as early as before 90 days of GA and even as early as 63 days, and subsequent initiation of treatment and follow-up treatment guidance.

Claims

1. A method for prognosis, prediction, risk assessment, and / or risk stratification of early-onset preeclampsia in a pregnant subject, comprising a. determining a first level of soluble fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof in a first sample isolated from the subject, wherein the first sample is isolated before 90 days of gestational age (GA), b. determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, wherein the second sample is isolated after the first sample, c. wherein a higher second level of sFlt-1 or a fragment thereof compared to the first level indicates the occurrence of early-onset preeclampsia before 231 days of GA.

2. The method according to any one of the preceding claims, wherein the second sample is isolated from the subject after 100 days of GA.

3. The method according to any one of the preceding claims, wherein the second sample is isolated after 140 days of GA, preferably between 140 days and 154 days of GA.

4. The method according to any one of the preceding claims, wherein the first level is below a reference level, and the reference level is preferably the population mean and / or median of a healthy population.

5. The method according to any one of the preceding claims, wherein the second level is equal to or above a reference level, and the reference level is preferably the population mean and / or median of a healthy population.

6. The method according to any one of the preceding claims, wherein: a. the first level is below a reference level, and the second level is equal to or above a reference level, and the reference level is preferably the population mean and / or median of a healthy population, or b. the first level is equal to or below a reference level, and the second level is above a reference level, and the reference level is preferably the population mean and / or median of a healthy population, or c. the first level is below a reference level, and the second level is above a reference level, and the reference level is preferably the population mean and / or median of a healthy population.

7. The method according to any one of the preceding claims, wherein the multiple of the median (MoM) of the first level is below 1.0, and the MoM of the second level is above 1.0, preferably above 2.0, more preferably above 3.0, indicating the occurrence of early-onset preeclampsia before 231 days of GA.

8. The method according to any one of the preceding claims, wherein a higher second level of sFlt-1 or a fragment thereof compared to the first level further indicates the subsequent occurrence of intrauterine fetal death (IUFD).

9. The method according to any one of the preceding claims, wherein the sample is a body fluid sample, such as a blood sample, such as a venous blood sample, a capillary blood sample, a serum sample, a plasma sample, a vaginal secretion sample, a saliva sample, or an amniotic fluid sample, preferably a blood sample, a serum sample, or a plasma sample.

10. The method according to any one of the preceding claims, wherein a second level of sFlt-1 or a fragment thereof that is higher compared to the first level indicates initiation or modification of treatment of the subject to reduce the risk of developing preeclampsia, delay the onset time point of preeclampsia, and / or reduce the severity of preeclampsia, for example by balancing the angiogenic / anti-angiogenic processes in placental development, reducing blood pressure, and / or protecting organ functions, such as the functions of the kidney and / or liver.

11. The method according to claim 10, wherein the indicated treatment comprises administration of acetylsalicylic acid.

12. The method according to any one of the preceding claims, the method further comprising determining the level of placental growth factor (PIGF) or a fragment thereof in the first sample and / or the second sample from the patient, wherein the combination of the level of sFlt-1 or a fragment thereof and the level of PIGF or a fragment thereof in the first sample and / or the second sample indicates early-onset preeclampsia before the end of week 33 of gestation.

13. The method according to any one of the preceding claims, the method further comprising determining or providing the maternal age, body mass index, mean arterial pressure (MAP), and / or uterine artery Doppler measurements of the subject, wherein the combination of the level of sFlt-1 or a fragment thereof in the first sample and / or the second sample with the maternal age, body mass index, mean arterial pressure (MAP), and / or uterine artery Doppler measurements of the subject, preferably in combination with the level of PIGF or a fragment thereof in the first sample and / or the second sample, indicates early-onset preeclampsia before the end of week 33 of gestation.

14. A kit for performing the method according to any one of the preceding claims, comprising - a detection reagent for determining the level of sFlt-1 or a fragment thereof in a sample from a subject, and - computer software in the form of a computer-readable medium and / or computer-executable code, the computer-readable medium and / or the computer software being configured to compare two determined levels of sFlt-1 or a fragment thereof, - wherein the computer-readable medium and / or the computer software optionally contains one or more reference levels of sFlt-1 or a fragment thereof, the reference levels preferably corresponding to the population mean and / or median of a healthy population, and the computer-readable medium and / or the computer software being configured to compare the two determined levels of sFlt-1 or a fragment thereof with the reference levels, and / or - wherein the computer-readable medium and / or the computer software is optionally configured to compare the maternal age, body mass index, and / or uterine artery Doppler measurements of the subject with one or more reference levels, the reference levels preferably corresponding to the population mean and / or median of a healthy population.

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