Multiple SFLT-1 measurements for the prognosis of early-onset preeclampsia

Abstract

The present invention relates to a method for prognosing, predicting, risk assessment, and / or risk stratifying preeclampsia in a pregnant subject, comprising determining the level of soluble fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof in a sample isolated from the pregnant subject. The present invention further relates to a method for prognosing, predicting, risk assessment, and / or risk stratifying early-onset preeclampsia in a pregnant subject. The present invention relates to a combined measurement of sFlt-1 and PlGF, optionally in combination taking into account one or more additional factors selected from maternal age, body mass index, uterine artery Doppler measurement, and / or mean arterial pressure (MAP). The present invention further relates to a kit for carrying out the method of the present invention, comprising detection reagents for determining the level of sFlt-1 or a fragment thereof in a sample from the subject, and optionally for determining the level of at least one additional biomarker described herein.

Description

[Technical Field]

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

[0002] Accordingly, the present invention relates to a method for the prognosis, prediction, risk assessment, and / or risk stratification of pre-eclampsia in a pregnant subject, comprising determining the level of sFlt-1 or a fragment thereof in a sample isolated from the pregnant subject, wherein the level of sFlt-1 or a fragment thereof is indicative of the likelihood of pre-eclampsia.

[0003] The present invention further relates to a method for prognosis, prediction, risk assessment, and / or risk stratification of 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, the first sample being isolated before the end of the 14th week of gestation; (b) determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, the second sample being isolated after the first sample; and (c) a higher second level of sFlt-1 or a fragment thereof compared to the first level is indicative of early onset preeclampsia occurring before the end of the 33rd week of gestation (before 231 days).

[0004] The present invention further relates to a method for prognosis, prediction, risk assessment, and / or risk stratification of early-onset pre-eclampsia 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 (PlGF) or a fragment thereof in a first sample and / or a second sample. The present invention further relates to the measurement of sFlt-1 and PlGF, optionally in combination taking into account 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 further relates to a kit for carrying out the method of the present invention, 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 described herein, such as PlGF. [Background technology]

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

[0007] The risk of maternal mortality is much higher in resource-limited settings. The most frequently recognized factor contributing to major maternal and fetal morbidity is failure to recognize preeclampsia in a timely manner. As a result, pregnant women do not receive effective monitoring or treatment until long after complications associated with the disorder, including elevated blood pressure and proteinuria, have developed. In addition, pregnant women who are at little or no risk of developing such disorders must undergo unnecessary testing for symptoms throughout their pregnancy because there are no effective means by which caregivers can exclude them from risk early in their pregnancy.

[0008] WO 2008 / 103202(A2) discloses a method for diagnosing pregnancy-associated hypertensive disorders by measuring COMT, HIF-1[α], EPO, LDH-A, ET-I, transferrin, transferrin receptor, and flk-I, free VEGF, total VEGF, sFlt-1, and PlGF. Altered expression of these polypeptides compared to reference levels is indicative of pregnancy-associated hypertensive disorders.

[0009] WO 2006 / 069373(A2) discloses a method for diagnosing whether a pregnant woman has or is susceptible to developing a hypertensive disorder. The levels of sFlt-1 and placental growth factor (PlGF) are measured in a urine sample. The ratio of sFlt-1 expression to PlGF expression is used as an indicator of whether the 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 administering a compound capable of binding to soluble fms-like tyrosine kinase-1 (sFlt-1). It was further disclosed that higher sFlt-1 concentrations in patients prior to the onset of preeclampsia are due to an acute elevation of sFlt-1 within 5 weeks before the onset of clinical disease.

[0011] Myatt et al. (BJOG: International Journal of Obstetrics and Gynaecology, vol. 120, no. 10, 2013) describe the measurement of PlGF, sFlt-1, and sEng during the first and second trimesters of pregnancy in low-risk patients. Samples were collected at 9-12, 15-18, and 23-26 weeks of gestation. Changes in biomarker levels from the first to second trimester and their association with the occurrence of early-onset preeclampsia were investigated.

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

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

[0014] Staff et al. (Cardiovascular Health, vol. 1, no. 1, 2010) disclose various biomarkers for predicting preeclampsia, including sFlt-1 and PlGF. This document cites conflicting studies regarding the prediction of preeclampsia based on the measurement of sFlt-1. This document includes several studies disclosing the prediction of early-onset preeclampsia by measuring sFlt-1, but also presents studies disclosing that measuring sFlt-1 at some time points during pregnancy is not suitable for predicting preeclampsia.

[0015] To date, the most invasive treatment for preeclampsia is termination of pregnancy, either by preterm vaginal delivery or cesarean section. As previously mentioned, maternal risk and fetal survival are significantly compromised in cases of preeclampsia before 34 weeks of gestation. Therefore, attempts should be made to delay delivery, thereby improving neonatal survival. Tsakiridis et al. (Volume 76, Number 10, Obstetric and Gynecological Survey) highlighted pregnancy guidelines describing early administration of low-dose aspirin in high-risk patients, ideally during the first trimester or up to 36–37 weeks of gestation (days 246–259) until delivery.

[0016] Improving the prognosis within the first stage of pre-eclampsia in women who are clinically asymptomatic or suspected of having or developing pre-eclampsia is of great clinical importance.

[0017] The most severe forms, such as premature PE, require treatment to be initiated as early as possible, ideally at the 11th week of pregnancy (71–77 days of gestation). Currently, approximately 60–66% of premature PE cases and approximately 70% of premature PE cases are detected using the Fetal Medicine Foundation (FMF) screening algorithm. The FMF screening algorithm involves consideration of multiple maternal characteristics and medical history, 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, a technology not universally available for all pregnant women.

[0018] Furthermore, sFlt-1 levels vary significantly at different gestational ages and correlate with preeclampsia, but both low (e.g., below healthy average levels) and high (e.g., above healthy average levels) levels have been observed, and the dynamics of sFlt-1 levels and their relationship to PE currently remain unclear. Findings regarding sFlt-1 concentrations in pregnancies related to PE risk are contradictory, with studies reporting decreased, increased, or unchanged sFlt-1 concentrations in pregnancies that develop PE (Pihl et al., Fetal Diagn Ther 2020;47:277-283; Akolekar et al., Prenat Diagn. 2010 Mar;30(3):191-7).

[0019] There is an urgent need in the art for improved and simplified means for determining the risk of pre-eclampsia, particularly early-onset pre-eclampsia, early in pregnancy. Summary of the Invention

[0020] In light of the prior art, the technical problem underlying the present invention is to provide an improved or alternative means for the prognosis, prediction, risk assessment, and / or risk stratification of pre-eclampsia in pregnant subjects. A further object of the present invention is to provide a means for early prognosis or risk assessment of pre-eclampsia. A further object of the present invention is to provide a prognostic approach to risk assessment of pre-eclampsia within the first trimester of pregnancy. A further object of the present invention relates to providing a means for improving and / or simplifying the screening or prognosis of early-onset pre-eclampsia in the early stages of pregnancy, which increases the sensitivity and preferably does not require uterine artery Doppler measurements.

[0021] This problem is solved by the features of the independent claims. Preferred embodiments of the invention are provided by the dependent claims.

[0022] Accordingly, the present invention provides a method for prognosis, prediction, risk assessment, and / or risk stratification of early-onset pre-eclampsia 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, the first sample being isolated before 90 days gestational age (GA); b. determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, the second sample being isolated after the first sample; c. A method wherein a second level of sFlt-1 or a fragment thereof that is higher compared to said first level is indicative of early-onset preeclampsia occurring before the end of 33 weeks gestation (before 231 days GA).

[0023] Therefore, the methods described herein enable EO-PE risk assessment early in pregnancy, thus providing clinicians with the possibility of initiating appropriate preventive treatment early in pregnancy. For the most severe forms of PE, such as EO-PE, treatment is recommended as early as possible, preferably at 16 weeks of gestation (GA 106-112 days), and more preferably at 11 weeks of gestation (GA 71-77 days). Until the present invention, diagnostic assays for PE prognosis were available, but prognosis of early-stage EO-PE primarily relied on FMF algorithms using uterine artery Doppler measurements. Thus, the present invention enables a simple and reliable molecular diagnostic and / or prognostic approach aimed at identifying subjects at risk for EO-PE at very early gestational age (GA).

[0024] Furthermore, sFlt-1 levels appear to vary significantly at different gestational ages. While sFlt-1 appears to correlate with preeclampsia, both low (e.g., below healthy average levels) and high (e.g., above healthy average levels) levels have been observed, and currently, the dynamics of sFlt-1 levels and their relationship to PE remain unclear. Therefore, another difficulty diagnosticians or physicians face in assessing EO-PE risk is that, although sFlt-1 appears to be a promising marker with some relationship to PE, the lack of understanding of sFlt-1 dynamics during pregnancy leads to difficulties in interpreting sFlt-1 levels and their meaning with respect to PE risk, particularly EO-PE risk. For example, if the exact date of conception is unknown or is falsely or incorrectly recorded, sFlt-1 levels obtained from routine testing may be misleading, resulting in either a false-negative or false-positive result with respect to PE or EO-PE risk. In particular, sFlit-1 levels obtained from a single sample within 12–13 weeks of gestation (GA of 78–91 days) can be misleading and most likely provide a false-negative result, as levels in subjects without EO-PE and subjects with EO-PE do not differ substantially within 12–13 weeks of gestation (GA of 78–91 days) (see Figure 15B).

[0025] In embodiments, the gestational age of a pregnant subject is determined by abdominal and / or vaginal ultrasound prior to obtaining a first sample from the subject.

[0026] For example, Pihl et al. (Fetal Diagn Ther 2020;47:277-283) review that results regarding sFlt-1 concentrations in pregnancy with respect to PE risk are conflicting, with studies reporting decreased, increased, or unchanged sFlt-1 concentrations in pregnancies that develop PE. Nevertheless, the present invention now enables a novel approach using sFlt-1 measurements in risk assessment of PE and EO-PE.

[0027] The inventors have identified that sFlt-1 levels are significantly but inversely correlated with EO-PE risk within the first 90 days of GA (low sFlt-1 levels correlate with an increased risk of EO-PE). However, assessment of sFlt-1 at 90 to 100 days does not show significant differences relative to the healthy population average, even in subjects who will develop EO-PE. Furthermore, after 100 days of GA, e.g., between 140 and 154 days of GA, sFlt-1 levels are significantly positively correlated with EO-PE risk (high sFlt-1 levels correlate with an increased risk of EO-PE). Thus, the inventors have developed a prognostic analysis scheme in which two samples are obtained from a pregnant subject, and an increase in sFlt-1 levels at a required time point indicates an elevated risk of EO-PE above the healthy population average. Obtaining a second sample after the first sample according to the present invention advantageously reduces false-negative results, particularly early in pregnancy, e.g., within the first trimester and at the beginning of the second trimester.

[0028] As can be seen from Figure 1 and the Examples below, before 90 days of gestation, women with EO-PE (N=10) had sFlt-1 levels below the median (p<0.01). Between 90-100 days of gestation, on average, women with EO-PE (N=24) had sFlt-1 levels. Between 140-154 days of gestation, women with EO-PE (N=4) had sFlt-1 levels above the median. Similar results can be seen from Figures 14 and 15B. These results allow us to conclude that sFlt-1 levels are abnormally decreased early in pregnancy (before 90 days of gestation) in women who will develop early preeclampsia (before 34 weeks of gestation, before 232 days of gestation), progressively increase to normal by the end of the first trimester (90-100 days), and then abnormally increase thereafter (after 140 days of gestation).

[0029] Various aspects of the present invention are based on and / or associated with the common discovery that levels of sFlt-1 or fragments thereof in samples from pregnant subjects are significantly lower than the healthy average in the first 90 days GA and increase to or exceed the healthy average after 100 days GA, such that increased sFlt-1 levels are indicative of a likelihood of early-onset preeclampsia. Advantageously, the prognostic analysis scheme of the present invention, in which two samples are obtained from a pregnant subject, thereby provides a reduced false negative rate compared to analysis schemes in which a prognostic statement is made based on a single sample obtained from the subject.

[0030] The present invention provides an efficient and reliable test for medical professionals, such as physicians, nurses, and emergency department personnel, to quickly and accurately assess the likelihood that a pregnant subject will develop PE (e.g., preterm PE, spontaneous abortion, EO-PE and severe PE, and associated complications such as preterm birth and short gestational age), particularly EO-PE. Typically, pregnant women who are at little or no risk of developing such disorders must undergo unnecessary testing for symptoms throughout their pregnancy because there are no effective means by which caregivers can exclude them from risk early in their pregnancy.

[0031] High levels of placental soluble fms-like tyrosine kinase (sFlt-1) are strongly associated with PE in the second trimester (14-27 weeks gestation, 92-189 days GA) and third trimester (28 weeks gestation to term, 190 days GA to term). Surprisingly, according to the present invention, levels of sFlt-1 or fragments thereof in the first 90 days of pregnancy are also associated with early-onset PE, with low sFlt-1 levels indicative of EO-PE.

[0032] Based on this surprising discovery, the present invention provides a means for identifying pregnant subjects at increased or high risk of developing EO-PE, and also for identifying patients who are less likely to develop such a complication or whose development can be substantially ruled out by determining the level of sFlt-1 or a fragment thereof in a sample isolated from the patient.

[0033] An additional benefit resulting from this surprising discovery is that, in addition to the conventional combination of biomarkers, clinical parameters, and imaging procedures for prognosing PE, the prognostic marker sFlt-1 can be used in any clinical setting, regardless of whether a device for measuring the uterine artery pulsatility index (UAPI) is available, which typically requires special ultrasound equipment and specialists to operate the device and perform the measurement, thus enabling a simple and minimally invasive test.

[0034] More notably, the Aspirin for Evidence-Based Preeclampsia Prevention (ASPRE) trial, a multicenter trial involving women identified as at high risk for preterm PE according to the FMF algorithm who were randomized to receive aspirin or placebo from 11 to 14 weeks gestation through 37 weeks gestation (71 to 98 days GA to 259 days GA), showed a 62% reduction in preterm PE with daily low-dose aspirin compared with the placebo group (relative risk, 0.38; 95% confidence interval [CI], 0.20 to 0.74).

[0035] If a high risk of developing EO-PE can be accurately prognosed in the first 12 or 14 weeks of pregnancy (78-98 days GA), this efficient treatment can be applied to pregnant subjects in the early stages of pregnancy. This provides another advantage based on the surprising discovery that a high risk of developing EO-PE can be prognosed in the first trimester of pregnancy, so that subjects who are prognosticated as having a high risk of developing PE can be given mild prophylactic treatment, medication, or even bed rest and frequent monitoring instead of any serious measures (e.g., early termination of pregnancy after developing PE in the later stages of pregnancy).

[0036] In one embodiment, the present invention provides a method for prognosis, prediction, risk assessment, and / or risk stratification of early-onset pre-eclampsia 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, the first sample being isolated before the end of 13 weeks gestation (before 90 days GA); b. determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, the second sample being isolated after the first sample; c. A method wherein a second level of sFlt-1 or a fragment thereof that is higher compared to said first level is indicative of early-onset preeclampsia occurring before the end of 33 weeks gestation (before 231 days GA).

[0037] In one embodiment, the present invention provides a method for prognosis, prediction, risk assessment, and / or risk stratification of early-onset pre-eclampsia 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, the first sample being isolated before the end of 13 weeks gestation (before 90 days GA); b. determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, the second sample being isolated after the first sample and being isolated after the end of 20 weeks gestation (after 140 days GA); c. A method wherein a second level of sFlt-1 or a fragment thereof that is higher compared to said first level is indicative of early-onset preeclampsia occurring before the end of 33 weeks gestation (before 231 days GA).

[0038] In one embodiment, the present invention provides a method for prognosis, prediction, risk assessment, and / or risk stratification of early-onset pre-eclampsia 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, the first sample being isolated prior to 90 days GA; b. determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, the second sample being isolated after the first sample and the second sample being isolated after 140 days GA; c. A method wherein a second level of sFlt-1 or a fragment thereof that is higher compared to said first level is indicative of early-onset preeclampsia occurring before GA of 231 days.

[0039] In an embodiment, the first sample is isolated from the subject before the end of the 12th or 14th week of gestation (before a GA of 100 days).

[0040] In an embodiment, the first sample is isolated from the subject before the end of the 7th or 9th gestational week (before a GA of 63 days).

[0041] In an embodiment, the first sample is isolated from the subject before the end of the 8th or 9th week of gestation (before a GA of 63 days).

[0042] Given the data presented herein, sFlt-1 levels appear to increase over time during gestation, with increases evident from 90 days GA to before 90-100 days GA, from 90-100 days GA to later time points, and from before 90 days GA to later time points, e.g., either 140-154 days or later (FIGS. 1, 14, and 15, and the Examples below). Thus, obtaining and evaluating a first sample before 100 days GA, then measuring a subsequent sample, and detecting an increased level of sFlt-1 in the second sample, indicates a risk of EO-PE in the subject. Furthermore, obtaining and evaluating a first sample before 90 days GA, then measuring a subsequent sample, and detecting an increased level of sFlt-1 in the second sample, indicates a risk of EO-PE in the subject. Furthermore, obtaining and evaluating a first sample before 63 days GA, then measuring a subsequent sample, and detecting an increased level of sFlt-1 in the second sample, indicates a risk of EO-PE in the subject.

[0043] In embodiments, the first sample was isolated from the subject before the end of the 13th week of gestation (before a GA of 90 days). In embodiments, the first sample was isolated from the subject before the end of the 9th week of gestation (before a GA of 63 days). In embodiments, the first sample was isolated from the subject before the end of the 8th week of gestation (before a GA of 56 days). In embodiments, the first sample was isolated from the subject before the end of the 7th week of gestation (before a GA of 49 days).

[0044] As outlined in detail below, levels of sFlt-1 in samples from pregnant subjects are significantly lower than the healthy average in the first 90 days GA and increase to or exceed the healthy average after 100 days GA, and thus increased levels of sFlt-1 indicate the likelihood of early-onset preeclampsia. In particular, levels prior to 90 days GA are significantly lower than the healthy average, and therefore obtaining the first sample within 90 days GA is a preferred embodiment of the present invention.

[0045] In embodiments, the level of sFlt-1 in a sample obtained in the first 90 days GA from a pregnant subject experiencing early-onset pre-eclampsia occurring before the end of 33 weeks gestation (before 231 days) is less than the 10th percentile of the levels of sFlt-1 in a healthy population in the first 90 days GA. In embodiments, the level of sFlt-1 in a sample obtained in the first 90 days GA from a pregnant subject experiencing early-onset pre-eclampsia occurring before the end of 33 weeks gestation (before 231 days GA) is less than the 15th percentile, e.g., less than the 15th, 14th, 13th, 12th, 11th, or 10th percentile, of the levels of sFlt-1 in a healthy population in the first 90 days GA.

[0046] In embodiments, the level of sFlt-1 in a second sample obtained after the first sample from a pregnant subject experiencing early-onset pre-eclampsia occurring before the end of 33 weeks gestation (before a GA of 231 days) is greater than the 90th percentile of levels of sFlt-1 in a healthy population. In embodiments, the level of sFlt-1 in a sample obtained after the first sample from a pregnant subject experiencing early-onset pre-eclampsia occurring before the end of 33 weeks gestation (before a GA of 231 days) is greater than the 95th percentile of levels of sFlt-1 in a healthy population, e.g., greater than the 95th, 96th, 97th, 98th, or 99th percentile.

[0047] In embodiments, the level of sFlt-1 in a second sample obtained after the end of the 13th week of gestation (after a GA of 90 days) from a pregnant subject experiencing early-onset pre-eclampsia occurring before the end of the 33rd week of gestation (before a GA of 231 days) is greater than the 90th percentile of levels of sFlt-1 in a healthy population. In embodiments, the level of sFlt-1 in a sample obtained after the end of the 13th week of gestation (after a GA of 90 days) from a pregnant subject experiencing early-onset pre-eclampsia occurring before the end of the 33rd week of gestation (before a GA of 231 days) is greater than the 95th percentile of levels of sFlt-1 in a healthy population, e.g., greater than the 95th, 96th, 97th, 98th, or 99th percentile.

[0048] In embodiments, the level of sFlt-1 in a second sample obtained between the end of the 20th week and the end of the 22nd week of gestation (between a GA of 140-154 days) from a pregnant subject experiencing early-onset pre-eclampsia occurring before the end of the 33rd week of gestation (before a GA of 231 days) is greater than the 90th percentile of sFlt-1 levels in a healthy population. In embodiments, the level of sFlt-1 in a sample obtained between the end of the 20th week and the end of the 22nd week of gestation (between a GA of 140-154 days) from a pregnant subject experiencing early-onset pre-eclampsia occurring before the end of the 33rd week of gestation (before a GA of 231 days) is greater than the 95th percentile of sFlt-1 levels in a healthy population, e.g., greater than the 95th, 96th, 97th, 98th, or 99th percentile.

[0049] In one embodiment, a healthy population refers to a population of pregnant subjects who do not develop early-onset pre-eclampsia, which occurs before the end of the 33rd week of gestation (before a GA of 231 days). In one embodiment, the sample is taken from a healthy population of the same gestational age as the pregnant subject from which the sample is obtained according to the methods of the present invention.

[0050] In one embodiment, the MoM of sFlt-1 levels in a first sample obtained before the end of the 13th week of gestation (before a GA of 90 days) from a pregnant subject experiencing early-onset preeclampsia occurring before the end of the 33rd week of gestation (before a GA of 231 days) is less than 1.0, preferably less than 0.9, more preferably less than 0.8, e.g., 0.99, 0.98, 0.97, 0.96, 0.95, 0.94, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 0.99, 0.98, 0.97, 0.96, 0.95, 0.94, 0.93 ... 2, 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.

[0051] In one embodiment, the MoM of sFlt-1 levels in a first sample obtained before the end of the 9th week of gestation (before a GA of 63 days) from a pregnant subject experiencing early-onset preeclampsia occurring before the end of the 33rd week of gestation (before a GA of 231 days) is less than 1.0, preferably less than 0.9, more preferably less than 0.8, e.g., 0.99, 0.98, 0.97, 0.96, 0.95, 0.94, 0.93, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98 ...9, 0.98, 0.99, 0.98, 0.99, 0.96, 0.95, 0 , 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.

[0052] In one embodiment, the MoM of sFlt-1 levels in a second sample obtained after the end of the 13th week of gestation (after a GA of 90 days) from a pregnant subject experiencing early-onset pre-eclampsia occurring before the end of the 33rd week of gestation (before a GA of 231 days) is greater than 1.0, preferably greater than 2.0, more preferably greater than 3.0, e.g., 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.

[0053] In one embodiment, the MoM of sFlt-1 levels in a second sample obtained after the end of the 13th week of gestation (after a GA of 90 days) from a pregnant subject experiencing early-onset pre-eclampsia occurring before the end of the 33rd week of gestation (before a GA of 231 days) is greater than 1.0, preferably greater than 2.0, more preferably greater than 3.0, e.g., 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.

[0054] In one embodiment, the MoM of sFlt-1 levels in a second sample obtained between the end of the 20th and 22nd gestational weeks (between 140 and 154 days GA) from a pregnant subject experiencing early-onset pre-eclampsia occurring before the end of the 33rd week of gestation (before a GA of 231 days) is greater than 1.0, preferably greater than 2.0, and more preferably greater than 3.0, e.g., 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.

[0055] In one embodiment, a healthy population that does not suffer from early-onset pre-eclampsia occurring before the end of the 33rd week of gestation (before a GA of 231 days) has an MoM of 1.0.

[0056] In one embodiment, a first level multiple of the median (MoM) of less than 1.0 and a second level MoM of greater than 1.0, preferably greater than 2.0, more preferably greater than 3.0 indicates early-onset preeclampsia occurring before a GA of 231 days.

[0057] In embodiments, the second sample was isolated from the subject after the end of the 13th week of gestation (after a GA of 90 days). In embodiments, the second sample was isolated from the subject after the end of the 14th week of gestation (after a GA of 100 days). In embodiments, the second sample was isolated from the subject between the 11th and 13th weeks of gestation (between a GA of 71 and 91 days). In embodiments, the second sample was isolated from the subject after the end of the 10th week of gestation (after a GA of 70 days). In embodiments, the second sample was isolated from the subject after the end of the 13th week of gestation (after a GA of 91 days). In embodiments, the second sample was isolated from the subject during the second trimester. Given the kinetics of sFlt-1 disclosed herein, isolating a second sample after the first sample and detecting an increase in sFlt-1 in the second sample is indicative of EO-PE risk. However, by obtaining and testing a second sample after 90 days, or after 100 days, or so, an even larger and more significant difference between the first and second sample can be determined, thus indicating EO-PE. Furthermore, obtaining and testing a first sample before 90 days, or even before 63 days, or even before 49 days of pregnancy provides an early indication of EO-PE risk, allowing for earlier initiation of treatment or increased monitoring rates, improving maternal and child health.

[0058] According to the present invention, the term "indicative," for example in the context of "indicating early-onset pre-eclampsia," is intended as a term of risk and / or likelihood. Preferably, an "indication" of the presence, absence, or subsequent development of early-onset pre-eclampsia (or other condition) is intended as a risk assessment and is typically not to be interpreted in a restrictive manner so as to clearly refer to the absolute presence or absence of the condition.

[0059] In other embodiments, the second sample can be obtained, but is not limited to, 1 day after the first sample, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 days after the first sample.

[0060] In one embodiment, the second sample is obtained at least one week after the first sample.

[0061] In one embodiment, the second sample is obtained at least two weeks after the first sample.

[0062] In one embodiment, the second sample is obtained at least three weeks after the first sample.

[0063] In an embodiment, the second sample was isolated after the end of the 20th week of gestation (after a GA of 140 days). In an embodiment, the second sample was isolated between the end of the 20th week and the end of the 22nd week of gestation (a GA of 140-154 days).

[0064] As outlined in the Examples herein, sFlt-1 levels observed at these time points during gestation allow for the determination of even greater and / or more significant differences between the first and second samples, thus indicating EO-PE risk.

[0065] In embodiments, the sFlt-1 level in the second sample is at least 1% higher than 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 in the first sample, even if the single measurement point is decreased or the same average as compared to a healthy population (reference level).

[0066] In an embodiment, the first level is below a reference level, preferably below the population mean and / or median of a healthy population.

[0067] In an embodiment, the second level is equal to or greater than a reference level, preferably the population mean and / or median of a healthy population.

[0068] As shown in Figure 1 and in the Examples below, subjects who do not acquire EO-PE exhibit significantly different sFlt-1 levels than subjects who will develop EO-PE. To determine the sFlt-1 level associated with risk, a comparison between the first and second samples and / or a comparison with a reference value can be made.

[0069] In method embodiments, the subject is at 9-11 weeks gestational age (57-77 days GA) when the first sample is obtained. In method embodiments, the subject is at 11 weeks gestational age (71-77 days GA) when the first sample is obtained. In method embodiments, the subject is at 50-90 days, preferably 60-90 days or 70-90 days gestational age (GA), e.g., 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 GA.

[0070] In method embodiments, the subject is at 7-9 weeks gestational age (43-63 days GA) when the first sample is obtained. In method embodiments, the subject is at 8-9 weeks gestational age (50-63 days GA) when the first sample is obtained. In method embodiments, the subject is at 43-63 days, preferably 50-63 days, gestational age (GA), e.g., 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63 days GA.

[0071] In embodiments of the method, the subject is between 7 and 11 weeks gestational age (43-77 days GA) when the first sample is obtained. In embodiments of the method, the subject is between 8 and 11 weeks gestational age (50-77 days GA) when the first sample is obtained. In embodiments of the method, the subject is at a gestational age (GA) of 43 to 77 days, preferably 50 to 77 days, e.g., 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 GA.

[0072] As previously mentioned, these early time points of sampling and testing allow for early initiation of treatment, which may be important in effectively treating and / or preventing the development of severe forms of PE.

[0073] In one embodiment of the method, a. the first level is less than a reference level and the second level is greater than or equal to the reference level, preferably the population mean and / or median of a healthy population; or b. the first level is below a reference level and the second level is above a reference level, preferably above 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, preferably above the population mean and / or median of a healthy population.

[0074] In embodiments, the present invention provides a method for prognosis, prediction, risk assessment, and / or risk stratification of early-onset pre-eclampsia 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, the first sample being isolated before the end of 13 weeks gestation (before 90 days GA); b. determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, the second sample being isolated after the first sample; c. A method wherein a first level below the reference level and a second level equal to or greater than the reference level, and wherein the second level of sFlt-1 or a fragment thereof higher than the first level indicates early-onset preeclampsia occurring before the end of 33 weeks gestation (before 231 days GA).

[0075] In embodiments, the present invention provides a method for prognosis, prediction, risk assessment, and / or risk stratification of early-onset pre-eclampsia 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, the first sample being isolated before the end of 13 weeks gestation (before 90 days GA); b. determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, the second sample being isolated after the first sample; c. A method wherein a first level below the reference level, a second level above the reference level, and a second level of sFlt-1 or a fragment thereof higher than the first level indicates early-onset preeclampsia occurring before the end of 33 weeks of gestation (before 231 days GA).

[0076] In embodiments, the present invention provides a method for prognosis, prediction, risk assessment, and / or risk stratification of early-onset pre-eclampsia 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, the first sample being isolated before the end of 13 weeks gestation (before 90 days GA); b. determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, the second sample being isolated after the first sample; c. A method wherein a first level below the reference level and a second level above the reference level, and a second level of sFlt-1 or a fragment thereof higher than the first level, is indicative of early-onset preeclampsia occurring before the end of 33 weeks gestation (before 231 days GA).

[0077] Further embodiments and aspects of the present invention relate to the methods described herein being used for indication of intrauterine fetal death (IUFD).

[0078] In one embodiment of the method, a higher second level of sFlt-1 or a fragment thereof compared to the first level is further indicative of the occurrence of subsequent intrauterine fetal death (IUFD). In an embodiment, the indication of the occurrence of subsequent intrauterine fetal death relates to an increased risk for the patient of subsequent IUFD above the average risk in a healthy population.

[0079] Embodiments of the present invention relate to the prognosis, prediction, risk assessment, and / or risk stratification of IUFD. The prognosis of IUFD can be independent of or combined with the prognosis of EO-PE. 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 with respect to the prognosis of EO-PE apply equally to the prognosis of IUFD, and vice versa.

[0080] As shown in more detail below, the combination of sFlt-1 and PlGF resulted in a prognosis of IUFD when samples were obtained between 90 and 100 days GA (Figure 11) and showed a statistically improved prognosis of IUFD when samples were obtained before 90 days GA (Figure 12).

[0081] Further embodiments relating to combinations with other biomarkers: In one embodiment, the method comprises: a. determining the level of placenta growth factor (PlGF) or a fragment thereof in the first sample and / or the second sample; b. The combination of levels of sFlt-1 or a fragment thereof and levels of said PlGF or a fragment thereof indicates the likelihood of early-onset pre-eclampsia occurring before the end of the 33rd week of gestation (before a GA of 231 days).

[0082] In embodiments, the methods of the present invention further comprise determining the level of placental growth factor (PlGF) 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 PlGF or a fragment thereof in the first sample and / or the second sample indicates early-onset preeclampsia occurring before the end of the 33rd week of gestation (before a GA of 231 days).

[0083] As shown in more detail below, the combination of sFlt-1 and PlGF results in a statistically improved prognosis for EO-PE when samples are obtained early in pregnancy, e.g., before the end of the 13th gestational week (before 90 days GA). Notably, while PlGF is typically effective in prognosing EO-PE when measured after 90 days GA, sFlt-1 does not appear to allow for reliable prognostic statements from a single measurement after 90 days GA (Figure 7). Surprisingly, while both sFlt-1 and PlGF allow for EO-PE prognosis when measured before the end of the 13th week (within 90 days) GA, sFlt-1 appears to provide greater sensitivity, with comparable specificity values, preferably greater than 0.6 (Figure 8). Also surprising, combined analysis of sFlt-1 and PlGF demonstrates an unexpected synergistic enhancement in EO-PE prognosis when measured before 90 days GA (Figure 9).

[0084] In an embodiment, the method comprises: a. determining or providing the subject's maternal age, body mass index, and / or uterine artery Doppler measurements; b. The level of sFlt-1 or a fragment thereof, preferably in combination with the level of PlGF or a fragment thereof, in combination with the subject's maternal age, body mass index, and / or uterine artery Doppler measurement is indicative of early-onset preeclampsia occurring before the end of the 33rd week of gestation (before a GA of 231 days).

[0085] In an embodiment, the method comprises: a. determining or providing a level of mean arterial pressure (MAP) in the subject; b. The level of sFlt-1 or a fragment thereof, preferably in combination with the level of PlGF or a fragment thereof, in combination with the level of MAP in the subject, is indicative of early-onset preeclampsia occurring before the end of the 33rd week of gestation (before GA of 231 days).

[0086] As shown in Figure 10 below, the combination of sFlt-1 and PlGF measurements shows further improvement in diagnostic ability when combined with additional uterine artery Doppler measurements.

[0087] In an embodiment, the method comprises: a. determining a level of sFlt-1 or a fragment thereof, and determining a level of PlGF or a fragment thereof in a first sample and / or a second sample isolated from a subject; b. determining or providing the subject's maternal age, body mass index (BMI), and uterine artery Doppler measurements, and optionally mean arterial pressure (MAP); c. The level of said sFlt-1 or fragment thereof, the level of said PGF or fragment thereof, in combination with the subject's maternal age, body mass index (BMI), and uterine artery Doppler measurement, and optionally mean arterial pressure (MAP), is indicative of the likelihood of early-onset pre-eclampsia occurring before the end of the 33rd week of gestation (before a GA of 231 days).

[0088] In an embodiment, the method comprises: a. determining a level of sFlt-1 or a fragment thereof, and determining a level of PlGF or a fragment thereof in a first sample and / or a second sample isolated from a subject; b. determining or providing the subject's maternal age, body mass index (BMI), and mean arterial blood pressure (MAP), and optionally a uterine artery Doppler measurement; c. The level of said sFlt-1 or fragment thereof, the level of said PlGF or fragment thereof, in combination with the subject's maternal age, body mass index (BMI), and mean arterial blood pressure (MAP), and optionally a uterine artery Doppler measurement, indicates the likelihood of early-onset pre-eclampsia occurring before the end of the 33rd week of gestation (before a GA of 231 days).

[0089] In embodiments, the method further comprises determining or providing the subject's maternal age, body mass index, and / or uterine artery Doppler measurement, wherein a combination of the level of sFlt-1 or a fragment thereof in the first sample and / or the second sample with the subject's maternal age, body mass index, and / or uterine artery Doppler measurement, preferably in combination with the level of PlGF or a fragment thereof in the first sample and / or the second sample, and / or preferably in combination with the subject's level of mean arterial pressure (MAP), indicates early-onset pre-eclampsia occurring before the end of 33 weeks gestation (before 231 days GA).

[0090] In an embodiment of the present invention, the level of sFlt-1 or a fragment thereof, preferably the level of said sFlt-1 or a fragment thereof, the level of said PlGF or a fragment thereof, in combination with the subject's maternal age, body mass index (BMI), and uterine artery Doppler measurement, and optionally mean arterial pressure (MAP), is indicative of early onset of pre-eclampsia occurring from the beginning of the 20th week of gestation and the end of the 33rd week of gestation (before a GA of 231 days).

[0091] In an embodiment of the present invention, the level of sFlt-1 or a fragment thereof, preferably the level of said sFlt-1 or a fragment thereof, the level of said PlGF or a fragment thereof, in combination with the subject's maternal age, body mass index (BMI), and uterine artery Doppler measurement, and optionally mean arterial pressure (MAP), is further indicative of the occurrence of subsequent intrauterine fetal death (IUFD).

[0092] In an embodiment of the invention, the first sample and / or the second sample is a body fluid sample, such as a blood sample, e.g., a venous blood sample, a capillary blood sample, a serum sample, a plasma sample, a vaginal fluid sample, a saliva sample, or an amniotic fluid sample, preferably a blood, serum, or plasma sample.

[0093] In embodiments of the present invention, a higher second level of sFlt-1 or a fragment compared to the first level indicates initiation or modification of a subject's treatment to reduce the risk of developing pre-eclampsia, delay the time of onset of pre-eclampsia, and / or reduce the severity of pre-eclampsia, for example, by balancing angiogenic / anti-angiogenic processes in placental development, lowering blood pressure, and / or protecting organ function such as the kidney and / or liver.

[0094] In embodiments of the invention, the treatment is selected from the group consisting of one or more diuretics, beta-blockers, ace inhibitors, angiotensin II receptor blockers, calcium channel blockers, alpha-blockers, methyldopa, central agonists, and vasodilators, VEGF, PLGF, statins, arginine vasopressin receptor antagonists, L-arginine, citrulline, inhibitors of arginase (nor-NOHA), iron chelators (deferoxamine), heparin, magnesium sulfate, diazepam, phenytoin, vitamin D, calcium, molecular selenium inhibitors, extracorporeal extraction (e.g., apheresis), lifestyle recommendations, outpatient monitoring, increased frequency of maternal and fetal monitoring, preferably low-dose acetylsalicylic acid or metformin.

[0095] In an embodiment of the invention, the treatment comprises administration of acetylsalicylic acid.

[0096] In an embodiment of the invention, the treatment comprises administration of metformin.

[0097] Accordingly, the present invention provides a method for treating a pregnant subject to reduce the risk of early onset pre-eclampsia, comprising: a. Prognosis, prediction, risk assessment, and / or risk stratification of early-onset pre-eclampsia in a pregnant subject, comprising: - 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, the first sample being isolated before the end of 14 weeks gestation (before 100 days GA); - determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, the second sample being isolated after the first sample; - prognosis, prediction, risk assessment, and / or risk stratification of early-onset pre-eclampsia in a pregnant subject, wherein a higher second level of sFlt-1 or a fragment thereof compared to said first level is indicative of early-onset pre-eclampsia occurring before the end of 33 weeks gestation (before 231 days GA); b. administering a treatment to a subject to reduce the risk of developing pre-eclampsia, delay the time of onset, and / or reduce the severity of pre-eclampsia.

[0098] Accordingly, the present invention provides a method for treating a pregnant subject to reduce the risk of early onset pre-eclampsia, comprising: a. Prognosis, prediction, risk assessment, and / or risk stratification of early-onset pre-eclampsia in a pregnant subject, comprising: - 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, the first sample being isolated before the end of 13 weeks gestation (before 90 days GA); - determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, the second sample being isolated after the first sample; - prognosis, prediction, risk assessment, and / or risk stratification of early-onset pre-eclampsia in a pregnant subject, wherein a higher second level of sFlt-1 or a fragment thereof compared to said first level is indicative of early-onset pre-eclampsia occurring before the end of 33 weeks gestation (before 231 days GA); b. administering a treatment to a subject to reduce the risk of developing pre-eclampsia, delay the time of onset, and / or reduce the severity of pre-eclampsia.

[0099] Accordingly, the present invention provides a method for treating a pregnant subject to reduce the risk of early onset pre-eclampsia, comprising: a. Prognosis, prediction, risk assessment, and / or risk stratification of early-onset pre-eclampsia in a pregnant subject, comprising: - 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, the first sample being isolated before the end of the 9th week of gestation (before 63 days GA); - determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, the second sample being isolated after the first sample; - prognosis, prediction, risk assessment, and / or risk stratification of early-onset pre-eclampsia in a pregnant subject, wherein a higher second level of sFlt-1 or a fragment thereof compared to said first level is indicative of early-onset pre-eclampsia occurring before the end of 33 weeks gestation (before 231 days GA); b. administering a treatment to a subject to reduce the risk of developing pre-eclampsia, delay the time of onset, and / or reduce the severity of pre-eclampsia.

[0100] In embodiments, the administered treatment relates to or includes balancing the angiogenic / anti-angiogenic processes in placental development, lowering blood pressure, and / or protecting organ function such as the kidney and / or liver.

[0101] In embodiments of the method of treatment, the treatment is selected from the group consisting of one or more diuretics, beta-blockers, ACE inhibitors, angiotensin II receptor blockers, calcium channel blockers, alpha-blockers, statins such as pravastatin, methyldopa, central agonists, and vasodilators, VEGF, PLGF, statins, arginine vasopressin receptor antagonists, L-arginine, citrulline, inhibitors of arginase (nor-NOHA), iron chelators (deferoxamine), anticoagulants such as acetylsalicylic acid (also known as CHO or aspirin) and heparin, magnesium sulfate, diazepam, phenytoin, vitamin D, calcium, molecular selenium inhibitors, extracorporeal extraction (e.g., apheresis), lifestyle recommendations, outpatient monitoring, increased frequency of maternal and fetal monitoring, preferably low-dose acetylsalicylic acid or metformin.

[0102] In an embodiment of the method of treatment, the treatment comprises administration of an anticoagulant.

[0103] In an embodiment of the method of treatment, the treatment comprises administration of acetylsalicylic acid.

[0104] In embodiments of the method of treatment, the treatment involves administration of a low dose of acetylsalicylic acid, preferably 75 to 150 mg daily, e.g., 75, 80, 81, 85, 90, 95, 100, 110, 120, 130, 140, 150 mg.

[0105] In one embodiment of the method of treatment, the treatment comprises administration of 81 mg of acetyl acid (aspirin) daily. In one embodiment, the treatment comprises administration of acetyl acid (aspirin). In one embodiment, the treatment comprising administration of 81 mg of acetyl acid (aspirin) is initiated before 16 weeks gestation (before 106 days GA).

[0106] In an embodiment of the method of treatment, the treatment comprises administration of heparin.

[0107] In an embodiment of the method of treatment, the treatment comprises administration of metformin.

[0108] In embodiments, a level of sFlt-1 or a fragment thereof that is at least 6% lower than the reference sample indicates initiation or modification of the subject's treatment to reduce the risk of developing PE, delay the time of onset, or at least reduce the severity of PE, such as by balancing the angiogenic / anti-angiogenic processes in placental development, lowering blood pressure, or protecting organ function, such as from the kidney or liver.

[0109] A gynecologist and / or doctor can determine the appropriate treatment for a subject according to their current condition, with or without risk factors.

[0110] In embodiments, aspirin treatment can be initiated before 16 weeks gestation (before 106 days GA), which has been associated with a significant reduction in early PE. The Evidence-Based Aspirin for Preeclampsia Prevention (ASPRE) trial, a multicenter trial involving women identified as at high risk for early PE according to the FMF algorithm who were randomized to receive aspirin or placebo from 11-14 weeks gestation through 37 weeks gestation (71-98 days GA through 259 days GA), demonstrated a 62% reduction in early PE with daily low-dose aspirin compared with the placebo group (relative risk, 0.38; 95% confidence interval [CI], 0.20-0.74).

[0111] In an embodiment of the invention, the subject has one or more risk factors selected from the group consisting of hypothyroidism, hyperthyroidism, BMI greater than 24, first pregnancy, history of pre-eclampsia, ethnicity with a risk disorder, multiple pregnancy, migraine, lupus, blood clotting disorder such as increased clotting, inflammatory disease, impaired cardiac reserve, diabetes, chronic kidney disease, and chronic hypertension.

[0112] In an embodiment of the invention, the method further comprises determining the level of at least one additional biomarker or fragment thereof in the first sample and / or the 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, thrombocyte / leukocyte, IL6, IL11, MR-proADM, VEGF, PAPP-A, PlGF, 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 II, LNPEP, ESM1, HGF, pikachurin, hemopexin, pp13, uE3, CT-proET1, ADAM12, sTNFαR. 1, 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 and a level of at least one additional biomarker selected from the group consisting of: urea, ornithine, citrulline, apolipoprotein H, argininosuccinic acid, ammonia), uterine artery Doppler (uterine artery Doppler, UtA-Pi), diastolic notch, MAP, blood pressure, smoking, leptin, genetic information, arginine, cervical length, and a level of sFlt-1 or a fragment thereof is indicative of early-onset preeclampsia occurring before the end of the 33rd week of gestation.

[0113] In embodiments, the additional markers PAPP-A and / or PlGF are used in combination with sFlt-1 in the first sample and / or the second sample.

[0114] In embodiments, the additional markers PAPP-A, PlGF, and / or βhCG are used in combination with sFlt-1 in the first sample and / or the second sample.

[0115] In embodiments, the additional markers MAP, PAPP-A, and / or PlGF are used in combination with sFlt-1 in the first sample and / or the second sample.

[0116] In embodiments, the additional markers MAP, PAPP-A, βhGC, and / or PlGF are used in combination with sFlt-1 in the first sample and / or the second sample.

[0117] In an embodiment of the invention, the subject is nulliparous.

[0118] In an embodiment of the invention, the subject has had one or more previous pregnancies.

[0119] In an embodiment of the invention, the subject has a multiple pregnancy.

[0120] In an embodiment of the invention, the subject is suspected of carrying a fetus with a chromosomal abnormality.

[0121] A further aspect of the present invention relates to kits for carrying out the methods described herein.

[0122] In embodiments, the kit comprises: a detection reagent for determining the level of sFlt-1 or a fragment thereof in a sample from a subject; and - a computer readable medium in the form of computer executable code and / or computer software configured to compare the two determined levels of sFlt-1 or a fragment thereof.

[0123] In embodiments, the computer-readable medium and / or computer software optionally includes one or more reference levels of sFlt-1 or a fragment thereof, preferably corresponding to a population mean and / or median of a healthy population, and is configured to compare the two determined levels of sFlt-1 or a fragment thereof with the reference levels.

[0124] In embodiments, the computer-readable medium and / or computer software is optionally configured to compare the subject's maternal age, body mass index, and / or uterine artery Doppler measurements with one or more reference levels, preferably corresponding to population means and / or medians of a healthy population, and in embodiments of the invention, the software in the kit, or software to which the kit is configured to connect, enables comparison of determined molecular markers such as those described herein and provides a prognostic statement regarding EO-PE risk based on samples obtained within or after 90 days GA, for example, a prognostic statement regarding EO-PE risk based on a first sample obtained within 90 days GA and a second sample obtained after 90 days GA.

[0125] In embodiments of the invention, the kit includes a physical disc or computer readable medium having the software; alternatively, the kit may provide a link or other code, such as a QR code, suitable for directing and / or providing a connection to a server via the internet where the appropriate software can be maintained and / or executed.

[0126] Additional aspects and embodiments of the present invention: The present invention provides a method for prognosis, prediction, risk assessment, and / or risk stratification of pre-eclampsia in a pregnant subject, comprising: a. determining the level of sFlt-1 or a fragment thereof in a sample isolated from said pregnant subject; b. A method wherein the level of said sFlt-1 or a fragment thereof indicates the likelihood of pre-eclampsia.

[0127] The present invention further relates to a kit for carrying out the method of the present invention, 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 described herein, the kit further comprising reference levels, such as one or more cut-off levels corresponding to reference levels indicating a high or low risk of pre-eclampsia.

[0128] Accordingly, the present invention further provides a method for prognosis, prediction, risk assessment, and / or risk stratification of pre-eclampsia in a pregnant subject, comprising: a. determining the level of sFlt-1 or a fragment thereof in a sample isolated from said pregnant subject; b. the level of sFlt-1 or a fragment thereof is indicative of possible preeclampsia; c. The method relates to a method wherein the sample is isolated from the subject by the end of the 12th week of pregnancy (by 84 days GA).

[0129] In one embodiment, the subject is between 9 and 11 weeks pregnant (GA of 57-77 days), preferably between 11 and 77 days pregnant (GA of 71-77 days). In one embodiment, the subject is between 7 and 9 weeks pregnant (GA of 43-63 days), preferably between 8 and 9 weeks pregnant (GA of 50-63 days).

[0130] Surprisingly, significant differences in the predictive value (AUC value) of sFlt-1 for premature PE were observed among women recruited at 11 weeks gestation (71-77 days GA), 12 weeks gestation (78-84 days GA), and 13 weeks gestation (85-91 days GA). The AUC value for predicting premature PE using sFlt-1 levels in samples from subjects at 11 weeks gestation (71-77 days GA) reached 0.82. The AUC value for predicting premature PE using sFlt-1 levels in samples from subjects at 12 weeks gestation (78-84 days GA) reached 0.62. The AUC value for predicting premature PE using sFlt-1 levels in samples from subjects at 13 weeks gestation (78-91 days GA) reached 0.50. A similar trend, surprisingly, was observed for the prediction of mid-term onset PE.

[0131] The surprising benefit of this finding is that determining the levels of sFlt-1 or its fragments early in pregnancy, particularly at 11 weeks gestation (71-77 days GA), allows for accurate prognosis of the likelihood of developing PE.

[0132] In an embodiment, the maternal age is 18 to 34 years.

[0133] In an embodiment, the maternal age is greater than 34 years.

[0134] Surprisingly, differences in the predictive value (AUC value) of sFlt-1 were observed for maternal ages between 18 and 34 years and for those over 34 years.

[0135] Pregnant subjects with a maternal age of 34 years or older may have a higher probability of preterm delivery, hypertension, complicated PE, severe PE, and a decreased risk of chorioamnionitis.

[0136] Pregnant subjects with a maternal age of 40 years or older may have an increased chance for mild PE, fetal distress, and poor fetal growth.

[0137] It would be highly advantageous for subjects in the indicated risk groups, including those under 18 years of age, 34 years and older, and maternal age 40 years and older, to be able to know an accurate prognosis of preeclampsia before or by the end of the 12th week of gestation (by 84 days GA).

[0138] In embodiments, the methods described herein include: a. the level of sFlt-1 or a fragment thereof determined in the sample is compared to a reference level derived from a reference sample; b. A level of sFlt-1 or a fragment thereof below the reference level indicates an increased risk of preeclampsia; or c. A level of sFlt-1 or a fragment thereof above the reference level indicates a low risk of pre-eclampsia.

[0139] In an embodiment, the reference level is derived from a reference sample isolated from a pregnant subject who does not have or is not affected by any pregnancy-related hypertensive disorder, such as PE or eclampsia.

[0140] In embodiments, a further risk parameter is the sex of the fetus. Surprisingly, significant differences in the predictive values ​​(AUC values) of sFlt-1 levels in samples from subjects having at least a female fetus or at least a male fetus can be observed, as well as significant decreases in sFlt-1 levels compared to the reference level. In embodiments, the level of sFlt-1 in the subject's sample is decreased 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 the reference level. Based on this surprising finding, subjects possessing such risk parameters can be prognosed as being at risk for PE.

[0141] The present invention further provides a method for identifying and treating a subject at risk for early-onset pre-eclampsia (occurring before the end of the 33rd week of gestation, before a GA of 231 days), the method comprising: (a) diagnosis, prognosis, prediction, risk assessment, and / or risk stratification of early-onset pre-eclampsia in a pregnant subject, comprising: - 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, the first sample being isolated before the end of 14 weeks gestation (before 100 days GA); - determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, the second sample being isolated after the first sample; - diagnosing, prognosing, predicting, risk assessment, and / or risk stratifying early-onset pre-eclampsia in a pregnant subject, wherein a higher second level of sFlt-1 or a fragment thereof compared to said first level is indicative of early-onset pre-eclampsia occurring before the end of 33 weeks gestation (before 231 days GA); (b) administering to said subject a treatment for or to reduce the risk of early-onset pre-eclampsia.

[0142] The present invention further provides a method for identifying and treating a subject at risk for early-onset pre-eclampsia (occurring before the end of the 33rd week of gestation, before a GA of 231 days), the method comprising: (a) diagnosis, prognosis, prediction, risk assessment, and / or risk stratification of early-onset pre-eclampsia in a pregnant subject, comprising: - 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, the first sample being isolated before the end of 13 weeks gestation (before 90 days GA); - determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, the second sample being isolated after the first sample; - diagnosing, prognosing, predicting, risk assessment, and / or risk stratifying early-onset pre-eclampsia in a pregnant subject, wherein a higher second level of sFlt-1 or a fragment thereof compared to said first level is indicative of early-onset pre-eclampsia occurring before the end of 33 weeks gestation (before 231 days GA); (b) administering to said subject a treatment for or to reduce the risk of early-onset pre-eclampsia.

[0143] The present invention further provides a method for identifying and treating a subject at risk for early-onset pre-eclampsia (occurring before the end of the 33rd week of gestation, before a GA of 231 days), the method comprising: (a) diagnosis, prognosis, prediction, risk assessment, and / or risk stratification of early-onset pre-eclampsia in a pregnant subject, comprising: - 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, the first sample being isolated before the end of the 9th week of gestation (before 63 days GA); - determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, the second sample being isolated after the first sample; - diagnosing, prognosing, predicting, risk assessment, and / or risk stratifying early-onset pre-eclampsia in a pregnant subject, wherein a higher second level of sFlt-1 or a fragment thereof compared to said first level is indicative of early-onset pre-eclampsia occurring before the end of 33 weeks gestation (before 231 days GA); (b) administering to said subject a treatment for or to reduce the risk of early-onset pre-eclampsia.

[0144] The present invention further provides 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: - providing a first sample from the subject, preferably a blood sample or a sample derived from a blood sample, having a complex comprising at least one binding agent for sFlt-1 or a fragment thereof, and providing a further sample from the subject, preferably a blood sample or a sample derived from a blood sample, having a complex comprising at least one binding agent for PlGF or a fragment thereof; providing a second sample from the subject, preferably a blood sample or a sample derived from a blood sample, having a complex comprising at least one binding agent for sFlt-1 or a fragment thereof, and providing a further sample from the subject, preferably a blood sample or a sample derived from a blood sample, having a complex comprising at least one binding agent for PlGF or a fragment thereof, - the second sample has a higher level of sFlt-1 than in the first sample, preferably the level of PlGF is higher in the second sample than in the first sample, or the level is below or above a threshold, such as any threshold disclosed herein, preferably the population mean and / or population median of sFlt-1 levels from normal pregnancies at any given time point in the respective patient population.

[0145] The present invention further provides a method for treating early-onset pre-eclampsia and / or reducing the risk of early-onset pre-eclampsia, or for administering a treatment for early-onset pre-eclampsia to a subject, the method comprising: administering to a subject a treatment for early-onset pre-eclampsia; - the subject has been determined to have a level of sFlt-1 in a second body fluid sample from the subject, preferably a blood sample or a sample derived from a blood sample, that is higher than the level in the first sample, or that is below or above a threshold value, such as any threshold value disclosed herein, preferably the population mean and / or population median sFlt-1 levels from normal pregnancies at any given time point in the respective patient population.

[0146] Embodiments describing methods of the invention may be used to describe kits of the invention, and vice versa. Features of any given embodiment of a method apply to other embodiments of the method, and features of any given method apply to other methods of the invention. The present invention is united by the novel and beneficial use of the prognostic marker sFlt-1 to indicate risk of developing EO-PE based on two samples, and therefore relevant features described herein for one embodiment may be used to describe any given embodiment of the invention, in a manner consistent with the understanding of one skilled in the art.

[0147] Alternative Embodiments In one embodiment, the present invention provides a method for prognosis, prediction, risk assessment, and / or risk stratification of early-onset pre-eclampsia 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, the first sample being isolated before the end of 14 weeks of gestation; b. determining a second level of sFlt-1 or a fragment thereof in a second sample isolated from the subject, the second sample being isolated after the first sample; c. A method wherein a second level of sFlt-1 or a fragment thereof that is higher compared to said first level is indicative of early-onset pre-eclampsia occurring before the end of 33 weeks of gestation.

[0148] In one embodiment, the first sample is isolated from the subject before the end of the 12th week of gestation.

[0149] In one embodiment, the second sample is isolated from the subject after the end of the 12th week of gestation.

[0150] In one embodiment, the second sample is isolated from the subject after the end of the 14th week of gestation.

[0151] In one embodiment, the second sample is isolated after the end of the 20th week of gestation, preferably between the end of the 20th week and the end of the 22nd week of gestation.

[0152] In one embodiment, the method further comprises determining the level of placental growth factor (PlGF) 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 PlGF or a fragment thereof in the first sample and / or the second sample indicates early-onset preeclampsia occurring before the end of the 33rd week of gestation.

[0153] In one embodiment, the method further comprises determining or providing the subject's maternal age, body mass index, mean arterial pressure (MAP), and / or uterine artery Doppler measurement, wherein a combination of the level of sFlt-1 or a fragment thereof in the first sample and / or the second sample with the subject's maternal age, body mass index, mean arterial pressure (MAP), and / or uterine artery Doppler measurement, preferably in combination with the level of PlGF or a fragment thereof in the first sample and / or the second sample, indicates early-onset pre-eclampsia occurring before the end of the 33rd week of gestation. DETAILED DESCRIPTION OF THE INVENTION

[0154] All cited references, both patent and non-patent literature, are incorporated herein by reference in their entirety.

[0155] The present invention relates to a method for the prognosis, prediction, risk assessment, and / or risk stratification of pre-eclampsia in a pregnant subject, comprising: a) determining the level of sFlt-1 or a fragment thereof in a sample isolated from the pregnant subject; and b) the level of sFlt-1 or a fragment thereof is indicative of the likelihood of pre-eclampsia.

[0156] As used herein, the term "subject" is intended to mean a mammal, including, but not limited to, a human or a non-human mammal (e.g., a cow, horse, dog, sheep, or cat). Included in this definition are pregnant mammals, postpartum mammals, and non-pregnant mammals.

[0157] In the present invention, the terms "risk assessment" and "risk stratification" relate to grouping subjects into different risk groups according to their further prognosis. Risk assessment also relates to stratification for applying preventive and / or therapeutic measures. The term "therapy stratification" particularly relates to grouping or classifying patients into different groups, such as risk groups or therapy groups that receive certain different therapeutic measures depending on the classification of the patient.

[0158] As used herein, "prognosis" relates to predicting the outcome or particular risk of a subject developing PE. It may also include estimating the likelihood of recovery or the likelihood of an adverse outcome for the subject. Assessment of the severity of PE may also be encompassed by the terms "prognosis" or "risk assessment" or "risk stratification."

[0159] As used herein, "preeclampsia" (PE) is used in its ordinary sense. PE can be defined according to well-established criteria, such as a blood pressure of at least 140 / 90 mmHg and a 24-hour urinary protein excretion of at least 0.3 grams of protein (or a dipstick test of at least +1 or greater) (two times, each 4-6 hours apart).

[0160] Preeclampsia is considered a multisystem disorder characterized by hypertension accompanied by proteinuria or edema, or both, glomerular dysfunction, cerebral edema, hepatic edema, or coagulation abnormalities due to pregnancy or the effects of a recent pregnancy. Preeclampsia typically occurs after the 20th week of gestation. Preeclampsia is generally defined as some combination of the following symptoms: (1) a systolic blood pressure (BP) of >140 mmHg and a diastolic BP of >90 mmHg (typically measured twice, 4 to 168 hours apart) after 20 weeks of gestation (after 140 days GA), (2) new-onset proteinuria (1+ by dipstick on urinalysis, >300 mg of protein in a 24-hour urine collection, or a single random urine sample with a protein / creatinine ratio >0.3), and (3) resolution of hypertension and proteinuria by 12 weeks postpartum. Severe preeclampsia is generally defined as (1) diastolic BP >110 mmHg (typically measured twice, 4–168 hours apart) or (2) proteinuria characterized by a measurement of 3.5 g or more of protein in a 24-hour urine collection or two random urine specimens with at least 3+ protein by dipstick.

[0161] In preeclampsia, hypertension and proteinuria generally occur within seven days of each other. In severe preeclampsia, severe hypertension, severe proteinuria, and HELLP syndrome (hemolysis, elevated liver enzymes, low platelets), or eclampsia, may occur simultaneously or with only one symptom at a time. In some cases, severe preeclampsia may result in the development of seizures. This severe form of the syndrome is called eclampsia. "Eclampsia" may also involve dysfunction or damage to several organs or tissues, such as the liver (e.g., hepatocellular damage, periportal necrosis) and the central nervous system (e.g., cerebral edema and cerebral hemorrhage). The etiology of seizures is thought to be secondary to the development of cerebral edema and focal spasms of small blood vessels in the kidneys.

[0162] "Severe preeclampsia" or "high severity preeclampsia" is also defined according to established criteria as a blood pressure of at least 160 / 110 mmHg on at least two occasions 6 hours apart and more than 5 grams of protein in a 24-hour urinary protein excretion or persistent +3 proteinuria on a dipstick test.

[0163] Severe preeclampsia may include the HELLP syndrome (hemolysis, elevated liver enzymes, and low platelet count). Other elements of severe preeclampsia may include intrauterine growth restriction (IUGR) below the 10th percentile for US demographics, persistent neurologic symptoms (headache, visual disturbances), epigastric pain, oliguria (<500 mL / 24 hours), serum creatinine >1.0 mg / dL, elevated liver enzymes (>2x normal), and thrombocytopenia (<100,000 cells / [mu]L).

[0164] As used herein, "preterm birth" is defined as birth before 37 weeks gestation (before 253 days GA).

[0165] As used herein, "early PE" is defined as PE delivered before 37 weeks gestation (before GA of 253 days).

[0166] As used herein, "early onset pre-eclampsia" shall mean that symptoms of pre-eclampsia occur between the beginning of the 20th week of gestation and the end of the 33rd week of gestation (GA of 148-231 days). In embodiments, early onset pre-eclampsia refers to cases where delivery occurs before 34 weeks of gestation (GA of 232 days).

[0167] As used herein, "mid-onset preeclampsia" shall mean that symptoms of preeclampsia occur between the beginning of the 34th week of gestation and the end of the 36th week of gestation (GA of 232-252 days).

[0168] As used herein, "late onset of pre-eclampsia" shall mean that symptoms of pre-eclampsia occur from 37 weeks gestation (from 253 days GA).

[0169] By way of example, "symptoms of preeclampsia" may refer to: (1) systolic blood pressure (BP) >140 mmHg and diastolic BP >90 mmHg after 20 weeks gestation, (2) new-onset proteinuria (urinalysis dipstick 1+, >300 mg of protein in a 24-hour urine collection, or random urine protein / creatinine ratio >0.3), and (3) resolution of hypertension and proteinuria by 12 weeks postpartum. Symptoms of preeclampsia may also include renal dysfunction and glomerular endotheliosis or hypertrophy.

[0170] As used herein, "symptoms of eclampsia" refers to the occurrence of any of the following symptoms due to pregnancy or the effects of a recent pregnancy: seizures, coma, thrombocytopenia, hepatic edema, pulmonary edema, and cerebral edema.

[0171] "At risk of developing" a pregnancy-related hypertensive disorder, such as preeclampsia or eclampsia, refers to a subject who does not currently have a pregnancy-related hypertensive disorder but has a higher-than-average likelihood of developing one. Such at-risk subjects include, but are not limited to, pregnant subjects with blood sFlt-1 levels that are at least 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% reduced compared to reference levels. The patient, in some embodiments, may not exhibit other symptoms of a pregnancy-related hypertensive disorder, such as preeclampsia.

[0172] As used herein, the term "indicating a likelihood of pre-eclampsia" refers to a subject who does not currently have pre-eclampsia but has a higher-than-average likelihood of developing pre-eclampsia. Such prognosis or risk assessment is based on the determined level of sFlt-1 or a fragment thereof. The level of sFlt-1 or a fragment thereof indicates the likelihood of developing pre-eclampsia. In one embodiment, the level of sFlt-1 or a fragment thereof determined in the sample is compared with a reference level, and a level of sFlt-1 or a fragment thereof below the reference level indicates a high risk of pre-eclampsia, or a level of sFlt-1 or a fragment thereof above the reference level indicates a low risk of pre-eclampsia.

[0173] As used herein, "high risk of pre-eclampsia" shall mean high risk of developing pre-eclampsia, but does not currently have pre-eclampsia.

[0174] As used herein, "low risk of pre-eclampsia" shall mean low risk of developing pre-eclampsia, but does not currently have pre-eclampsia.

[0175] "Pregnancy-related hypertensive disorder" means any condition or disease during pregnancy associated with or characterized by elevated blood pressure. These conditions and diseases include preeclampsia (including preterm preeclampsia and severe preeclampsia), eclampsia, gestational hypertension, HELLP syndrome (hemolysis, elevated liver enzymes, low platelets), placental abruption, chronic hypertension during pregnancy, pregnancies with intrauterine growth restriction, and pregnancies with small for gestational age (SGA) infants.

[0176] As used herein, the term "soluble Flt-1 (sFlt-1)" (also known as soluble fms-like tyrosine kinase 1, 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 registration name VGFR1_HUMAN and possesses sFlt-1 biological activity. The biological activity of the sFlt-1 polypeptide can be assayed using any standard method, for example, by assaying sFlt-1 binding to VEGF. sFlt-1 lacks the transmembrane and cytoplasmic tyrosine kinase domains of the Flt-1 receptor. Although sFlt-1 can bind VEGF and PlGF with high affinity, it is unable to induce proliferation or angiogenesis and is therefore functionally distinct from the Flt-1 and KDR receptors. sFlt-1 was first purified from human umbilical endothelial cells and later shown to be produced by trophoblast cells in vivo. As used herein, sFlt-1 includes any sFlt-1 family member or isoform.

[0177] As used herein, the term "specifically binds" refers to a compound or antibody or any detection reagent that 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 to sFlt-1 does not bind to Flt-1.

[0178] As used herein, a "detection reagent" or the like refers to a reagent suitable for determining a marker described herein, e.g., sFlt-1, PAPP-A, or PlGF. Such an exemplary detection reagent is, for example, a ligand, e.g., an antibody or fragment thereof, that specifically binds to a peptide or epitope of a marker described herein. Such a ligand can be used in an immunoassay, as described above. Additional reagents used in an immunoassay to determine the level of a marker can also be included in the kit and are considered detection reagents herein. Detection reagents can also refer to reagents used to detect a marker or fragments thereof by mass spectrometry-based methods. Thus, such detection reagents can also be reagents used to prepare a sample for MS analysis, e.g., enzymes, chemicals, buffers, etc. A mass spectrometer can also be considered a detection reagent.

[0179] A detection reagent according to the present invention can also be, for example, a calibration solution that can be used to determine and compare levels of markers.

[0180] According to the present invention, the antibody may be a monoclonal or polyclonal antibody, and in particular, an antibody that specifically binds at least to sFlt-1 or a fragment thereof is used.

[0181] An antibody is considered specific if its affinity for a molecule of interest, e.g., sFlt-1 or a fragment thereof, is at least 50-fold higher, preferably 100-fold higher, and most preferably at least 1000-fold higher than 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 preferred as detection reagents. The antibody or antibody-binding fragment specifically binds to a marker or fragment thereof defined herein. In particular, the antibody or antibody-binding fragment binds to a peptide of sFlt-1 defined herein. Therefore, the peptide defined herein can also be the epitope to which the antibody specifically binds. Furthermore, the methods and kits of the present invention use antibodies or antibody-binding fragments that specifically bind to sFlt-1 or a fragment thereof.

[0182] Additionally, the methods and kits of the invention use antibodies or antibody-binding fragments that specifically bind to sFlt-1 or a fragment thereof, and optionally to other markers of the invention, such as PAPP-A or PIGF.

[0183] Exemplary immunoassays can be luminescence immunoassays (LIA), radioimmunoassays (RIA), chemiluminescence- and fluorescence-immunoassays (ENZYME immunoassays, EIA), enzyme-linked immunoassays (ELISA), luminescence-based bead arrays, magnetic bead-based arrays, protein microarray assays, rapid test formats, and rare earth cryptate assays. Additionally, assays suitable for point-of-care testing and rapid test formats, such as immunochromatographic strip tests, can be used. Automated immunoassays, such as the BRAHMS KRYPTOR assay, are also contemplated.

[0184] Alternatively, instead of antibodies, other capture molecules or molecular scaffolds that specifically and / or selectively recognize sFlt-1 may be included within the scope of the present invention. As used herein, the terms "capture molecule" or "molecular scaffold" include molecules that can be used to bind target molecules or molecules of interest, i.e., analytes (e.g., sFlt-1), from a sample. To this end, the capture molecule must be appropriately shaped, both spatially and with respect to surface features such as surface charge, hydrophobicity, hydrophilicity, and the presence or absence of Lewis donors and / or acceptors, to specifically bind to the target molecule or molecule of interest. Thus, binding may be mediated, for example, by ionic, van der Waals, π-π, sigma-π, hydrophobic, or hydrogen-bonding 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 may be selected from the group consisting of, for example, nucleic acid molecules, carbohydrate molecules, PNA molecules, proteins, peptides, and glycoproteins. Capture molecules or molecular scaffolds include, for example, aptamers, DAR pins (Designed Ankyrin Repeat Proteins), affimers, and the like.

[0185] The method according to the present invention can be further embodied as a homogeneous method, in which the sandwich complex formed by the antibody / antibodies to be detected and the marker, sFlt-1 or a fragment thereof, remains suspended in a liquid phase. In this case, when two antibodies are used, it is preferred that both antibodies are labeled as part of a detection system, thereby resulting in the generation or induction of a signal when both antibodies are combined into a single sandwich. Such techniques should be embodied, in particular, as fluorescence enhancement or fluorescence quenching detection methods. Particularly preferred embodiments relate to the use of paired detection reagents, such as those described in U.S. Pat. No. 4,882,733, European Patent No. 0,180,492, or European Patent No. 0,539,477, and the prior art cited therein. In this way, measurements are possible in which only reaction products containing both labeled components are detected directly in a single immune complex in the reaction mixture. For example, such technology is provided under the trade name TRACE™ (Time Resolved Amplified Cryptate Emission), or KRYPTOR™, which implements the teachings of the above-cited applications. Accordingly, in particularly preferred embodiments, a diagnostic device is used to perform the methods provided herein. For example, the level of sFlt-1 or a fragment thereof, and / or the level of any additional marker of the methods provided herein, such as PAPP-A, PlGF, etc., is determined. In particularly preferred embodiments, the diagnostic device is a BRAHMS KRYPTOR.

[0186] In embodiments, quantitative determination of sFlt-1 can be performed by an automated immunofluorescence assay, the BRAHMS sFlt-1 KRYPTOR Assay, preferably in conjunction with the BRAHMS PlGF+KRYPTOR Assay. The lower and upper detection limits of 22 and 90,000 pg / mL BRAHMS sFlt-1 KRYPTOR provide the measurement range necessary for reliable detection of clinical sFlt-1 levels throughout pregnancy. Only an 8 μL serum sample isolated from the subject is required for the assay.

[0187] Those skilled in the art will be able to obtain or develop means for identifying, measuring, determining, and / or quantifying any one of the above sFlt-1 molecules, or fragments or variants thereof, as well as other markers of the present invention in accordance with standard molecular biological practices.

[0188] The level of a marker of the invention, e.g., sFlt-1 or a fragment thereof, PAPP-A or a fragment thereof, 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, PAPP-A, or PlGF, in the biological sample or, e.g., a protein digest (e.g., a tryptic digest) from the sample, optionally separating the sample using a chromatographic method, and subjecting the prepared and optionally separated sample to MS analysis. For example, selected reaction monitoring (SRM), multiple reaction monitoring (MRM), or parallel reaction monitoring (PRM) mass spectrometry can be used in the MS analysis, particularly to determine the amount of sFlt-1 or a fragment thereof.

[0189] As used herein, the terms "mass spectrometry" or "MS" refer to an analytical technique for identifying compounds by their mass. To increase the mass resolution and mass determination capabilities of mass spectrometry, samples can be treated prior to MS analysis.

[0190] The present invention therefore relates to immunoenrichment techniques, methods for sample preparation, and / or MS detection methods that can be combined with chromatographic methods, preferably liquid chromatography (LC), more preferably high performance liquid chromatography (HPLC) or ultra-high performance liquid chromatography (UHPLC).

[0191] Sample preparation methods include techniques for lysis, fractionation, digestion of the sample to peptides, depletion, concentration, dialysis, desalting, alkylation, and / or peptide reduction. However, these steps are optional. Selective detection of analyte ions can be performed using tandem mass spectrometry (MS / MS). Tandem mass spectrometry is characterized by a mass selection step (as used herein, the term "mass selection" refers to the isolation of ions with a specific m / z or narrow range of m / z / s), followed by fragmentation of the selected ions and mass analysis of the resulting product (fragment) ions.

[0192] As used herein, the term "detection reagent that specifically binds to sFlt-1 and fragments thereof" means that the detection reagent recognizes and binds to sFLT-1 polypeptides and fragments thereof, but does not substantially recognize or bind to other molecules in a sample, e.g., a biological sample that naturally contains sFlt-1 polypeptides.

[0193] Detection reagents for determining the level of sFlt-1 or a fragment thereof, and optionally for determining the level of PAPP-A, PIGF and / or a fragment thereof, are preferably selected from those necessary to carry out the method, such as an antibody directed against sFlt-1, a suitable label such as a fluorescent label, preferably two separate fluorescent labels suitable for use in the KRYPTOR assay, and a sample collection tube.

[0194] As used herein, the term "determining the level of sFlt-1 or a fragment thereof in a sample" refers to any means of determining sFlt-1 or a fragment thereof.

[0195] As used herein, a "fragment" is intended to 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 entire 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.

[0196] 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 what constitutes an abnormal result. In practice, receiver operating characteristic curves (ROC curves) are typically calculated by plotting the value of a variable against its relative frequency in a "normal" (i.e., apparently healthy individuals without infection) and a "disease" population, e.g., subjects with an infection. For any particular marker (such as sFlt-1), the distributions of marker levels for subjects with and without disease / condition may overlap. Under such conditions, the test will not perfectly distinguish between normal and disease with 100% accuracy, and the area of ​​overlap may indicate where the test is unable to distinguish between normal and disease. A threshold is selected below which the test is considered abnormal and above which the test is considered normal, or below or above which the test indicates a particular condition, e.g., infection. The area under the ROC curve is a measure of the probability that the perceived measurement will allow for correct identification of a condition. ROC curves can be used even when test results do not necessarily provide an accurate number. As long as the results can be ranked, ROC curves can be created. For example, the test results for "disease" samples can be ranked according to degree (e.g., 1 = low, 2 = normal, and 3 = high). This ranking can be correlated with the results of a "normal" population, and ROC curves 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 value is selected to provide an ROC curve area greater than about 0.5, more preferably greater than about 0.7, even more preferably greater than about 0.8, even more preferably greater than about 0.85, and most preferably greater than about 0.9. The term "about" in this context refers to + / - 5% of a given measurement value.

[0197] The horizontal axis of the ROC curve represents (1-specificity), which increases with the false positive rate. The vertical axis of the curve represents sensitivity, which increases with the true positive rate. Therefore, for a particular cutoff selected, the value of (1-specificity) may be determined, and the corresponding sensitivity may be obtained. The area under the ROC curve is a measure of the probability that the measured marker level will allow for the correct identification of a disease or condition. Therefore, the area under the ROC curve can be used to determine the validity of a 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 logistic regression.

[0198] As used herein, multiple of the mean (MoM) is a measure of how much an individual test result, such as a subject's SFI-1 level, deviates from the median of a reference population, e.g., from the median SFI-1 level of a healthy population, e.g., from the median sFlt-1 level of a population of pregnant subjects who did not develop EO-PE. MoM is calculated by dividing the individual test result by the median of the reference population. Thus, the MoM value describes how high or low a measurement is relative to the median of the reference population. In one embodiment, MoM is adjusted for one or more additional factors, such as gestational age and / or BMI.

[0199] As used herein, terms such as "marker," "surrogate," "prognostic marker," "factor," or "biomarker" or "biological marker" are used interchangeably and refer to a measurable and quantifiable biological marker (e.g., a specific protein or enzyme concentration, or fragment thereof, a specific hormone concentration, or fragment thereof, or the presence of a biological substance, or fragment thereof) that serves as an index of health- and physiology-related assessments such as disease / disorder / clinical condition risk, preferably adverse events. A marker or biomarker is defined as a characteristic that can be objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions. Biomarkers can be measured in samples (blood, plasma, urine, or tissue tests).

[0200] The stage of pregnancy at which the methods described herein can be performed depends on various clinical factors, including the subject's overall health and the severity of the symptoms of pre-eclampsia.

[0201] In certain embodiments, the method is performed on a subject by the end of 12 weeks of gestation (before 84 days GA), which shall mean the last day of 12 weeks of gestation (84 days GA), or the second to last day (83 days GA), the third to last day (82 days GA), or the fourth to last day (81 days GA) of 12 weeks of gestation.

[0202] In certain embodiments, the method is performed on the subject by the end of the 13th week of gestation (prior to a GA of 90 days).

[0203] In some embodiments, the method is performed on a subject at 9 weeks gestational age (GA of 57-63 days). In one embodiment, the method is performed at 10 weeks gestational age (GA of 64-70 days). In one embodiment, the method is performed at 11 weeks gestational age (GA of 71-77 days). In an embodiment, the method is performed after 12 weeks gestational age (GA of 84 days). In an embodiment, the method is performed at 13 weeks gestational age (GA of 85-91 days). In an embodiment, the method is performed at 14 weeks gestational age (GA of 92-98 days). In an embodiment, the method is performed between, inclusive, 15 and 20 weeks gestational age (between 99-140 days GA).

[0204] As used herein, "sample" is intended to mean a body fluid sample, for example a blood sample, such as a venous blood sample, a capillary blood sample, a serum sample, a plasma sample, a vaginal fluid sample, a saliva sample or an amniotic fluid sample, cerebrospinal fluid, preferably a blood, serum or plasma sample.

[0205] "Plasma" in the context of the present invention refers to the substantially cell-free supernatant of blood containing an anticoagulant obtained after centrifugation. Exemplary anticoagulants include calcium ion-binding compounds such as EDTA or citrate, and thrombin inhibitors such as heparinate or hirudin. Cell-free plasma can be obtained by centrifuging anticoagulated blood (e.g., citrate-treated, EDTA- or heparin-treated blood) at, for example, 2000-3000 g for at least 15 minutes.

[0206] "Serum" in the context of the present invention is the liquid fraction of whole blood that is collected after the blood has been allowed to clot. When the clotted blood (clot) is centrifuged, serum can be obtained as the supernatant.

[0207] "Sample" also refers to a tissue biopsy (e.g., placental tissue), chorionic villus sample, cells, or other specimen obtained from a subject. Desirably, the biological sample contains an sFlt-1 nucleic acid molecule or polypeptide, or both.

[0208] As used herein, the term "reference sample" refers to any sample, standard, or level used for comparison purposes. A "normal reference sample" can be a previous sample taken from the same subject, a sample from a pregnant subject who does not have any pregnancy-related hypertensive disorder such as pre-eclampsia or eclampsia, a subject who is pregnant but the sample was taken early in pregnancy (e.g., in the first or second trimester, or before a pregnancy-related hypertensive disorder such as pre-eclampsia or eclampsia is detected), a subject who is pregnant and does not have a history of a pregnancy-related hypertensive disorder such as pre-eclampsia or eclampsia, a subject who is not pregnant, or a sample of a purified reference polypeptide with a known normal concentration (i.e., not exhibiting a pregnancy-related hypertensive disorder such as pre-eclampsia or eclampsia).

[0209] 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. Desirably, all reference samples, standards, and levels are matched to the sample subject by at least one of the following criteria: fetal gestational age, maternal age, maternal blood pressure before pregnancy, maternal blood pressure during pregnancy, maternal BMI, fetal weight, previous diagnosis of pregnancy-related hypertensive disorder, and family history of pregnancy-related hypertensive disorder.

[0210] In one embodiment, the reference level relates to a value derived from a pregnant subject who does not develop pre-eclampsia or a pregnancy-related hypertensive disorder (e.g., in the first or second trimester or before a pregnancy-related hypertensive disorder such as pre-eclampsia or eclampsia is detected).

[0211] In an embodiment, the reference level refers to a value derived from a pregnant subject with no history of a pregnancy-related hypertensive disorder, such as pre-eclampsia or eclampsia.

[0212] In one embodiment, the terms "reference level" and "determined level from a subject" used herein refer to the measurement of sFlt-1 or a fragment thereof protein levels in a blood sample, preferably a whole blood sample or a plasma or serum sample, obtained from a pregnant subject without preeclampsia, preferably by the Thermo Scientific BRAHMS KRYPTOR assay. Thus, the values ​​disclosed herein may vary to some extent depending on the detection / measurement method used, and specific values ​​disclosed herein are intended to read corresponding values ​​determined by other methods. In embodiments of the present invention, a decrease in the level of sFlt-1 or a fragment thereof compared to a reference level that may define a transition from low to high risk of developing PE may be any decrease in the range of 6% to 20% compared to the reference level. Any value within this range may be considered an appropriate reference level for high and low-risk sFlt-1 levels. Furthermore, values ​​below such a reference level may indicate a high risk of preeclampsia, while values ​​above such a reference level may indicate a low risk of preeclampsia. Suitable cut-off levels that may be used in the context of the present invention include, but are not limited to, a change of 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.

[0213] As used herein, the term "positive reference" sample, standard, or value refers to a sample, value, or number derived from a subject known to have or have had a pregnancy-related hypertensive disorder, such as preeclampsia or eclampsia. A reference standard or level may also reflect the average or mean level of a nucleic acid, polypeptide, or small molecule from a normal reference subject or a positive reference subject, depending on the context. A reference may also be a chart, graph, or standard curve representing normal reference levels of a polypeptide, nucleic acid, or small molecule at any and / or all stages (e.g., weekly) of pregnancy. Desirably, all reference samples, standards, and levels are matched to the sample subject by at least one of the following criteria: fetal gestational age, maternal age, maternal blood pressure before pregnancy, maternal blood pressure during pregnancy, maternal BMI, fetal weight, previous diagnosis of a pregnancy-related hypertensive disorder, and family history of a pregnancy-related hypertensive disorder.

[0214] As used herein, the term "history of a pregnancy-related hypertensive disorder" is intended to mean a previous diagnosis of a pregnancy-related hypertensive disorder (e.g., pre-eclampsia or eclampsia or gestational hypertension) in the subject himself or herself or in a relevant family member.

[0215] As used herein, "gestational age" refers to the age of the fetus, counting from the first day of the mother's last menstrual period.It also refers to the corresponding gestational age estimated by more accurate methods in the art.In the case of in vitro fertilization, it is the known period from conception plus 14 days.Gestational age can be determined by obstetric ultrasound.

[0216] As used herein, "maternal age" shall mean the age of the pregnant subject at the time of birth.

[0217] As used herein, the term "gestational hypertension" is intended to mean the onset of hypertension without proteinuria after 20 weeks of gestation (after 140 days GA).

[0218] As used herein, the term "polypeptide" refers to a polymer of amino acids and not to a specific length. Thus, peptides, oligopeptides, and proteins are included within the definition of a polypeptide.

[0219] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0220] The term "such as" is used herein to mean, and is used interchangeably with, the phrase "such as but not limited to."

[0221] As used herein, the term "risk parameter" or "risk factor" refers to a health condition that predisposes a pregnant subject to developing preeclampsia. One of the risk parameters is the parent's blood type, preferably the blood type AB of the biological father or biological mother. A more preferred blood type is the parent's Rh factor, especially when the pregnant subject is Rh-negative and the fetus's biological father is Rh-positive. Risk parameters include, but are not limited to, hypothyroidism, hyperthyroidism, BMI greater than 24, primigravidity, a history of preeclampsia, ethnicity with a risk disorder, multiple pregnancy, migraine, lupus, blood clotting disorders such as increased coagulation, inflammatory disease, impaired cardiac reserve, diabetes, chronic kidney disease, and chronic hypertension.

[0222] In the methods described herein, a level of sFlt-1 below the reference level indicates initiation or modification of a subject's treatment to reduce the risk of developing preeclampsia, delay the onset of preeclampsia, or at least reduce the severity of preeclampsia, such as by balancing the angiogenic / anti-angiogenic processes in placental development, lowering blood pressure, or protecting organ function, such as the kidney or liver.

[0223] In embodiments, a level of sFlt-1 below the reference level indicates initiation or modification of a subject's treatment to reduce the risk of developing preeclampsia, delay the onset of preeclampsia, or at least reduce the severity of preeclampsia, such as by balancing the angiogenic / antiangiogenic processes in placental development, lowering blood pressure, protecting organ function such as the kidney or liver, etc. Such treatment also involves prenatal monitoring, lifestyle modifications, nutritional measures such as nutritional supplementation, bed rest, restricted activity or regular exercise, reduced salt intake, and antioxidants such as vitamins C and E, garlic, and fish oil.

[0224] As used herein, the term "nulliparous" refers to a subject that has never given birth. "Primuliparous" shall mean that a subject has given birth once, and "biparous" shall mean that a subject has given birth twice. "Multiparous" shall mean that a subject has given birth more than twice.

[0225] As used herein, the term "chromosomal abnormality" refers to a difference in chromosomes that can occur during fetal development. They can be inherent to the fetus or inherited from a parent. Abnormalities are divided into two categories: numerical, which refers to a number of chromosomes that is different from what is expected, such as monosomy or trisomy, and structural, which refers to translocations, deletions, duplications, the formation of rings resulting from the splitting of a portion of a 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 syndrome, and Cri-Du-Chat syndrome.

[0226] The present invention further relates to kits, uses of kits, and methods in which such kits are used. The present invention relates to kits for carrying out the methods provided herein above and below. The definitions provided herein, such as those provided with respect to the methods, also apply to the kits of the present invention. The kits may be part of a medical device that also contains calibrators, controls, buffer reagents, and can be used in conjunction with diagnostic equipment and / or software. In particular, the present invention relates to kits for therapeutic monitoring, including prognosis, risk assessment, or risk stratification of subsequent adverse events in a patient's health, comprising detection reagents for determining the level of sFlt-1 or a fragment thereof, optionally additional reagents for determining the level of a further biomarker in a sample from the subject, and reference data, such as reference levels corresponding to the sFlt-1 risk level and, optionally, the further biomarker level, preferably stored on a computer-readable medium and / or used in the form of computer-executable code configured to compare the determined level of sFlt-1 or a fragment thereof and, optionally, further determined levels of a further biomarker or fragment thereof, with the reference data.

[0227] In one embodiment of the methods described herein, the method further comprises comparing the determined level of sFlt-1 or a fragment thereof with a reference level, threshold, and / or population mean corresponding to sFlt-1 or a fragment thereof in patients at risk of suffering from PE, wherein the comparing is performed on a computer processor using computer-executable code.

[0228] The methods of the present invention can be partially computer-implemented. For example, the step of comparing the detected level of a biomarker, such as sFlt-1 or a fragment thereof, with a reference level can be performed by a computer system. In the computer system, the determined level of the biomarker can be combined with other biomarker levels and / or clinical parameters of the subject to calculate a score indicative of prognosis, risk assessment, and / or risk stratification. For example, the determined values ​​can be entered into the computer system (either manually by a medical professional or automatically from a device on which the respective marker level is determined). The computer system can be directly at the point of care (e.g., in a primary care, hospital, or home environment) or can be at a remote location connected via a computer network (e.g., via the Internet or a specialized medical cloud system, which can optionally be combined with other IT systems or platforms, such as hospital information systems (HIS)). Typically, the computer system will store values ​​(e.g., biomarker levels, or clinical parameters such as age, blood pressure, weight, sex, etc., or pregnancy parameters such as UAPI, FMF algorithm, VOCAL score, BMI, etc.) in a computer-readable medium and calculate a score based on predefined and / or pre-stored reference levels or values. The resulting score will be displayed and / or printed for a user (typically a healthcare professional such as a physician or patient). Alternatively, or in addition, an associated prognosis, assessment, treatment guideline, patient management guideline, or stratification will be displayed and / or printed for a user (typically a healthcare professional such as a physician or patient).

[0229] In one embodiment of the present invention, a software system can be used in which a machine learning algorithm, preferably using data from an electronic health record (EHR), is used to identify patients at risk for PE. The machine learning approach can be trained with a random forest classifier using EHR data from patients (e.g., lab results, biomarker expression, vitals, demographics, etc.). Machine learning, unlike simple rule-based systems, is a type of artificial intelligence that provides computers with the ability to learn complex patterns in data without being explicitly programmed. Previous studies have used EHR data to trigger alerts and detect general 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 be processed in machine learning software to aid in the prognosis of PE development.

[0230] "PAPP-A" is pregnancy-associated plasma protein A, papalysin-1, and refers to a plasma protein used as a screening test between 8 and 14 weeks of gestation. Decreased levels of the protein indicate an increased risk of Down syndrome, intrauterine growth retardation, pre-eclampsia, and stillbirth.

[0231] "PlGF" refers to placental growth factor (UniprotKB-Q6IB04), a member of the vascular endothelial growth factor (VEGF) family. PlGF is involved in the glycosylphosphatidylinositol-alcohol biosynthetic pathway, which is part of glycolipid biosynthesis. PlGF levels are decreased in pregnant subjects who are destined to develop preeclampsia.

[0232] As used herein, reference data refers to, for example, reference levels corresponding to patient groups with maternal ages up to 18, between 18 and 34, and over 34, and, optionally, additional markers described herein, preferably PAPP-A and / or PIGF levels, preferably stored on a computer-readable medium and / or used in the form of computer-executable code configured to compare the determined levels of sFlt-1 or a fragment thereof, and optionally, further determined levels of PAPP-A and / or PIGF or a fragment thereof, with the reference data. A "reference date" includes additional reference levels corresponding to patient groups with AB blood type, Rh-negative blood type, Rh-negative blood type and Rh-positive biological father of the fetus, pregnant with at least one female fetus, pregnant with at least one male fetus, nulliparous, with one or more previous pregnancies, and / or suspected of being pregnant with a fetus with a chromosomal abnormality. The reference data may also include instructions for use of the kit of the present invention.

[0233] The kit can further include items useful for obtaining a sample, such as a blood sample; for example, the kit can include a container including a device for attaching the container to a cannula or syringe, e.g., a syringe suitable for blood isolation and exhibiting an internal pressure less than atmospheric, suitable for drawing a predetermined volume of sample into the container; and / or a filter system comprising a surfactant, a chaotropic salt, a ribonuclease inhibitor, a chelating agent such as guanidinium isothiocyanate, guanidinium hydrochloride, sodium dodecyl sulfate, polyoxyethylene sorbitan monolaurate, an RNAse inhibitor protein, and mixtures thereof; and a filter system comprising nitrocellulose, a silica matrix, ferromagnetic spheres, a cup collection spillover, trehalose, fructose, lactose, mannose, polyethylene glycol, glycerol, EDTA, TRIS, limonene, xylene, benzoyl, phenol, mineral oil, aniline, pyrrole, citrate, and mixtures thereof.

[0234] The present invention is further illustrated by the drawings, which are not intended to limit the scope of the invention. [Brief explanation of the drawings]

[0235] [Figure 1] Box plots depicting sFlt-1 levels before 90 days gestation, between 90 and 100 days gestation, and between 140 and 154 days gestation, comparing levels in subjects with and without EO-PE. The study included 11,952 women recruited in the first trimester; 11,918 had no PE and 34 had early-onset PE. Before 90 days gestation, all cases of women with EO-PE (N = 10) had sFlt-1 levels below the median (p < 0.01). On average, between 90 and 100 days gestation, women with EO-PE (N = 24) had sFlt-1 levels. On average, between 140 and 154 days gestation, all women with EO-PE (N = 4) had sFlt-1 levels above the median. [Figure 2] Statistical evaluation of sFlt-1 levels in subjects whose samples were obtained in the first trimester. The study included 11,952 women recruited in the first trimester, of whom 11,918 did not have PE and 34 had early-onset PE. As can be seen, sFlt-1 in the first trimester was not associated with EO-PE (EO-PE: 0.98 MoM vs. control: 1.00 MoM, p=0.09). [Figure 3] Statistical evaluation of sFlt-1 levels in subjects whose samples were obtained after 12 6 / 7 weeks GA. The study involved 7,409 women recruited after 90 days gestation, of whom 7,385 did not have PE and 24 had early-onset PE. As can be seen, sFlt-1 levels after 12 6 / 7 weeks (after 90 days GA) were not associated with EO-PE (EO-PE: 1.00 MoM vs. control: 1.00 MoM, p=0.40). [Figure 4] 12 ROC curve for the data presented in Figure 3 showing that sFlt-1 after 6 / 7 weeks (after 90 days GA) is not associated with EO-PE. [Figure 5] Statistical evaluation of sFlt-1 levels in subjects whose samples were obtained before 12 6 / 7 weeks GA (before 90 days GA). The study involved 4,543 women recruited before 90 days gestation, of whom 4,533 were PE-free and 10 had early-onset PE. As can be seen, sFlt-1 before 12 6 / 7 weeks (before 90 days GA) is inversely associated with EO-PE (EO-PE: 0.94 MoM vs. control: 1.00 MoM, p=0.003). [Figure 6] ROC curve for data presented in Figure 5 showing that sFlt-1 before 12 6 / 7 weeks (before 90 days GA) correlates with EO-PE. Measurement of sFlt-1 (adjusted for gestational age - MoM) and sFlt-1 (not adjusted for gestational age - Raw) both predict early-onset PE and can be used as markers of early-onset PE (AUC: 0.74 (95% CI: 0.64-0.84), p<0.01). [Figure 7] ROC curves for data on sFlt-1 and PlGF levels in subjects whose samples were obtained after 12 6 / 7 weeks GA (after 90 days GA). After 90 days gestation, PlGF, but not sFlt-1, is a strong marker of EO-PE. [Figure 8] ROC curves for data on sFlt-1 and PlGF levels in subjects whose samples were obtained before 12 6 / 7 weeks GA (before 90 days GA). Before 90 days gestation: sFlt-1 is a strong marker of EO-PE, but PlGF is not as good as after 90 days. [Figure 9] Receiver operating characteristic curves for data on sFlt-1 and PlGF levels in subjects whose samples were obtained before 12 6 / 7 weeks GA (before 90 days GA). In addition, ROC curves are shown using combined data for both sFlt-1 and PlGF levels. The ROC curves show AUC values ​​for PlGF: 0.70 (95% CI: 0.56-0.85), sFlt-1: 0.74 (95% CI: 0.64-0.84), and both markers combined: 0.85 (95% CI: 0.78-0.92). [Figure 10] ROC curves for data on sFlt-1 and PlGF levels in subjects whose samples were obtained before 12 6 / 7 weeks GA (before 90 days GA). In addition, data from uterine artery Doppler measurements obtained in this study were combined in the analysis. ROC curves are shown using combined data for both sFlt-1 and PlGF levels, and these combined levels are further combined with Doppler data. The ROC curve shows an AUC value of 0.87 (95% CI: 0.80-0.94) for the Doppler combination. [Figure 11] ROC curve for data on sFlt-1 and PlGF levels in subjects whose samples were obtained between 90 and 100 days GA. The combination of sFlt-1 and PlGF predicts some cases of IUFD between 90 and 100 days. [Figure 12] ROC curve for data on sFlt-1 and PlGF levels in subjects whose samples were obtained before 90 days GA. The combination of sFlt-1 and PlGF predicted IUFD, whereby the association was stronger before 90 days gestation compared with between 90 and 100 days gestation (AUC: 0.72 95%, CI: 0.60-0.84). [Figure 13] Box plots and ROC curves showing the correlation between sFlt-1 levels (measured before the 90-day GA) in subjects with estimated high or low risk as determined using the FMF algorithm. As can be seen, patients with high risk as determined by the FMF algorithm have significantly lower sFlt-1 levels before the 90-day GA. [Figure 14-1](A) sFlt-1 levels in subjects whose samples were obtained between 9 and 12 6 / 7 weeks GA (57 and 90 days GA). Each dot represents the sFlt-1 level of a single subject at the respective time point. Larger dots represent the sFlt-1 level of subjects who developed early-onset preeclampsia (EO-PE), and smaller dots represent the sFlt-1 level of subjects who did not develop EO-PE (no EO-PE). (B) sFlt-1 MoM in subjects whose samples were obtained between 9 and 12 6 / 7 weeks GA (57 and 90 days GA). Each dot represents the sFlt-1 MoM of a single subject at the respective time point. Larger dots represent the sFlt-1 MoM of subjects who developed early-onset preeclampsia (EO-PE), and smaller dots represent the sFlt-1 MoM of subjects who did not develop EO-PE (no EO-PE). Subjects who did not develop EO-PE had a median sFlt-1 (MoM) of 1.00 (IQR: 0.76-1.34). Subjects who developed EO-PE had a median sFlt-1 (MoM) of 0.78 (IQR: 0.60-0.87). (C) ROC curve for data on sFlt-1 levels in subjects whose samples were obtained at 9-12 6 / 7 weeks gestation (57-90 days GA). N=1108; 4 cases of EO-PE. The ROC curve shows an AUC value of 0.75 for sFlt-1 (95% Cl: 0.63-0.87). [Figure 14-2](A) sFlt-1 levels in subjects whose samples were obtained between 9 and 12 6 / 7 weeks GA (57 and 90 days GA). Each dot represents the sFlt-1 level of a single subject at the respective time point. Larger dots represent the sFlt-1 level of subjects who developed early-onset preeclampsia (EO-PE), and smaller dots represent the sFlt-1 level of subjects who did not develop EO-PE (no EO-PE). (B) sFlt-1 MoM in subjects whose samples were obtained between 9 and 12 6 / 7 weeks GA (57 and 90 days GA). Each dot represents the sFlt-1 MoM of a single subject at the respective time point. Larger dots represent the sFlt-1 MoM of subjects who developed early-onset preeclampsia (EO-PE), and smaller dots represent the sFlt-1 MoM of subjects who did not develop EO-PE (no EO-PE). Subjects who did not develop EO-PE had a median sFlt-1 (MoM) of 1.00 (IQR: 0.76-1.34). Subjects who developed EO-PE had a median sFlt-1 (MoM) of 0.78 (IQR: 0.60-0.87). (C) ROC curve for data on sFlt-1 levels in subjects whose samples were obtained at 9-12 6 / 7 weeks gestation (57-90 days GA). N=1108; 4 cases of EO-PE. The ROC curve shows an AUC value of 0.75 for sFlt-1 (95% Cl: 0.63-0.87). [Figure 14-3](A) sFlt-1 levels in subjects whose samples were obtained between 9 and 12 6 / 7 weeks GA (57 and 90 days GA). Each dot represents the sFlt-1 level of a single subject at the respective time point. Larger dots represent the sFlt-1 level of subjects who developed early-onset preeclampsia (EO-PE), and smaller dots represent the sFlt-1 level of subjects who did not develop EO-PE (no EO-PE). (B) sFlt-1 MoM in subjects whose samples were obtained between 9 and 12 6 / 7 weeks GA (57 and 90 days GA). Each dot represents the sFlt-1 MoM of a single subject at the respective time point. Larger dots represent the sFlt-1 MoM of subjects who developed early-onset preeclampsia (EO-PE), and smaller dots represent the sFlt-1 MoM of subjects who did not develop EO-PE (no EO-PE). Subjects who did not develop EO-PE had a median sFlt-1 (MoM) of 1.00 (IQR: 0.76-1.34). Subjects who developed EO-PE had a median sFlt-1 (MoM) of 0.78 (IQR: 0.60-0.87). (C) ROC curve for data on sFlt-1 levels in subjects whose samples were obtained at 9-12 6 / 7 weeks gestation (57-90 days GA). N=1108; 4 cases of EO-PE. The ROC curve shows an AUC value of 0.75 for sFlt-1 (95% Cl: 0.63-0.87). [Figure 15-1](A) sFlt-1 levels in subjects whose samples were obtained between 21 and 40 weeks of gestation (141-280 days GA). Each dot represents the sFlt-1 level of a single subject at the respective time point. Larger dots represent sFlt-1 levels in subjects who developed early-onset preeclampsia (EO-PE), while smaller dots represent sFlt-1 levels in subjects who did not develop EO-PE (no EO-PE). The dotted lines represent the overall trend in sFlt-1 levels for each population (no EO-PE and EO-PE) between 21 and 40 weeks of gestation (141-280 days GA). Top: EO-PE; bottom: no EO-PE. Subjects who developed EO-PE show increased sFlt-1 levels between 21 and 40 weeks of gestation (141-280 days GA) compared with subjects who did not develop EO-PE (no EO-PE). (B) sFlt-1 levels in subjects whose samples were obtained between 9 and 40 weeks GA (57 to 280 days GA). Each dot represents the sFlt-1 level of a single subject at the respective time point. Larger dots represent sFlt-1 levels in subjects who developed early-onset preeclampsia (EO-PE), while smaller dots represent sFlt-1 levels in subjects who did not develop EO-PE (no EO-PE). The dotted lines represent the overall trend of sFlt-1 levels for each cohort (no EO-PE and EO-PE) between 9 and 40 weeks GA (57 to 280 days GA). Top: 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 90 days GA) and increased sFlt-1 levels after the end of 13 weeks of gestation (after 90 days GA) compared with healthy subjects who did not develop EO-PE. Subjects who developed EO-PE showed a substantially greater increase in sFlt-1 levels over the course of pregnancy (weeks 9-40, 57-280 days GA) compared with patients who did not develop EO-PE. [Figure 15-2](A) sFlt-1 levels in subjects whose samples were obtained between 21 and 40 weeks of gestation (141-280 days GA). Each dot represents the sFlt-1 level of a single subject at the respective time point. Larger dots represent sFlt-1 levels in subjects who developed early-onset preeclampsia (EO-PE), while smaller dots represent sFlt-1 levels in subjects who did not develop EO-PE (no EO-PE). The dotted lines represent the overall trend in sFlt-1 levels for each population (no EO-PE and EO-PE) between 21 and 40 weeks of gestation (141-280 days GA). Top: EO-PE; bottom: no EO-PE. Subjects who developed EO-PE show increased sFlt-1 levels between 21 and 40 weeks of gestation (141-280 days GA) compared with subjects who did not develop EO-PE (no EO-PE). (B) sFlt-1 levels in subjects whose samples were obtained between 9 and 40 weeks GA (57 to 280 days GA). Each dot represents the sFlt-1 level of a single subject at the respective time point. Larger dots represent sFlt-1 levels in subjects who developed early-onset preeclampsia (EO-PE), while smaller dots represent sFlt-1 levels in subjects who did not develop EO-PE (no EO-PE). The dotted lines represent the overall trend of sFlt-1 levels for each cohort (no EO-PE and EO-PE) between 9 and 40 weeks GA (57 to 280 days GA). Top: 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 90 days GA) and increased sFlt-1 levels after the end of 13 weeks of gestation (after 90 days GA) compared with healthy subjects who did not develop EO-PE. Subjects who developed EO-PE showed a substantially greater increase in sFlt-1 levels over the course of pregnancy (weeks 9-40, 57-280 days GA) compared with patients who did not develop EO-PE. [Example]

[0236] Example 1: Study design: Pregnant women were recruited at 11–14 weeks gestational age (71–98 days GA) and followed until delivery. sFlt-1 was measured at recruitment using a Thermo Scientific BRAHMS KRYPTOR and reported as a multiple of the median adjusted for gestational age (MoM). Median sFlt-1 levels were compared among women who developed early-onset PE (<34 weeks, <231 days GA), mid-onset PE (34–36 weeks, 232–252 days GA), late-onset PE (≥37 weeks, ≥253 days GA), and no PE (controls). The area under the receiver operating characteristic curve (AUC) was used to estimate the potential predictive value of sFlt-1 for PE.

[0237] result: We included 12,383 participants who delivered after 16 weeks gestation (after 112 days GA), of whom 33 (0.3%) developed early-onset PE, 65 (0.5%), and 400 (4.0%) developed late-onset PE. We observed that first-trimester sFlt-1 was lower in participants who developed early- or mid-onset PE (p=0.02), but more importantly, the difference was primarily present in participants recruited early in pregnancy.

[0238] Significant differences in the predictive value of early-onset PE were observed between women recruited at 11 weeks (71-77 days GA) (AUC: 0.82, 95% CI: 0.72-0.92, p<0.001), 12 weeks (AUC: 0.62, 95% CI: 0.49-0.74, p=0.06), and 13 weeks (AUC: 0.50, 95% CI: 0.32-0.67, p=0.97). A similar trend was observed for the prediction of mid-onset PE.

[0239] Conclusion: First-trimester maternal sFlt-1 is decreased in women who will develop PE before term (<37 weeks, <253 days GA). Its predictive value is significantly improved when collected at or before 12 weeks gestation (<78 days GA), and this peculiarity may explain the conflicting results among previous studies.

[0240] Example 2: Study design: We conducted a secondary analysis of a prospective cohort study of nulliparous women recruited at 11–14 weeks gestational age (71–98 days GA). Maternal characteristics, mean arterial blood pressure, maternal serum biomarkers (pregnancy-associated plasma protein-A, placental growth factor, two risk cutoffs (1 / 70 and 1 / 100 from the FMF algorithm), sFlt-1, and mean uterine artery pulsatility index levels were obtained to calculate the risk of early and term PE compared with a reference group that did not develop early or term PE. Detection rates, false positive rates, and positive and negative predictive values ​​were calculated for term and early PE as placental-mediated complications. Women who reported taking aspirin daily were excluded. PAPP-A, PlGF, and sFlt-1 concentrations were measured using the Thermo Scientific BRAHMS KRYPTOR automated assay. Early PE was defined as PE delivered before 37 weeks' gestational age (before 253 days' GA), and early-onset PE referred to cases delivered before 34 weeks' gestational age (before 231 days' GA). Analyses were performed using the SAS statistical software package (version 9.3, SAS Institute All analyses were performed using a 5% type I error margin of error (SIR).

[0241] result: We included 4575 participants with complete observations. 29 patients developed early PE, while 194 women developed term PE, and 3705 women did not develop placenta-mediated complications (reference group). The median sFlt-1 level in the reference group was 1023 pg / mL, and Q1–Q3 levels ranged from 771 to 1373 pg / mL. For women with onset PE, the median sFlt-1 and Q1–Q3 levels were 933 pg / mL (range, 726–1221 pg / mL). Thus, pregnant women who developed term PE showed a median sFlt-1 level decrease of 8.8% and a median fold change (FC) of 0.91.

[0242] The median sFlt-1 level and Q1-Q3 values ​​in women developing early PE were 852 pg / mL (658-1095 pg / mL). Thus, pregnant women who developed early PE showed a higher sFlt-1 decrease compared to the reference group, as did the term PE group with a median sFlt-1 decrease of 16.7% at 11-14 weeks gestation (71-98 days GA) and a median fold change (FC) of 0.83.

[0243] Q1 of sFlt-1 levels in early PE compared with the reference group showed an FC of 0.85 with a 14.7% decrease.

[0244] Q1 of sFlt-1 levels in end-stage PE compared with the reference group showed an FC of 0.94 with a 5.8% decrease.

[0245] Q3 of sFlt-1 levels in early PE compared with the reference group showed an FC of 0.8 with a 20% reduction.

[0246] Q3 of sFlt-1 levels in end-stage PE compared with the reference group showed an FC of 0.89 with an 11% decrease.

[0247] Conclusion: Nulliparous women with a median sFlt-1 decline of at least 8.8% detected between 11 and 14 weeks gestation (71–98 days GA) were at high risk of developing term PE, and a median sFlt-1 decline of at least 16.7% was at high risk of developing early PE.

[0248] Example 3: Prediction of early preeclampsia and preterm birth in the first trimester (prospective study): Preeclampsia is a complication of pregnancy that affects 2-5% of pregnant women. It is one of the leading causes of maternal and neonatal mortality and morbidity worldwide. Early-onset preeclampsia requires delivery before the 34th week of gestation (before 232 days GA) and is associated with significant perinatal morbidity.

[0249] Online software (Fetal Medicine Foundation) is now available that combines biophysical factors (age, BMI, BP, medical history), ultrasound factors (uterine artery Doppler), and biochemical factors (PlGF and PAPP-A) measured during the first trimester of pregnancy, suggesting that more than 90% of early preeclampsia can be predicted with a false positive rate of less than 10%.

[0250] To explore the correlation between biomarkers such as sFlt-1, PAPP-A, or PlGF and pre-eclampsia, additional patient groups such as patients under the age of 18 years, with some rare blood types such as AB, Rh negative, with or with multiple pregnancies, with one or more previous pregnancies, with or with dysplasia or polydysplasia syndrome, and / or suspected of carrying or with a fetus with a chromosomal abnormality (highly translucent nuchal region) will be further included and investigated in this discovery study.

[0251] the purpose: 1) To validate the FMF screening tool between 11 and 13 weeks of gestation (71–91 days GA) for early preeclampsia and other placenta-related pregnancy disorders (early preeclampsia, IUGR <3rd percentile, perinatal death). 2) To compare screening tools that use uterine artery Doppler measurements with those that do not. 3) To investigate the efficacy of potential markers (serum sFlt-1, serum ADAM-12, serum PP-13, placental and subplacental volume, placental vascularity) for predicting preeclampsia.

[0252] Methodology: Prospective observational study. Nulliparous women with a single pregnancy and a fetus without lethal anomalies were recruited between 11 3 / 7 and 13 6 / 7 weeks of gestation (73-90 days GA). Lifestyle questionnaires were completed. BMI and BP measurements and blood samples (20 mL) were collected. Doppler ultrasound of the uterine arteries and 3D evaluation of the placenta completed the clinic visit. Medical record follow-up was performed approximately 1 month after the expected delivery date.

[0253] Serum was analyzed for PAPP-A, PIGF, sFIt1, fbHCG, and AFP within 10 days of recruitment using the Thermo Scientific BRAHMS KRYPTOR automated assay. The remaining serum was stored at -80°C for additional analyses at the end of the project (PP-13, ADAM-12, vitamin D) to evaluate the possibility of improving the predictive model using promising markers, including placental volume and vascularity assessed by 3D ultrasound. Calculations of the risk of early and overall preeclampsia for each participant were calculated using FMF software and were disclosed to the participant. The optimal sensitivity and specificity of the tool were assessed using receiver operating characteristic (ROC) curves.

[0254] We estimated a 0.7% incidence of early-onset preeclampsia (GA <34 weeks, <231 days) in our nulliparous population (Quebec and Montreal). We recruited 7554 women and demonstrated that the FMF screening tool was at least 80% sensitive and 90% specific when expected to be 95% sensitive and 92% specific.

[0255] the purpose Main purpose To validate the FMF early preeclampsia screening tool during 11-13 weeks of gestation (71-91 days GA).

[0256] secondary purpose 1) To evaluate the performance of the FMF test in predicting all cases of preeclampsia and other placenta-related pregnancy disorders (early and severe preeclampsia, IUGR <3rd percentile, perinatal death). 2) Compare screening tools with and without uterine artery Doppler. 3) If the FMF screening tool is not positively validated in our population, evaluate the predictive value of each biomarker individually and assess whether they can be used in different predictive models. 4) To evaluate the predictive value of potential biomarkers of early preeclampsia (sFlt-1, PP13, ADAM-12, 25-OH-vitamin D, placental volume and placental / subplacental vascularity, sFlt-1). 5) To assess the predictive value of first trimester cervical measurements for preterm birth.

[0257] I. Research citation This was a prospective observational study in nulliparous pregnant women recruited in the first trimester of pregnancy, at which point a number of biomarkers were collected and analyzed, and followed until delivery to validate the presence of our primary and secondary outcomes.

[0258] II. Population and Selection Criteria Study set of inclusion criteria: ■ Women with a live singleton pregnancy between 11 3 / 7 weeks and 13 6 / 7 weeks (GA of 80-97 days). ■ Nulliparous women (no history of pregnancy up to 20.7 weeks gestation (up to 147 days GA), regardless of the reason)

[0259] Exclusion criteria study set: ■ Women under 18 years old ■Women who are unable to give informed consent (e.g., do not understand English or French). ■ Women who planned to give birth outside the participating centers (excluding women who were giving birth at the Hotel-Dieu de Levis and who were eligible for the project because they consented via a consent form to have their files inspected at this institution). ■ Women who are HIV positive, Hepatitis C positive, or Chronic Hepatitis B positive (not cured)

[0260] Further inclusion criteria study set: Multiple pregnancies (women with two fetuses, one of which has stopped growing, are not eligible) ■ Presence of fetal dysplasia syndrome or polydysplasia syndrome ■ The presence of a nuchal translucency measurement of 3.5 mm or greater, which is associated with a very high risk of chromosomal abnormalities and / or cardiac malformations and may affect serum PAPP-A levels. ■Fetal heart negative on the day of recruitment visit

[0261] III. Conduct of Research 11 March 7th - 13 June 7th (GA 80-97 days) Visit to nurse (Weeks 11 3 / 7 to 13 6 / 7 (GA 80-97 days)) First, a research nurse collected blood samples by venipuncture (2 × 5 mL tubes, BD Vacutainer SST). The tubes were gently inverted five times to thoroughly mix the reagent with the blood. The tubes were left in an upright position in a dark box until treatment (minimum 30 minutes, maximum 2 hours). In the second step, a research nurse measured the patient's blood pressure. The patient had to sit and rest with uncrossed legs for 5 minutes without speaking before the measurement. Blood pressure was measured simultaneously in both arms (sleeveless vest) three times using a preprogrammed Microlife electronic blood pressure monitor (model 33603).

[0262] A questionnaire was administered to the patients to learn about their medical and obstetric family history, as well as their socio-economic background, including date of birth, anthropometry, tagabism, etc. The total meeting with the nurse lasted a maximum of 30 minutes.

[0263] Visit to the technician (11 / 3 / 7-13 / 6 / 7 week, GA 80-97 days) Following the nurse appointment, all patients were interviewed by a radiologist from the research team and certified for nuchal translucency measurements to perform ultrasound acquisitions using a Voluson E8 Expert (GE Medical Systems, Milwaukee, WI, USA) equipped with a 4-8 MHz transducer. The equipment settings were the same for all patients: "Angiomode" = 100, "Smooth" = 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.

[0264] An ultrasound was performed to confirm the project's eligibility criteria: 1) If the cephalocaudal length (CCL) is 77 mm or greater, confirm the gestational age using the cephalocaudal length (CCL) and parietal diameter. 2) Nuchal translucency measurements were performed according to the Fetal Medicine Foundation standards. If participants had a prescription for nuchal translucency, they were provided with a report with the results. 3) In the presence of malformation syndromes, multiple pregnancies, nuchal translucency ≥ 3.5 mm and / or negative fetal heart, the patient was informed and possibly examined by one of the physicians in charge of the project or their representative. The ultrasound report with the results was sent to the patient's referring physician.

[0265] Technicians completed the eligibility sheet accordingly. Participants who met at least one of the exclusion criteria were treated as any other participant; their blood samples were analyzed, and all data already collected or to be collected until delivery were retained. All data from these participants were excluded from the primary analysis.

[0266] The visit window between 11 3 / 7 weeks and 13 6 / 7 weeks of gestation (80-97 days GA) is important and was respected to ensure the validity of certain data (biochemistry and ultrasound). Therefore, if an ultrasound date test confirms gestational age, it is as follows: a)11 3 / 7 Less than 1 week (GA less than 80 days) (LCC less than 45 mm): The visit was rescheduled for a day between 11 3 / 7 weeks and 13 6 / 7 weeks (GA between 80 and 97 days). Blood sampling and ultrasound were repeated at this time. Both samples were retained, but only the second sample was used for the primary analysis. If a minimum number of these patients (n ≥ 5) showed early preeclampsia, the first sample was analyzed and compared to the second sample in a case-control study.

[0267] Participants were free to return for a repeat visit. If participants declined a second visit, the collected data were retained but excluded from the primary analysis. b) >14 weeks (GA >98 days) (CCL >84 mm): Blood samples, ultrasound data and questionnaire data were kept intact. No further visits were scheduled. These patients were excluded from the analysis for the primary purpose.

[0268] The following ultrasound measurements were then performed for research purposes: 4) Using Doppler ultrasound, the left and right uterine arteries were visualized and pulsatility index measurements were performed according to the FMF criteria. The uterine arteries were examined at the level of the internal jugular vein, and the pulsatility index was automatically calculated by the machine using the pulsatile flow curves of three subsequent similar cardiac cycles. Measurements were performed in the sagittal and transverse directions, and the differences between the two techniques were evaluated on a sample of approximately 1,000 cases to assess their reproducibility, duration, and efficiency. The presence or absence of a notch was recorded bilaterally (a notch was considered present if early diastolic dissection occurred in each cycle). 5) 3D ultrasound of the placenta and subplacental area with and without Doppler. This examination takes approximately 30 seconds.

[0269] At the end of recruitment, for the case-control analysis, a technician blinded to the clinical data performed the following volumetric measurements and calculations: using VOCAL (Virtual Organ Computer-aided Analysis) and a series of six sections of the placenta, each rotated 30 degrees from the previous one horizontally on planes A and B, the placental contour was manually delineated, taking care to exclude the uterine wall. Similarly, the volume of the subplacental myometrium was assessed from the border between the placenta and the duodenal myometrium to the full thickness of the myometrium (maximum 1 cm thick). These volumetric measurements were measured for the following variables: a. Placental volume b. Placental quotient (PQ=1 / 4 placental volume / LCC). c. The vascularity index (VI), flow index (FI), and vascular flow index (VFI) of the placenta and duodenum-myometrium region are assessed using VOCAL software. VFI represents the number of stained voxels in the studied volume (expressed as a percentage). FI is the mean color value of all stained voxels representing the mean intensity of blood flow (expressed as an absolute value from 0 to 100). VIF is the mean color value of all voxels in the studied region (gray and colored, expressed as an absolute value from 0 to 100). 6) Cervical length: vagina (intravaginal probe). This test, performed only in the Quebec City CHU, validated the prediction of preterm birth that is also present in the FMF algorithm. It was not systematically performed in all participants, but was performed based on the doctor's prescription. 7) An ultrasound acquisition of the abdominal region was also performed to inductively measure the thickness of visceral adipose tissue between the medial border of the rectus abdominis muscle and the anterior wall of the abdominal aorta. This ultrasound measurement may have greater predictive power than BMI in predicting preeclampsia. The acquisition takes just a few seconds or more.

[0270] The total time required for all ultrasound acquisitions ranged from 15 to 35 minutes. Study technicians were certified and approved by the Fetal Medicine Foundation (FMF) and PQDT21 for nuchal transillumination and cervical measurements. Participants received a DVD or USB stick with fetal images as a thank you for their participation.

[0271] Monitoring during pregnancy (34 0 / 7 weeks to 35 6 / 7 weeks, 232 to 251 days GA) The survey (electronic and emailed to participants) was administered during the 34th trimester of pregnancy. eThe survey was conducted weekly. This verified whether the patient's medications had changed during pregnancy and whether the pregnancy was progressing normally, had moved, or had experienced any complications to date. If the survey was not completed, an email reminder was automatically sent one week later. Those who did not respond to the survey were then contacted by phone.

[0272] Postpartum follow-up (6 weeks after DPA) A second electronic survey (sent via email to participants) was administered approximately 6 weeks after the participant's expected due date. This allowed us to verify whether the participant or their infant experienced any difficulties after birth. Specifically, we wanted to learn about cases of postpartum preeclampsia and rare cases of perinatal or maternal death (regardless of the hospital in which these events occurred). If the survey was not completed, an email reminder was automatically sent one week later. Those who did not respond to the survey were subsequently contacted by phone. Procedures were in place to ensure that participants who had terminations, intrauterine deaths, or other adverse complications (mentioned in the 34-week survey) were not contacted again at 46 weeks unless necessary, to avoid inconvenience.

[0273] Follow-up at the end of pregnancy (1 month after DPA) Data on pregnancy (e.g., fetal hypertension, preeclampsia, perinatal death), delivery (e.g., gestational age), and neonates (e.g., sex, birth weight) were collected from patients' medical records via CristalNet software by research nurses after delivery. In the rare event that a patient delivered at a non-participating center (not CHUL, HSFA, or Levis), the patient was contacted to request permission to obtain this information at the place of delivery. For all cases of suspected preeclampsia and cases delivered before 37 weeks (<10%, i.e., <532 records retrieved from the CHU-Q archive and <228 records retrieved from the CHUSJ archive), a second review of the data was conducted by a physician (EB, KG, FA, or their representative) directly from the medical records.

[0274] blood sample Blood specimens were transported to the laboratory in boxes at room temperature and centrifuged less than 2 hours after puncture but more than 30 minutes after puncture.

[0275] Serum collected after centrifugation (1200 × g, 10 min at room temperature) was transferred to 1 mL aliquots and then used to measure PAPP-A, PlGF, sFlt-1, free bHCG, and maternal serum AFP using commercially available kits validated by the FMF. For CHU de Quebec (CHUL and HSFA), samples were stored at 4°C until assays were performed within 24 hours. For CHU Ste-Justine, aliquots were stored at -20°C and sent twice a month by registered mail on dry ice to CHU de Quebec for analysis and storage. After primary analysis (serum PAPP-A, PlGF, and sFlt-1 assays), the remaining serum was frozen and stored at -80°C and used to measure PP13, ADAM12, and vitamin D in a case-cohort substudy conducted at the end of the main study.

[0276] Case-cohort study A case-cohort study nested within the main cohort evaluated the predictive effect of potential biomarkers for the prediction of early preeclampsia. Maternal serum sFlt-1, ADAM-12, PP-13, and vitamin D were measured using commercially available kits in a randomly selected subgroup of women (approximately 236 women) and in all cases of early preeclampsia (approximately 45 women) at the end of the study. This same case cohort was analyzed for the following variables: placental volume (PV), placental and subplacental vascularity index (IV), flow index (FI), and perfusion index (VFI).

[0277] IV. Judgment criteria Primary endpoint: Preeclampsia requiring delivery before 34 weeks of gestation (before 231 days GA) based on gestational age determined by last menstrual period (DMD) or 11-13 weeks (71-91 days GA) ultrasound (if the latter showed a difference of 5 days or more with the DMD method). Preeclampsia diagnosed according to the following criteria: 1) gestational hypertension ≥ 140 systolic and / or ≥ 90 diastolic (twice within 4 hours) with the following conditions: growth retardation < 10 e percentile, thrombocytopenia <100, AST and / or ALT greater than twice normal, diastolic blood pressure ≥110 mmHg, and / or proteinuria (≥2+ on stick or greater than 300 mg / 24 h).

[0278] Secondary endpoints: Delivery before 37 weeks (GA less than 253 days) (spontaneous birth or PPROM before 37 weeks of gestation) Delivery before 34 weeks (GA less than 231 days) (spontaneous birth or PPROM before 34 weeks of gestation)

[0279] Preeclampsia: Preterm preeclampsia at <37 weeks (GA <253 days) Severe preeclampsia (having any of the following conditions: 1) systolic ≥ 160 mmHg and diastolic ≥ 110 mmHg after 4 hours of rest, 2) proteinuria ≥ 5 g / 24 hours or ≥ 3+ on the rod, 3) oliguria ≤ 400 mL / 24 hours, visual or encephalopathy, epigastric pain, pulmonary edema or cyanosis, thrombocytopenia < 100,000 mm 3 . Perinatal deaths (before delivery, up to 7 days after birth) Growth retardation (<10 based on Canadian reference values) e percentile) Severe syncope (<3 e percentile) Birth weight <2500 grams Birth weight <1500 grams average birth weight

[0280] V. Data Analysis Plan Main Analyses: At the end of the study, calculations of risk of early pre-eclampsia and pre-eclampsia were performed automatically by the FMF software using all necessary collected data, including uterine Doppler measurements. Risk calculations were repeated automatically for all eligible participants, excluding uterine Doppler measurements.

[0281] The area under the curve, sensitivity, specificity, positive predictive value, and negative predictive value of the two screening methods were reported and calculated using ROC curves and different cut-off values.

[0282] result: As can be seen from Figures 1-12, data from the PREDICTION study support the prognostic ability of sFlt-1 in identifying patients at risk for EO-PE and / or IUFD when analyzing samples obtained before 90 days GA.

[0283] As can be seen from Figure 1, 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, women with EO-PE (N = 24) had average sFlt-1 levels. Between 140 and 154 days of gestation, women with EO-PE (N = 4) had sFlt-1 levels above the median. From this study, we can conclude that sFlt-1 levels are abnormally decreased early in pregnancy (before 90 days of gestation) in women who develop early preeclampsia (before 34 weeks, before 231 days of gestation), progressively increase to normal by the end of the first trimester (between 90 and 100 days), and then abnormally increase thereafter (after 140 days of gestation).

[0284] Thus, an increase in sFlt-1 levels between the first sample and the second sample allows for the determination of risk patterns in a subject and the potential initiation of appropriate treatment.

[0285] However, sFlt-1 measurements do not provide a statistically relevant correlation with EO-PE when determined from samples obtained throughout trimester 1 (Figure 2).More notably, sFlt-1 measurements also do not provide a statistically relevant correlation with EO-PE when determined from multiple samples obtained after 12 6 / 7 weeks (>90 days) of GA (Figures 3 and 4).

[0286] Surprisingly, measurement of sFlt-1 within 12 weeks or 90 days of gestation shows a significant correlation with EO-PE, particularly an inverse correlation with EO-PE (Figures 5 and 6). Thus, determining sFlt-1 within 90 days of GA allows for reliable prognosis of EA-PE at an early time point.

[0287] The combination of sFlt-1 and PlGF results in a statistically improved prognosis for EO-PE when samples are obtained early in pregnancy, e.g., before the end of the 12th gestational week (before 90 days of gestational age). Notably, while PlGF is typically effective in prognosing EO-PE when measured after 90 days of gestational age, sFlt-1 does not appear to allow for reliable prognostic statements from a single measurement after 90 days of gestational age (Figure 7). Surprisingly, both sFlt-1 and PlGF allow for EO-PE prognosis when measured before the end of 12 weeks of gestational age (within 90 days). However, sFlt-1 appears to provide greater sensitivity, with comparable specificity values, preferably greater than 0.6 (Figure 8). Also surprising, combined analysis of sFlt-1 and PlGF demonstrates an unexpected synergistic enhancement in EO-PE prognosis when measured before 90 days of gestational age (Figure 9).

[0288] Also, throughout this study, it was found that the combination of uterine artery Doppler measurements, preferably in combination with sFlt-1, PlGF, maternal age and BMI, showed improved predictive power for EO-PE in subjects compared to sFlt-1 or PlGF alone (Figure 10).

[0289] The combination of sFlt-1 and PlGF also provided a prognosis for IUFD when samples were obtained between 90 and 11 days of GA (Figure 11) and showed a statistical improvement in IUFD prognosis when samples were obtained before 90 days of GA (Figure 12).

[0290] In addition, comparison of datasets obtained using the FMF algorithm and sFlt-1 analysis described herein reveals that the two prognostic procedures exhibit a strong correlation with each other. As can be seen from Figure 13, patients with high risk as determined by the FMF algorithm have significantly lower sFlt-1 levels before 90 days of gestational age. Given that the FMF screening algorithm involves consideration of multiple maternal characteristics and medical history, including factors such as blood pressure, pregnancy-associated plasma protein A and placental growth factor, crown-rump length, and uterine artery pulsatility index, this finding represents a significant simplification in the prognostic approach, allowing for comparable risk assessments, thereby potentially avoiding the more complex FMF approach. Analysis of sFlt-1 before 90 days may actually enable prediction of pregnant women who will have a positive FMF test without the need for Doppler uterine artery evaluation.

[0291] In conclusion, this study allowed us to conclude that in the majority of pregnant women who will develop early preeclampsia, sFlt-1 decreases before 90 days of gestation but then increases and becomes abnormally high in the second trimester in these same women. Thus, sFlt-1 before 90 days of gestation makes it possible to predict early PE, especially when combined with PlGF and / or uterine artery Doppler (40% detection rate for a 10% FPR). Furthermore, we also observed that the combination of the two markers can predict UFDI in approximately 35% of cases for a 10% false positive rate.

[0292] The importance of this information is important because the earlier aspirin or similar therapy is initiated in an attempt to address and potentially avoid EO-PE, the more effective the treatment. Thus, the present invention allows for an alternative and improved diagnostic approach to identify patients at risk for EO-PE by using biomarker analysis of samples obtained as early as first trimester, before 90 days GA, and subsequent treatment initiation and guidance.

[0293] Example 4: Pregnant women were recruited at 7-9 weeks gestational age (43-63 days GA) and followed until delivery. sFlt-1 and, optionally, PAPP-α, PlGF, and / or βhCG were measured using a Thermo Scientific BRAHMS KRYPTOR in serum samples obtained between 11-13 and 20-22 weeks gestational age (71-91 and 140-154 days GA) at recruitment and reported as multiples of the median (MoM), preferably adjusted for gestational age and / or BMI. Median levels of sFlt-1 were compared among women with early-onset PE (<34 weeks, <232 days GA), mid-onset PE (34-36 weeks, 232-252 days GA), late-onset PE (≥37 weeks GA), and no PE (controls). The area under the receiver operating characteristic curve (AUC) is used to estimate the potential predictive value of sFlt-1, PAPP-α, PlGF, and βhCG for PE. Analyses, including univariate and multivariate regression analyses, are performed using the SAS statistical software package (version 9.3, SAS Institute Inc, Cary, NC). A 5% type I error is considered in all analyses.

[0294] Additionally, parental and maternal characteristics (height, smoking, BMI, parity, chromosomal or anatomical fetal abnormalities identified by, for example, nuchal translucency, method of conception for blood test performance, poor FMF risk score, etc.), mean arterial blood pressure, levels of maternal serum biomarkers (pregnancy-associated plasma protein A, placental growth factor), two risk cutoffs (1 in 70 and 1 in 100 from the FMF algorithm), sFlt-1, and mean uterine artery pulsatility index are obtained to calculate the risk of early PE (including early-onset PE and mid-onset PE) and term PE compared to a reference group that does not develop early or term PE. Detection rates, false positive rates, and positive and negative predictive values ​​are calculated and assessed for term and early PE as placental-mediated complications.

[0295] When analyzing samples obtained before 90 days GA and even before 63 days GA (before the end of the 9th week of gestation, before 63 days GA), the prognostic ability of sFlt-1 in identifying patients at risk for EO-PE can be determined. sFlt-1 levels decrease early in pregnancy (before 90 days gestation, even before 63 days gestation) in women who will develop early preeclampsia (before 34 weeks GA, before 232 days GA), progressively increase, become normal at the end of the first trimester (between 90 and 100 days), and then become abnormally elevated thereafter (after 140 days gestation). The increase in sFlt-1 levels between the first and second samples allows for the determination of risk patterns in the subject and the potential initiation of appropriate treatment.

[0296] The combination of sFlt-1 with PlGF, PAPP-A, and / or βhGC may result in a statistically improved prognosis for EO-PE when the first sample is obtained early in pregnancy, for example, before the end of the 12th week of gestation (before 90 days GA), and even before the end of the 9th week of gestation (before 63 days GA).

[0297] sFlt-1, together with PlGF, PAPP-α, and / or βhCG, allows for EO-PE prognosis when measured before the end of 12 weeks GA (within 90 days) and even before the end of 9 weeks GA (within 63 days). Combined analysis of sFlt-1, together with PlGF, PAPP-α, and / or βhCG, allows for synergistic enhancement of EO-PE prognosis when measured before 90 days GA and even before 63 days.

[0298] The importance of early prognosis is important because the earlier aspirin or similar therapy is initiated in an attempt to address and potentially avoid EO-PE, the more effective the treatment. Thus, the present invention enables an alternative and improved diagnostic approach to identify patients at risk for EO-PE by using biomarker analysis of samples obtained early in pregnancy, before 90 days GA, and even before 63 days, and subsequent treatment initiation and guidance.

Claims

1. A method for prognosis, prediction, risk assessment, and / or risk stratification of premature eclampsia in pregnant subjects, a. To determine a first level of soluble fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof in a first sample isolated from the subject, which was isolated before 90 days of gestation (GA). b. A second sample isolated from the subject, which is isolated after the first sample, comprising determining a second level of sFlt-1 or a fragment thereof in the second sample, c. A method in which a second level of sFlt-1 or a fragment thereof, higher than the first level, indicates pre-eclampsia that occurs before GA at 231 days.

2. The method according to claim 1, wherein the second sample is isolated from the subject after 100 days of GA.

3. The method according to claim 1, wherein the second sample was isolated after 140 days of GA.

4. The method according to claim 3, wherein the second sample is isolated during GA 140 to 154 days.

5. The method according to claim 1, wherein the first level is less than a reference level selected from the population mean and / or median of a healthy population.

6. The method according to claim 1, wherein the second level is greater than or equal to a reference level selected from the group mean and / or median of a healthy population.

7. a. The first level is below the reference level, and the second level is above the reference level selected from the population mean and / or median of a healthy population, or b. The first level is below the reference level, and the second level is above the reference level selected from the population mean and / or median of a healthy population, or c. The method according to claim 1, wherein the first level is below a reference level, and the second level is above a reference level selected from the population mean and / or median of a healthy population.

8. The method according to claim 1, wherein a multiple of the median (MoM) of the first level less than 1.0 and a MoM of the second level greater than 1.0 indicate pre-eclampsia occurring before GA at 231 days.

9. The method according to claim 1, wherein a second level of sFlt-1 or a fragment thereof, higher than the first level, further indicates the occurrence of subsequent intrauterine fetal death (IUFD).

10. The method according to claim 1, wherein the sample is a bodily fluid sample.

11. The sample is A blood sample selected from venous blood samples, capillary blood samples, serum samples, or plasma samples, or The sample is a vaginal fluid sample, a saliva sample, or an amniotic fluid sample. The method according to claim 10.

12. The method according to claim 1, wherein a second level of sFlt-1 or a fragment thereof, higher than the first level, is used to initiate or modify the treatment of the subject in order to reduce the risk of developing pre-eclampsia, delay the time of onset of pre-eclampsia, and / or reduce the severity of pre-eclampsia.

13. The method according to claim 12, wherein the treatment comprises balancing the angiogenesis / anti-angiogenesis processes in placental development, lowering blood pressure, and / or protecting organ function.

14. The method according to claim 12, wherein the indicated treatment comprises the administration of acetylsalicylic acid and / or metformin.

15. The method according to claim 1, further comprising determining the level of placental growth factor (PlGF) or a fragment thereof in the first sample and / or the second sample from the subject, wherein the combination of the level of sFlt-1 or a fragment thereof in the first sample and / or the second sample and the level of PlGF or a fragment thereof indicates premature eclampsia occurring before 231 days of GA.

16. The method according to claim 1, further comprising determining or providing the maternal age, body mass index, mean arterial pressure (MAP), and / or uterine artery Doppler measurement of the subject, wherein the combination of the level of sFlt-1 or fragment thereof in the first sample and / or the second sample with the maternal age, body mass index, MAP, and / or uterine artery Doppler measurement of the subject indicates pre-eclampsia occurring before 231 days of GA.

17. A kit for carrying out the method described in claim 1, - A detection reagent for determining the level of sFlt-1 or its fragments in a sample from the target, A computer-readable medium and / or computer software in the form of computer executable code, configured to compare two determined levels of -sFlt-1 or a fragment thereof, - The computer-readable medium and / or computer software is configured to optionally include one or more reference levels of sFlt-1 or its fragments that correspond to the population mean and / or median of a healthy population, and to compare two determined levels of sFlt-1 or its fragments with the reference levels, and / or - A kit in which the computer-readable medium and / or computer software is configured to optionally compare the subject's maternal age, body mass index, and / or uterine artery Doppler measurement to one or more reference levels corresponding to the population mean and / or median of a healthy population.

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