Biomarkers for prognosis of early-onset preeclampsia

Abstract

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 a level of soluble fms-like tyrosine kinase-1 (sFlt-1) or a fragment thereof in a sample isolated from said pregnant subject.The present invention further relates to a method for the prognosis, prediction, risk assessment and / or risk stratification of early onset pre-eclampsia in a pregnant subject, comprising determining a level of (sFlt-1) or a fragment thereof in a sample isolated from said pregnant subject, wherein the sample is isolated from the subject before the end of the 12th week of gestation, and wherein the level of sFlt-1 or a fragment thereof is indicative of the likelihood of early onset pre-eclampsia occurring before the end of the 33rd week of gestation.The present invention further relates to a combined measurement of sFlt-1 and PlGF, optionally in combination with consideration of one or more further 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 a 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 of medical conditions, in particular preeclampsia (PE).

[0002] Accordingly, the present invention relates to a method for 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 said pregnant subject, wherein said 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 determining a level of soluble fms-like tyrosine kinase-1 (sFlt-1), or a fragment thereof, in a sample isolated from the pregnant subject, wherein the sample is isolated from the subject before the end of the 12th week of gestation, and wherein the level of sFlt-1, or a fragment thereof, is indicative of the likelihood of early onset preeclampsia occurring before the end of the 33rd week of gestation.

[0004] The present invention further relates to a method for the 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 sample isolated from said pregnant subject. The present invention further relates to the measurement of sFlt-1 and PlGF in combination with consideration of one or more further factors selected from maternal age, body mass index, uterine artery Doppler measurement 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 16% of global maternal mortality (approximately 63,000 maternal deaths per year) is due to PE alone. Infants are also at risk. Preeclampsia complicates approximately 2-8 percent of all pregnancies and is the leading cause of maternal and fetal deaths worldwide (Duley 2009,Semin Perinatal:33:130-37). Preeclampsia is generally defined as pregnancy-associated or induced hypertension and proteinuria with onset after the 20th week 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 intra-uterine fetal death (IUFD) and high perinatal mortality.

[0007] The risk of maternal death 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 are not effectively monitored or treated until long after complications associated with the disorder, including elevated blood pressure and proteinuria, have developed. Furthermore, pregnant women who are at little or no risk of developing such disorders must undergo unnecessary testing for symptoms throughout their pregnancy, as there are no effective measures by which caregivers can exclude them from risk in the early stages of pregnancy.

[0008] WO 2008 / 103202(A2) discloses a method for diagnosing pregnancy-associated hypertensive disorders by measuring COMT, HIF-1α, EPO, LDH-A, ET-I, transferrin, transferrin receptor, and Flk-I, free VEGF, total VEGF, sFlt-1, 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 a pregnant woman as having or 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 as to whether the woman is at risk of developing a hypertensive disorder.

[0010] WO 2004 / 008946(A2) discloses a method of treating or preventing pre-eclampsia 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 pre-eclampsia are due to an acute elevation of sFlt-1 within 5 weeks prior to the onset of clinical disease.

[0011] The most invasive treatment for preeclampsia to date is the termination of the pregnancy, either by preterm vaginal or cesarean delivery. As mentioned above, in the case of preeclampsia before gestational age 34, maternal risk and fetal survival are significantly compromised. Therefore, attempts should be made to delay delivery and thereby improve neonatal survival. Tsakiridis et al. (Volume 76, Number 10, Obstetric and Gynecological Survey) highlighted the content of pregnancy-related guidelines describing early administration of low-dose aspirin in high-risk patients, ideally in the first trimester or at gestational age 36-37 weeks until delivery.

[0012] 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.

[0013] In the most severe forms, such as early onset PE, it is necessary to start treatment as early as possible, ideally at the 11th week of gestation, although early treatment may be started later, such as at the 12th to 15th week of gestation. Currently, approximately 60-66% of early onset PE and approximately 70% of early onset PE are detected with the FMF algorithm. The Fetal Medicine Foundation (FMF) screening algorithm includes 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 that is not universally available to all pregnant subjects. There is an urgent need in the art for improved and simplified means to determine risk of pre-eclampsia, especially early onset pre-eclampsia, at an early stage of pregnancy. Summary of the Invention

[0014] In light of the prior art, the technical problem underlying the present invention is to provide 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 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 means for improving and / or simplifying the screening or prognosis of early onset pre-eclampsia in the early stages of pregnancy, increasing the sensitivity and preferably not requiring uterine artery Doppler measurements.

[0015] This problem is solved by the features of the independent claims. Preferred embodiments of the invention are provided by the dependent claims.

[0016] 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 the level of soluble fms-like tyrosine kinase-1 (sFlt-1), or a fragment thereof, in a sample isolated from the pregnant subject; b. the sample is isolated from the subject before the end of the 12th week of gestation (before 90 days GA); c. A method wherein the level of said sFlt-1 or a fragment thereof indicates the likelihood of early onset pre-eclampsia occurring before the end of the 33rd week of gestation.

[0017] Thus, the method described herein allows EO-PE risk assessment at an early stage of pregnancy, thus providing clinicians with the possibility to start appropriate preventive treatment at a very early stage of pregnancy. In the most severe forms of PE, such as EO-PE, it is recommended to start treatment as early as possible, preferably as early as the 11th week of pregnancy. Until the time of the present invention, although molecular analysis was available for PE prognosis, prognosis of early stages of EO-PE mainly relied on FMF algorithms using uterine artery Doppler measurements. Thus, the present invention allows a simple and reliable molecular diagnostic and / or prognostic approach aimed at identifying subjects at risk of EO-PE at very early gestational age (GA).

[0018] Of note, sFlt-1 measurements do not provide a statistically relevant correlation with EO-PE when determined from multiple samples obtained throughout the first trimester (Figure 2, below). Even more noteworthy, 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 of GA (Figures 3, 4, below). Surprisingly, measurements of sFlt-1 within 12 weeks or within 90 days of gestation show a significant correlation with EO-PE, and in particular, an inverse correlation with EO-PE (Figures 5, 6). Thus, determining sFlt-1 within 90 days of GA allows for reliable prognosis of EO-PE at an early time point.

[0019] The various aspects of the present invention are unified by, benefit from, are based on and / or are related by the common finding that levels of sFlt-1 or a fragment thereof in a sample from a pregnant subject with a GA before the end of the 12th week, e.g., before 90 days GA, are indicative of the likelihood of early onset pre-eclampsia.

[0020] The present invention provides an efficient and reliable test for medical personnel, such as physicians, nurses, and emergency department personnel, to quickly and accurately assess the likelihood that a pregnant subject will develop PE. Typically, pregnant women who have little or no risk of developing such a disorder must undergo unnecessary testing for the condition throughout their pregnancy, because there is no effective means by which caregivers can eliminate them from risk in the early stages of pregnancy.

[0021] High levels of placental soluble fms-like tyrosine kinase (sFlt-1) are strongly associated with PE in the second (14-27 weeks gestation) and third trimesters (28 weeks gestation to term) of pregnancy. 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 being indicative of EO-PE.

[0022] Based on this surprising discovery, the present invention provides means for identifying pregnant subjects who are at increased or high risk of developing EO-PE, and also for identifying patients who are less likely to develop such a complication or who can substantially exclude the development of such a complication by determining the level of sFlt-1 or a fragment thereof in a sample isolated from the patient.

[0023] A further advantage arising as a consequence of this surprising discovery is that in addition to the conventional combination of biomarkers, clinical parameters and imaging procedures for prognosticating 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 or not, for which special ultrasound examination devices and specialists to operate the device and perform the measurements are typically required, thus allowing a simple and minimally invasive test.

[0024] More notably, the Aspirin for Evidence-Based Preeclampsia Prevention (ASPRE) trial, a multicenter trial that included women identified as at high risk for early PE according to the FMF algorithm who were randomized to receive aspirin or placebo from gestational ages 11 to 14 weeks until gestational age 37 weeks, showed a 62% reduction in early PE with daily low-dose aspirin compared with the placebo group (relative risk, 0.38; 95% confidence interval [confidence interval, CI], 0.20-0.74).

[0025] If it is possible to accurately predict the high risk of developing EO-PE in the first 12 weeks of pregnancy, this efficient treatment can be applied to pregnant subjects in early pregnancy.This brings another advantage based on the surprising discovery that the high risk of developing EO-PE can be predicted in the first trimester of pregnancy, so that subjects predicted to have high risk of developing PE can be given mild treatment for prevention, medication, or even bed rest, and frequent monitoring instead of any serious measures (such as early termination of pregnancy after developing PE in the later stages of pregnancy).

[0026] In one embodiment, the method comprises: a. determining the level of placenta growth factor (PlGF) or a fragment thereof in a sample isolated from the subject; b. The combination of levels of sFlt-1 or a fragment thereof and levels of PlGF or a fragment thereof indicates the likelihood of early onset pre-eclampsia occurring before the end of the 33rd week of gestation.

[0027] As shown in more detail below, the combination of sFlt-1 and PlGF results in a statistically improved prognosis of EO-PE when samples are obtained early in pregnancy, for example, before the end of the 12th 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 a reliable prognostic statement from a single measurement after 90 days GA (Figure 7). Surprisingly, both sFlt-1 and PlGF allow for EO-PE prognosis when measured before the end of 12 weeks (within 90 days) GA. However, sFlt-1 appears to provide greater sensitivity with comparable specificity values, preferably values ​​above 0.6 (Figure 8). Also surprising is that the combined analysis of sFlt-1 and PlGF shows an unexpected synergistic enhancement in EO-PE prognosis when measured before 90 days GA (Figure 9).

[0028] In an embodiment of this method, the subject is between 9 and 11 weeks of gestation.

[0029] In an embodiment of this method, the subject is in the 11th week of gestation.

[0030] In an embodiment of this method, the subject has a gestational age (GA) of 50 to 90 days, preferably 60 to 90 days, or 70 to 90 days, 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.

[0031] As discussed above, these early time points of sampling and testing allow for early initiation of treatment, which may be important in effectively combating and / or preventing the development of severe forms of PE.

[0032] 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 measurements, is indicative of early onset pre-eclampsia occurring before the end of the 33rd week of gestation.

[0033] 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 a subject, is indicative of early onset pre-eclampsia occurring before the end of the 33rd week of gestation.

[0034] As shown in FIG. 10 below, the combination of sFlt-1 and PlGF measurements shows further improvement in diagnostic ability when combined with additional uterine artery Doppler measurements.

[0035] 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 sample isolated from a subject; b. determining or providing the subject's maternal age, body mass index (BMI), and uterine artery Doppler measurement, and optionally, mean arterial pressure (MAP); c. The combination of the level of sFlt-1 or a fragment thereof, the level of PlGF or a fragment thereof, and 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.

[0036] In an embodiment, the method comprises: a. determining a level of sFlt-1 or a fragment thereof, and determining a level of PIGF or a fragment thereof in a 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 the sFlt-1 or a fragment thereof, the level of the PlGF or a fragment thereof, in combination with the subject's maternal age, body mass index (BMI) and mean arterial pressure (MAP), and optionally, a uterine artery Doppler measurement, is indicative of the likelihood of early onset pre-eclampsia occurring before the end of the 33rd week of gestation.

[0037] In an embodiment of the invention, the level of sFlt-1 or a fragment thereof determined in a sample is compared to a reference level, preferably the population mean and / or median for a healthy population, and a level of sFlt-1 or a fragment thereof below the reference level indicates a risk, e.g., a high risk, of early onset pre-eclampsia.

[0038] In an embodiment, the reference level is determined from a population of healthy pregnancies from the same GA, or pregnancies that do not develop EO-PE.

[0039] In an embodiment of the present invention, the level of PlGF or a fragment thereof determined in a sample is compared with a reference level, preferably the population mean and / or median for a healthy population, and a level of PlGF or a fragment thereof above the reference level indicates a risk, e.g. a high risk, of early onset pre-eclampsia.

[0040] In an embodiment of the 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.

[0041] Further embodiments and aspects of the present invention relate to the methods described herein which are used for indication of intrauterine fetal death (IUFD).

[0042] Embodiments of the present invention relate to the prognosis, prediction, risk assessment and / or risk stratification of IUFD. The prognosis of IUFD may be independent of or combined with the prognosis of EO-PE. In other words, EO-PE may occur in combination with IUFD or IUFD may occur independently of EO-PE. The 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.

[0043] As shown in more detail below, the combination of sFlt-1 and PlGF provided prognosis for IUFD when samples were obtained between 90 and 100 days GA (Figure 11) and showed a statistical improvement in prognosis for IUFD when samples were obtained prior to 90 days GA (Figure 12).

[0044] In an embodiment of the 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 subsequent occurrence of intrauterine fetal death (IUFD).

[0045] In an embodiment of the invention, the sample is a body fluid sample, such as a blood sample, such as a venous blood sample, a capillary blood sample, a serum sample, a plasma sample, a vaginal fluid sample, a saliva sample or an amniotic fluid sample, preferably a blood, serum or plasma sample.

[0046] In embodiments of the invention, a subject's level of sFlt-1 or a fragment thereof, and optionally, level of PlGF or a fragment thereof, maternal age, body mass index (BMI), uterine artery Doppler measurements, and / or mean arterial pressure (MAP) indicate initiation or modification of a subject's treatment to reduce the risk of developing pre-eclampsia, delay the time of onset, and / or reduce the severity of pre-eclampsia, and / or protect organ function, such as the kidney and / or liver, e.g., by balancing angiogenic / anti-angiogenic processes in placental development, lowering blood pressure.

[0047] In an embodiment 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.

[0048] In an embodiment of the invention, the treatment comprises administration of acetylsalicylic acid.

[0049] In an embodiment of the invention, the treatment comprises administration of metformin.

[0050] Accordingly, the present invention provides a method of treating a pregnant subject to reduce the risk of early onset pre-eclampsia, comprising: a. - determining a level of soluble fms-like tyrosine kinase-1 (sFlt-1), or a fragment thereof, in a sample isolated from the pregnant subject; - the sample is isolated from the subject before the end of the 12th week of gestation (before 90 days GA); - prognosing, predicting, risk assessment and / or risk stratifying early onset pre-eclampsia in a pregnant subject, wherein the level of said sFlt-1 or a fragment thereof is indicative of the likelihood of early onset pre-eclampsia occurring before the end of the 33rd week of gestation; b. administering to the subject a treatment to reduce the risk of developing pre-eclampsia, delay the time of onset, and / or reduce the severity of pre-eclampsia.

[0051] In embodiments, the treatment administered relates to or includes balancing angiogenic / anti-angiogenic processes in placental development, lowering blood pressure, and / or protecting organ function such as the kidney and / or liver.

[0052] In embodiments of the method of treatment, the treatment is selected from the group consisting of one or more diuretics, beta blockers, acetonitrile 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.

[0053] In an embodiment of the method of treatment, the treatment comprises administration of acetylsalicylic acid.

[0054] In an embodiment of the method of treatment, the treatment comprises administration of metformin.

[0055] In embodiments, a level of sFlt-1 or a fragment thereof that is at least 6% lower than the reference sample indicates initiating or modifying a 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.

[0056] In embodiments, a level of sFlt-1 or a fragment thereof that is at least 8% lower than the reference sample, or 12%, 15%, or 20% lower than the reference sample indicates initiating or modifying a 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, protecting organ function such as the kidney or liver.

[0057] The gynecologist and / or physician may determine the appropriate treatment for the subject according to the current condition with or without risk factors. In embodiments, a level of sFlt-1 or a fragment thereof at least 8% lower than the reference sample, or 12%, 15%, or 20% 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 balancing the angiogenic / antiangiogenic process in placental development, lowering blood pressure, protecting organ function such as kidney or liver.

[0058] A gynecologist and / or doctor may determine a suitable treatment for a subject according to the current condition, with or without risk factors.

[0059] In embodiments, aspirin treatment may be initiated prior to the 16th week of gestation, which has been associated with a significant reduction in early PE. The Evidence-Based Aspirin for Preeclampsia Prevention (ASPRE) trial, a multicenter study 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 of gestation until 37 weeks of gestation, 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).

[0060] 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 headaches, lupus, blood clotting disorders such as increased clotting, inflammatory disease, impaired cardiac reserve, diabetes, chronic kidney disease, and chronic hypertension.

[0061] In an embodiment of the invention, the method further comprises determining a level of at least one additional biomarker or a fragment thereof in a sample from the patient, the at least one additional biomarker being βhCG, copeptin, vasopressin, troponin, BNP, ANP, CRP, trombocyte / leukocyte, IL6, IL11, MR-proADM, VEGF, PAPP-A, PIGF, endoglin, pro-Epil, PP-13, ADAM-12, vitamin D, inhibin-a, activin-a, pentraxin-3, p-selectin, free fetal hemoglobin, α-1-microglobulin, unconjugated estriol, α-fetoprotein, GDF15, neurophysin II, L NPEP, ESM1, HGF, pikachurin, hemopexin, pp13, uE3, CT-proET1, ADAM12, sTNFαR1, RBP4, ICAM, cell-free fetal DNA, FSTL3, visfatin, AFP, MMP9, TIMP1, Flt1, PCT, SHGB, creatinine, GBP1, IGFALS, urinary protein, PAI1 / PAI2, catechol-o-methyltransferase (COMT), heme breakdown products (bilirubin, biliverdin, carbon monoxide, ferritin), arginine breakdown products (urea, ornithine, citrulline, apolipoprotein H, argininosuccinic acid, ammonia), uterine artery Doppler The level of at least one additional biomarker selected from the group consisting of UtA-Pi, diastolic notch, MAP, blood pressure, smoking, leptin, genetic information and arginine, and the level of sFlt-1 or a fragment thereof are indicative of early onset preeclampsia before the end of the 33rd week of gestation.

[0062] In embodiments, the additional markers PAPP-A and / or PIGF are used in combination with sFlt-1.

[0063] In embodiments, the additional markers MAP, PAPP-A and / or PlGF are used in combination with sFlt-1.

[0064] In embodiments, the additional markers MAP, PAPP-A, βhGC and / or PlGF are used in combination with sFlt-1.

[0065] In an embodiment of the invention, the subject is nulliparous.

[0066] In an embodiment of the invention, the subject has had one or more previous pregnancies.

[0067] In an embodiment of the invention, the subject has a multiple pregnancy.

[0068] In an embodiment of the invention, the subject is suspected of carrying a fetus having a chromosomal abnormality.

[0069] A further aspect of the invention relates to kits for carrying out the methods described herein.

[0070] In embodiments, the kit comprises: - a detection reagent for determining the level of sFlt-1 or a fragment thereof and / or for determining the level of PIGF or a fragment thereof in a sample from a subject; - a computer readable medium in the form of computer executable code and / or computer software configured to perform an analysis useful for determining EO-PE risk.

[0071] In an embodiment, the computer readable medium and / or computer software in the form of computer executable code comprises: i. comparing the determined level of sFlt-1 or a fragment thereof and the determined level of PlGF or a fragment thereof with one or more reference levels, preferably corresponding to a population mean and / or median for a healthy population; ii. comparing the subject's maternal age, body mass index, MAP and / or uterine artery Doppler measurement to one or more reference levels, preferably corresponding to population means and / or medians for a healthy population.

[0072] In an embodiment of the invention, software in the kit, or to which the kit is configured to connect, allows for comparison of determined molecular markers, such as those described herein, and provides a prognostic statement regarding EO-PE risk based on samples obtained within 90 days GA.

[0073] In an embodiment of the invention, the kit may include a physical disk or computer readable medium having the software thereon, or 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 may be maintained and / or executed.

[0074] Further 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 is indicative of the likelihood of pre-eclampsia.

[0075] 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 indicative of high or low risk of pre-eclampsia.

[0076] 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 in which the sample is isolated from the subject by the end of the 12th week of pregnancy.

[0077] In one embodiment, the subject is in the 9th to 11th week of pregnancy, preferably the 11th week of pregnancy.

[0078] Surprisingly, significant differences in the predictive values ​​(AUC values) of sFlt-1 for early onset PE were observed between women recruited at 11, 12 and 13 weeks of gestation. The AUC value for predicting early onset PE of sFlt-1 levels in samples from subjects at 11 weeks of gestation reaches 0.82. The AUC value for predicting early onset PE of sFlt-1 levels in samples from subjects at 12 weeks of gestation reaches 0.62. The AUC value for predicting early onset PE of sFlt-1 levels in samples from subjects at 13 weeks of gestation reaches 0.50. A similar trend was surprisingly observed for the prediction of mid-onset PE.

[0079] A surprising advantage of the discovery is that by determining levels of sFlt-1 or fragments thereof early in pregnancy, particularly at 11 weeks gestation, one can accurately predict the likelihood of developing PE.

[0080] In an embodiment, the maternal age is less than 18.

[0081] In an embodiment, the maternal age is 18-34.

[0082] In an embodiment, the maternal age is greater than 34.

[0083] Surprisingly, differences in the predictive values ​​(AUC values) of sFlt-1 for maternal ages 18-34 and >34 were observed.

[0084] Pregnant subjects with a maternal age of 34 or greater may have a greater probability of reduced risk of preterm delivery, hypertension, complicated PE, severe PE, and chorioamnionitis.

[0085] Pregnant subjects with a maternal age of 40 or greater may have increased odds for mild PE, fetal distress, and poor fetal growth.

[0086] It would be highly advantageous for subjects in the indicated risk groups, including maternal ages under 18, 34 and over, and 40 and over, to be able to know an accurate prognosis of pre-eclampsia before or by the end of the 12th week of gestation.

[0087] In embodiments, the methods described herein include: 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. Wherein a level of sFlt-1 or a fragment thereof below the reference level indicates an increased risk of pre-eclampsia; or c. A level of sFlt-1 or a fragment thereof above the reference level indicates a lower risk of pre-eclampsia.

[0088] In an embodiment, the reference level is derived from a reference sample isolated from a pregnant subject that is free of or unaffected by any pregnancy-related hypertensive disorder, such as PE or eclampsia.

[0089] In embodiments, a level of sFlt-1 or a fragment thereof that is at least 2-5% lower than the reference sample indicates a high risk of PE, hi embodiments, a level of sFlt-1 or a fragment thereof that is less than 2% lower than the reference sample indicates a low risk of PE.

[0090] In embodiments, a level of sFlt-1 or a fragment thereof that is at least 5.8% lower than the reference sample indicates a high risk of PE, hi embodiments, a level of sFlt-1 or a fragment thereof that is less than 5.8% lower than the reference sample indicates a low risk of PE.

[0091] In embodiments, a level of sFlt-1 or a fragment thereof that is at least 8% lower than the reference sample indicates a high risk of PE, hi embodiments, a level of sFlt-1 or a fragment thereof that is less than 8% lower than the reference sample indicates a low risk of PE.

[0092] In embodiments, a level of sFlt-1 or a fragment thereof that is at least 12% lower than the reference sample indicates a high risk of PE, hi embodiments, a level of sFlt-1 or a fragment thereof that is less than 12% lower than the reference sample indicates a low risk of PE.

[0093] In embodiments, a level of sFlt-1 or a fragment thereof that is at least 15% lower than the reference sample indicates a high risk of PE, hi embodiments, a level of sFlt-1 or a fragment thereof that is less than 15% lower than the reference sample indicates a low risk of PE.

[0094] In embodiments, a level of sFlt-1 or a fragment thereof that is at least 20% lower than the reference sample indicates a high risk of PE, hi embodiments, a level of sFlt-1 or a fragment thereof that is less than 20% lower than the reference sample indicates a low risk of PE.

[0095] In embodiments, a level of sFlt-1 or a fragment thereof that is at least 5.8% lower than the reference sample indicates a high risk of terminal PE, hi embodiments, a level of sFlt-1 or a fragment thereof that is less than 5.8% lower than the reference sample indicates a low risk of terminal PE.

[0096] In embodiments, a level of sFlt-1 or a fragment thereof that is at least 8.8% lower than the reference sample indicates a high risk of terminal PE, hi embodiments, a level of sFlt-1 or a fragment thereof that is less than 8.8% lower than the reference sample indicates a low risk of terminal PE.

[0097] In embodiments, a level of sFlt-1 or a fragment thereof that is at least 11% lower than the reference sample indicates a high risk of terminal PE, hi embodiments, a level of sFlt-1 or a fragment thereof that is less than 11% lower than the reference sample indicates a low risk of terminal PE.

[0098] In embodiments, a level of sFlt-1 or a fragment thereof that is at least 14.7% lower than the reference sample indicates a high risk of early onset PE, hi embodiments, a level of sFlt-1 or a fragment thereof that is less than 14.7% lower than the reference sample indicates a low risk of early onset PE.

[0099] In embodiments, a level of sFlt-1 or a fragment thereof that is at least 16.7% lower than the reference sample indicates a high risk of early onset PE, hi embodiments, a level of sFlt-1 or a fragment thereof that is less than 16.7% lower than the reference sample indicates a low risk of early onset PE.

[0100] In embodiments, a level of sFlt-1 or a fragment thereof that is at least 20% lower than the reference sample indicates a high risk of early onset PE, hi embodiments, a level of sFlt-1 or a fragment thereof that is less than 20% lower than the reference sample indicates a low risk of early onset PE.

[0101] In embodiments, the level of sFlt-1 or a fragment thereof is indicative of early onset of PE, occurring from the beginning of the 20th week of gestation to the end of the 33rd week of gestation, or mid-onset of PE, occurring from the beginning of the 34th week of gestation to the end of the 36th week of gestation.

[0102] In embodiments, a level of sFlt-1 or a fragment thereof in the sample that is at least 15%, preferably 20% lower than the reference level is indicative of early onset of PE, occurring between the beginning of the 20th week of gestation and the end of the 33rd week of gestation.

[0103] In embodiments, a level of sFlt-1 or a fragment thereof in the sample that is less than 15% below the reference level indicates a low risk of early onset of PE.

[0104] In embodiments, a level of sFlt-1 or a fragment thereof in the sample that is at least 6%, preferably 12%, lower than the reference level indicates an early onset of PE occurring between the beginning of the 20th gestational week and the end of the 33rd gestational week, or an intermediate onset of PE occurring between the beginning of the 34th gestational week and the end of the 36th gestational week.

[0105] In an embodiment, a further risk parameter is the sex of the fetus.Surprisingly, a significant difference in the predictive value (AUC value) of sFlt-1 level in samples of subjects having at least a female fetus or at least a male fetus can be observed, as well as a significant decrease in sFlt-1 level compared to the reference level.In an embodiment, the level of sFlt-1 in the samples of the subjects 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 discovery, subjects carrying such risk parameters can be predicted to have a risk of PE.

[0106] One aspect of the invention is a kit for carrying out the method of the invention, comprising: - a detection reagent for determining the level of sFlt-1 or a fragment thereof in a sample from the subject, and optionally for determining the level of at least one additional biomarker described herein, preferably PAPP-A and / or PIGF; - a reference level, such as one or more cut-off levels, corresponding to a reference level indicative of a high or low risk of pre-eclampsia.

[0107] In one embodiment, the present invention provides a kit for carrying out the methods described herein, comprising: - a detection reagent 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, preferably PAPP-A and / or PIGF; - a reference level, e.g. one or more cut-off levels, Levels of sFlt-1 or fragments thereof in samples isolated from subjects up to 18 years of age that are at risk for preeclampsia; a level of sFlt-1 or a fragment thereof in a sample isolated from a subject aged 18-34 years that is at risk for preeclampsia; or a reference level corresponding to the level of sFlt-1 or a fragment thereof in a sample isolated from a subject over 34 years of age that is at risk for pre-eclampsia; The reference level is stored in 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, the determined level of at least one additional biomarker, to the reference level.

[0108] In one embodiment, a kit for carrying out the methods described herein comprises: - a detection reagent 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, preferably PAPP-A and / or PIGF; - a reference level, e.g. one or more cut-off levels, the level of sFlt-1 or a fragment thereof in a sample isolated from a subject having blood type AB, which is indicative of a risk of pre-eclampsia; or the level of sFlt-1 or a fragment thereof in a sample isolated from a subject having an Rh-negative blood type, which is indicative of a risk of pre-eclampsia; or the level of sFlt-1 or a fragment thereof in a sample isolated from a subject who has Rh-negative blood type and is the biological father of an Rh-positive fetus that is at risk for preeclampsia; or the level of sFlt-1 or a fragment thereof in a sample isolated from a subject carrying at least one female fetus at risk for pre-eclampsia; or the level of sFlt-1 or a fragment thereof in a sample isolated from a subject carrying at least one male fetus that is at risk for preeclampsia; or a level of sFlt-1 or a fragment thereof in a sample isolated from a nulliparous subject that is at risk for pre-eclampsia; or the level of sFlt-1 or a fragment thereof in a sample isolated from a subject having one or more previous pregnancies indicative of a risk for pre-eclampsia, or the level of sFlt-1 or a fragment thereof in a sample isolated from a subject having a multiple pregnancy indicative of a risk for pre-eclampsia, or a reference level corresponding to the level of sFlt-1 or a fragment thereof in a sample isolated from a subject suspected of carrying a fetus having a chromosomal abnormality indicative of risk of pre-eclampsia; The reference level is stored in 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, the determined level of at least one additional biomarker, to the reference level.

[0109] In one embodiment, a kit for carrying out the methods described herein comprises: - a detection reagent 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, preferably PAPP-A and / or PIGF; - a reference level, e.g. one or more cut-off levels, a level of sFlt-1 or a fragment thereof in a sample isolated from the subject, which is indicative of a risk of early onset of pre-eclampsia occurring between the beginning of the 20th week of gestation and the end of the 33rd week of gestation; the level of sFlt-1 or a fragment thereof in a sample isolated from a subject at risk for mid-onset pre-eclampsia occurring between the beginning of the 34th week of gestation and the end of the 36th week of gestation; or a reference level corresponding to the level of sFlt-1 or a fragment thereof in a sample isolated from a subject exhibiting a low risk of developing pre-eclampsia; The reference level is stored in 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, the determined level of at least one additional biomarker, to the reference level.

[0110] The present invention provides a method for identifying a subject at risk for early onset pre-eclampsia (occurring before the end of the 33rd week of gestation) and treating said subject, comprising: (a) diagnosis, prognosis, prediction, risk assessment and / or risk stratification of early onset pre-eclampsia in a pregnant subject, comprising: - determining a level of soluble fms-like tyrosine kinase-1 (sFlt-1), or a fragment thereof, in a sample isolated from the pregnant subject; - the sample is isolated from the subject before the end of the 12th week of gestation (before 90 days GA); - diagnosis, prognosis, prediction, risk assessment and / or risk stratification, wherein the level of said sFlt-1 or a fragment thereof is indicative of the likelihood of early onset pre-eclampsia occurring before the end of the 33rd week of gestation; (b) administering to the subject a treatment for or a treatment for reducing the risk of early onset pre-eclampsia.

[0111] The present invention provides a method for detecting soluble fms-like tyrosine kinase-1 (sFlt-1), or a fragment thereof, in a sample derived from a subject, comprising the steps of: providing a sample from a subject, preferably a blood sample or a sample derived from a blood sample, with a complex comprising at least one binding agent for sFlt-1 or a fragment thereof; and providing a sample from a subject, preferably a blood sample or a sample derived from a blood sample, with a complex comprising at least one binding agent for PlGF or a fragment thereof; - The method further relates to a method in which the sample has a level of sFlt-1, preferably PlGF, below or above a threshold value, e.g., any threshold value disclosed herein, preferably the population mean and / or population median of sFlt-1 levels from normal pregnancies at any given time in the respective patient population.

[0112] The present invention relates to a method for treating and / or reducing the risk of early onset pre-eclampsia occurring before the end of the 33rd week of gestation, or for administering to a subject a treatment for early onset pre-eclampsia, comprising: administering to a subject a treatment for early onset pre-eclampsia, The method further relates to a method in which the subject has been determined to have, in a body fluid sample, preferably a blood sample or a sample derived from a blood sample, a level of sFlt-1, preferably PlGF, below or above a threshold level of sFlt-1, preferably PlGF, from normal pregnancies at any given time in the respective patient population, such as any threshold disclosed herein, preferably the population mean and / or population median.

[0113] An embodiment describing a method of the invention may be used to describe a kit of the invention, and vice versa. Features disclosed in some embodiments of the method may also be used to characterize other embodiments of the method or other methods of the invention. The present invention is united by the novel and beneficial use of the prognostic marker sFlt-1 to indicate the risk of developing EO-PE in samples obtained by 12 weeks (90 days) GA. Thus, the relevant features described herein for one aspect may be used to describe any given aspect of the invention in a manner according to the understanding of the skilled artisan. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0114] All cited references, both patent and non-patent literature, are incorporated herein by reference in their entirety.

[0115] The present invention relates to 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 the pregnant subject, and b) the level of sFlt-1 or a fragment thereof is indicative of the likelihood of pre-eclampsia.

[0116] As used herein, the term "subject" means a mammal, including, but not limited to, a human or a non-human mammal, such as a cow, horse, dog, sheep, or cat. Included in this definition are pregnant mammals, postpartum mammals, and non-pregnant mammals.

[0117] In the present invention, the terms "risk assessment" and "risk stratification" refer to grouping subjects into different risk groups according to the further prognosis of the subject. 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 treatment groups that receive certain different therapeutic measures depending on the classification of the patient.

[0118] As used herein, "prognosis" relates to predicting the outcome or particular risk of a subject developing PE. It may also include estimating the chances of recovery or chances of adverse outcome for the subject. Assessment of the severity of PE may also be encompassed by the term "prognosis" or "risk assessment" or "risk stratification."

[0119] 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 urinary excretion of at least 0.3 grams of protein in a 24-hour urinary protein excretion (or a dipstick test of at least +1 or greater) on two occasions 4-6 hours apart.

[0120] 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 the effects of pregnancy or recent pregnancy. Preeclampsia typically occurs after the 20th week of gestation. Preeclampsia is generally defined as some combination of the following symptoms: (1) systolic blood pressure (BP) >140mmHg and diastolic BP >90mmHg after the 20th week of gestation (typically measured twice, 4-168 hours apart), (2) new-onset proteinuria (1+ by dipstick on urinalysis, >300mg 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 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.

[0121] In preeclampsia, hypertension and proteinuria generally occur within 7 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 only one symptom at a time. Occasionally, severe preeclampsia may result in the development of seizures. This severe form of the syndrome is called eclampsia. "Eclampsia" may also include 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 kidney.

[0122] "Severe pre-eclampsia" or "high severity pre-eclampsia" is also defined according to established criteria as a blood pressure of at least 160 / 110 mmHg on at least two occasions six hours apart and more than 5 grams of protein in a 24-hour urinary protein excretion or persistent +3 proteinuria on a dipstick test.

[0123] Severe preeclampsia may include the HELLP syndrome (hemolysis, elevated liver enzymes, low platelet count). Other elements of severe preeclampsia may include in-utero growth restriction (IUGR) below the 10th percentile for US population statistics, 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).

[0124] As used herein, "preterm birth" is defined as birth before the 37th week of gestation.

[0125] As used herein, "early onset PE" is defined as PE delivered before the 37th week of gestation.

[0126] As used herein, "early onset of pre-eclampsia" is intended to mean that symptoms of pre-eclampsia occur beginning at the 20th week of gestation and ending at the 33rd week of gestation. In embodiments, early onset of pre-eclampsia refers to cases where delivery occurs before the 34th week of gestation.

[0127] As used herein, "mid-onset pre-eclampsia" shall mean that symptoms of pre-eclampsia occur between the beginning of the 34th week of gestation and the end of the 36th week of gestation.

[0128] As used herein, "opportunistic onset of pre-eclampsia" is intended to mean that symptoms of pre-eclampsia occur from the 37th week of gestation.

[0129] By way of example, "symptoms of preeclampsia" may refer to: (1) systolic blood pressure (BP) >140 mmHg and diastolic BP >90 mmHg after the 20th week of gestation, (2) new-onset proteinuria (1+ by urinalysis dipstick, >300 mg 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.

[0130] 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.

[0131] "At risk of developing" a pregnancy-associated hypertensive disorder, such as pre-eclampsia or eclampsia, refers to a subject who does not currently have a pregnancy-associated hypertensive disorder, but has a higher than average chance of developing such a disorder. Such at-risk subjects include pregnant subjects who have blood sFlt-1 levels that are decreased by at least, but not limited to, 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. The patient may, in some embodiments, not exhibit other symptoms of a pregnancy-associated hypertensive disorder, such as pre-eclampsia.

[0132] As used herein, the term "indicative of a likelihood of pre-eclampsia" refers to a subject who does not currently have pre-eclampsia but has a greater 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 is indicative of the likelihood of developing pre-eclampsia. In one embodiment, the level of sFlt-1 or a fragment thereof determined in the sample is compared to 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.

[0133] As used herein, "high risk of pre-eclampsia" means high risk of developing pre-eclampsia, but does not currently have pre-eclampsia.

[0134] As used herein, "low risk of pre-eclampsia" means low risk of developing pre-eclampsia, but does not currently have pre-eclampsia.

[0135] "Pregnancy-related hypertensive disorder" is intended to mean any condition or disease during pregnancy associated with or characterized by elevated blood pressure. These conditions and diseases include pre-eclampsia (including preterm pre-eclampsia, severe pre-eclampsia), 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.

[0136] As used herein, the term "soluble Flt-1 (sFlt-1)" (also known as soluble fms-like tyrosine kinase 1, sVEGF-RI) refers to a soluble form of the Flt-1 receptor that is homologous to the protein defined by GenBank Accession No. U01134 or UniProt P17948 or the accession name VGFR1_HUMAN and has sFlt-1 biological activity. The biological activity of the sFlt-1 polypeptide can be assayed by assaying sFlt-1 binding to VEGF using any standard method. sFlt-1 lacks the transmembrane and cytoplasmic tyrosine kinase domains of the Flt-1 receptor. sFlt-1 can bind to VEGF and PlGF with high affinity, but 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.

[0137] As used herein, the term "specifically binds" refers to a compound or antibody or any detection reagent that recognizes and binds a polypeptide (i.e., sFlt-1 or any fragment thereof) but does not substantially recognize and bind other molecules in a sample (e.g., a biological sample that naturally contains a sFLt-1 polypeptide or any fragment thereof). In one embodiment, an antibody that specifically binds to sFlt-1 does not bind to Flt-1.

[0138] As used herein, a "detection reagent" or the like is a reagent suitable for determining a marker described herein, e.g., sFlt-1, PAPP-A, PIGF. Such an exemplary detection reagent is, for example, a ligand, e.g., an antibody or a fragment thereof, that specifically binds to a peptide or epitope of a marker described herein. Such a ligand may be used in an immunoassay as described above. Additional reagents used in an immunoassay to determine the level of a marker may also be included in the kit and are considered detection reagents herein. Detection reagents may also refer to reagents used to detect a marker or fragments thereof by mass spectrometry-based methods. Thus, such detection reagents may also be reagents used to prepare a sample for MS analysis, e.g., enzymes, chemicals, buffers, etc. A mass spectrometer may also be considered a detection reagent. A detection reagent according to the present invention may also be, for example, a calibration solution that may be used to determine and compare the level of a marker.

[0139] According to the present invention, the antibody may be a monoclonal or polyclonal antibody. In particular, an antibody that specifically binds at least to sFlt-1 or a fragment thereof is used.

[0140] 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 times higher, preferably 100 times higher, and most preferably at least 1000 times higher, than for other molecules contained in the sample containing the molecule of interest. It is well known in the art how to develop and select an antibody with a given specificity. In the context of the present invention, monoclonal antibodies as detection reagents are preferred. The antibody or antibody-binding fragment specifically binds to a marker or a fragment thereof as defined herein. In particular, the antibody or antibody-binding fragment binds to a peptide of sFlt-1 as defined herein. Thus, the peptide as defined herein may also be an epitope to which the antibody specifically binds. Furthermore, in the methods and kits of the present invention, an antibody or antibody-binding fragment that specifically binds to sFlt-1 or a fragment thereof is used.

[0141] 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, other markers of the invention, such as PAPP-A or PIGF.

[0142] 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, 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 B·R·A·H·M·S KRYPTOR assay, are also contemplated.

[0143] 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 term "capture molecule" or "molecular scaffold" includes 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, presence or absence of Lewis donors and / or acceptors, to specifically bind to the target molecule or molecule of interest. Thereby, the binding may be mediated, for example, by ionic, van der Waals forces, pi-pi, sigma-pi, hydrophobic or hydrogen bond interactions, or a combination of two or more of the foregoing interactions or covalent interactions between the capture molecule or molecular scaffold and the target molecule or molecule of interest. In the context of the present invention, the capture molecule or molecular scaffold may be selected, for example, from the group consisting of 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.

[0144] The method according to the invention can further be 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, it is preferred that when two antibodies are used, both antibodies are labeled as part of the detection system, which results in the generation of a signal or the induction of a signal when both antibodies are integrated into a single sandwich. Such a technique should be embodied in particular as a fluorescence enhancement or fluorescence quenching detection method. A particularly preferred embodiment relates to the use of detection reagents used in pairs, such as those described in US4882733, EP0180492, or EP0539477, and the prior art cited therein. In this way, measurements are possible in which only reaction products containing both labeling components are detected directly in a single immune complex in the reaction mixture. For example, such a technique is provided under the trade name TRACE™ (Time-Resolved Amplified Cryptate Release), or KRYPTOR™, which implements the teachings of the above-cited applications. Thus, in a particularly preferred embodiment, a diagnostic device is used to carry out the method 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, PIGF, etc. In a particularly preferred embodiment, the diagnostic device is a B·R·A·H·M·S KRYPTOR.

[0145] In an embodiment, quantitative determination of sFlt-1 can be performed by an automated immunofluorescence assay B·R·A·H·M·S sFlt-1 KRYPTOR Assay, preferably together with the B·R·A·H·M·S PlGF+KRYPTOR Assay. The lower and upper detection limits of 22000 and 90000 pg / mL B·R·A·H·M·S sFlt-1 KRYPTOR provide the measurement range required for reliable detection of clinical sFlt-1 levels throughout pregnancy. Only 8 μL of serum sample isolated from the subject is required for the assay.

[0146] One 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.

[0147] 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 may include detecting the presence, amount or concentration of one or more modified or unmodified fragment peptides, e.g., sFlt-1 or PAPP-A, PIGF, in the biological sample or, e.g., in 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 may be used in the MS analysis, particularly to determine the amount of sFlt-1 or a fragment thereof.

[0148] As used herein, the term "mass spectrometry" or "MS" refers 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.

[0149] The present invention therefore relates to MS detection methods, methods relating to sample preparation and / or chromatographic methods that can be combined with immunoenrichment techniques, preferably methods using liquid chromatography (LC), more preferably methods using high performance liquid chromatography (HPLC) or ultra-high performance liquid chromatography (UHPLC).

[0150] 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 a narrow range of m / z / s), followed by fragmentation of the selected ions and mass analysis of the resulting product (fragment) ions.

[0151] As used herein, the term "a detection reagent that specifically binds to sFlt-1 and fragments thereof" is intended to mean 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.

[0152] 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, a sample collection tube.

[0153] 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.

[0154] As used herein, a "fragment" is intended to mean a portion of a polypeptide or nucleic acid molecule. The portion preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of the reference nucleic acid molecule or polypeptide. A fragment may 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.

[0155] The sensitivity and specificity of diagnostic and / or prognostic tests do not only depend on the analytical "quality" of the test, but they also depend 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" population (i.e., apparently healthy individuals with no apparent 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 will likely overlap. Under such conditions, the test will not perfectly distinguish normal from disease with 100% accuracy, and the area of ​​overlap may indicate where the test cannot distinguish normal from 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 a perceived measurement will allow for correct identification of a condition. ROC curves can be used even when the test results do not necessarily give an exact number. As long as the results can be ranked, a ROC curve can be created. For example, the results of a test on a "disease" sample 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 a ROC curve can be created. These advantages 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 a 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.

[0156] 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. Thus, 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 correct identification of a disease or condition. Thus, 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. ROC curves with a larger AUC represent logistic regression.

[0157] 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 a fragment thereof, a specific hormone concentration, or a fragment thereof, or the presence of a biological substance, or a fragment thereof) that serves as an index of a health- and physiology-related assessment, such as a disease / disorder / clinical condition risk, preferably an adverse event. A marker or biomarker is defined as a characteristic that can be objectively measured and evaluated as an indicator of a normal biological process, a pathogenic process, or a pharmacological response to a therapeutic intervention. Biomarkers can be measured in samples (blood, plasma, urine, or as tissue tests).

[0158] The stage of pregnancy at which the methods described herein may be practiced will depend on a variety of clinical factors, including the subject's overall health and the severity of the symptoms of pre-eclampsia.

[0159] In certain embodiments, the method is performed on the subject by the end of the 12th week of gestation, which is intended to mean the last day of the 12th week of gestation, or the second, third or fourth last day of the 12th week of gestation.

[0160] In some embodiments, the method is performed on a subject at 9 weeks gestation. In one embodiment, the method is performed at 10 weeks gestation. In one embodiment, the method is performed at 11 weeks gestation. In an embodiment, the method is performed after 12 weeks gestation. In an embodiment, the method is performed at 13 weeks gestation. In an embodiment, the method is performed at 14 weeks gestation. In an embodiment, the method is performed between, including, 15 and 20 weeks gestation.

[0161] As used herein, "sample" is intended to mean 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, cerebrospinal fluid, preferably a blood, serum or plasma sample.

[0162] "Plasma" in the context of the present invention is the substantially cell-free supernatant of blood containing 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.

[0163] "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.

[0164] "Sample" also refers to a tissue biopsy (e.g., placental tissue), chorionic villus sample, cells, or other specimen obtained from a subject. Desirably, a biological sample contains an sFlt-1 nucleic acid molecule or polypeptide, or both.

[0165] 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 that does not have any pregnancy-related hypertensive disorder such as pre-eclampsia or eclampsia, a subject that is pregnant but the sample is taken early in pregnancy (e.g., first or second trimester or before detection of pregnancy-related hypertensive disorder such as pre-eclampsia or eclampsia), a subject that is pregnant and does not have a history of pregnancy-related hypertensive disorder such as pre-eclampsia or eclampsia, a subject that is not pregnant, a sample of a purified reference polypeptide with a known normal concentration (i.e., does not show pregnancy-related hypertensive disorder such as pre-eclampsia or eclampsia).

[0166] 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.

[0167] 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).

[0168] 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.

[0169] In one embodiment, the reference level and the level from a subject determined as used herein preferably refer to the measurement of the protein level of sFlt-1 or a fragment thereof in a blood sample, preferably a whole blood sample or a plasma or serum sample, obtained from a pregnant subject not suffering from pre-eclampsia, by the Thermo Scientific B·R·A·H·M·S KRYPTOR assay. Thus, the values ​​disclosed herein may vary to some extent depending on the detection / measurement method used, and the particular values ​​disclosed herein are also intended to read corresponding values ​​determined by other methods. In an embodiment of the invention, the reduction in the level of sFlt-1 or a fragment thereof compared to the reference level that may define a transition from low to high risk of developing PE may be any reduction in the range of 6% to 20% compared to the reference level. Any value within this range may be considered as a suitable reference level for high and low risk sFlt-1 levels. Furthermore, values ​​below such reference levels may indicate a high risk of pre-eclampsia, and values ​​above such reference levels may indicate a low risk of pre-eclampsia. 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.

[0170] As used herein, the term "positive reference" sample, standard or value is a sample or value or number derived from a subject who is known to have or has had a pregnancy-related hypertensive disorder, such as pre-eclampsia or eclampsia. The reference standard or level may also reflect the average or mean value of the levels of a nucleic acid, polypeptide or small molecule from a normal reference subject or a positive reference subject, depending on the context. The reference may also be a chart, graph or standard curve representing the normal reference levels of a polypeptide, nucleic acid or small molecule at any and / or all stages (e.g., every week) 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 pregnancy-related hypertensive disorder, and family history of pregnancy-related hypertensive disorder.

[0171] 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.

[0172] 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 fertilization plus 14 days.Gestational age can be determined by obstetric ultrasound examination.

[0173] As used herein, "maternal age" is intended to mean the age of the pregnant subject at the time of birth.

[0174] As used herein, the term "gestational hypertension" is intended to mean the development of hypertension without proteinuria after the 20th week of gestation.

[0175] 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.

[0176] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0177] The term "such as" is used herein to mean, and is used interchangeably with, the phrase "such as, but not limited to."

[0178] 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 biological father of the fetus is Rh positive. Risk parameters include, but are not limited to, hypothyroidism, hyperthyroidism, BMI greater than 24, first pregnancy, history of preeclampsia, ethnicity with 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.

[0179] 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 pre-eclampsia, delay the time of onset, or at least reduce the severity of pre-eclampsia, such as by balancing the angiogenic / anti-angiogenic processes in placental development, lowering blood pressure, protecting organ function such as the kidney or liver.

[0180] 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 pre-eclampsia, delay the time of onset, or at least reduce the severity of pre-eclampsia, such as balancing the angiogenic / anti-angiogenic processes in placental development, lowering blood pressure, protecting organ function such as 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, fish oil, etc.

[0181] 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.

[0182] As used herein, the term "chromosomal abnormality" is intended to mean differences in chromosomes that may occur during fetal development. They may be inherent to the fetus or inherited from the parents. Abnormalities are divided into two categories: numerical, which refers to a number of chromosomes different from that expected, such as monosomy or trisomy, and structural, which refers to translocations, deletions, duplications, the formation of rings as a result of the division of a part of a chromosome, and inversion of a chromosome. Chromosomal abnormalities include, but are not limited to, Down's syndrome, Turner's syndrome, Klinefelter's syndrome, trisomy 13, trisomy 14, triple X syndrome, XYY syndrome, fragile X syndrome, and Cri-Du-Chat syndrome.

[0183] The present invention further relates to kits, uses of the 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 for 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, and optionally additional reagents for determining the level of a further biomarker in a sample from a subject, as well as reference data such as reference levels corresponding to the sFlt-1 risk level and, optionally, the further biomarker level, said reference data being preferably stored on a computer readable medium and / or used in the form of a computer executable code configured for comparing the determined level of sFlt-1 or a fragment thereof and, optionally, the determined level of a further biomarker or a fragment thereof with said reference data.

[0184] In one embodiment of the methods described herein, the method further comprises comparing the determined level of sFlt-1 or a fragment thereof to a reference level, threshold, and / or population average 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.

[0185] The method of the present invention may 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 may be performed in a computer system. In the computer system, the determined level of the biomarker may be combined with other biomarker levels and / or clinical parameters of the subject to calculate a score that is indicative for prognosis, risk assessment, and / or risk stratification. For example, the determined values ​​may be entered into the computer system (either manually by a medical professional or automatically from a device where the respective marker level was determined). The computer system may be directly at the point-of-care (e.g., primary care, hospital or home environment) or may be at a remote location connected via a computer network (e.g., via the Internet or a specialized medical cloud system that can optionally be combined with other IT systems or platforms such as hospital information systems (HIS)). Typically, the computer system will store the values ​​(e.g., biomarker levels, or clinical parameters such as age, blood pressure, weight, sex, 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 prestored reference levels or values. The resulting score will be displayed and / or printed for the user (typically a medical professional such as a physician or patient). Alternatively or in addition, an associated prognosis, evaluation, treatment guideline, patient management guideline, or stratification will be displayed and / or printed for the user (typically a medical professional such as a physician or patient).

[0186] In one embodiment of the present invention, a software system in which machine learning algorithms exist can be used to identify patients at risk for PE, preferably using data from electronic health records (EHR). The machine learning approach can be trained with a random forest classifier using EHR data from the patient (lab results, biomarker expression, vitals, demographics, etc.). Machine learning is a type of artificial intelligence that gives computers the ability to learn complex patterns of data without being explicitly programmed, unlike simple rule-based systems. Previous studies have used electronic health record data to trigger alerts to 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 data sets, for example, sFlt-1 levels can be processed in machine learning software to aid in the prognosis of the occurrence of PE.

[0187] "PAPP-A" is pregnancy associated plasma protein A, papalysin-1, and refers to a plasma protein used as a screening test between the 8th and 14th weeks of gestation. Decreased levels of the protein indicate an increased risk of Down's syndrome, intrauterine growth retardation, pre-eclampsia and stillbirth.

[0188] "PlGF" is 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.

[0189] As used herein, reference data, e.g., reference levels corresponding to patient groups with maternal age up to 18, between 18-34, and above 34, and optionally additional markers as described herein, preferably PAPP-A and / or PIGF levels, preferably stored in a computer readable medium and / or used in the form of a computer executable code configured to compare the determined levels of sFlt-1 or a fragment thereof, and optionally, in addition, the determined levels of PIGF or a fragment thereof, and optionally, further, the determined levels of PAPP-A and / or PIGF or a fragment thereof, to the reference data. The "reference date" includes further reference levels corresponding to patient groups having blood type AB, having blood type Rh negative, having blood type Rh negative and the biological father of the fetus Rh positive, carrying at least one female fetus, carrying at least one male fetus, being nulliparous, having one or more previous pregnancies, and / or suspected of carrying a fetus with a chromosomal abnormality. The reference data may also include instructions on how to use the kit of the invention.

[0190] The kit may further include items useful for obtaining a sample, such as a blood sample, for example, the kit may include a container, where the container includes 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 pressure, 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, RNAse inhibitor protein, and mixtures thereof, and a filter system comprising nitrocellulose, silica matrix, ferromagnetic spheres, cup collection spillover, trehalose, fructose, lactose, mannose, polyethylene glycol, glycerol, EDTA, TRIS, limonene, xylene, benzoyl, phenol, mineral oil, aniline, pyrrole, citrate, and mixtures thereof. The invention is further illustrated by the drawings, which are not intended to limit the scope of the invention. [Brief description of the drawings]

[0191] [Figure 1] Box plots depicting levels of sFlt-1 before 90 days GA, between 90 and 100 days GA, or between 140 and 154 days GA, comparing levels in subjects with or without EO-PE. The study included 11,952 women recruited in the first trimester, 11,918 without PE, and 34 with 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 above the median. On average, between 140 and 154 days gestation, all women with EO-PE (N=4) had sFlt-1 levels above the median. [Diagram 2] Statistical evaluation of sFlt-1 levels in subjects whose samples were obtained in trimester 1. The study included 11,952 women recruited in trimester 1, 11,918 without PE and 34 with early-onset PE. As can be observed, sFlt-1 in trimester 1 is not associated with EO-PE (EO-PE: 0.98 MoM vs. control: 1.00 MoM, p=0.09). [Diagram 3] Statistical evaluation of sFlt-1 levels in subjects whose samples were obtained after 12 6 / 7 weeks GA. The study included 7409 women recruited after 90 days gestation, 7385 without PE and 24 with early-onset PE. As can be observed, sFlt-1 after 12 6 / 7 weeks is not associated with EO-PE (EO-PE: 1.00 MoM vs. control: 1.00 MoM, p=0.40). [Figure 4] 12 ROC curve of the data presented in FIG. 3 showing that sFlt-1 after 6 / 7 weeks is not associated with EO-PE. [Diagram 5]Statistical evaluation of sFlt-1 levels in subjects whose samples were obtained before 12 6 / 7 weeks GA (90 days GA). The study included 4543 women recruited before 90 days gestation, 4533 without PE and 10 with early-onset PE. As can be observed, sFlt-1 before 12 6 / 7 weeks is inversely correlated with EO-PE (EO-PE: 0.94MoM vs. control: 1.00MoM, p=0.003). [Figure 6] ROC curve for data presented in Figure 5 showing that sFlt-1 before 6 / 7 weeks correlates with EO-PE. Measurement of sFlt-1 (adjusted for gestational age-MoM) and sFlt-1 (not adjusted for gestational age-birth) 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] 1 is a ROC curve of data for sFlt-1 and PlGF levels in subjects whose samples were obtained after 12 6 / 7 weeks GA (after 90 days GA). After 90 days of gestation, PlGF, but not sFlt-1, is a strong marker of EO-PE. [Figure 8] 3 is a ROC curve 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] 1 is a ROC curve for data for sFlt-1 and PlGF levels in subjects whose samples were obtained 12 6 / 7 weeks prior to GA (90 days prior to GA). Additionally, the ROC curve is shown using combined data for both sFlt-1 and PlGF levels. The ROC curve shows AUC values ​​for PlGF: 0.70 (95% CI: 0.56-0.85), sFlt-1: 0.74 (95% CI: 0.64-0.84), and the combination of both markers: 0.85 (95% CI: 0.78-0.92). [Figure 10]1 is a ROC curve for data on sFlt-1 and PlGF levels in subjects whose samples were obtained 12 6 / 7 weeks pre-GA (90 days pre-GA). Additionally, data from uterine artery Doppler measurements obtained in this study were combined in the analysis. The ROC curve is shown using the combined data for both sFlt-1 and PlGF levels, and in addition, these combined levels are further combined with the Doppler data. The ROC curve shows an AUC value of 0.87 (95% CI: 0.80-0.94) for the Doppler combination. [Figure 11] 1 is a ROC curve of data for sFlt-1 and PlGF levels in subjects whose samples were obtained between 90-100 days GA. The combination of sFlt-1 and PlGF predicts some cases of IUFD between 90-100 days. [Figure 12] Receiver operating characteristic curves 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 to between 90-100 days gestation (AUC: 0.72 95%, CI: 0.60-0.84). [Figure 13] 1 is a box plot and ROC curve showing the correlation between sFlt-1 levels (measured prior to 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 prior to 90-day GA. EXAMPLES

[0192] Example 1: Study design: Pregnant women were recruited at 11-14 weeks gestation and followed until delivery. sFlt-1 was measured at recruitment using a Thermo Scientific BRAHMS KRYPTOR and reported as multiple of the median (MoM) adjusted for gestational age. Median levels of sFlt-1 were compared among women who developed early-onset PE (<34 weeks), mid-onset PE (34-36 weeks), late-onset PE (≥37 weeks), 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.

[0193] result: We included 12,383 participants who delivered after 16 weeks of gestation, 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-onset or mid-onset PE (p=0.02), but more importantly, the difference was observed to be present primarily in participants recruited early in pregnancy.

[0194] Significant differences in predictive value of early-onset PE were observed between women recruited at 11th (AUC: 0.82, 95% CI: 0.72-0.92; p<0.001), 12th (AUC: 0.62, 95% CI: 0.49-0.74; p=0.06) and 13th gestational weeks (AUC: 0.50, 95% CI: 0.32-0.67; p=0.97). A similar trend was observed for predicting mid-onset PE.

[0195] Conclusion: First trimester maternal sFlt-1 is decreased in women who develop PE before term (<37 weeks). Its predictive value is significantly improved when collected at or before 12 weeks of gestation, and this peculiarity may explain the conflicting results between previous studies.

[0196] Example 2: Study design: We conducted a secondary analysis of a prospective cohort study of nulliparous women recruited between 11 and 14 weeks' gestation. Maternal characteristics, mean arterial blood pressure, maternal serum biomarkers (pregnancy associated plasma protein-A, placental growth factor, two risk cut-offs (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 to a reference group that did not develop early or term PE. Detection rates, false positive rates, and positive and negative predictive values ​​were calculated and estimated for term and early PE as placental mediated complications. Women who reported taking aspirin daily were excluded. PAPP-A, PIGF, sFlt-1 concentrations were measured by using the Thermo Scientific BRAHMS KRYPTOR automated assay. Early PE was defined as PE delivered before 37 weeks of gestation, and premature PE refers to cases delivered before 34 weeks of gestation. Analyses were performed using the SAS statistical software package (version 9.3, SAS Institute All analyses were performed using the National Institute of Standards and Technology (NIST, Cary, NC). A type I error margin of error of 5% was considered in all analyses.

[0197] result: We included 4575 participants with complete observations. Twenty-nine patients developed premature PE, whereas 194 women developed term PE and 3705 women did not develop placenta-mediated complications (reference group). The median sFlt-1 value in the reference group was 1023 pg / mL and Q1-Q3 values ​​ranged from 771 to 1373 pg / mL. The median sFlt-1 value and Q1-Q3 values ​​for women with term PE were 933 pg / mL (726 to 1221 pg / mL). Thus, pregnant women who developed term PE showed a median decrease in sFlt-1 levels of 8.8% and a median fold change (FC) of 0.91.

[0198] The median sFlt-1 value and Q1-Q3 values ​​in women developing early-onset PE were 852 pg / mL (range 658-1095 pg / mL). Thus, pregnant women who developed early-onset PE showed a higher sFlt-1 decline compared to the reference group as the term PE group with a median sFlt-1 decline of 16.7% between gestational weeks 11-14 and a median fold change (FC) of 0.83.

[0199] Q1 of sFlt-1 levels in early-onset PE compared to the reference group showed an FC of 0,85 with a reduction of 14.7%.

[0200] Q1 of sFlt-1 levels in end-stage PE compared to the reference group showed an FC of 0,94 with a decrease of 5.8%.

[0201] Q3 of sFlt-1 levels in early-onset PE compared with the reference group showed an FC of 0.8 with a 20% reduction.

[0202] Q3 of sFlt-1 levels in end-stage PE compared to the reference group showed an FC of 0,89 with a reduction of 11%.

[0203] Conclusion: Nulliparous women with a median sFlt-1 decline of at least 8.8% detected between 11 and 14 weeks of gestation 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-onset PE.

[0204] 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 in the world. Early onset preeclampsia requires delivery before the 34th week of gestation and is associated with significant perinatal morbidity.

[0205] Online software (Fetal Medicine Foundation) is now available that combines biophysical (age, BMI, BP, medical history), ultrasound (uterine artery Doppler) and biochemical (PlGF and PAPP-A) factors measured during the first trimester of pregnancy and suggests that more than 90% of early preeclampsia can be predicted with a false positive rate of less than 10%.

[0206] To explore the correlation between biomarkers such as sFlt-1, PAPP-A or PlGF and pre-eclampsia, additional patient groups such as patients under 18 years of age, having some rare blood types such as AB, Rh negative, having or having multiple pregnancies, having one or more previous pregnancies, having dysplasia or polydysplasia syndrome, and / or suspected of or having a fetus with chromosomal abnormality (high nuchal translucency) will be further included and investigated in this discovery study.

[0207] the purpose: 1) To validate the FMF screening tool during weeks 11-13 of pregnancy for early preeclampsia and other placenta-related pregnancy disorders (early onset 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 volumes, placental vascularity) for predicting preeclampsia.

[0208] Methodology: Prospective observational study. Nulliparous women with one pregnancy and a fetus without lethal anomalies were recruited between the 11th 3 / 7th and 13th 6 / 7th weeks of gestation. A lifestyle questionnaire was completed. BMI and BP measurements and blood samples (20 mL) were taken. Doppler ultrasound of the uterine arteries and 3D evaluation of the placenta completed the clinic visit. Follow-up of medical records was performed approximately 1 month after expected delivery date.

[0209] Serum was analyzed within 10 days of recruitment for PAPP-A, PIGF, sFIt1, fbHCG and AFP using the Thermo Scientific B·R·A·H·M·S KRYPTOR automated assay. The remaining serum was stored at -80°C for further analysis (PP-13, ADAM-12, vitamin D) at the end of the project to evaluate the possibility of improving the predictive model with promising markers including placental volume and vascularity assessed by 3D ultrasound. For each participant, calculation of the risk of early pre-eclampsia and overall pre-eclampsia was calculated using FMF software, but was disclosed to the participant. The optimal sensitivity and specificity of the tool will be evaluated using ROC curves.

[0210] We estimated a 0.7% incidence of early-onset preeclampsia (<34 weeks) 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.

[0211] the purpose Main purpose To validate the FMF early preeclampsia screening tool during weeks 11-13 of pregnancy.

[0212] 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 could be used in different predictive models.Evaluate the predictive value of each biomarker individually and assess whether they could be used in different predictive models. 4) To evaluate the predictive value of potential biomarkers for early onset preeclampsia (sFlt-1, PP13, ADAM-12, 25-OH-vitamin D, placental volume and placental / subplacental vascularity, sFlt-1). 5) To evaluate the predictive value of first trimester cervical measurements for preterm delivery.

[0213] 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 up until delivery to validate the presence of our primary and secondary outcomes.

[0214] II. Population and Selection Criteria Study set of inclusion criteria: Females with a live singleton pregnancy between 3 / 7 weeks 11 and 6 / 7 weeks 13. Nulliparous women (no previous pregnancy for any reason up to 20 / 7 weeks of gestation)

[0215] Study set of exclusion criteria: Girls under 18 Women unable to give informed consent (e.g., do not understand English or French). Women who planned to give birth outside the participating centres (excluding women who were planning to give birth at the Hotel-Dieu de Levis and who were eligible for the project because they agreed via a consent form to have their file consulted at this institution). Women who are HIV positive or Hepatitis C positive or Chronic Hepatitis B positive (not cured)

[0216] Further inclusion criteria for the study set: Multiple pregnancies (women with two fetuses, one of which has stopped growing, are not eligible) Presence of fetal malformation syndrome or polymorphism syndrome The presence of a nuchal translucency measurement greater than 3.5mm, which is associated with a very high risk of chromosomal and / or cardiac anomalies and may affect serum PAPP-A levels. Negative fetal heart on the day of recruitment visit

[0217] III. Conducting the Research 11 3 / 7 week~13 6 / 7 week Visit to the nurse (Week 3 / 7 of 11 to Week 6 / 7 of 13) First, the research nurse collected blood specimens by venipuncture (2 × 5 mL tubes, BD Vacutainer SST). The tubes were gently inverted 5 times to mix the reagents well with the blood. The tubes were left in an upright position in a dark box until treatment (minimum 30 min, maximum 2 h). In a second step, the research nurse measured the patient's blood pressure. The patient had to sit and rest with uncrossed legs for 5 min without speaking before the measurement. Blood pressure was measured in triplicate in both arms (sleeveless vest) simultaneously by a preprogrammed Microlife electronic blood pressure monitor (model 33603).

[0218] A questionnaire was administered to the patients (see attached questionnaire) to know their medical and obstetric family history, as well as their socio-economic background including date of birth, anthropometric measurements, tagabism etc. The total encounter with the nurse lasted a maximum of 30 minutes.

[0219] Visit for technicians (11 / 3 / 7~13 / 6 / 7) Following the meeting with the nurse, all patients were interviewed by a radiology technician from the study 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, i.e., "Angiomode" = 100, "Smooth" = 4 / 5, FRQ = low, quality = normal, density = 6, "Enhancement" = 16, balance = 175, WMF = low 1, "Real power" = 2 dB, "Pulse repetition frequency" = 0.6 kHz, gain color = -7.2 dB.

[0220] Ultrasound was performed to confirm the project 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 was 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 lucency ≥ 3.5 mm and / or negative fetal heart, the patient was informed and potentially 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.

[0221] Technicians completed the eligibility sheet accordingly. Participants who met at least one of the exclusion criteria were treated as any other participant, i.e., 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.

[0222] The visit window between the 11th 3 / 7th and 13th 6 / 7th weeks of gestation is important and was respected to guarantee the validity of certain data (biochemistry and ultrasound). Therefore, when the ultrasound date test confirms gestational age, it is as follows:

[0223] a)<11 3 / 7 Weeks (LCC<45mm): The visit was rescheduled to a day between 11 3 / 7 weeks and 13 6 / 7 weeks. Blood sampling and ultrasound were repeated at this time. Both samples were kept, but only the second sample was used for the primary analysis. If a minimum number (n>5) of these patients showed early pre-eclampsia, the first sample was analyzed and compared to the second sample in a case-control study.

[0224] Participants' wishes to return for a repeat visit were respected. If participants declined a second visit, the data collected were retained but excluded from the primary analysis. b) >14 weeks (CCL>84mm): 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.

[0225] Next, the following ultrasonic measurements were 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 cartilage 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 were evaluated on a sample of about 1000 cases to evaluate the reproducibility, duration and efficiency of the two techniques. The presence and absence of a notch was recorded bilaterally (a notch is considered to be present if an early diastolic incision occurs in each cycle). 5) 3D ultrasound of the placenta and subplacental area with and without Doppler. This examination takes approximately 30 seconds.

[0226] At the end of recruitment, for 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 thickness). These volumetric acquisitions 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-myometrial region are assessed using VOCAL software. VFI represents the number of stained voxels in the studied volume (expressed as a percentage). FI is the average color value of all stained voxels representing the mean intensity of blood flow (expressed as an absolute value between 0 and 100). VIF is the average color value of all voxels in the studied region (gray and colored, expressed as an absolute value between 0 and 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 according to the prescription of the attending physician. 7) An ultrasound acquisition of the abdominal region was also performed to inductively measure the thickness of the 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 only a few seconds.

[0227] 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 thanks for their participation.

[0228] Monitoring during pregnancy (34 0 / 7 weeks to 35 6 / 7 weeks) The survey (in electronic form and emailed to participants) was administered to participants during their 34th trimester of pregnancy. eA follow-up survey was conducted weekly to verify whether the patient's medications had changed during pregnancy and to ascertain whether the pregnancy was progressing normally, had moved, or had had any complications to date. If the survey was not completed, an email reminder was sent automatically one week later. Those who had not responded to the survey were then contacted by phone.

[0229] Postpartum follow-up (6 weeks after DPA) A second electronic survey (sent by 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. In particular, we wanted to know 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 after 1 week. Those who did not respond to the survey were then contacted by phone. Procedures were taken 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 if not necessary, to avoid inconvenience.

[0230] Follow-up at the end of pregnancy (1 month after DPA) Data on pregnancy (e.g. gestational hypertension, pre-eclampsia, perinatal death), delivery (e.g. gestational age) and neonates (e.g. sex, birth weight) were collected from the patients' medical records via CristalNet software by the study nurses after delivery. In the rare event that a patient delivered at a non-participating centre (not CHUL, HSFA or Levis), the patient was contacted and permission to obtain this information at the place of delivery was requested. A second review of the data was performed by physicians (EB, KG, FA or their representatives) directly from the medical records for all cases of suspected pre-eclampsia and cases where delivery occurred before 37 weeks (<10%, i.e. <532 records retrieved from the CHU-Q archive and <228 records retrieved from the CHUSJ archive).

[0231] Blood samples 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.

[0232] 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 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 the primary analysis (serum PAPP-A, PlGF and sFlt-1 assays), the remaining serum was stored frozen at −80°C and used to measure PP13, ADAM12 and vitamin D in a case-cohort substudy performed at the end of the main study.

[0233] Case-cohort study A case-cohort study nested within the main cohort evaluated the predictive effect of potential biomarkers on the prediction of early pre-eclampsia. Maternal serum sFlt-1, ADAM-12, PP-13 and vitamin D were measured using commercial kits in a randomly selected subgroup of women (approximately 236 women) and in all cases of early pre-eclampsia (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 blood flow index (VFI).

[0234] IV. Judgment criteria Primary endpoint: Preeclampsia requiring delivery before 34 weeks of gestation based on gestational age as determined by date of last menstrual period (DMD) or 11-13 weeks ultrasound (if the latter showed a difference of ≥ 5 days by 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, combined with thrombocytopenia <100, AST and / or ALT >2x normal, diastolic blood pressure ≥110mmHg, and / or proteinuria (≥2+ on stick or >300mg / 24hr).

[0235] Secondary endpoints: Delivery < 37 weeks (spontaneous delivery or PPROM before the 37th week of pregnancy) Delivery < 34 weeks (spontaneous delivery or PPROM before the 34th week of pregnancy)

[0236] Pre-eclampsia: Preterm preeclampsia < 37 weeks Severe preeclampsia (having any of the following conditions: 1) systolic ≥ 160mmHg and diastolic ≥ 110mmHg after 4 hours of rest, 2) proteinuria ≥ 5g / 24 hours or ≥ 3+ on rod, 3) oliguria ≤ 400mL / 24 hours, visual or encephalopathy, epigastric pain, pulmonary edema or cyanosis, thrombocytopenia < 100,000mm3. Perinatal deaths (before birth, up to 7 days after birth) Growth retardation based on Canadian norms (<10 e percentile) Severe syncope (<3 e percentile) Birth weight < 2500 grams Birth weight < 1500 grams average birth weight

[0237] 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.

[0238] 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.

[0239] 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.

[0240] 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 90-100 days of gestation, on average, women with EO-PE (N=24) had sFlt-1 levels above the median. Between 140-154 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), progressively increase to normal at the end of the first trimester (90-100 days), and then abnormally increase thereafter (after 140 days of gestation).

[0241] 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 of GA (Figures 3, 4).

[0242] Surprisingly, measurement of sFlt-1 within 12 weeks or 90 days of gestation shows a significant correlation with EO-PE, especially an inverse correlation with EO-PE (Figures 5, 6). Thus, determining sFlt-1 within 90 days of GA allows for reliable prognosis of EA-PE at an early time point.

[0243] The combination of sFlt-1 and PlGF results in a statistical improvement in the prognosis of EO-PE when samples are obtained early in pregnancy, for example, before the end of the 12th 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 a reliable prognostic statement from a single measurement after 90 days GA (Figure 7). Surprisingly, both sFlt-1 and PlGF allow for EO-PE prognosis when measured before the end of 12 weeks (within 90 days) GA. However, sFlt-1 appears to provide greater sensitivity with comparable specificity values, preferably values ​​above 0.6 (Figure 8). Also surprising is that the combined analysis of sFlt-1 and PlGF shows an unexpected synergistic enhancement in EO-PE prognosis when measured before 90 days GA (Figure 9).

[0244] It was also found throughout this study that the combination of uterine artery Doppler measurements, preferably in combination with sFlt-1, PlGF, maternal age and BMI, showed improved predictive power of EO-PE in subjects compared to sFlt-1 or PlGF alone (Figure 10).

[0245] The combination of sFlt-1 and PlGF also provided prognosis for IUFD when samples were obtained between 90 and 11 days GA (Figure 11) and shows a statistical improvement in prognosis for IUFD when samples were obtained before 90 days GA (Figure 12).

[0246] Moreover, comparisons on data sets obtained using the FMF algorithm and sFlt-1 analysis described herein reveal that the two prognostic procedures show strong correlation with each other. As can be seen from FIG. 13, patients with high risk as determined by the FMF algorithm have significantly lower sFlt-1 levels prior to 90 days GA. 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 that allows for comparable risk assessments, thereby potentially avoiding the more complex FMF approach. Analysis of sFlt-1 prior to 90 days may in fact allow prediction of pregnant women who will have a positive FMF test without the need for Doppler uterine artery evaluation.

[0247] In conclusion, this study allowed us to conclude that in the majority of pregnant women who develop early-onset preeclampsia, sFlt-1 decreases before 90 days of gestation, but then increases and becomes abnormally high in the second trimester of pregnancy in the same pregnant women. Thus, sFlt-1 before 90 days of gestation allows to predict early-onset 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 is able to predict about 35% UFDIs for a 10% false positive rate.

[0248] The importance of this information is important because the earlier aspirin or similar therapy is initiated in the first trimester 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 early in pregnancy, before 90 days GA, and subsequent initiation and guidance of treatment.

Claims

1. 1. 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, in a sample isolated from the pregnant subject; b. the sample is isolated from the subject before the end of the 12th week of gestation; c) A method wherein the level of sFlt-1 or a fragment thereof is indicative of the likelihood of early onset pre-eclampsia occurring before the end of the 33rd week of gestation.

2. Furthermore, d. determining the level of Placenta Growth Factor (PlGF) or a fragment thereof in said sample isolated from said subject; e. The method of claim 1, wherein a combination of the level of sFlt-1 or a fragment thereof and the level of PlGF or a fragment thereof is indicative of the likelihood that early onset pre-eclampsia will occur before the end of the 33rd week of gestation.

3. 3. The method of claim 1 or 2, wherein the subject is in the 9th to 11th week of gestation.

4. 4. The method of claim 3, wherein the subject is in the 11th week of gestation.

5. Furthermore, a. determining or providing the subject's maternal age, body mass index, and / or uterine artery Doppler measurement; b. The method of any one of claims 1 to 4, wherein the combination of levels of sFlt-1 or a fragment thereof, preferably in combination with levels of PlGF or a fragment thereof, with the subject's maternal age, body mass index and / or uterine artery Doppler measurements is indicative of early onset pre-eclampsia occurring before the end of the 33rd week of gestation.

6. Furthermore, a. determining or providing a level of mean arterial pressure (MAP) of the subject; b. The method of any one of claims 1 to 5, wherein the combination of levels of sFlt-1 or a fragment thereof, preferably in combination with levels of PlGF or a fragment thereof, with levels of MAP in the subject is indicative of early onset pre-eclampsia occurring before the end of the 33rd week of gestation.

7. a. determining a level of sFlt-1 or a fragment thereof, and determining a level of PIGF or a fragment thereof in a sample isolated from the subject; b. determining or providing the subject's maternal age, body mass index (BMI) and uterine artery Doppler measurement, and optionally, mean arterial pressure (MAP); c) The method of any one of claims 1-6, wherein a combination of the level of sFlt-1 or a fragment thereof, the level of PlGF or a fragment thereof, and 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 that early onset pre-eclampsia will occur before the end of the 33rd week of gestation.

8. a. determining a level of sFlt-1 or a fragment thereof, and determining a level of PIGF or a fragment thereof in a 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; 8. The method of any one of claims 1 to 7, wherein the level of sFlt-1 or a fragment thereof, the level of PlGF or a fragment thereof, in combination with the subject's maternal age, body mass index (BMI) and mean arterial pressure (MAP), and optionally, a uterine artery Doppler measurement, is indicative of the likelihood that early onset pre-eclampsia will occur before the end of the 33rd week of gestation.

9. The method of any one of claims 1 to 8, wherein the level of sFlt-1 or a fragment thereof determined in the sample is compared to a reference level, preferably a population mean and / or median for a healthy population, and a level of sFlt-1 or a fragment thereof below the reference level indicates an increased risk of early onset pre-eclampsia.

10. The method according to any one of claims 2 to 8, wherein the level of PlGF or a fragment thereof determined in the sample is compared with a reference level, preferably a population mean and / or median for a healthy population, and a level of PlGF or a fragment thereof above the reference level indicates an increased risk of early onset pre-eclampsia.

11. 11. The method of any one of claims 1 to 10, wherein the level of sFlt-1 or a fragment thereof, preferably the level of sFlt-1 or a fragment thereof, the level of 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.

12. The method of any one of claims 1 to 11, wherein the level of sFlt-1 or a fragment thereof, preferably the level of sFlt-1 or a fragment thereof, the level of 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 subsequent occurrence of intrauterine fetal death (IUFD).

13. 13. The method of any one of claims 1 to 12, wherein the maternal age is between 18 and 34, or greater than 34.

14. The method according to any one of claims 1 to 13, wherein the sample is a body fluid sample, such as a blood sample, such as a venous blood sample, a capillary blood sample, a serum sample, a plasma sample, a vaginal fluid sample, a saliva sample or an amniotic fluid sample, preferably a blood, serum or plasma sample.

15. 15. The method of any one of claims 1 to 14, wherein the level of sFlt-1 or a fragment thereof, and optionally the level of PlGF or a fragment thereof, maternal age, body mass index (BMI), uterine artery Doppler measurements, and / or mean arterial pressure (MAP) in the subject indicate to initiate or modify treatment of the subject, e.g., to reduce the risk of developing pre-eclampsia, delay the time of onset, and / or reduce the severity of pre-eclampsia, and / or protect organ function, such as the kidney and / or liver, e.g., by balancing angiogenic / anti-angiogenic processes in placental development, reducing blood pressure.

16. 16. The method according to any one of claims 1 to 15, wherein 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, ambulatory monitoring, increased frequency of maternal and fetal monitoring, preferably low dose acetylsalicylic acid.

17. 15. The method of claim 14, wherein the treatment comprises administration of acetylsalicylic acid.

18. The method of any one of claims 1 to 17, wherein the subject is nulliparous.

19. The method of any one of claims 1 to 18, wherein the subject has had one or more previous pregnancies.

20. The method of any one of claims 1 to 19, wherein the subject has a multiple pregnancy.

21. The method of any one of claims 1 to 20, wherein the subject is suspected of carrying a fetus having a chromosomal abnormality.

22. 22. The method of any one of claims 1 to 21, wherein the subject has one or more risk factors selected from the group consisting of hypothyroidism, hyperthyroidism, BMI greater than 24, primigravidity, history of pre-eclampsia, ethnicity with a risk disorder, multiple pregnancy, migraine headaches, lupus, blood clotting disorders such as increased clotting, inflammatory disease, impaired cardiac reserve, diabetes, chronic kidney disease, and chronic hypertension.

23. The method further comprises determining a level of at least one additional biomarker or fragment thereof in a sample from the patient, the at least one additional biomarker being βhCG, copeptin, vasopressin, troponin, BNP, ANP, CRP, thrombocytes / leukocytes, IL6, IL11, MR-proADM, VEGF, PAPP-A, PIGF, endoglin, pro-Epil, PP-13, A DAM-12, vitamin D, inhibin-a, activin-a, pentraxin-3, p-selectin, free fetal hemoglobin, α-1-microglobulin, unconjugated estriol, α-fetoprotein, GDF15, neurophysin II, LNPEP, ESM1, HGF, pikachurin, hemopexin, pp13, uE3, CT-proET1, ADAM12, sTNFαR1, RBP4, IC 23. The method of any one of claims 1 to 22, wherein the level of the at least one additional biomarker and the level of sFlt-1 or a fragment thereof are indicative of early onset pre-eclampsia before the end of the 33rd week of gestation.

24. A kit for carrying out the method according to any one of claims 1 to 23, comprising: - detection reagents for determining the level of sFlt-1 or a fragment thereof and for determining the level of PIGF or a fragment thereof in a sample from a subject; - a computer executable code, i. comparing the determined level of sFlt-1 or a fragment thereof and the determined level of PlGF or a fragment thereof to one or more reference levels, preferably corresponding to a population mean and / or median for a healthy population; ii. a computer readable medium in the form of computer executable code and / or computer software configured to: compare the subject's maternal age, body mass index, MAP and / or uterine artery Doppler measurements to one or more reference levels, preferably corresponding to population means and / or medians for a healthy population.

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