Methods for predicting and monitoring spontaneous preterm birth - Patents.com

Abstract

This is a prospective multicenter cohort study including 200 pregnant patients, with 5 serum samplings per patient. We investigated protein biomarker concentrations in the plasma of high-risk pregnant women in the second and third trimesters of pregnancy to predict spontaneous preterm birth. We showed that PROK1 (Prokineticin 1), also called EG-VEGF (endocrinoline-derived vascular endothelial growth factor), secreted by the placenta, was elevated in serum in sPTB patients less than 37 weeks pregnant compared to uncomplicated pregnant women. More precisely, women with spontaneous preterm birth showed higher serum PROK1 / EG-VEGF concentrations at 20, 24, 28 and 32 weeks than uncomplicated patients. Thus, serum PROK1 / EG-VEGF concentrations can be considered as biomarkers of spontaneous preterm birth in high-risk pregnant women.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a method and a kit for predicting and monitoring spontaneous preterm birth. More specifically, the present invention relates to a method for the prevention of spontaneous preterm birth by detecting a specific serum protein biomarker in patients (PROK1 / Prokineticin 1), also called EG-VEGF (Endocrine-derived Vascular Endothelial Growth Factor).

[0002] 2. Background of the Invention Preterm birth is defined by the Women's Health Organization as any birth occurring before 37 weeks of gestation (1). Preterm births were responsible for 10.6% of births worldwide in 2014 (2). Spontaneous preterm birth (sPTB) is due to spontaneous vaginal delivery and represents 40-45% of preterm births, while premature rupture of membranes (PROM) represents 25-30% of preterm births (3). To date, sPTB is the leading cause of neonatal mortality and morbidity (4, 5). Risk factors for sPTB include a history of preterm birth, black race, periodontal disease, and low maternal body mass index (3). Predicting spontaneous preterm birth (sPTB) in asymptomatic women remains a major challenge for public health systems. Short cervical length and elevated cervical-vaginal fetal fibronectin concentrations are predictors of spontaneous preterm birth, but their performance remains very poor (6). Effective and safe screening tools are not yet available in clinical practice, such as the use of amniocentesis-based predictive risk models, which are still in clinical development. (7, 8) Early identification of women who may exhibit sPTB would allow decisions regarding intensified patient monitoring, prescription of tocolytic or vaginal progestational agents, placement of a pessary and / or cervical cerclage, and antenatal corticosteroid therapy.

[0003] Prokineticins (PROKs) are secreted peptides that have the ability to regulate both angiogenesis and inflammatory processes in humans and other species (9-11). The PROK family consists of two members, PROK1 and PROK2 (9). PROK1, the canonical member of this family, is also known as endocrine gland-derived vascular endothelial growth factor (EG-VEGF). EG-VEGF and PROK2 act through specific G protein-coupled receptors, PROK receptor 1 (PROKR1) and PROK receptor 2 (PROKR2), to regulate multiple biological functions such as angiogenesis, circadian rhythm, olfactory bulb neurogenesis, neuronal survival, reproduction, and inflammation (11). EG-VEGF and PROKR1 are highly expressed in the first and term placenta and immunolocalized to different cell types, including syncytiotrophoblasts, cytotrophoblasts, fetal endothelial cells, and macrophages (11). Several studies have reported that EG-VEGF and its receptors are directly involved in the pathogenesis of recurrent pregnancy loss (RPL), gestational trophoblastic disease (GTD), and placenta-mediated complications (PMC).(12) These data strongly suggest that elevated EG-VEGF levels contribute to the development of PMC or rather participate in an overall compensatory mechanism that occurs to allow pregnancy to progress.

[0004] In the context of chorioamnionitis and parturition, reports from our group and from Jabbour's group strongly suggest that dysregulation of the expression levels of members of the prokineticin family, including their receptors, may be related to the pathogenesis of this condition (13-16). However, no prospective studies have been performed to date in women with high-risk pregnancies to determine EG-VEGF levels from the early second trimester with a view to exploring their usefulness as a new biomarker of sPTB.

[0005] Summary of the Invention A first object of the present invention relates to an in vitro method for assessing the risk of a subject to have or develop spontaneous preterm birth at an early stage, comprising the steps of: i) determining the level of the protein PROK1 marker in a blood sample obtained from the subject; ii) comparing the level determined in step i) with a reference value; and iii) concluding that if the level of the PROK1 marker determined in step i) is higher than the reference value, it predicts an increased risk of having or developing severe or spontaneous preterm birth.

[0006] A further object of the present invention relates to an in vitro method for monitoring the risk of a subject having or developing spontaneous preterm birth, comprising the steps of: i) determining the level of the protein PROK1 marker in a blood sample obtained from the subject at a first specific time of the disease; ii) determining the level of the protein PROK1 marker in a blood sample obtained from the subject at a second specific time of the disease; iii) comparing the level determined in step i) with the level determined in step ii) and iv) concluding that the risk of having or developing spontaneous preterm birth has worsened if the level determined in step ii) is higher than the level determined in step i).

[0007] A further object of the present invention relates to an in vitro method for monitoring a treatment of spontaneous preterm birth comprising the steps of: i) determining the level of the protein PROK1 marker in a blood sample obtained from the subject before the treatment, ii) determining the level of the protein PROK1 marker in a blood sample obtained from the subject after the treatment, iii) comparing the level determined in step i) with the level determined in step ii) and iv) concluding that the treatment is efficient if the level determined in step ii) is lower than the level determined in step i).

[0008] Detailed Description of the Invention In this study, using a prospective multicenter cohort study including 200 pregnant women with 5 serum samples per woman, we investigated protein biomarker concentrations in the plasma of high-risk pregnant women in the second and third trimesters of pregnancy to predict spontaneous preterm birth. We demonstrated that PROK1 (prokineticin 1), also called EG-VEGF (endocrinoline-derived vascular endothelial growth factor), is secreted by the placenta and has elevated serum levels in sPTB patients less than 37 weeks of gestation compared to uncomplicated pregnant women. More precisely, women with spontaneous preterm birth had higher serum PROK1 / EG-VEGF concentrations at 20, 24, 28, and 32 weeks than uncomplicated patients. Finally, serum PROK1 / EG-VEGF concentrations may be biomarkers of spontaneous preterm birth in asymptomatic pregnant women long before the onset of sPTB.

[0009] Prediction method for inventions The present invention relates to an in vitro method for assessing the risk of a subject having or developing spontaneous preterm birth, comprising the steps of: i) determining the level of the protein PROK1 marker in a blood sample obtained from the subject; ii) comparing the level determined in step i) with a reference value; and iii) concluding that if the level of the protein PROK1 marker determined in step i) is higher than the reference value, then an increased risk of having or developing spontaneous preterm birth is predicted.

[0010] In another aspect, the present invention relates to an in vitro method for predicting having or developing spontaneous preterm birth in a subject, comprising the steps of: i) determining the level of the protein PROK1 marker in a blood sample obtained from the subject; ii) comparing the level determined in step i) with a reference value; and iii) concluding that a subject is predicted to have or develop severe or spontaneous preterm birth if the level of the protein PROK1 marker determined in step i) is higher than the reference value.

[0011] The term "prediction" is the medical term for predicting the likelihood or expected onset of a disease. Predictive scoring is also used to predict disease outcomes.

[0012] In the context of the present invention, high levels of PROK1 / EG-VEGF may constitute both a predictive marker and a risk factor for developing spontaneous preterm birth.

[0013] The term "subject" as used herein refers to a mammal, such as a rodent (e.g., a mouse or rat), a feline, a canine, or a primate. In a preferred embodiment, the subject is a human subject. The subject of the present invention may be a healthy subject or a subject suffering from a particular disease.

[0014] In certain embodiments, the subjects of the present invention do not suffer from and / or have not been previously diagnosed with pre-eclampsia.

[0015] The term "preterm birth" is commonly defined by the Women's Health Organization as birth occurring before 37 weeks of gestation. Preterm birth affected 10.6% of births worldwide in 2014.

[0016] "Spontaneous preterm birth" (or "sPTB"), resulting from the natural onset of labor, accounts for 40-45% of all preterm births, while "premature rupture of membranes" (or "PROM") accounts for 25-30%. To date, sPTB is the number one cause of neonatal mortality and morbidity, and predicting sPTB in asymptomatic women remains a major challenge for clinicians. Effective and safe screening tools for sPTB are not yet available in clinical practice. However, early identification of women at high risk for sPTB could allow for enhanced monitoring during pregnancy, including prescription of tocolytic or vaginal progesterone medications, placement of a pessary and / or cervical cerclage, and antenatal corticosteroid therapy.

[0017] As used herein, the term "sample" or "biological sample" refers to any biological sample from a subject, and may include, by way of example and not limitation, body fluids and / or tissue extracts, such as homogenates or lysed tissues, obtained from a subject. Tissue extracts are routinely obtained from tissue biopsies. In certain embodiments of the method of predicting the prognosis of spontaneous preterm birth according to the present invention, the biological sample is a body fluid sample (such as blood) or tissue biopsy (placenta) from the subject.

[0018] In a preferred embodiment, the liquid sample is a blood sample. The term "blood sample" refers to a whole blood sample obtained from a subject (e.g., an individual in whom it is of interest to determine whether a population of biomarkers can be identified).

[0019] As used herein, the term "prokineticin 1" or "PROK1" is also known as "endocrine gland-derived vascular endothelial growth factor" (EG-VEGF), a protein that in humans is encoded by the OLR1 gene (human gene: Gene ID: 84432 / Entrez Gene: PROK1 prokineticin 1: www.ncbi.nlm.nih.gov / gene=84432). Prokineticins (PROKs) are secreted peptides that have the ability to regulate both angiogenesis and inflammatory processes in humans and other species (9-11). PROK1 and PROK2 are the two major members of the prokineticin (PROK) family (9). PROK1 (EG-VEGF) and PROK2 act through specific G protein-coupled receptors, PROK receptor 1 (PROKR1) and PROK receptor 2 (PROKR2), to regulate multiple biological functions, including angiogenesis, circadian rhythm, olfactory bulb neurogenesis, neuronal survival, reproduction, and inflammation (11). PROK1 (EG-VEGF) and PROKR1 are highly expressed in the placenta during the first and second trimester of pregnancy and are immunolocalized in different cell types, including syncytiotrophoblasts, cytotrophoblasts, fetal endothelial cells, and macrophages (11). Several studies have reported that EG-VEGF and its receptors are directly involved in the pathogenesis of recurrent pregnancy loss (RPL), gestational trophoblastic disease (GTD), and placenta-mediated complications (PMC) (12, 24, 25, 26, 27). These data strongly suggest that elevated EG-VEGF levels contribute to the development of PMC or rather participate in an overall compensatory mechanism that occurs to allow pregnancy to progress.

[0020] An example of the PROK1 human amino acid sequence (UniProtKB-P58294) is provided in the NCBI database: NCBI Reference Sequence: NP_115790 (Prokineticin 1 precursor).

[0021] An example of a nucleotide sequence encoding wild-type human PROK1 is provided in the NCBI database: NCBI Reference Sequence: NM_032414 (Prokinetin 1 precursor).

[0022] Of course, variant sequences of PROK1 may be used (as biomarkers) in the context of the present invention, including, but not limited to, functional homologs, paralogs or orthologs, transcriptional variants of such sequences.

[0023] The level of PROK1 can be determined using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reactions such as immunohistochemistry, or sandwich-type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays such as ELISA; biotin / avidin-type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, etc. The reaction generally involves revealing a label such as a fluorescent, chemiluminescent, radioactive, enzyme-labeled or dye molecule, or other method for detecting the formation of a complex between an antigen and an antibody or antibodies reacting therewith.

[0024] For example, the determination of PROK1 levels can be carried out by various techniques and any of the methods well known in the art: ELISA kit (PeproTech, France, Invitrogen™ Kit ELISA human EG-VEGF / PROK1) RIA kit immunochemiluminescence method.

[0025] In certain embodiments, the methods of the invention comprise contacting a blood sample with a binding partner.

[0026] As used herein, a binding partner refers to a molecule that can selectively interact with PROK1.

[0027] The binding partner is generally a polyclonal or monoclonal antibody, preferably a monoclonal antibody. Polyclonal antibodies against PROK1 can be produced according to known methods by administering an appropriate antigen or epitope to a host animal selected from, for example, pigs, cows, horses, rabbits, goats, sheep, and mice. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in the practice of the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against PROK1 can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation are disclosed above. Antibodies useful in the practice of the invention also include anti-PROK1, including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of intact antibody molecules, and Fab fragments, which can be generated by reducing disulfide bridges of F(ab')2 fragments. Alternatively, Fab and / or scFv expression libraries can be constructed to allow rapid identification of fragments with the desired specificity for PROK1. For example, phage display of antibodies can be used. In such methods, single chain Fv (scFv) or Fab fragments are expressed on the surface of a suitable bacteriophage, for example M13. Briefly, the spleen cells of a suitable host, for example a mouse immunized with the protein, are removed. The coding regions for the VL and VH chains are obtained from a cell that produces the desired antibody against the protein. These coding regions are then fused to the ends of the phage sequence. When the phage is inserted into a suitable carrier, for example bacteria, the phage displays the antibody fragment. Phage display of antibodies can also be performed by combinatorial methods known to those skilled in the art. The antibody fragments displayed by the phage can be used as part of an immunoassay.

[0028] In another embodiment, the binding partner may be an aptamer, as described above.

[0029] A binding partner of the invention, such as an antibody or aptamer, may be labeled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule, or other label known in the art. Labels are generally known in the art to provide (directly or indirectly) a signal.

[0030] As used herein, the term "labeled" with respect to a binding partner is intended to encompass direct labeling of an antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorescent dye (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE), indocyanine (Cy5)), and indirect labeling of a probe or antibody by reactivity with a detectable substance. The antibodies or aptamers of the invention can be labeled with a radioactive molecule by any method known in the art. For example, radioactive molecules include, but are not limited to, radioactive atoms for scintigraphy studies, such as I123, I124, In111, Re186, Re188.

[0031] The above-mentioned assays generally involve binding a binding partner (i.e., an antibody or an aptamer) to a solid support. Solid supports that can be used in the practice of the present invention include nitrocellulose (e.g., in the form of a membrane or microtiter well); polyvinyl chloride (e.g., in the form of a sheet or microtiter well); polystyrene latex (e.g., beads or microtiter plates); polyvinylidene fluoride; diazotized paper; nylon membrane; activated beads, magnetically responsive beads, and other substrates. More specifically, an ELISA method can be used, in which the wells of a microtiter plate are coated with a set of antibodies against PROK1 protein. A blood sample containing or suspected of containing PROK1 is added to the coated wells. After a period of incubation sufficient for the formation of a binding partner-PROK1 complex, the plate is washed to remove unbound material and a labeled secondary binding molecule is added. The secondary binding molecule is reacted with the marker protein of the captured sample, the plate is washed, and the presence of the secondary binding molecule is detected by methods known in the art.

[0032] As a binding partner, the secondary binding molecule may be labelled.

[0033] Different immunoassays, such as radioimmunoassay or ELISA, are described in the art.

[0034] With or without immunoassay-based methods, measuring the level of PROK1 protein can also include separating proteins by: centrifugation based on the molecular weight of the protein; electrophoresis based on mass and charge; HPLC based on hydrophobicity; size exclusion chromatography based on size; and solid phase affinity based on the affinity of the protein for the particular solid phase used. Once separated, PROK1 is identified based on the known "separation profile" of the protein, e.g., retention time, and measured using standard techniques. Alternatively, separated proteins can be detected and measured, e.g., by mass spectrometry.

[0035] In a preferred embodiment, the method for measuring the level of PROK1 comprises the step of contacting a blood sample with a binding partner capable of selectively interacting with PROK1 to allow formation of a binding partner-PROK1 complex.

[0036] In a more preferred embodiment, the method of the present invention further comprises the steps of separating any unbound material of the blood sample from the binding partner-PROK1 complex, contacting the binding partner-PROK1 complex with a labeled secondary binding molecule, separating any unbound secondary binding molecule from the secondary binding molecule-PROK1 complex, and measuring the level of the secondary binding molecule in the secondary binding molecule-PROK1 complex.

[0037] Typically, high or low levels of PROK1 are intended by comparison to a control reference value.

[0038] Thus, in a particular embodiment, the prognostic method of the invention comprises the step of comparing said level of PROK1 to a control reference value, A high level of the PROK1 marker is predictive of a high risk of having or developing spontaneous preterm birth, A low level of the PROK1 marker is predictive of a low risk of having or developing spontaneous preterm birth.

[0039] The control reference value may be determined with respect to the level of PROK1 present in a blood sample taken from one or more healthy subjects or the distribution of PROK1 in a control population.

[0040] In one embodiment, the method of the present invention comprises the step of comparing the level of PROK1 with a control reference value, wherein a high level of the PROK1 marker compared to the control reference value is predictive of a high risk of having spontaneous preterm birth and a low level of the PROK1 marker compared to the control reference value is predictive of a low risk of having spontaneous preterm birth.

[0041] The control reference value may depend on various parameters, such as the method used to measure the PROK1 level in the subject.

[0042] Typically, using marker dose Elisa technology for the reference values ​​of "PROK1 marker" at 32 weeks of gestation shown in the Examples section (Figure 2 and Table 2); serum levels of EG-VEGF greater than 195 pg / ml predict having or a high risk of having or developing spontaneous preterm birth, and serum levels of EG-VEGF less than 195 pg / ml predict not having or developing spontaneous preterm birth, or a low risk of having spontaneous preterm birth.

[0043] The control reference value can be readily determined by one skilled in the art by using techniques similar to those for determining PROK1 levels in a blood sample previously taken from the patient under test.

[0044] The "reference value" can be a "threshold" or "cut-off value". Typically, the "threshold" or "cut-off value" can be determined experimentally, empirically, or theoretically. The threshold can also be arbitrarily selected based on existing experimental and / or clinical conditions, as the skilled person will recognize. The threshold must be determined to obtain optimal sensitivity and specificity according to the function of the test and the benefit / risk balance (false positive and false negative clinical outcomes). Typically, the optimal sensitivity and specificity (and therefore the threshold) can be determined using a receiver operating characteristic (ROC) curve based on experimental data (see FIG. 2). Preferably, the skilled person can compare the level of PROK1 (protein or nucleic acid sequence (mRNA)) of the present invention with a defined threshold. In one embodiment of the present invention, the threshold is derived from a PROK1 protein level (or ratio, or score) determined in a blood sample from one or more subjects who are responders (to the method of the present invention). In one embodiment of the present invention, the threshold may be derived from a PROK1 protein level (or ratio, or score) determined in a blood sample from one or more subjects who are non-responders or subjects who are non-responders. Furthermore, retrospective measurements of PROK1 protein levels (or ratios, or scores) in appropriately banked historical subject samples can be used to establish these thresholds.

[0045] For example, after determining the expression level of PROK1 protein (protein or nucleic acid sequence (mRNA)) in the reference group, algorithm analysis can be used for the statistical processing of the expression level determined in the blood sample to be tested, and a classification criterion with significance for blood sample classification can be obtained. The full name of the ROC curve is the receiver operator characteristic curve, and it is also called the receiver operation characteristic curve. It is mainly used in clinical biochemistry diagnostic tests. The ROC curve is a comprehensive index that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). The relationship between sensitivity and specificity is revealed by the image synthesis method. A series of different cutoff values ​​(thresholds or critical values, boundary values ​​of normal and abnormal results of diagnostic tests) are set as continuous variables, and a series of sensitivity and specificity values ​​are calculated. Then, the curve is drawn with sensitivity as the ordinate and specificity as the abscissa. The larger the area under the curve (AUC), the higher the diagnostic accuracy. In the ROC curve, the point closest to the top left of the coordinate diagram is the critical point with high sensitivity and high specificity values. The AUC value of the ROC curve is 1.0-0.5. When AUC>0.5, the closer the AUC is to 1, the better the diagnostic result. When AUC is 0.5-0.7, the accuracy is low. When AUC is 0.7-0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is high. The algorithm method is preferably implemented by a computer. Existing software or systems in the art can be used to plot the ROC curve, such as MedCalc 9.2.0.1 medical statistics software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VI0.0 (Dynamic Microsystems, Silver Spring, Md.USA).

[0046] In some embodiments, the method of the present invention includes the use of a classification algorithm, typically selected from linear discriminant analysis (LDA), topological data analysis (TDA), neural networks, support vector machine (SVM) algorithms, and random forest algorithms (RF). In some embodiments, the method of the present invention includes a step of determining a subject response using a classification algorithm. As used herein, the term "classification algorithm" has its general meaning in the art and refers to classification and regression tree methods and multivariate classifications well known in the art, such as those described in US8,126,690; WO2008 / 156617. As used herein, the term "support vector machine (SVM)" refers to a universal learning machine useful for pattern recognition, whose decision boundary is parameterized by a collection of support vectors and a corresponding set of weights, and which processes multiple variables simultaneously, rather than separately. Thus, support vector machines are useful as statistical tools for classification. Support vector machines nonlinearly map an n-dimensional input space into a high-dimensional feature space, and present the optimal interface (optimal splitting surface) between the features. A support vector machine includes two phases: a training phase and a testing phase. In the training phase, support vectors are generated, and in the testing phase, estimation is performed according to certain rules. In general, an SVM provides a model for use in classifying each of n subjects into two or more disease categories based on one k-dimensional vector (called a k-tuple) of biomarker measurements for each subject. An SVM first transforms the k-tuple into a space of the same or higher dimension using a kernel function. The kernel function projects the data into a space where the categories can be better separated using a hyperplane than was possible in the original data space. To determine the hyperplane that discriminates between the categories, a set of support vectors closest to the boundary between the disease categories is selected. The hyperplane is then selected by known SVM techniques such that the distance between the support vectors and the hyperplane is maximized within a cost function that penalizes inaccurate predictions.This hyperplane is the one that optimally separates the data from a predictive perspective (Vapnik, 1998 Statistical Learning Theory. New York: Wiley). Any new observation is then classified as belonging to one of the categories of interest based on where it lies relative to the hyperplane. If more than one category is considered, the process is run pairwise for all categories and the results are combined to create a rule that discriminates between all categories. As used herein, the term "random forest algorithm" or "RF" has its common meaning in the art and refers to classification algorithms such as those described in US8,126,690; WO2008 / 156617. Random forests are decision tree-based classifiers built using an algorithm originally developed by Leo Breiman (Breiman L, "Random forests," Machine Learning 2001, 45:5-32). This classifier uses a large number of individual decision trees and determines the class by selecting the mode of the class determined by the individual trees. Each tree is constructed using the following: (1) an algorithm that assumes the number of cases in the training set is N and the number of variables in the classifier is M; (2) an algorithm that selects the number of input variables used to determine the decision at the nodes of the tree; (3) an algorithm that selects, with replacement, N samples from the training set; (4) an algorithm that, for each node of the tree, randomly selects m out of the M variables based on the decision at that node; and (5) an algorithm that calculates the best split based on the m variables in the training set. In some embodiments, the scores are generated by a computer program.

[0047] In some embodiments, the method of the present invention comprises: a) quantifying the level of PROK1 expression (protein or nucleic acid sequence (mRNA)) in a blood sample; b) performing a classification algorithm on the data comprising the quantified PROK1 protein to obtain an algorithm output; and c) determining the probability that the subject will experience spontaneous preterm birth from the algorithm output of step b).

[0048] The algorithms used with the methods of the present invention can be executed by one or more programmable processors executing one or more computer programs, which operate on input data to generate output to perform functions. The algorithms can also be executed by special purpose logic circuitry, such as FPGAs (field programmable gate arrays) or ASICs (application specific integrated circuits), and devices can be implemented as well. Processors suitable for executing computer programs include, by way of example, both general purpose and special purpose microprocessors, and any one or more processors of any kind of digital computer. Generally, a processor receives instructions and data from a read-only memory or a random access memory or both. The essential elements of a computer are a processor for executing instructions and one or more memory devices for storing instructions and data. Generally, a computer includes or is operatively connected to one or more mass storage devices (e.g., magnetic disks, magneto-optical disks, optical disks, etc.) for storing data, for receiving data from, transferring data to, or both, the mass storage devices. Furthermore, a computer can be incorporated into another device. Computer-readable media suitable for storing computer program instructions and data include all forms of non-volatile memory, media and memory devices, including, for example, semiconductor memory devices, such as EPROM, EEPROM and flash memory devices; magnetic disks, such as internal hard disks or removable disks; magneto-optical disks; and CD-ROM and DVD-ROM disks. The processor and memory may be supplemented by or incorporated with special purpose logic circuitry. To provide for interaction with a user, embodiments of the present invention may be implemented on a computer having a display device, such as, in non-limiting examples, a CRT (cathode ray tube) or LCD (liquid crystal display) monitor, for displaying information to the user, and a keyboard and pointing device, such as a mouse or trackball, by which the user can provide input to the computer.Other types of devices can be used to provide interaction with a user as well; for example, feedback provided to the user can be any form of sensory feedback, e.g., visual feedback, auditory feedback, or haptic feedback, and input from the user can be received in any form, including acoustic, speech, or haptic input. Thus, in some embodiments, the algorithms can be implemented in a computing system including a back-end component, e.g., a data server, or a middleware component, e.g., an application server, or a front-end component, e.g., a client computer having a graphical user interface or a web browser through which a user can interact with an embodiment of the invention, or any combination of one or more such back-end, middleware, or front-end components. The components of the system can be interconnected by any form or medium of digital data communication, e.g., a communications network. Examples of communications networks include local area networks ("LANs") and wide area networks ("WANs"), e.g., the Internet. The computing system can include clients and servers. Clients and servers are generally remote from each other and typically interact through a communications network. The relationship of client and server arises by virtue of computer programs running on the respective computers, which have a client-server relationship to each other.

[0049] "Risk" in the context of the present invention relates to the probability that an event will occur over a certain period of time, such as conversion to spontaneous preterm birth, and can refer to the "absolute" or "relative" risk of a subject. Absolute risk can be measured by referring to actual observational measurements of the relevant time cohort, or by referring to index values ​​developed from statistically valid historical cohorts followed for the relevant period of time. Relative risk refers to the ratio of the absolute risk of a subject compared to either the absolute risk of a low-risk cohort or the average population risk, and depends on how clinical risk factors are evaluated. Odds ratio is the ratio of positive events to negative events in a test result, and is commonly used (odds are calculated according to the formula p / (lp), where p is the probability of the event and (1-p) is the probability of no event). Other continuous measurements that can be evaluated in the context of the present invention include time to spontaneous preterm birth and conversion risk reduction ratio.

[0050] "Risk evaluation" or "evaluation of risk" in the context of the present invention encompasses making a prediction of the probability, odds or likelihood of an event or disease state occurring, the incidence of an event or conversion from one disease state to another, i.e., conversion from a normal state to a state of spontaneous preterm birth or a state at risk of developing spontaneous preterm birth. Risk evaluation can include prediction in absolute or relative terms based on previously measured populations, such as future clinical parameters, traditional laboratory risk factor values ​​or other indices of spontaneous preterm birth, e.g. cell population determinations in peripheral tissues, serum or other body fluids. The method of the present invention can be used to measure the risk of conversion to spontaneous preterm birth continuously or categorically, thereby prognosticating and defining the risk spectrum of categories of subjects defined as at risk of spontaneous preterm birth. In a categorical scenario, the present invention can be used to distinguish between healthy subjects and other cohorts of subjects at high risk of spontaneous preterm birth. In another embodiment, the present invention can be used to help distinguish between those with spontaneous preterm birth and healthy subjects.

[0051] The present invention further relates to the use of PROK1 in blood samples as a predictive biomarker for spontaneous preterm birth, particularly at different stages of pregnancy, where "different stages" according to the present invention refers to the stages of pregnancy at least at 20, 21, 22, 23, 24; 25, 26; 27, 28, 29, 30, 31, 32, 33, 34; 35, 36; 37 weeks of pregnancy.

[0052] Therefore, the detection method of the present invention is useful for in vitro prediction of spontaneous preterm birth from a blood sample. In particular, the detection method of the present invention is useful for in vitro prediction of spontaneous preterm birth at an early stage (at least 20 weeks of pregnancy) from a blood sample.

[0053] Monitoring methods and management A further object of the present invention relates to an in vitro method for monitoring spontaneous preterm birth comprising the steps of: i) determining the level of the protein PROK1 marker in a blood sample obtained from the subject at a first specific time of the disease; ii) determining the level of the protein PROK1 marker in a blood sample obtained from the subject at a second specific time of the disease; iii) comparing the level determined in step i) with the level determined in step ii); and iv) concluding, if the level determined in step ii) is higher than the level determined in step i), that the risk of suffering or developing spontaneous preterm birth is developing in an aggravated manner.

[0054] A further object of the present invention relates to an in vitro method for monitoring a treatment of spontaneous preterm birth comprising the steps of: i) determining the level of the protein marker PROK1 in a blood sample obtained from the subject before the treatment; ii) determining the level of the protein marker PROK1 in a blood sample obtained from the subject after the treatment; iii) comparing the level determined in step i) with the level determined in step ii); and iv) concluding that the treatment is efficient if the level determined in step ii) is lower than the level determined in step i).

[0055] In certain embodiments, spontaneous preterm birth is detected at an early stage (at least 20 weeks gestation).

[0056] The reduction rate is, for example, at least 5%, or at least 10%, or at least 20%, more preferably at least 50%, and even more preferably at least 100%.

[0057] Treatment strategies for specific populations The present invention further relates to methods for treating spontaneous preterm birth with prenatal tocolytic or vaginal progestational drugs, placement of a pessary and / or cervical cerclage, in a subject, wherein the level of the protein PROK1 marker obtained from said patient is detected by any of the methods of the present invention.

[0058] In the context of the present invention, the terms "treating" or "treatment" as used herein mean reversing, alleviating, inhibiting the progression of, or preventing the disorder or condition to which such term applies, or reversing, alleviating, inhibiting the progression of, or preventing one or more symptoms of the disorder or condition to which such term applies.

[0059] Another object of the present invention is a method for treating / preventing spontaneous preterm birth in a subject, comprising: a) providing a blood sample containing neutrophils from a subject; b) detecting the level of the protein PROK1 marker; c) comparing the level determined in step b) with a reference value; and If the level determined in step b) is higher than the reference value, treating the subject with prenatal progesterone and / or tocolytic therapy.

[0060] As mentioned above, the current main treatments for spontaneous preterm birth are antenatal tocolytic or vaginal progesterone medications, placement of a pessary and / or cervical cerclage.

[0061] Therefore, the present invention further relates to a method for the treatment of spontaneous preterm birth using antenatal tocolytic or vaginal progesterone drugs, placement of a pessary and / or cervical cerclage, in which the level of the protein PROK1 marker obtained from said subject, detected by one of the methods of the present invention, is indicative of a prognosis for the disease.

[0062] 1. A method of treating spontaneous preterm birth in a subject, comprising: a) providing a blood sample containing neutrophils from a subject; b) detecting the level of the protein PROK1 marker; c) comparing the level determined in step b) with a reference value; and If the level determined in step b) is higher than the reference value, treating the subject with a tocolytic or vaginal progestational agent, placement of a pessary and / or cervical cerclage.

[0063] In certain embodiments, spontaneous preterm birth is detected at an early stage (at least 20 weeks gestation).

[0064] The term "progesterone" refers to a drug and naturally occurring steroid hormone. Progesterone therapy is a "progestogen" (also called progestagen, gestagen, or gestogen, a type of drug that mimics the effects of progesterone) that is primarily used in combination with estrogen in hormone therapy for menopausal symptoms and low sex hormone levels in women. It is also used to support pregnancy and infertility and treat gynecological disorders. Progesterone can be taken orally, vaginally, and by other routes such as injection into muscle or fat. Many synthetic progestogens or progestins derived from progesterone are also used as drugs (Kuhl H (2005). Climacteric.8 Suppl1:3-63). Examples include medroxyprogesterone acetate and norethisterone.

[0065] The term "tocolytics", also referred to as "anticontractile drugs" or "tocolytics", are drugs used to prevent premature birth. Thus, tocolytic treatment is offered when premature birth is imminent, to delay birth long enough to allow administration of glucocorticoids to promote fetal lung maturation, but may take 1-2 days to be effective.

[0066] Commonly used tocolytics include oxytocin receptor antagonists (e.g., atosiban), beta2 agonists (see below), calcium channel blockers (e.g., nifedipine (Procardia, Adalat)), nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., indomethacin, sulindac), and magnesium sulfate. These can help delay preterm birth by inhibiting uterine muscle contractions, and their use aims to reduce fetal morbidity and mortality associated with preterm birth (Mayer C. et al (2021), “Tocolysis”, StatPearls, Treasure Island (FL): StatPearls Publishing). Inhibition of uterine contractions is often partial, and tocolytics can only be relied upon for a few days to delay birth. Depending on the tocolytic used, monitoring of the pregnant woman or fetus may be required (e.g., blood pressure monitoring if nifedipine is used to reduce blood pressure; electrocardiogram to assess fetal well-being, etc.).

[0067] Examples of beta2 agonists used as tocolytics can be selected from the list consisting of salbutamol (INN) or albuterol (USAN), fenoterol, terbutaline (Brethine), ritodrine (Yutopar), hexoprenaline (Gynipral).

[0068] In certain embodiments, the tocolytic agent is nifedipine, one of the most commonly used tocolytic agents.

[0069] The present invention is further illustrated by the following figures and examples, which should not, however, be construed in any way as limiting the scope of the present invention. [Brief description of the drawings]

[0070] [Figure 1] Circulating PROK1 / EG-VEGF concentrations at four gestational stages in uncomplicated and spontaneously preterm pregnant women. The central bar is the median. The lower and upper limits of the box are the 1st and 3rd quartiles. *p<0.05 **<0.001.. [Diagram 2] ROC curve analysis of circulating PROK1 / EG-VEGF concentrations for predicting spontaneous preterm birth at 32 weeks. AUC: area under the curve. EXAMPLES

[0071] method Study design and population The present study is based on data from the previously described AngioPred study (17). The AngioPred study is a prospective multicenter cohort study carried out at the Departments of Obstetrics and Gynecology of Saint-Étienne and Nimes University Hospital and at the Hematology Institute of Nimes University Hospital from June 2008 to October 2010. Patients included in the study were those seen within 20 weeks and at high risk for the development or recurrence of PMC.

[0072] All patients were at high risk for the occurrence or recurrence of PMC, including diabetes, chronic hypertension, obesity, maternal age <18 or >38 years, chronic kidney disease, systemic lupus erythematosus, antiphospholipid syndrome, family history of first-degree cardiovascular disease or venous thromboembolism, biological thrombosis without a history of venous thromboembolism or PMC, and one or more previous episodes of PMC or venous thromboembolism. Exclusion criteria were twin pregnancy, patients with a history of fetal death, IUGR of chromosomal, genetic or infectious etiology, and the presence of PMC or venous thromboembolism at the time of inclusion.

[0073] The Ethics Committee and Institutional Review Board of the Saint-Étienne University Hospital approved the protocol in March 2008. The study was registered at clinicaltrials.gov (identifier NCT00695942). The clinical trial was performed in accordance with the Declaration of Helsinki of 1975, as revised in 1996. All patients were included at less than 20 weeks' gestation and provided written informed consent.

[0074] Outcomes The primary outcome was the occurrence of spontaneous preterm birth, defined as the number of babies born after 20 weeks 0 days of gestation and before 37 weeks 0 days of gestation with spontaneous onset of labor, either intact or with rupture of membranes before labor.

[0075] blood sampling Blood samples were taken at the collection center of the Saint-Étienne-Nimes University Hospital at weeks 20, 24, 28, 32 and 36 of gestation, a total of five samples per patient. Samples were immediately sent to the laboratory for analysis. Samples were centrifuged, aliquoted and stored at -80°C.

[0076] biological analysis Each analysis was performed in a blinded manner to the other. All samples from the same patient were grouped into the same series of assays. The analyses were performed after thawing for 10 min in a 37°C water bath and centrifugation at 2500 g. Serum EG-VEGF levels were measured at 20, 24, 28, 32 and 36 weeks by enzyme-linked immunosorbent assay (ELISA) kits (PeproTech, Neuilly-Sur-Seine, France), with a standard range of 16-1000 pg / mL. Two separate standard curves were constructed to allow accurate measurement of samples at the upper and lower limits of the assay.

[0077] statistical analysis Statistical analysis was performed using XlSTAT®. Qualitative data were presented as absolute and relative frequencies (expressed as %). Qualitative variables were compared by chi-square test or, in cases of insufficient numbers, by Fisher's exact test. Quantitative variables were presented as means and standard deviations. Normal distribution of data was tested by Shapiro-Wilk test. Results were reported as box plots. The threshold value of EG-VEGF plasma levels for predicting spontaneous preterm birth was determined at each gestational age through receiver operating characteristic (ROC) curves calculating the area under the curve with 95% confidence intervals (95% CI) (18). All hypothesis tests were performed at a significance level of 0.05, with p<0.05 considered significant.

[0078] result Clinical Features Two hundred consecutive pregnant women were included in the study between June 2008 and October 2010. Demographics and inclusion criteria are summarized in Table 1.

[0079] During the study period, 45 women developed PMC and were excluded from the analysis. Seven presented with spontaneous preterm birth. All demographic characteristics and inclusion criteria were similar between women with uncomplicated and sPTB.

[0080] Categorical variables were reported as frequencies (percentages) and continuous variables as means (standard deviations). Abbreviations: BMI: body mass index, MAP: mean arterial pressure, UARI: mean uterine artery resistive index, VTE: venous thromboembolism, PMC: placenta-mediated complications.

[0081] Serum EG-VEGF concentration and the incidence of spontaneous preterm birth Women with sPTB had higher EG-VEGF concentrations than patients without complications at weeks 24 (244.1 vs. 144.3 pg / mL), 28 (247.5 vs. 146.2 pg / mL), and 32 (328.5 vs. 152.7 pg / mL) (p=0.03, 0.02, and <0.001). Results are summarized in Figure 1.

[0082] The receiver operating characteristic curve was used to calculate the threshold serum EG-VEGF level with optimal sensitivity and specificity for predicting sPTB. The area under the curve (AUC) reached 0.9, and the sensitivity for predicting spontaneous preterm birth was 100% at 32 weeks (Figure 2).

[0083] Consideration Conclusions: Serum EG-VEGF levels increased as early as 24 weeks of gestation and were predictive of spontaneous preterm birth. Circulating EG-VEGF levels were higher in women with spontaneous preterm birth and showed good predictive ability at 32 weeks.

[0084] Previous reports from our group showed that in normal pregnancy, circulating EG-VEGF concentrations increase during the third trimester compared with the second trimester, but decrease at the time of labor compared with non-laboring patients (19). EG-VEGF is also expressed in mouse fetal membranes (FM) by the end of pregnancy, suggesting that this protein plays a local role in the mechanism of labor (19). These data suggested that EG-VEGF is a cytokine that acts locally to ensure fetal membrane protection during late pregnancy. Thus, reduced expression of EG-VEGF, as revealed by the sudden drop in expression levels as well as that of its receptor, may contribute to the initiation of the labor process in humans.

[0085] To date, no study has evaluated the predictive ability of EG-VEGF levels for spontaneous preterm birth. The increase in EG-VEGF levels in sPTB substantiated the assumption of a direct involvement of this factor in the pathogenesis of pregnancy-related pathology. A compensatory role in this pathology is likely. It can therefore be speculated that EG-VEGF concentrations increase not only to reactivate angiogenesis but also to suppress inflammation associated with labor. Microbiological studies suggest that intrauterine infections may account for 25–40% of preterm births (20). Importantly, Jabbour et al. (15) tested the possibility of its involvement in preterm birth induction by injecting EG-VEGF in an animal model. EG-VEGF was compared with lipopolysaccharide (LPS), a component of the cell wall of Escherichia coli (E. coli), a gram-negative bacterium widely known to induce labor. Injection of LPS induced preterm birth within the following 20 h, whereas EG-VEGF did not. Injection of EG-VEGF increased the mRNA expression of the inflammatory mediators IL-6, IL-1, tumor necrosis factor (TNF), CXCL2, and CXCL5 in cells of the fetal membrane (21). EG-VEGF also increased the same inflammatory mediators in human myometrium (22). Furthermore, monocytes treated with EG-VEGF for 24 h released the chemokines CXCL1, CXCL8, and CCL4, and costimulation with LPS synergized the production of CCL18 and CCL20 (23). These findings strongly suggest that EG-VEGF is involved in processes associated with preterm labor, although to date it is unknown whether its elevated concentrations are a cause or a consequence. Further in vivo studies are underway to elucidate the significance of elevated EG-VEGF in this pathology.

[0086] Overall, this study demonstrates for the first time that EG-VEGF concentrations may constitute a powerful biomarker for the development of sPTB. The significance of this study is that a patient population at high risk for PMC was prospectively recruited and followed from 20 weeks until delivery.

[0087] The association between EG-VEGF levels and spontaneous preterm birth will be investigated in a larger cohort to validate its informative value and to propose its use in the routine evaluation of patients at high pathological risk.

[0088] [Table 1]

[0089] [Table 2]

[0090] References Throughout this application, various references are described that describe the state of the art to which this invention pertains, the disclosures of which are incorporated by reference into this disclosure. [Table 3] TIFF2025505035000004.tif234165 TIFF2025505035000005.tif170165

Claims

1. An in vitro method for assessing a subject's risk of having or developing spontaneous preterm birth, comprising: i) determining the level of the protein marker PROK1 in a blood sample obtained from the subject; ii) comparing the level determined in step i) with a reference value; and iii) indicating that if the level of the protein PROK1 marker determined in step i) is higher than the reference value, the subject is predicted to be at high risk of having or developing spontaneous preterm birth. A method comprising:

2. The in vitro method described in claim 1, wherein the prediction is for spontaneous preterm birth in the early stages of at least the 20th week of pregnancy.

3. 1. An in vitro method for monitoring spontaneous preterm birth, comprising: i) determining the level of the protein marker PROK1 in a blood sample obtained from the subject at a first specific stage of pregnancy; ii) determining the level of protein marker PROK1 in a blood sample obtained from said subject at a second specific time during said pregnancy; iii) comparing the level determined in step i) with the level determined in step ii); and iv) if the level determined in step ii) is higher than the level determined in step i), it indicates that the risk of having or developing spontaneous preterm birth has progressed in a worse manner. A method comprising:

4. 1. An in vitro method for monitoring a treatment of spontaneous preterm birth, comprising: i) determining the level of the protein marker PROK1 in a blood sample obtained from said subject before treatment; ii) determining the level of the protein PROK1 marker in a blood sample obtained from said subject after said treatment; iii) comparing the level determined in step i) with the level determined in step ii); and iv) indicating that the treatment is effective if the level determined in step ii) is lower than the level determined in step i). A method comprising:

5. A pharmaceutical composition for treating spontaneous preterm labor, comprising a tocolytic agent or a vaginal progestational agent, A pharmaceutical composition characterized in that the tocolytic agent or the vaginal progesterone agent is administered to a subject from whom the level of the protein PROK1 marker has been determined by the method of any one of claims 1 to 4.

6. A method for determining whether a subject requires treatment for spontaneous preterm birth, comprising: a) obtaining a blood sample from said subject; b) determining the level of the protein marker PROK1 in said blood sample; c) comparing the level determined in step b) with a reference value; and d) if the level determined in step b) is higher than the reference value, indicating that the subject requires treatment with tocolytics, vaginal progestational agents, pessary placement, and / or cervical cerclage. A method comprising:

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