Early prediction method for preterm birth using retinoid metabolome

Abstract

The present invention relates to a method for early prediction of premature birth using retinoid metabolome.

Description

[Technical field]

[0001] The present invention relates to a method for early prediction of premature birth using retinoid metabolome. [Background technology]

[0002] Preterm birth (PTB) refers to birth before 37 weeks of gestation, and according to recent statistics from 107 Asian countries, it occurs at a frequency of approximately 10.6%.

[0003] In South Korea, the premature birth rate was approximately 4.8% in 2006, but nearly doubled to 7.2% in 2016, showing an upward trend. In the short term, premature birth poses the risk of increasing the mortality rate of newborns and children under the age of five, as well as raising the likelihood of problems with neurodevelopment, and in the long term, it poses the risk of increasing the prevalence of type 2 diabetes, obesity, high blood pressure, and other conditions.

[0004] The pathogenesis of preterm birth has not been clearly elucidated, and risk factors commonly associated with spontaneous labor include reproductive tract infection, multiple pregnancy, second and third trimester bleeding, and previous preterm birth. Approximately 75% of preterm births occur with preterm labor and amniotic rupture, with associated cervical incompetence and chorionitis.

[0005] Although Korea has a low birth rate, the premature birth rate (<37 weeks) is steadily increasing, threatening the health of mothers and newborns and incurring economic losses. To date, fetal fibronectin, CRP measurement, cervical length measurement, etc. have been used to predict premature birth, but the sensitivity of the prediction rate of premature birth is still low.

[0006] Therefore, when signs of preterm labor appear in the early stages, delaying labor through appropriate treatment and promoting neonatal maturity are key to the health, quality of life, and costs of mothers and neonates. Summary of the Invention [Problem to be solved by the invention]

[0007] One example of the present invention is to provide a method for predicting preterm birth or providing information for predicting preterm birth, in which retinoid metabolomic levels in a biological sample can be used to predict preterm birth.

[0008] Another embodiment of the present invention is to provide a composition for predicting preterm birth, which can utilize retinoid metabolomic levels in a biological sample to predict preterm birth.

[0009] Yet another example of the present invention relates to a kit for predicting preterm birth, which can utilize retinoid metabolome levels in a biological sample to predict preterm birth.

[0010] Yet another example of the present invention relates to a system for predicting preterm birth, which can utilize retinoid metabolomic levels in a biological sample to predict preterm birth. [Means for solving the problem]

[0011] One example of the present invention relates to a method for predicting preterm birth or providing information for predicting preterm birth, comprising quantifying the retinoid metabolome in a biological sample.

[0012] Another example of the present invention relates to a composition for predicting preterm birth, comprising a formulation capable of quantifying the retinoid metabolome in a biological sample.

[0013] Yet another example of the present invention relates to a kit for predicting preterm birth, comprising a quantitative preparation or a quantitative device for quantifying the retinoid metabolome in a biological sample.

[0014] Yet another example of the present invention relates to a system for predicting preterm birth, comprising a quantification unit that quantifies the retinoid metabolome in a biological sample.

[0015] The present invention will now be described in further detail.

[0016] One example of the present invention relates to a method for providing information for predicting, predicting, diagnosing preterm birth or diagnosing preterm birth, comprising the step of quantifying the retinoid metabolome in a biological sample.

[0017] As used herein, the terms "premature birth," "preterm birth," "PTB," and the like, have the same meaning and refer to the birth of a fetus before 37 weeks.

[0018] According to the examples of the present application, the retinoid metabolomic marker for premature birth according to the present invention was analyzed using blood samples from parturient women and showed a significant difference between the premature birth group and the full-term delivery group. Therefore, the retinoid metabolomic marker according to one example of the present invention can be used to predict premature birth.

[0019] In the present invention, the term "diagnosis of risk of premature birth" or "prediction of risk of premature birth" means: Pregnant women Preterm birth is when a woman is at risk of having a preterm birth, has a relatively high risk of having a preterm birth, or is likely to show signs of preterm birth.

[0020] The present invention relates to a method for treating Pregnant women With specific and appropriate management, the present invention can be used to delay or prevent preterm birth. The present invention can also be used clinically to diagnose preterm birth early and make treatment decisions by selecting the most appropriate treatment regime.

[0021] As used herein, the term "or" is intended to include "and / or" unless the context clearly dictates otherwise.

[0022] As used herein, the term "marker," "diagnostic marker," "marker for diagnosing," "diagnosis marker," or "indicator" refers to a marker for predicting preterm birth. Pregnant women It is a standard substance for distinguishing between normal and preterm births, and is predicted to be preterm compared to the control group. Pregnant womenAccording to one embodiment of the present invention, the markers may include the metabolome in a biological sample.

[0023] The term "metabolite" refers to a substance produced as a result of metabolism in vivo, and may be expressed as a metabolite, an intermediate metabolite, a metabolic intermediate, or the like, and mainly refers to the entire metabolites of small molecules generated during cellular processes. For example, according to one embodiment of the present invention, the metabolome may be a retinoid metabolome. Specifically, the metabolome may include one or more selected from the group consisting of all trans-retinal, 13-cis-retinoic acid, and retinyl palmitate. As an example, the metabolome may include one or more selected from the group consisting of all trans-retinal and 13-cis-retinoic acid.

[0024] As used herein, the term "biological sample" refers to a sample obtained from a test subject or test individual, e.g. Pregnant women The biological sample means a sample isolated or collected from Pregnant women The sample origin, specifically Pregnant women The blood sample may be one selected from the group consisting of whole blood, plasma, and serum.

[0025] The method of providing information for predicting premature birth, the method of predicting premature birth, the method of providing information for diagnosing premature birth, or the method of diagnosing premature birth according to one embodiment of the present invention may further include a step of comparing the quantified metabolome level with a control group.

[0026] For example, if the metabolome is one or more selected from the group consisting of all trans-retinal, 13-cis-retinoic acid, and retinyl palmitate, and the quantified metabolome level is higher than that of a control group, the biological sample is isolated. Pregnant women The method may further include predicting that the patient is at risk of premature birth. The control group may be women who have had a normal delivery without premature birth, women who have delivered at 37 weeks or more of pregnancy, or women who have delivered at full term.

[0027] As used herein, the term "increase" refers to an increase in quantity, level, distribution, distribution ratio, or concentration from a comparative control group, for example, an established control group or a standard control group (e.g., the average level of a marker metabolome shown in biological samples from women who have had a normal delivery or biological samples from women who have delivered at 37 weeks or more of pregnancy). Specifically, the distribution ratio may be the relative content or relative concentration of a specific metabolome in a biological sample, such as the results of an analysis using a liquid chromatography mass spectrometer or the like.

[0028] For example, the increase may be greater than 1-fold, 1.1-fold or more, 1.15-fold or more, 1.2-fold or more, 1.25-fold or more, 1.3-fold or more, 1.35-fold or more, 1.4-fold or more, 1.45-fold or more, or 1.5-fold or more increase or a statistically significant increase over the control group value, although one of ordinary skill in the art would be able to clearly understand a significant increase over the control group even if the absolute amount of increase is not specified. The control group value may be, for example, an average of at least 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, or 35 or more control group values.

[0029] For example, when the concentration of all-trans-retinal in a biological sample is 1.5 times or more the control value, the biological sample is isolated. Pregnant women can predict the risk of preterm birth.

[0030] For example, when the concentration of 13-cis-retinoic acid in a biological sample is 1.4 times or more the control value, the biological sample is isolated. Pregnant women can predict the risk of preterm birth.

[0031] For example, when the concentration of retinyl palmitate in a biological sample is 1.5 times or more the control value, the biological sample is isolated. Pregnant women can predict the risk of preterm birth.

[0032] For example, when the concentration of all-trans-retinal in a biological sample is 2.48 ppb or more, the biological sample is separated. Pregnant women can predict the risk of preterm birth.

[0033] For example, when the concentration of 13-cis-retinoic acid in a biological sample is 5.56 ppb or more, the biological sample is separated. Pregnant women can predict the risk of preterm birth.

[0034] For example, when the concentration of retinyl palmitate in a biological sample is 100.33 ppb or more, the biological sample is separated. Pregnant women can predict the risk of preterm birth.

[0035] The terms indicating such an increase, such as "high," "more," "higher," "significantly higher," "statistically significantly higher," etc., are used interchangeably as described above. In contrast, the terms "non-significantly," "comparably," "substantially the same," "substantially unchanged," etc., are meant to indicate little or no significant change from the standard control group value, such as within ±10%, within ±5%, within ±3%, or within ±2% of the standard control group.

[0036] As used herein, the term "control group" refers to a subject for comparing the level of the metabolome according to the present invention to predict preterm birth, and may be used interchangeably with terms such as comparative control group, standard control group, or normal control group. For example, the control group may be women who did not have a preterm birth, women who had a normal birth, or women who gave birth at 37 weeks or more of pregnancy.

[0037] The step of quantifying the metabolome may be performed using one or more of the following: liquid chromatography mass spectrometry (LC / MS), nuclear magnetic resonance spectrometry, ultraviolet spectrometry, infrared spectrometry, fluorescence spectrometry, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry.

[0038] The premature birth according to one embodiment of the present invention may not be premature birth due to liver dysfunction. According to the present example, the premature birth group and the normal control group that showed a significant difference in the retinoid metabolome premature birth marker according to the present invention did not show a significant difference in the liver function-related indicators, specifically, the levels of hepatocellular damage factors such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and more specifically, did not show a significant difference in the levels of the hepatocellular damage factors in the third trimester of pregnancy. Therefore, the premature birth according to one embodiment of the present invention may not be premature birth due to liver dysfunction.

[0039] That is, a subject for predicting premature birth by one embodiment of the present invention Pregnant womendoes not have liver dysfunction Pregnant women Specifically, Pregnant women does not have liver dysfunction during the third trimester of pregnancy Pregnant women It could be.

[0040] For example, Pregnant women There was no significant difference in liver function-related indicators or hepatocellular injury factor levels compared with the normal control group. Pregnant women Specifically, Pregnant women In the third trimester of pregnancy, there was no significant difference in liver function-related indicators or hepatocellular injury factor levels compared with the normal control group. Pregnant women It could be.

[0041] For example, Pregnant women The levels of alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST) were not significantly different from those of normal controls. Pregnant women For example, Pregnant women is AST ≤ 50U / L, ≤ 45U / L, ≤ 40U / L, ≤ 35U / L, ≤ 30U / L, ≤ 25U / L, or ≤ 20U / L Pregnant women It could be.

[0042] For example, Pregnant women ALT 50U / L or less, 45U / L or less, 40U / L or less, 35U / L or less, 30U / L or less, 25U / L or less, 20U / L or less, or 15U / L or less Pregnant women It could be.

[0043] For example, Pregnant women AST / ALT ratio of 0.1–2, 0.1–1.8, 0.1–1.5, 0.3–2, 0.3–1.8, 0.3–1.5, 0.5–2, 0.5–1.8, 0.5–1.5, 0.7–2, 0.7–1.8, 0.7–1.5, 1–2, 1–1.8, or 1–1.5 Pregnant women It could be.

[0044] Yet another example of the present invention relates to a composition for predicting preterm birth, comprising a formulation capable of quantifying the retinoid metabolome in a biological sample.

[0045] The retinoid metabolome is as described above, and may include, for example, one or more selected from the group consisting of all trans-retinal, 13-cis-retinoic acid, and retinyl palmitate. For example, the metabolome may include one or more selected from the group consisting of all trans-retinal and 13-cis-retinoic acid.

[0046] The formulation may quantify the quantity, level, distribution, distribution ratio, or concentration of the metabolome in a biological sample.

[0047] For example, the formulation may be one that allows for quantifying an increase, for example, an increase in the amount, level, distribution, distribution ratio, or concentration, of one or more selected from the group consisting of all trans-retinal, 13-cis-retinoic acid, and retinyl palmitate, for example, one or more selected from the group consisting of all trans-retinal and 13-cis-retinoic acid.

[0048] The metabolome is quantified by one or more selected from the group consisting of liquid chromatography mass spectrometry (LC / MS), nuclear magnetic resonance spectrometry, ultraviolet spectrometry, infrared spectrometry, fluorescence spectrometry, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry, but is not limited thereto.

[0049] Yet another embodiment of the present invention relates to a kit for predicting preterm birth, comprising a quantitative preparation or a quantitative device for quantifying retinoid metabolome in a biological sample. The retinoid metabolome is as described above.

[0050] The kit may be capable of quantifying an increase, for example, an increase in the amount, level, distribution, distribution ratio, or concentration, of one or more selected from the group consisting of all trans-retinal, 13-cis-retinoic acid, and retinyl palmitate, for example, one or more selected from the group consisting of all trans-retinal and 13-cis-retinoic acid.

[0051] The quantitative device is one or more selected from the group consisting of a liquid chromatography mass spectrometer (LC / MS), a nuclear magnetic resonance spectrometer, an ultraviolet spectrometer, an infrared spectrometer, a fluorescence spectrometer, an ELISA (enzyme-linked immunosorbent assay), and a mass spectrometer, but is not limited thereto.

[0052] Yet another example of the present invention relates to an apparatus, device, or system for predicting or diagnosing premature birth. Specifically, the present invention relates to an apparatus, device, or system capable of performing all or part of the steps of the above-described method for predicting premature birth, method for diagnosing premature birth, or method for providing information for diagnosing premature birth. The apparatus, device, or system may perform all or part of the steps using a composition for predicting premature birth or a kit for predicting premature birth according to an example of the present invention.

[0053] For example, the device, equipment, or system may obtain a biological sample of a test subject, such as a blood sample separated from a test subject pregnant woman, quantify the amount or concentration of the marker metabolome according to the present invention in the sample, compare the amount or concentration with the value of a control group, such as a control group of women who delivered normally, and display a result indicating the presence or absence of a risk of premature birth in the test subject based on the comparison result. Thus, the device, equipment, or system according to one example of the present invention may include a quantification unit that quantifies the retinoid metabolome in a biological sample.

[0054] Specifically, the device, equipment, or system may include an input unit for receiving an input of a biological sample of a test subject; a quantification unit for quantifying a marker metabolome according to the present invention in the sample; a calculation unit for comparing the level of the quantified metabolome with a value of a control group; and a display unit for displaying a result indicating whether or not the test subject is at risk of premature birth based on the comparison result.

[0055] The marker metabolome is as described above, and may include, for example, one or more selected from the group consisting of all trans-retinal, 13-cis-retinoic acid, and retinyl palmitate. As an example, the marker metabolome may include one or more selected from the group consisting of all trans-retinal and 13-cis-retinoic acid.

[0056] When the calculation unit calculates that the level of one or more selected from the group consisting of all trans-retinal, 13-cis-retinoic acid, and retinyl palmitate is higher than that of a control group, the display unit may display that the test subject is at risk of premature birth.

[0057] The biological sample comprises: Pregnant women The biological sample may be a blood sample, and may be, for example, one or more selected from the group consisting of whole blood, plasma, and serum.

[0058] The quantification unit quantifies the metabolome using one or more devices selected from the group consisting of a liquid chromatography mass spectrometer (LC / MS), a nuclear magnetic resonance spectrometer, an ultraviolet spectrometer, an infrared spectrometer, a fluorescence spectrometer, an ELISA (enzyme-linked immunosorbent assay), and a mass spectrometer, but is not limited to these. Effect of the Invention

[0059] By using a retinoid metabolome premature birth marker according to one embodiment of the present invention, premature birth can be predicted early, and premature birth can be predicted non-invasively and with high accuracy. [Brief description of the drawings]

[0060] [Figure 1] 1 is a flowchart showing a process for selecting test subjects according to an embodiment of the present invention. [Diagram 2] RPC positivity showed a clearly demarcated category for PTB compared with TB (Q2=0.072; R2=0.085). [Diagram 3] Scatter plot of the path influence analysis is shown, with the y-axis showing p-value. [Figure 4] FIG. 3 shows retinoid metabolome changes in plasma samples from women who gave birth preterm, as determined by retinoid metabolite analysis (39TB, 20PTB). [Diagram 5] Figure 1 shows ROC curve analysis for metabolome, predictive performance of preterm birth biomarkers using ROC curves of sensitivity and specificity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] The present invention will now be described in more detail with reference to the following examples, although it should be noted that these examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0062] Experimental Materials and Methods

[0063] Example 1. Study subjects Visited Ewha Women's University Medical Center for prenatal checkups and delivery Pregnant women The study recruited 10 asymptomatic singleton fetuses between 16 and 35 weeks and 5 days of gestation. The subjects were divided into a discovery group (10 TB and 11 PTB) for the analysis of metabolomic profiles and a validation group (39 TB and 20 PTB) for the analysis of retinoid metabolome. Pregnant women(asymptomatic singleton pregnant women with asymptomatry) or hospitalization with preterm labor (PTL) and / or premature rupture of membrane (pPROM) Pregnant women It was.

[0064] Patients were diagnosed with preterm labor (PTL) if they had regular uterine contractions and 4 or more contractions within 20 minutes or 8 or more contractions within 60 minutes as detected by electrocardiography. To diagnose premature rupture of the membranes (pPROM), a sterile speculum examination to detect intravaginal amniotic fluid fixation and a nitrazine test to detect membrane rupture were performed. Gestational age was determined using the first day of the last menstrual period and ultrasound examination. Pregnant women When women visited for antenatal checkups or were admitted to the hospital for delivery, blood samples were collected and stored at -80°C until the time of metabolomic analysis.

[0065] This study was approved by the Ewha Women's University Hospital Institutional Review Board (EUMC2018-07-007-010). All experiments were performed in accordance with the approved guidelines, and prior informed consent was obtained from all subjects.

[0066] Example 2. Pregnant women Blood sample preparation 200 μL of plasma samples were precipitated with 600 μL of a solvent mixture (acetonitrile / methanol / acetone; 1:1:1, v / v), mixed on a vortex mixer for 3 min, and stored at −20° C. for 30 min. The precipitated samples were centrifuged at 10,000 g for 10 min at 4° C., after which 600 μl of the supernatant was transferred to another tube and evaporated to dryness under a gentle nitrogen stream. After reconstitution of the residue in 200 μl of 50% methanol, 5 μl (RPC separation mode) of the sample was analyzed by UPLC-LTQ-Orbitrap MS.

[0067] Example 3. Metabolomic profiling by LTQ-Orbitrap MS Metabolomic profiling was performed using an Ultimate 3000 UHPLC system (Thermo Fisher Scientific, San Jose, CA, USA) coupled to an LTQ-OrbitrapVelos Pro hybrid mass spectrometer (Thermo Fisher Scientific) equipped with an electrospray source operated in positive mode (ESI+). TM Reversed-phase separation was performed on a UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm, Waters, Milford, MA, USA) UPLC analytical column. The mobile phase solvents used were 95% water, 5% ACN, and 0.1% formic acid (mobile phase A) and 95% ACN, 5% water, and 0.1% formic acid (mobile phase B). The elution gradient was as follows: 100% mobile phase A from 0 to 3 min; linear increase to 50% mobile phase B from 3 to 10 min; linear increase of mobile phase B from 50 to 90% from 10 to 12 min; linear increase of 100% mobile phase A from 12 to 15 min; re-equilibration to 100% mobile phase A from 15 to 18 min. The column was maintained at 40 °C; the total run time was 18 min. A 5 μl aliquot of each sample was injected for analysis. Samples were maintained at 4° C. in an autosampler during analysis.

[0068] Example 4. Preparation of standard stock solutions for retinoid metabolome validation To analyze the metabolic pathways of TB and PTB, we decided to analyze the retinoid metabolome. Stock solutions of all listed analytical reference standards and internal standards of retinoids for retinol (ROH), retinyl acetate (RAc), retinyl palmitate (RP), all-trans retinal (at-RAL), all-trans retinoic acid (at-RA), 13cis-retinoic acid (13cis-RA), retinyl acetate-D6 (RAc-D6), all-trans retinal-D6 (at-RAL-D6), and all-trans retinoic acid-D6 (at-RA-D6) were homogenously dissolved in methanol to a final concentration of 2000 ppm. Retinol-D6 (ROH-D6) was prepared at a final concentration of 1000 ppm as an exception. The solutions were stored at -20°C until use. Calibration curves for the final quantification of target retinoids were prepared by four-fold serial dilution of such standard stock solutions in the range of 800 to 0.05 ppb. ROH, RAc, at-RAL and at-RA standards were obtained from Cayman Chemicals, Inc. (Ann Arbor, Michigan), and RP and 13cis-RA were obtained from Sigma Aldrich Corp. (St. Louis, Missouri, USA). All four isotope-labeled internal standards mentioned above were purchased from Cambridge Isotopes Laboratories, Inc. (Tewksbury, Massachusetts, USA).

[0069] Example 5. Sample preparation A liquid-liquid extraction method was used to effectively extract the target retinoids in the plasma samples. 200 μL of each plasma sample was added with an isotopically labeled internal standard mixture and vortexed briefly before precipitating serum proteins with 200 μL of acetonitrile. After vortexing for 1 min, an additional 1.2 mL of methyl-tert-butylether was added to each tube and vortexed again for 1 min. The samples were then centrifuged at 13,000 rpm at 4°C for 10 min, and the upper organic layer was finally transferred to a new tube. The transferred supernatant was dried with nitrogen gas at room temperature and the residue was reconstituted in 20 μL of methanol. After vortexing for 5 min, the supernatant was finally transferred to a clean glass MS vial tube and capped for further LC-MS / MS analysis.

[0070] Example 6. LC-MS / MS analysis Chromatographic separation of plasma sample extracts was performed using an Accucore C18 column (2.1 × 100 mm, 2.6 μm particle size, Thermo Scientific TMThe experiments were carried out at a 30°C centrifuge at 100°C for 2 min and the temperature was maintained at 30°C throughout the experiment. The sample injection volume was fixed at 2 μL. The target retinoids were separated within 22 min under a mobile phase composition consisting of 80% acetonitrile (A) and 100% methanol (B), both of which were modified with 0.1% formic acid. The mobile phase system was initially maintained in isosolvent mode at 100% of phase A for 7 min, and the flow rate was linearly increased from 0.2 mL / min to 0.4 mL / min. It was then immediately changed to 100% of phase B and maintained until 15 min, at a flow rate of 0.4 mL / min. For another 2 min, the system was gradually linearly gradient back to 100% phase A, and the flow rate was reduced again to 0.2 mL / min. The conditions were maintained for 5 min to allow for re-equilibration. The column effluent was then analyzed using an Agilent 6460C Triple Quadrupole LC-MS / MS system fitted with an electrospray ionization (ESI) source. The mass spectrometer was operated in cation mode using nitrogen as the nebulizing gas. The temperature of the heated atomizer was set at 300 °C with an ion spray voltage of 4,500. Quantification of retinoids was performed in multiple reaction monitoring (MRM) mode by selecting the precursor ion of [M+H-fatty acid-H2O]+ for retinol (ROH), retinol-D6 (ROH-D6), retinyl acetate (RAc), retinyl acetate-D6 (RAc-D6) and retinyl palmitate (RP) and [M+H]+ for at-RAL, at-RAL-D6, at-RA, 9cis-RA, 13cis-RA and at-RA-D6. The optimized MRM transitions, MS parameters and LC retention times of all the aforementioned analytes are summarized in Table 1. For final quantification, each peak area of ​​the target retinoid was normalized to the response of the corresponding internal standard (0.5 ppm ROH-D6 for ROH; 0.25 ppm RAc-D6 for RAc and RP; 0.25 ppm at-RAL-D6 for at-RAL; 1 ppm at-RA-D6 for at-RA, 9cis-RA, 13cis-RA).

[0071] [Table 1]

[0072] Example 7. Statistical Analysis Baseline characteristics of the study groups were compared using Student's t-test for continuous variables and chi-square test for categorical variables. Pregnant women Multivariate analysis was performed to obtain a list of variables from the variation in metabolic profile in plasma of patients.

[0073] Multivariate analyses were performed using SIMCA-P software v14.0 from Umetrics (Umea, Sweden).

[0074] Pathway influence analysis and heatmap visualization were performed by Metaboanalyst 3.0 (Montreal, QC, Canada), a web-based metabolomics data processing tool and visualizing metabolomics.

[0075] Pathway mapping and chemical similarity analysis were generated by R version 3.2.2, MetaMapp and CytoScape 3.4.0 (Boston, MA, USA).

[0076] For validation using LC-MS / MS, differential metabolomic levels were analyzed using a generalized linear model adjusted for gestational age at sampling time.

[0077] The diagnostic accuracy of PTB was summarized by applying the AUC of the receiver operating characteristic curve. Statistically, p<0.05 was considered significant.

[0078] The Statistical Package for Social Sciences (SPSS, version 2.0 Chicago, IL, USA) and online MEDCALC software were used for statistical analysis.

[0079] Experimental Results

[0080] 1. Clinical characteristics of experimental subjects To confirm the metabolomic signature of preterm birth (PTB) (exploration group; TB, n = 10; PTB, n = 11; Table 2 ) and to validate the targeted metabolome (validation group; TB, n = 39; PTB, n = 20; Table 3 ), we performed a randomized controlled trial of 16- to 40-week 5-day pregnancies. Pregnant women Peripheral blood was collected from the 100-mL donor mice (Figure 1). Figure 1 is a flowchart showing the process of selecting study subjects. The clinical characteristics of the preterm and term women in the exploration group are shown in Table 2, and the clinical characteristics of the preterm and term women in the validation group are shown in Tables 3 and 4.

[0081] Clinical factors such as gestational age at sampling and delivery were significantly different between the preterm and term groups in both the discovery and validation groups (p<0.05, Table 3). Table 3 shows the clinical characteristics of the subjects for validation. White blood cell count and C-reactive protein were significantly different between the two groups in the validation group (p<0.05). Third trimester Pregnant women Liver function enzymes such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and lipid profiles were not significantly different between the preterm and term birth groups (Table 4). There were no cases of chorioamnionitis in the women who delivered at term, but two cases were identified in the women who delivered preterm.

[0082] [Table 2]

[0083] Data are presented as median (interquartile range; IQR). *Mann Whitney test, p < 0.05; †χ 2 test, p < 0.05 GAS: gestational age at sampling; PregBMI: body mass index at delivery; GAD: gestational age at delivery.

[0084]

Table 3

[0085] Data of continuous variables were presented as mean ± SE. *Student’s t-test, p < 0.05; †χ 2 test, p < 0.05 TB: term birth; PTB: preterm birth; GAS: gestational age at sampling; PreBMI: body mass index before pregnancy; PregBMI: body mass index at delivery; WBC: white blood cell; GAD: gestational age at delivery; APGAR: Appearance, Pulse, Grimace, Activity, Respiration, Apgar score

[0086]

Table 4

[0087] Data are presented as mean ± SE. *Student's t-test

[0088] 2. Metabolomic profiling of maternal plasma samples To elucidate the metabolome and metabolic pathways specific to preterm birth, untargeted metabolomic profiling was performed using ultra-high performance liquid chromatography (UPLC) coupled with an LTQ-Orbitrap Velos Pro hybrid mass spectrometer. Parturient women Multivariate analysis was performed to obtain a list of variables from the variations in metabolic profiles in plasma of women. Partial least-squares discriminant analysis (PLS-DA) score plots were applied to identify metabolomic patterns that could be used to distinguish between PTB and TB groups. Figure 2 shows a clear demarcation category for PTB compared with TB in RPC positivity (Q2 = 0.072; R2 = 0.085). As shown in Figure 2, PLS-DA score plots classified plasma metabolomes of preterm (red) and term (blue) women differently. Based on variable importance in projection (VIP) values ​​greater than 1 and p-values ​​less than 0.05, 15 metabolomes out of 235 variable ions were determined. Specific data are shown in Table 5.

[0089] [Table 5]

[0090] 3. Metabolic pathways and preterm birth To better understand how the metabolomic changes in maternal plasma are associated with preterm birth, metabolic pathways were analyzed. Pathway impact analysis was performed based on the KEGG database and MetaboAnalyst 3.0 for comprehensive metabolic data analysis, visualization and interpretation. The results of the impact pathway analysis are shown in Figure 3. Figure 3 shows the scatter plot of the pathway impact analysis, where the y-axis shows the p-value. The x-axis shows the pathway topology analysis, and the y-axis shows the pathway enrichment analysis. Metabolic pathways including retinol metabolism, linoleic acid metabolism, D-arginine and D-ornithine metabolism were shown to be associated with preterm birth.

[0091] 4. Targeted Plasma Metabolome Analysis We targeted the analysis of the metabolome involved in retinoid metabolism to investigate the pathological mechanisms underlying preterm birth. The retinoid metabolome was analyzed using LC-MS (TB, n = 39; PTB, n = 20). Pregnant women The retinoid metabolome was analyzed in plasma from preterm women (39 TB, 20 PTB) with gestational age adjusted. When controlling for gestational age, the retinoid metabolome was significantly altered in PTB compared to TB (Figure 4). Figure 4 shows the altered retinoid metabolome in plasma samples from preterm women (39 TB, 20 PTB) as determined by retinoid metabolite analysis. Retinol levels were significantly decreased in PTB compared to TB, whereas retinyl palmitate, all-trans (AT)-retinal, and 13-cis-retinoic acid (13cis-RA) were significantly increased (p<0.05).

[0092] 5. Prediction performance for preterm birth The diagnostic accuracy of preterm birth was verified by applying the AUC (area under the curve) of the receiver operating characteristic (ROC) curve. The retinoid metabolome showed a significant predictive value of 0.6 or more for predicting preterm birth (p<0.01; Table 6, Figure 5). Figure 5 shows the ROC curve analysis for the metabolome, and the predictive performance of preterm birth biomarkers using the ROC curves of sensitivity and specificity. Among the metabolome, AT-retinal (AUC0.808, 95%CI:0.683-0.933) and 13cis-RA (AUC0.826, 95%CI:0.723-0.930) showed improved predictive ability among the retinol metabolome. The cut-off value, which is the standard for the metabolome concentration value that distinguishes between the preterm birth risk group and the normal group, was derived as the metabolome concentration value equivalent to the highest data value among the values ​​obtained by adding the sensitivity and specificity.

[0093] [Table 6]

[0094] For statistical analysis, ROC (Receiver Operating Characteristics) curve analysis was performed and p<0.05 was considered significant. AUC: Area under the curve; SENS: Sensitivity; SPEC: Specificity; PPV: Positive predictive value; NPV: Negative predictive value; OR: Odds ratio; AT: all trans; RA: Retinoic acid; Cut-off value: critical value

[0095] conclusion The present invention identifies plasma metabolomic profiles and metabolic pathways to support the pathogenetic mechanism of preterm birth, Pregnant women We have elucidated biomarkers for predicting preterm birth. According to the present invention, the metabolomic profile of the preterm birth group was significantly different from that of the full-term birth group, and all-trans-retinal (AT-retinal) and 13-cis-retinoic acid (13cis-RA) showed improved prediction of preterm birth in the retinol metabolome.

[0096] In the present invention, maternal blood was collected during the second trimester of pregnancy or at delivery. Despite adjusting for gestational age, the plasma metabolomic profile of the preterm group was different from that of the term group, and alterations in the retinoid metabolome were associated with preterm birth.

[0097] Vitamin A in blood is transported to plasma in a 1:1 complex with retinol-binding protein RBP. According to the present invention, plasma retinol levels were decreased in PTB compared to TB, whereas RBP levels associated with AT-retinal and 13cis-RA levels were increased in PTB, and these metabolomes were positively correlated. RBP can bind not only retinol but also AT-retinal and 13cis-RA, and can be delivered to target tissues via Stra6 receptor. High levels of certain metabolites of retinoic acid (ATRA and 13cis-RA) can affect gene activity and induce teratogenicity during critical organogenesis and embryonic development periods.

[0098] In summary, the present invention elucidated the metabolomic profile and target metabolomic changes between TB and PTB maternal plasma. In particular, all-trans-retinal (AT-retinal) and 13cis-retinoic acid (13cis-RA) had excellent predictive accuracy for preterm birth.

Claims

1. A method for providing information for predicting preterm birth, comprising a step of quantifying one or more metabolomes selected from the group consisting of all trans-retinal, 13-cis-retinoic acid, and retinyl palmitate in a biological sample.

2. The method of claim 1, further comprising comparing the quantified metabolome level to a control group.

3. The method of claim 2, further comprising predicting that there is a risk of preterm birth if the quantified metabolome level is higher than that of a control group.

4. The method according to claim 3, wherein the control group is normal parturient women.

5. The method of claim 1 , wherein the biological sample is isolated from a pregnant woman.

6. The method of claim 1 , wherein the biological sample is a blood sample.

7. 2. The method of claim 1, wherein the biological sample is one or more selected from the group consisting of whole blood, plasma, and serum.

8. 2. The method of claim 1, wherein the step of quantifying the metabolome is performed by one or more selected from the group consisting of a liquid chromatography mass spectrometer, a nuclear magnetic resonance spectrometer, an ultraviolet spectrometer, an infrared spectrometer, a fluorescence spectrometer, an enzyme-linked immunosorbent assay (ELISA), and a mass spectrometer.

9. 2. The method of claim 1, wherein the preterm birth is not due to liver dysfunction.

10. The method of claim 1 , wherein the biological sample is isolated from a pregnant woman without liver dysfunction.

11. A composition for predicting preterm birth, comprising a preparation capable of quantifying one or more metabolomes selected from the group consisting of all trans-retinal, 13-cis-retinoic acid, and retinyl palmitate in a biological sample.

12. The composition of claim 11 , wherein the biological sample is isolated from a pregnant woman.

13. The composition of claim 11 , wherein the biological sample is a blood sample.

14. The composition of claim 11, wherein the biological sample is one or more selected from the group consisting of whole blood, plasma, and serum.

15. A kit for predicting premature birth, comprising a preparation capable of quantifying one or more metabolomes selected from the group consisting of all trans-retinal, 13-cis-retinoic acid, and retinyl palmitate in a biological sample.

16. 16. The kit of claim 15, wherein the biological sample is isolated from a pregnant woman.

17. The kit of claim 15 , wherein the biological sample is a blood sample.

18. The kit according to claim 15, wherein the biological sample is one or more selected from the group consisting of whole blood, plasma, and serum.

19. an input section for receiving an input of a biological sample to be tested; a quantification unit for quantifying one or more metabolomes selected from the group consisting of all trans-retinal, 13-cis-retinoic acid, and retinyl palmitate in the sample; A computing unit that compares the quantified metabolomic level with a control group; and A system for predicting premature birth comprising a display unit that displays a result indicating whether or not the test subject is at risk of premature birth depending on the comparison result.

20. The system according to claim 19 , wherein when the calculation unit calculates that the metabolome level is higher than that of a control group, the display unit displays that the test subject is at risk of premature birth.

21. 20. The system of claim 19, wherein the biological sample is isolated from a pregnant woman.

22. 20. The system of claim 19, wherein the biological sample is a blood sample.

23. 20. The system of claim 19, wherein the biological sample is one or more selected from the group consisting of whole blood, plasma, and serum.

24. The system according to claim 19, wherein the quantification unit quantifies the metabolome using one or more selected from the group consisting of a liquid chromatography mass spectrometer, a nuclear magnetic resonance spectrometer, an ultraviolet spectrometer, an infrared spectrometer, a fluorescence spectrometer, an ELISA (enzyme-linked immunosorbent assay), and a mass spectrometer.

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