KR102216004B1 - Method for Predicting Premature Birth less than 34 weeks using Alcohol Metabolites levels - Google Patents

Abstract

The present invention relates to a method for predicting premature birth, a composition for predicting premature birth, a kit for predicting premature birth, or a system for predicting premature birth and, more specifically, to predict premature birth by quantifying metabolites in a biological sample. The method for predicting premature birth less than 34 weeks includes the step of quantifying one or more metabolites selected from a group consisting of acetone, ethanol, ethylene glycol, formate, glycolate, isopropanol, methanol, and trimethylamine oxide.

Description

Method for Predicting Premature Birth less than 34 weeks using Alcohol Metabolites levels

The present invention relates to a composition for predicting preterm birth, a kit for predicting preterm birth, a method for predicting preterm birth, a method for providing information for predicting preterm birth, or a system for predicting preterm birth.

Preterm birth (PTB) refers to giving birth at less than 37 weeks. According to statistics from 107 countries in Asia, it occurs at a frequency of about 10.6%. In Korea, the preterm birth rate was about 4.8% in 2006, but it nearly doubled to 7.2% in 2016, and the preterm birth rate is increasing. Preterm birth has a risk of increasing the mortality rate of newborns and children under 5 years of age in the short term, increasing the likelihood of problems such as neurodevelopment, and increasing the prevalence of type 2 diabetes, obesity, and high blood pressure in the long term. .

The mechanisms of preterm birth are not yet clear, and risk factors commonly associated with spontaneous labor include reproductive system infections, multiple pregnancy, bleeding in the second and third trimesters, and previous preterm birth history. In addition, about 75% of preterm births are accompanied by premature labor, amniotic membrane rupture, and related cervical asthenia and chorionitis.

Despite Korea's low birth rate, the preterm birth rate under 37 weeks is steadily increasing, threatening maternal and newborn health, and further incurring economic losses. To overcome premature birth, premature birth has been predicted through fetal fibronectin, CRP measurement, and local neck length measurement, but the sensitivity of the preterm birth rate is still low.

Therefore, when the signs of premature birth come in the early weeks, delaying the delivery weeks through appropriate treatment to increase the maturity of the newborn is a critical factor in the health, quality of life, and cost of the mother and newborn.

JP 2019505815 A US 20140221236 A1

An example of the present invention is to analyze metabolites using H-NMR for early diagnosis of preterm birth to find metabolites that showed significant differences in preterm and term delivery mothers, and based on this, in biological samples of mothers in the middle of pregnancy It is intended to provide a method for predicting preterm birth less than 34 weeks through comparison of marker metabolite levels.

Specifically, acetone, ethanol, ethylene glycol, formate, glycolate, isopropanol, in the preterm delivery group of less than 34 weeks and delivery control of 34 weeks or more. At least one metabolite selected from the group consisting of methanol and trimethylamine N-oxide showed a significant level difference.

An example of the present invention relates to a composition for predicting preterm birth, including an agent capable of quantifying metabolites in a biological sample.

Another example of the present invention relates to a kit for predicting preterm birth, including a preparation for detection of metabolites, a preparation for quantification, or a quantification device in a biological sample.

Another example of the present invention relates to a method for providing information for predicting preterm birth, a method for predicting preterm birth, a method for providing information for diagnosing preterm birth, or a method for diagnosing preterm birth, including quantifying metabolites in a biological sample. .

Another example of the present invention relates to an apparatus, equipment, or system for predicting or diagnosing preterm birth.

Hereinafter, the present invention will be described in more detail.

An example of the present invention relates to a method for providing information for predicting preterm birth, a method for predicting preterm birth, a method for providing information for diagnosing preterm birth, or a method for diagnosing preterm birth, including the step of quantifying metabolites in a biological sample.

In the present specification, the terms "premature birth", "premature birth", "preterm birth", "PTB" and the like have the same meaning, and mean birth of a fetus in less than 37 weeks. Preferably, the preterm birth in the present invention may be a preterm birth of less than 34 weeks with a particularly high risk.

According to the present example, the metabolic marker for premature birth according to the present invention was analyzed using vaginal secretions of mothers who visited the hospital during the second trimester (between 14 weeks and 28 weeks), and premature births less than 34 weeks and more than 34 weeks. A significant difference was shown in the delivery group, and accordingly, the composition for predicting preterm birth according to an example of the present invention can predict preterm delivery of less than 34 weeks.

The complications of preterm infants are similar to those of normal delivery newborns after 34 weeks of pregnancy, and since pulmonary maturation occurs until 34 weeks of pregnancy, newborns born under 34 weeks are likely to fall into fetal respiratory distress syndrome, and medical help is required. Among premature babies born before 34 weeks of pregnancy, babies with a weight of less than 1.5 kg use an incubator, and they come out of the incubator when they are able to control their own body temperature after 34 weeks of pregnancy and when their weight is over 1.8 kg. For this reason, it is very important to predict preterm birth less than 34 weeks gestation.

In the present invention, the term "diagnosing the risk of preterm birth" or "predicting the risk of preterm birth" means whether a pregnant woman is likely to have preterm birth within 34 weeks, whether the likelihood of preterm birth is relatively high, or whether there is a possibility of showing signs of preterm birth. It means to determine whether or not.

The present invention can be used to delay or prevent preterm birth through special and appropriate management for pregnant women with high risk of preterm birth. In addition, the present invention can be used clinically to make treatment decisions by early diagnosis of preterm birth and selecting the most appropriate treatment method.

In this specification, the term "or" is used as a meaning including "and/or" unless the content is clearly specified.

In the present invention, the terms "marker", "diagnosis marker", "diagnosis marker", "diagnosis marker" or "marker" predict premature pregnant women less than 34 weeks and distinguish them from delivery groups over 34 weeks As a reference material, it refers to biological organic molecules that show a significant difference in a sample of a pregnant woman predicted to be prematurely compared to the sample of the control group. According to an example of the present invention, the marker and the like may include metabolites in a biological sample.

The "metabolite" is a substance produced as a result of metabolism in a living body, and can be expressed as a metabolite, an intermediate metabolite, a metabolic intermediate, etc., and the whole metabolome of a small molecule mainly generated during cellular processes Means. For example, according to an example of the present invention, the metabolite is acetone, ethanol, ethylene glycol, formate, glycolate, isopropanol, methanol (methanol), and trimethylamine oxide (trimethylamine oxide) selected from the group consisting of at least one, two or more, three or more, four or more, 5 or more, 6 or more, 7 or more, or 8 The trimethylamine oxide may be trimethylamine N-oxide For example, the metabolite may include at least one selected from the group consisting of ethanol and formate. .

In the present specification, the term "biological sample" refers to a sample isolated or collected from a test subject, or a test subject, for example, a pregnant woman. The biological sample may be a sample derived from a pregnant woman, specifically a sample derived from the vagina of a pregnant woman, for example, a vaginal sample, for example, a vaginal fluid sample.

The biological sample may be isolated from a subject whose cervix length is less than 25 mm. According to the present example, when the metabolite level of the biological sample and the length of the cervix are combined, the probability of predicting premature birth was significantly increased. Specifically, the accuracy of the prediction of preterm birth using the metabolite according to the present invention was significantly increased in subjects with a cervical length of less than 25 mm. Metabolites that increase the accuracy of predicting preterm birth less than 34 weeks in combination with cervical length are acetone, ethanol, ethylene glycol, formate, glycolate, and isopropanol ( isopropanol), methanol (methanol), and trimethylamine oxide (trimethylamine oxide) may be one containing one or more, two or more, or three or more selected from the group consisting of, preferably acetone, isopropanol, and methanol. The cervix length may include at least one selected from the group, and more preferably acetone, The cervix length refers to the length of the cervix, which is a region connected to the upper end of the vagina at the lower end of the uterus, and a person skilled in the art If so, you will be able to clearly understand the length of the cervix by clearly identifying the cervix.

Even if the lower limit of the cervical length is not specified, a person skilled in the art will be able to clearly implement the present invention, but for example, the lower limit of the cervical length is more than 0 mm, more than 0.001 mm, more than 0.01 mm, more than 0.1 mm , Or 1 mm or more, but is not limited thereto.

The method for providing information for predicting preterm birth, a method for predicting preterm birth, a method for providing information for diagnosing preterm birth, or a method for diagnosing preterm birth may further include comparing the level of the quantified metabolite with a mother who delivered at least 34 weeks of gestation. It may be included. In addition, when the level of the compared metabolites is higher than that of the control group delivered at 34 weeks of pregnancy or more, the pregnant woman from which the biological sample has been separated may further include predicting that there is a risk of premature birth.

For example, the metabolites are acetone, ethanol, ethylene glycol, formate, glycolate, isopropanol, methanol, and trimethylamine At least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 or 8 selected from the group consisting of oxides (trimethylamine oxide), and the quantified level of metabolites If it is higher than that of a mother who delivered at least 34 weeks of pregnancy, it may be an additional step of predicting that there is a risk of preterm birth. The trimethylamine oxide may be trimethylamine N-oxide. For example, the metabolite may include one or more selected from the group consisting of ethanol and formate.

According to the present example, the metabolic marker for preterm birth according to the present invention showed a significant difference in the preterm delivery group less than 34 weeks gestation and the delivery group over 34 weeks gestation, thus predicting preterm birth according to an example of the present invention. A method of providing information for, a method for predicting preterm birth, a method for providing information for diagnosing preterm birth, or a method for diagnosing preterm birth may be capable of predicting preterm birth less than 34 weeks gestation.

The method of providing information for predicting preterm birth, a method for predicting preterm birth, a method for providing information for diagnosing preterm birth, or a method for diagnosing preterm birth, further comprises comparing the level of the quantified metabolite with a control group delivered at 34 weeks of gestation or more. It may be included. In addition, when the level of the compared metabolites is higher than the control group delivered at 34 weeks of gestation or more, the pregnant woman from which the biological sample has been separated may further include predicting that there is a risk of preterm delivery at less than 34 weeks of pregnancy. I can.

For example, the metabolites are acetone, ethanol, ethylene glycol, formate, glycolate, isopropanol, methanol, and trimethylamine At least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 or 8 selected from the group consisting of oxides (trimethylamine oxide), and the quantified level of metabolites If the pregnancy is higher than that of the control group at 34 weeks of pregnancy or more, it may be to further include a step of predicting that there is a risk of premature birth at less than 34 weeks of pregnancy. For example, the metabolite may include one or more selected from the group consisting of ethanol and formate.

As used herein, the term "increased" is a marker that appears in a comparative control, for example, an established control or a standard control (eg, a biological sample from a mother who has delivered a normal delivery, or a biological sample from a mother who is pregnant at least 34 weeks) It means a change in quantity, change in level, change in distribution, change in distribution ratio, change in concentration, etc. from the average level of metabolite). Specifically, the distribution ratio means the ratio of the content of the specific metabolite to the total metabolite in the sample as a result of analysis such as nuclear magnetic resonance, or the relative concentration ratio.

For example, the increase is greater than 1 times, 1.1 times or more, 1.15 times or more, 1.2 times or more, 1.25 times or more, 1.3 times or more, 1.35 times or more, 1.4 times or more, 1.45 times or more, 1.5 times or more of the control value, 1.7 times or more, 1.75 times or more, 1.8 times or more, 2 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times or more, 4.5 times or more, 5 times or more, 5.5 times or more, 6 times or more, 6.5 times It may be an increase of more than, 7 times or more, 7.5 times or more, 8 times or more, 9 times or more, 10 times or more, 10.5 times or more, 11 times or more, 12 times or more, 13 times or more, 14 times or more, or 15 times or more. , Even if an absolute increase amount is not specified, those skilled in the art will be able to clearly understand the significantly increased change compared to the control group.

Terms denoting the increase, for example “more”, “higher”, “significantly higher”, “statistically significantly higher”, and the like are used interchangeably as described above. In contrast, the terms “not significantly”, “equal”, “substantially identical”, “substantially unchanged”, etc. are within ±10%, within ±5%, within ±3%, or ±2 of the standard control. It means showing little or no significant change from the standard control value, such as within %.

An example of the present invention relates to a composition for predicting preterm birth, including an agent capable of quantifying metabolites in a biological sample.

The metabolites are as described above, for example, acetone, ethanol, ethylene glycol, formate, glycolate, isopropanol, methanol ), and trimethylamine oxide (trimethylamine oxide) selected from the group consisting of one or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8 types can be included. have. For example, the metabolite may include one or more selected from the group consisting of ethanol and formate.

The formulation may be one that quantifies the quantity, level, distribution, distribution ratio, or concentration of metabolites in a biological sample.

For example, the agent is acetone (acetone), ethanol (ethanol), ethylene glycol (ethylene glycol), formate (formate), glycolate (glycolate), isopropanol (isopropanol), methanol (methanol), and trimethylamine oxide (trimethylamine oxide) at least one, two or more, three or more, four or more, five or more, six or more, seven or more, or eight kinds of quantity, level, distribution, distribution ratio, Alternatively, the increase in concentration may be quantified. For example, the formulation may be one capable of quantifying an increase in the amount, level, distribution, distribution ratio, or concentration of one or more selected from the group consisting of ethanol and formate.

The metabolite may be quantified as one or more selected from the group consisting of nuclear magnetic resonance spectroscopy, chromatography, ultraviolet spectroscopy, infrared spectroscopy, fluorescence spectroscopy, ELISA (enzyme-linked immunosorbent assay), and mass spectrometry, but limited thereto. It does not become.

Another example of the present invention relates to a kit for predicting preterm birth, including a preparation for detection of metabolites in a biological sample, a preparation for quantification, or a quantification device. The metabolites are as described above, for example, acetone, ethanol, ethylene glycol, formate, glycolate, isopropanol, methanol ), and trimethylamine oxide (trimethylamine oxide) selected from the group consisting of one or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8 I can. For example, the metabolite may include one or more selected from the group consisting of ethanol and formate.

The kit includes acetone, ethanol, ethylene glycol, formate, glycolate, isopropanol, methanol, and trimethylamine oxide. 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8 types selected from the group consisting of, e.g., amount, level, distribution, distribution ratio , Or an increase in concentration may be quantified. For example, the kit may be one capable of quantifying an increase in one or more selected from the group consisting of ethanol and formate, for example, an increase in amount, level, distribution, distribution ratio, or concentration.

The quantification device may be one or more selected from the group consisting of nuclear magnetic resonance spectroscopy, chromatography, ultraviolet spectroscopy, infrared spectroscopy, fluorescence spectroscopy, ELISA (enzyme-linked immunosorbent assay), and mass spectrometry, but is not limited thereto. .

According to the examples of the present invention, the metabolic markers for preterm birth according to the present invention showed a significant difference in the preterm delivery group and the delivery group of 34 weeks or more by collecting a sample in the middle of pregnancy and delivering less than 34 weeks. The kit for predicting preterm birth according to an example may be capable of distinguishing between preterm delivery less than 34 weeks and delivery more than 34 weeks from samples of pregnant women collected in the middle of pregnancy.

Another example of the present invention relates to an apparatus, equipment, or system for predicting or diagnosing preterm birth. Specifically, it relates to an apparatus, equipment, or system capable of performing all or part of the above-described method for predicting preterm birth, a method for providing information for predicting preterm birth, a method for diagnosing preterm birth, or a method for providing information for diagnosing preterm birth. The apparatus, equipment, or system may perform all or part of the above steps using a composition for predicting preterm birth or a kit for predicting preterm birth according to an example of the present invention.

For example, the device, equipment, or system obtains a biological sample of a test subject, for example, a vaginal fluid sample isolated from a pregnant woman to be tested in the middle of pregnancy, and determines the amount or concentration of the marker metabolite according to the present invention in the sample. Quantify, and compare the amount or concentration with the numerical value of a control, for example, a control mother who delivered at least 34 weeks of gestation, and display a result indicating the presence or absence of the risk of premature birth of the test subject according to the comparison result. have. Accordingly, the device, equipment, or system may include an input unit for receiving a biological sample of a test subject; A quantification unit for quantifying the marker metabolite according to the present invention among the samples; An operation unit for comparing the quantified level of metabolites with a numerical value of a control group; And a display unit displaying a result indicating whether the test subject is at risk of premature birth according to the comparison result.

The marker metabolites are as described above, for example, acetone, ethanol, ethylene glycol, formate, glycolate, isopropanol, methanol ( methanol), and trimethylamine oxide selected from the group consisting of at least one, two or more, three or more, four or more, five or more, six or more, seven or more, or eight I can. For example, the marker metabolite may include one or more selected from the group consisting of ethanol and formate.

In the operation part, acetone, ethanol, ethylene glycol, formate, glycolate, isopropanol, methanol, and trimethylamine oxide When the level of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8 selected from the group consisting of is calculated to be higher than the control, the display unit It may be that the test subject is marked as having a risk of preterm birth.

When analyzing the level of marker metabolites according to an example of the present invention in a vaginal fluid sample of a pregnant woman in the middle of pregnancy, premature birth less than 34 weeks can be predicted early, and it can be helpful in managing the health of a premature baby. Since it is usually performed, even if the method for predicting preterm birth according to an example of the present invention is additionally performed, there is little increase in test cost, so it can be said that the marketability is very bright. Accordingly, the present invention can provide a new preterm birth prediction method that is superior to the past preterm birth prediction technology, and according to the present invention, it is expected that non-invasive and accurate preterm birth prediction is possible.

1 is a diagram showing the results of confirming metabolites in vaginal fluid of pregnant women using ¹ H NMR-nuclear magnetic resonance spectroscopy.

Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Example 1: Sample collection

Ewha Womans University Mokdong Hospital recruited 63 pregnant women from 2018 to 2019 who were at risk of preterm birth due to early labor or early amniotic membrane rupture or healthy pregnant women in the middle of pregnancy. The study included 5 gestational diabetes, 7 preeclampsia, 5 twin pregnancy, 1 placenta previa, and 1 other underlying disease. During the second trimester of pregnancy, samples were taken from the vaginal support dogs of each pregnant woman with a cotton swab and stored in a -80 °C freezer until analysis. The clinical information of pregnant women was collected on an electronic chart. According to the number of delivery weeks of pregnant women, preterm delivery groups less than 34 weeks (<34 weeks; n=16) and delivery groups over 34 weeks (>=34 weeks, n=27) were classified.

Example 2: Nuclear magnetic resonance spectroscopy analysis

(1) Preparation of vaginal fluid sample

The vaginal liquid sample stored in the -80 °C freezer was dissolved at room temperature and mixed uniformly. A 200uL vaginal liquid sample was mixed with 450uL of 0.2 M sodium phosphate buffer (pH 7.00). After adjusting the pH to 7.00 ± 0.05, centrifugation was performed at 17,000 rpm for 20 minutes at 4 °C, and the final 600 uL was transferred to a 5 mm NMR tube.

(2) Nuclear magnetic resonance spectroscopy analysis

The 1H nuclear magnetic resonance spectrum of the vaginal fluid sample was obtained on a Bruker Avance III HD 800 MHz FT-NMR (Bruker BioSpin Co., Billerica, Massachusetts, Middlesex) spectrometer using a triple resonance 5 mm CPTIC cryogenic probe. To obtain a one-dimensional 1H spectrum of the vaginal fluid sample, relaxation delay (RD) = 2.0s, CPMG echo delay (τ) = 0.2 ms, number of repetitions (n) = 256, dummy scan = 16, loop = 160 and acquisition time (Acq ) = 2.0 s with a Bruker standard 1D 1H T2 filter (Car-Purcell-Meiboom-Gill [CPMG]) pulse sequence was used. Water signal was suppressed at the water peak during RD. Free Induction Decay (FID) was obtained with a spectral width of 20 ppm for 64,000 data points. As a reference sample, an ERETIC (electronic reference to approach concentration in vivo) reference (ER) sample was used, and in the ER sample valine was included as a reference molecule. Thus, ER samples were prepared by mixing 150 μL of vaginal fluid, 12 μL of 100 mM valine, and 438 μL of D2O to a final concentration of 2 mM valine.

(3) 1H nuclear magnetic resonance spectrum and data processing of multivariate analysis

All 1H nuclear magnetic resonance spectra were adjusted using TopSpin software (ver. 3.1, Bruker BioSpin, Rheinstetten, Germany). The spectrum of the vaginal fluid sample was calibrated using the chemical shift of formate at 8.445 ppm. A synthetic electronic reference signal (ERETIC) was used instead of trimethyl silyl propionic acid (TSP) in the vaginal fluid sample. Relative concentration was determined using the ERETIC peak of the vaginal fluid sample and the intensity of the identified metabolites. The ERETIC peak parameters were as follows: ERETIC peak position m = line0.0 ppm, line width = 0.4 Hz, integral = correction2.2e + 008 and correction factor = 1.

The processed nuclear magnetic resonance spectrum was analyzed for identification and quantification of metabolites using Chenomx (version 7.1, Edmonton, Canada, Albert). Individual compounds were identified and quantified using the 800-MHz Chenomx library, and the quantification results were normalized by the total spectral area. Ambiguous peaks due to overlapping or slight shifts were confirmed by spiking experiments.

The quantified data was then imported into SIMCA-P + version 12.0 (Umetrics, Umea, Sweden), and the data were adjusted to unit variance for multivariate statistical analysis. PCA (Principal Components Analysis) was performed to confirm the variation between groups. The quality of the model was described using the R2 and Q2 values. R2 was defined as the ratio of variance of the data described by the model. Q2 was defined as the variance ratio of the data predictable by the model.

(4) statistical analysis

Clinical characteristics and analyzed metabolites were compared using the Mann whitney test. Pearson's correlation was used to derive a correlation between clinical (age, pre-pregnancy BMI, CL, GAB, WBC and CRP) and increased metabolite profile. Metabolites that were significantly increased in the delivery group within 7 days after sample collection compared to the other groups were generated by using Receiver Operating Characteristic (ROC) curve analysis to generate the area under the curve (AUC). Statistically p <0.05 was considered significant. SPSS (version 2.0; Chicago, IL, USA) and online MEDCALC software were used for all statistical analysis.

Example 3: Analysis of metabolites in vaginal fluid

(1) Identification of metabolites in vaginal fluid

The vaginal fluid of pregnant women was analyzed by ¹ H Nuclear Magenetic Resonance spectroscopy ( ¹ H NMR-nuclear magnetic resonance spectroscopy), and 28 metabolites were identified (Table 1 and Fig. 1). In Table 1, the letters in parentheses indicate peak multiplicities: s is a singlet; d is double; t is triplet; dd is the doublet of doublet, m is the multiplets, and q is the quartet. Peaks 1 to 28 in FIG. 1 indicate the following: 1. leucine; 2. valine; 3. isoleucine; 4. isopropanol; 5. ethanol; 6. lactate; 7. alanine; 8. acetate; 9. acetone; 10. glutamate; 11. pyruvate; 12. succinate; 13. aspartate; 14. creatine; 15. choline; 16. glucose; 17. trimethylamine N-oxide; 18. taurine; 19. methanol; 20. glycine; 21. ethylene glycol; 22. glycolate; 23. threonine; 24. tyrosine; 25. histidine; 26. phenylalanine; 27. hypoxanthine; 28. formate.

¹ H chemical shift and relative concentrations of identified metabolites in CVF (CervicoVaginal Fluid) from pregnancy Metabolites Chemical shifts (δ ^One Hppm) Acetate 1.90 (s) Acetone 2.22 (s) Alanine 1.47 (d), 3.78 (q) Aspartate 2.67 (dd), 2.80 (dd), 3.89 (dd) Choline 3.19(s) Creatine 3.02 (s), 3.92 (s) Ethanol 1.17 (t), 3.65 (q) Ethylene glycol 3.66 (s) Formate 8.44 (s) Glucose 3.23 (t), 3.40-3.90 (m), 4.64 (d), 5.23 (d) Glutamate 2.05 (m), 2.12 (m), 2.30-2.38 (m), 3.75 (dd) Glycine 3.55 (s) Glycolate 3.93(s) Histidine 7.11 (s), 7.94 (s) Hypoxanthine 8.18 (s), 8.20 (s) Isoleucine 0.93 (t), 1.00 (d) Isopropanol 1.16 (d), 4.02 (m) Lactate 1.32 (d), 4.10 (q) Leucine 0.95 (dd), 1.65-1.75 (m) Methanol 3.34 (s) Phenylalanine 7.32 (d), 7.37 (t), 7.42 (t) Pyruvate 2.36 (s) Succinate 2.39 (s) Taurine 3.25 (t), 3.41 (t) Threonine 3.58 (d), 4.24 (m) Trimethylamine N-oxide 3.25 (s) Tyrosine 6.89 (d), 7.18 (d) Valine 0.98 (d), 1.03 (d), 2.26 (m), 3.60 (d)

The clinical data of the samples are shown in Table 2. Table 2 compares the characteristics of the preterm delivery group less than 34 weeks and the delivery group more than 34 weeks. There was no difference in blood tests, but there was a significant difference between the two groups in the number of delivery weeks and baby weight.

division <34 weeks (n=16) >=34 weeks (n=27) p value Median IQR Median IQR Age 32.5 4.5 33.0 6.0 0.930 Pre-BMI 21.2 4.2 20.7 3.1 0.474 GAS (weeks) 27.2 8.9 23.0 4.3 0.163 GAD (weeks) 28.3 6.8 39.2 1.6 0.000 Paridity 0.0 1.0 0.0 1.0 0.882 WBC (1x10 ³ /μL) 9.4 5.6 9.6 2.7 0.685 CRP (mg/dL) 0.4 0.6 0.3 0.3 0.317 Cervical length (mm) 3.9 28.8 28.5 5.8 0.052 Birth weight (g) 1210.0 961.0 3150.0 440.0 0.000 Mann Whitney test was used for statistical analysis. Data are presented as median and interquirtile range (IQR).
p<0.05 considered as significant.
Pre-BMI: pre-pregnancy body mass index;
GAS: gestational age at sampling;
GAD: gestational age at delivery

(2) Quantitative analysis of metabolites in vaginal fluid

Table 3 shows the relative concentrations of metabolites according to the 1H nuclear magnetic resonance spectrum of Example 2 and the data processing of the multivariate analysis.

Metabolites <34 weeks (n=16) >=34 weeks (n=27) p value Median IQR Median IQR Acetate 1.174 1.285 1.007 0.770 0.070 Acetone 0.097 0.317 0.031 0.038 0.003 Alanine 0.526 0.199 0.614 0.169 0.010 Aspartate 0.200 0.158 0.269 0.155 0.063 Choline 0.021 0.026 0.012 0.007 0.067 Creatine 0.083 0.071 0.082 0.041 0.940 Ethanol 2.190 4.889 0.766 0.803 0.004 Ethylene glycol 0.189 0.370 0.012 0.021 0.000 Formate 1.234 2.187 0.113 0.195 0.004 Glucose 2.410 6.570 2.030 8.036 0.802 Glutamate 0.676 0.443 1.078 0.775 0.079 Glycine 0.289 0.290 0.548 0.266 0.012 Glycolate 0.167 0.198 0.016 0.015 0.001 Histidine 0.073 0.045 0.079 0.038 0.706 Hypoxanthine 0.061 0.028 0.045 0.043 0.191 Isoleucine 0.163 0.255 0.338 0.114 0.007 Isopropanol 0.076 0.139 0.010 0.012 0.003 Lactate 7.729 8.108 13.377 7.382 0.008 Leucine 0.366 0.311 0.487 0.165 0.018 Methanol 0.673 0.982 0.120 0.096 0.001 Phenylalanine 0.174 0.128 0.242 0.106 0.025 Pyruvate 0.102 0.263 0.200 0.341 0.108 Succinate 0.151 0.387 0.260 0.540 0.980 Taurine 0.446 0.382 0.415 0.159 0.920 Threonine 0.265 0.110 0.281 0.111 0.669 Trimethylamine N-oxide 0.017 0.022 0.005 0.005 0.003 Tyrosine 0.126 0.082 0.153 0.079 0.024 Valine 0.275 0.161 0.376 0.107 0.033 Mann Whitney test was used for statistical analysis. Data are presented as median and interquartile range (IQR).
p<0.05 considered as significant.

As shown in Table 3, among metabolites in vaginal fluid, acetone, ethanol, ethylene glycol, formate, glycolate, isopropanol, methanol , And trimethylamine N-oxide showed a significant increase in the preterm delivery group less than 34 weeks.

Example 4: Verification of metabolic markers for prediction of preterm birth less than 34 weeks

(1) Accuracy analysis of prediction of preterm birth less than 34 weeks

To predict preterm birth less than 34 weeks, ROC (area under the curve) was analyzed in SPSS (ver 26) using quantitative data of each metabolite, and MEDCALC® ( https://www.medcalc.org/) calc/diagnostic_test.php ) The sensitivity, specificity, positive rate, false positive rate, and accuracy were analyzed using web-based software. As shown in Table 4, acetone, ethanol, ethylene glycol, formate, glycolate, isopropanol, methanol, and trimethylamine oxide ( The AUC of trimethylamine oxide) was more than 0.7, which was useful in predicting preterm birth, and the prediction accuracy was all over 70%.

In addition, in the case of acetone, isopropanol, and methanol, when combined with cervical length data, AUC of 0.7 or more and accuracy of 80% or more were found to be useful in predicting preterm birth. In particular, in the case of acetone, when combined with cervical length, premature birth of less than 34 weeks could be significantly predicted. This was a significant increase in both AUC and accuracy compared to the case of predicting preterm birth with cervical length alone (Cervical length).

division AUC 95% CI p value cut-off Sensitivity Specificity Positive Predictive Value Negative
Predictive Value Accuracy lower upper Acetone 0.778 0.628 0.928 0.003 0.045 60.0% 81.8% 75.0% 69.2% 71.4% Ethanol 0.766 0.615 0.918 0.004 1.190 61.1% 80.0% 68.8% 74.1% 72.1% Ethylene glycol 0.854 0.727 0.981 0.000 0.031 75.0% 84.0% 75.0% 84.0% 80.5% Formate 0.764 0.607 0.921 0.004 0.382 70.6% 84.6% 75.0% 81.5% 79.1% Glycolate 0.796 0.622 0.970 0.001 0.044 85.7% 85.7% 75.0% 92.3% 85.7% Isopropanol 0.778 0.613 0.942 0.003 0.028 78.6% 82.8% 68.8% 88.9% 81.4% Methanol 0.813 0.659 0.966 0.001 0.439 91.7% 83.9% 68.8% 96.3% 86.1% TrimethylamineN-oxide 0.778 0.620 0.936 0.003 0.009 66.7% 78.6% 62.5% 81.5% 74.4% Cervical length 0.304 0.093 0.516 0.052 32.2 3.1% 83.3% 42.9% 66.7% 24.3% Acetone +
Cervical length 0.827 0.697 0.957 0.000 0.290 76.5% 88.0% 81.3% 84.6% 83.3% Isopropanol +Cervical length 0.785 0.597 0.974 0.005 0.108 71.4% 87.0% 76.9% 83.3% 81.1% Methanol+Cervical length 0.816 0.639 0.993 0.002 1.055 76.9% 87.5% 76.9% 87.5% 83.8%

(2) Logistic regression analysis

Logistic regression analysis was performed using quantitative data of each metabolite to predict preterm birth less than 34 weeks. Tables 5 and 6 are the results of logistic regression analysis of ethanol and formate levels, respectively, and odds ratios for predicting preterm birth using ethanol and formate levels are about 1.5 times (confidence interval 1.028-2.236), respectively, about It was twice (confidence interval 1.072-3.931). As ethanol and formate concentrations increased, the probability of premature birth increased significantly.

Metabolite β-value SE Odds ratio 95% CI p- value Ethanol 0.416 0.198 1.516 1.028-2.236 0.036 Constant -1.364 0.474 0.256 0.004

Metabolite β-value SE Odds ratio 95% CI p- value Formate 0.719 0.332 2.053 1.072-3.931 0.030 Constant -1.161 0.432 7.220 0.007

Tables 7 to 9 are the results of logistic regression analysis by combining the levels of acetone, isopropanol, and methanol and the length of the cervix to predict premature birth. Specifically, if the length of the cervix is less than 25 mm, it was substituted with 1, and if the length of the cervix was more than 25 mm, it was substituted with 0 to generate a new value in addition to each metabolite value, and logistic regression analysis was performed on preterm birth. The odds ratio of premature birth using acetone and uterine joint length is about 13 times (confidence interval, 2.915-56.243), and the odds ratio using isopropanol and cervical length is about 10 times (confidence interval 2.349 -46.218), and the odds ratio using methanol and cervical length was about 8 times (confidence interval: 1.945-30.061). In pregnant women with a cervix length of less than 25 mm, as the concentration of acetone, isopropanol, and methanol increased, the probability of premature birth was significantly increased.

Metabolite β-value SE Odds ratio 95% CI p- value Acetone and
Cervical length 2.550 0.755 12.805 2.915-56.243 0.001 Constant -1.837 0.569 0.159 0.001

Metabolite β-value SE Odds ratio 95% CI p- value Isopropanol and
Cervical length 2.344 0.760 10.419 2.349-46.218 0.002 Constant -1.748 0.572 0.174 0.002

Metabolite β-value SE Odds ratio 95% CI p- value Methanol and
Cervical length 2.034 0.698 7.646 1.945-30.061 0.004 Constant -2.298 0.702 0.100 0.001

Claims (35)

In biological samples, acetone, ethanol, ethylene glycol, formate, glycolate, isopropanol, methanol, and trimethylamine oxide ), comprising the step of quantifying one or more metabolites selected from the group consisting of, information providing method for predicting premature birth less than 34 weeks. The method of claim 1, further comprising comparing the level of the quantified metabolite to a control. The method of claim 2, further comprising predicting that there is a risk of premature birth when the level of the quantified metabolite is higher than that of the control group. The method of claim 3, wherein the control group is a mother who delivered at least 34 weeks. The method of claim 1, wherein the biological sample is isolated from a pregnant woman. The method of claim 1, wherein the biological sample is isolated from a subject having a cervical length of less than 25 mm. The method of claim 6, wherein the metabolite is at least one selected from the group consisting of acetone, isopropanol, and methanol. The method of claim 1, wherein the biological sample is a vaginal fluid sample. According to claim 1, wherein the step of quantifying the metabolite is one selected from the group consisting of nuclear magnetic resonance spectroscopy, chromatography, ultraviolet spectroscopy, infrared spectroscopy, fluorescence spectroscopy, ELISA (enzyme-linked immunosorbent assay), and mass spectrometer. The method that is carried out above. In biological samples, acetone, ethanol, ethylene glycol, formate, glycolate, isopropanol, methanol, and trimethylamine oxide A composition for predicting preterm birth less than 34 weeks, comprising a formulation capable of quantifying at least one metabolite selected from the group consisting of). 11. The composition of claim 10, wherein the agent is capable of quantifying an increase in the level of the metabolite. The composition of claim 11, wherein the increase is compared to the level of metabolites in the biological sample of the control group. The composition of claim 12, wherein the control group is a mother who has delivered at least 34 weeks. The composition of claim 10, wherein the biological sample is isolated from a pregnant woman. The composition of claim 10, wherein the biological sample is isolated from a subject having a cervical length of less than 25 mm. The composition of claim 15, wherein the metabolite is at least one selected from the group consisting of acetone, isopropanol, and methanol. The composition of claim 10, wherein the biological sample is a vaginal fluid sample. The method of claim 10, wherein the metabolite is quantified by one or more selected from the group consisting of nuclear magnetic resonance spectroscopy, chromatography, ultraviolet spectroscopy, infrared spectroscopy, fluorescence spectroscopy, ELISA (enzyme-linked immunosorbent assay), and mass spectrometry. Which, composition. In biological samples, acetone, ethanol, ethylene glycol, formate, glycolate, isopropanol, methanol, and trimethylamine oxide A kit for predicting preterm birth less than 34 weeks, comprising a preparation for detection of one or more metabolites selected from the group consisting of, a preparation for quantification, or a quantification device. The kit of claim 19, wherein the kit quantifies the increase in the level of the metabolite. The kit of claim 20, wherein the increase is compared to the level of metabolites in the biological sample of the control group. The kit of claim 21, wherein the control group is a mother who has delivered at least 34 weeks. The kit of claim 19, wherein the biological sample is isolated from a pregnant woman. The kit of claim 19, wherein the biological sample is isolated from a subject having a cervical length of less than 25 mm. The kit of claim 24, wherein the metabolite is at least one selected from the group consisting of acetone, isopropanol, and methanol. The kit of claim 19, wherein the biological sample is a vaginal fluid sample. The kit of claim 19, wherein the quantification device is at least one selected from the group consisting of a nuclear magnetic resonance spectroscopy, chromatography, ultraviolet spectroscopy, infrared spectroscopy, fluorescence spectroscopy, ELISA (enzyme-linked immunosorbent assay), and mass spectrometer. An input unit for receiving a biological sample to be tested;
In the sample, acetone, ethanol, ethylene glycol, formate, glycolate, isopropanol, methanol, and trimethylamine oxide ) A quantification unit for quantifying at least one metabolite selected from the group consisting of;
An operation unit comparing the quantified level of metabolites with a control group; And
A system for predicting preterm birth less than 34 weeks, including a display unit for displaying a result indicating whether the test subject is at risk of preterm birth according to the comparison result. 29. The system of claim 28, wherein when the operation unit calculates that the level of the metabolite is higher than the control, the display unit indicates that the test subject is at risk of premature birth. The system of claim 28, wherein the control group is a mother who has delivered at least 34 weeks. The system of claim 28, wherein the biological sample is isolated from a pregnant woman. The system of claim 28, wherein the test subject has a cervical length of less than 25 mm. The system of claim 32, wherein the metabolite is at least one selected from the group consisting of acetone, isopropanol, and methanol. The system of claim 28, wherein the biological sample is a vaginal fluid sample. The method of claim 28, wherein the quantification unit comprises one or more metabolites selected from the group consisting of nuclear magnetic resonance spectroscopy, chromatography, ultraviolet spectroscopy, infrared spectroscopy, fluorescence spectroscopy, ELISA (enzyme-linked immunosorbent assay), and mass spectrometry. The system of quantification.

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