Biomarker for predicting preterm birth and analysis method therefor

Abstract

Provided are a novel biomarker for predicting preterm birth and an analysis method therefor. This biomarker for predicting preterm birth is at least one selected from the group consisting of oleic acid, myristic acid, lauric acid, margaric acid, and batyl alcohol in amniotic fluid.

Description

Biomarkers for predicting preterm birth and methods for analyzing same

[0001] The present invention relates to biomarkers for predicting preterm birth and methods for analyzing the same.

[0002] Preterm birth (premature birth) generally refers to delivery after 22 weeks or more but less than 37 weeks of gestation. Preterm birth may affect the health and development of the newborn. Therefore, predicting preterm birth is important because if a high risk of preterm birth can be identified or determined during pregnancy, prevention and countermeasures can be implemented early. Examples of biomarkers for predicting preterm birth include those described in Patent Document 1.

[0003] Special Table 2021-501343

[0004] However, there are still few biomarkers that can predict preterm birth, so there is a need to develop alternative new biomarkers.

[0005] An object of the present invention is to provide a novel biomarker for predicting preterm birth and an analytical method therefor.

[0006] The analytical method for predicting preterm birth according to the first aspect of the present invention comprises measuring at least one component selected from the group consisting of oleic acid, myristic acid, lauric acid, margaric acid and batyl alcohol in amniotic fluid.

[0007] According to the biomarker for predicting preterm birth and the method for analyzing the same of the first aspect, it is possible to predict preterm birth in a subject.

[0008] 1. First Embodiment The analytical method of the first embodiment of the present invention is an analytical method for predicting preterm birth, and includes a step of measuring at least one component selected from the group consisting of oleic acid, myristic acid, lauric acid, margaric acid, and batyl alcohol in amniotic fluid. In other words, this analytical method uses at least one component selected from the group consisting of oleic acid, myristic acid, lauric acid, margaric acid, and batyl alcohol as a biomarker for predicting preterm birth.

[0009] In the analysis method of the first embodiment, first, as a preparation step, amniotic fluid is collected from a pregnant woman who is a subject by a known method such as using a puncture needle.

[0010] Next, in the measurement step, amniotic fluid is used as a measurement sample and at least oleic acid, myristic acid, lauric acid, margaric acid, or batyl alcohol (3-octadecyloxy-1,2-propanediol) is measured. Only one of these components may be measured, or multiple components may be measured.

[0011] The measurement method is not limited as long as it can detect the amount of the above-mentioned component (biomarker), and examples thereof include gas chromatography, liquid chromatography, mass spectrometry, immunological analysis, spectrophotometry, electrochemical analysis, and combinations thereof. From the viewpoint of being able to measure the amount of biomarker in amniotic fluid more reliably and simply, preferred are gas chromatography, liquid chromatography, mass spectrometry, and combinations thereof, more preferred are gas chromatography, mass spectrometry, and combinations thereof, and most preferred is gas chromatography mass spectrometry. Examples of mass spectrometers used in mass spectrometry include triple quadrupole, Q-TOF, TOF-TOF, ion trap, and ion trap time-of-flight.

[0012] When detecting amniotic fluid using the above-described measurement method, it is preferable to perform a predetermined pretreatment method on the amniotic fluid. This removes impurities from the amniotic fluid and makes it easier to detect target components, thereby enabling accurate measurement of the amount of biomarkers. The pretreatment method can be appropriately determined depending on the measurement device used in the measurement method, and may be, for example, in accordance with the manual recommended by the measurement device. Specifically, when gas chromatography, liquid chromatography, mass spectrometry, or the like is employed, examples of the pretreatment method include protein removal treatment, hydrophilic substance removal treatment, and derivatization treatment (silylation, esterification, etc.). Other treatments such as washing, purification, and drying may also be performed as appropriate. These treatments may be used alone or in combination.

[0013] Among the biomarkers to be measured, it is preferable to measure at least one selected from oleic acid, myristic acid, and lauric acid, as this is statistically significant and allows for more accurate prediction of preterm birth; it is even more preferable to measure at least one selected from oleic acid and myristic acid, and it is most preferable to measure oleic acid.

[0014] In this analysis method, after the measuring step, a determination step may be performed in which the risk of preterm birth is determined based on the measured value (for example, peak area value of a chromatogram, concentration, etc.).

[0015] An example of the determination step is a method in which a biomarker measurement value is compared with a predetermined threshold value, and if the measurement value exceeds the predetermined threshold value, the risk of preterm birth is determined to be high. In this case, the predetermined threshold value may be set in advance based on biomarker measurement data obtained from amniotic fluid of multiple subjects who have delivered preterm and multiple subjects who have delivered full-term. Another example is a method in which amniotic fluid is periodically collected from the same subject, biomarkers are measured, and the latest biomarker measurement value is compared with previous biomarker measurements for the same subject to determine a high risk of preterm birth. Specifically, if the measurement value at a certain stage of pregnancy is significantly higher than the measurement value at an earlier stage of pregnancy, the risk of preterm birth is determined to be high. Note that the determination step may involve performing a known correction on the measurement value, and the above-mentioned determination may be made using the corrected value.

[0016] If it is determined that there is a high risk of preterm birth, it is preferable to take steps to prepare for preterm birth or to prevent preterm birth.

[0017] According to the biomarkers and analytical methods of the present invention, prediction can be made from amniotic fluid using known measurement methods, and therefore preterm birth can be predicted easily. In particular, by using at least one method selected from gas chromatography and mass spectrometry, the biomarkers of the present invention can be measured accurately and easily, and therefore preterm birth can be predicted accurately and easily.

[0018] 2. Aspects It will be appreciated by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0019] (Item 1) In one embodiment, the biomarker for predicting preterm birth may include at least one selected from the group consisting of oleic acid, myristic acid, lauric acid, margaric acid, and batyl alcohol in amniotic fluid.

[0020] (Item 2) In one embodiment, the method for analyzing a biomarker for predicting preterm birth may measure at least one component selected from the group consisting of oleic acid, myristic acid, lauric acid, margaric acid, and batyl alcohol in amniotic fluid.

[0021] (Item 3) In the analytical method according to item 2, the components may be measured using at least one method selected from gas chromatography and mass spectrometry.

[0022] The present invention will now be described in detail with reference to examples and comparative examples, but the scope of the present invention is not limited thereto.

[0023] Example 1: Amniotic fluid samples (n=3) collected from pregnant women who delivered preterm and those (n=3) collected from pregnant women who delivered full-term were used as samples. Approximately 400 components, including organic acids, sugars, nucleic acids, fatty acids, and amino acids, were measured using a gas chromatograph mass spectrometer (GCMS-TQ8040 NX, manufactured by Shimadzu Corporation; the system used was the Smart Metabolites Database). The peak area values ​​of the chromatograms for each measured component were subtracted by the area value measured for a blank (a sample consisting of water) and corrected using an internal standard (2-isopropylmalic acid).

[0024] Before analysis, the samples were pretreated. Specifically, a 2.5:1:1 mixed solvent of methanol, water, and chloroform was added to the amniotic fluid for protein removal, followed by shaking and recovery of the supernatant. Next, ultrapure water was added to the supernatant for hydrophilic substance removal, followed by centrifugation and recovery of the supernatant, which was then dried in a freeze-dryer. Next, a 20 mg / mL methoxyamine / pyridine solution and a silylating agent (MSTFA: N-methyl-N-trimethylsilyltrifluoroacetamide) were added to the dried supernatant for derivatization, followed by shaking. The pretreated sample was then loaded into a gas chromatograph mass spectrometer. The analytical conditions were as follows:

[0025] [Gas chromatograph section] Column: BPX5 (30 m x 0.25 mm I.D. df = 0.25 μm) Insert: Split insert with wool Vaporizer temperature: 280°C Open temperature: 100°C (4 min), increased at 10°C / min, 320°C (11 min) Injection mode: Split (10:1) Carrier gas control: Constant linear velocity (39.0 cm / sec) Injection volume: 1 μL [Mass spectrometer section] Mass spectrometer: Triple quadrupole Interface temperature: 280°C Ion source temperature: 200°C Measurement mode: Scan, MRM Loop time: 0.4 sec

[0026] Volcano analysis was performed by integrating the results of approximately 400 measured components with 16 delivery data items (newborn weight, DNA content, ddPCR results, amniocentesis birth interval, CRP at amniocentesis, WBC at amniocentesis, WBC at amniocentesis (ex <4d after steroid), BT at WBC at amniocentesis, AMF IL-6, AMF Cre, AP1, AP2, UA ph, UA BE, UA lactate, BW).

[0027] As a result, it was found that the five components shown in the table below were specifically found in higher concentrations in the amniotic fluid of pregnant women who had given birth prematurely compared to other components. Therefore, it was found that measuring the amounts of these five components in amniotic fluid can predict premature birth.

[0028]

Claims

1. A biomarker for predicting preterm birth in amniotic fluid, comprising at least one selected from the group consisting of oleic acid, myristic acid, lauric acid, margaric acid and batyl alcohol.

2. An analytical method for predicting preterm birth, which comprises measuring at least one component selected from the group consisting of oleic acid, myristic acid, lauric acid, margaric acid and batyl alcohol in amniotic fluid.

3. The analytical method according to claim 2, wherein the components are measured using at least one method selected from gas chromatography and mass spectrometry.

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