JP2019027885A - Diagnostic biomarker of onset risk of pregnancy diabetes mellitus - Google Patents

Abstract

To provide a biomarker for judgement of an onset risk of a pregnancy diabetes mellitus which is detected in a biological specimen collectable by a method having a relatively less burden on the pregnant woman who is a subject and capable of accurately judging whether or not the risk for developing a pregnancy diabetes mellitus is high by a pregnant woman at a relatively initial stage after the pregnancy.SOLUTION: It is possible to determine whether or not the content of ethanolamine or methionine in the urine collected from a pregnant woman who is not developing a pregnancy diabetes mellitus is low as compared with a subject who does not develop the pregnancy diabetes mellitus, and whether or not the pregnant woman incurs a risk of developing the pregnancy diabetes mellitus by analysing whether or not the content of 1,3-diphosphoglycerate, shikimate-3-phosphate or N-acetyl-L-alanine is high or low as compared with the subject.SELECTED DRAWING: None

Description

  The present invention relates to a group of metabolites of [Group A] and [Group B] described later in urine collected from a pregnant woman who has not developed gestational diabetes (hereinafter collectively referred to as “the present group of metabolites”). Including a step (a) of measuring the concentration of one or more metabolites selected from 1) or 2) selected from the metabolite group of this case The present invention relates to a biomarker for determining the risk of developing gestational diabetes, comprising more than one kind of metabolite.

  Gestational diabetes mellitus (GDM) is a state of symptomatic glucose metabolism that has developed during pregnancy and has not led to diabetes. It has been reported that GDM is associated with various complication risks in a mother, embryo or fetus such as a giant, premature birth, preeclampsia (Non-patent Document 1). GDM also affects the growth of children after childbirth and has been reported to cause, for example, obesity and metabolic abnormalities (Non-Patent Documents 2 to 4). For this reason, in Japan, almost all pregnant women have a GDM screening, that is, a random blood glucose level test and a 50 g glucose challenge test (GCT) for 24 to 28 weeks of pregnancy, or the second random blood glucose level. Implementation with value testing is recommended. For pregnant women who are positive in GDM screening, a 75 g oral glucose tolerance test (OGTT) is further performed to diagnose GDM (Non-patent Document 5).

  In order to manage the maternal and fetal health of pregnant women, in addition to early detection of GDM, it is important to determine the risk of developing GDM and prevent the onset of GDM. In recent years, it has been reported that diabetes and glucose metabolism abnormality are detected at an early stage using metabolome analysis (Non-Patent Documents 6 and 7). In addition, in order to identify metabolites related to GDM, it has been reported that amniotic fluid, urine, or blood was collected from pregnant women and metabolic profiling analysis was performed (Non-Patent Documents 8 to 10). In addition, sex hormone-binding globulin (SHBG) and placental growth factor (PIGF) are reported to be biomarkers that can determine the onset risk of gestational diseases such as preeclampsia and gestational diabetes (Patent Document 1). ). However, it has not been known so far that this metabolite group is a biomarker that can determine the risk of developing GDM.

Special table 2007-506979 gazette

HAPO Study Cooperative Research Group et al. N Engl J Med. 2008; 358 (19): 1991-2002. Ouyang F. et al. Obesity (Silver Spring). 2016; 24 (4): 938-46. Kim SY. Et al. Exp Diabetes Res. 2011; 2011: 541308. Boney CM. Et al. Pediatrics. 2005; 115 (3): e290-6. Minakami H. et al. J Obstet Gynaecol Res. 2011; 37 (9): 1174-97. Wang TJ. Et al. Nat Med. 2011; 17 (4): 448-53. Menni C. et al. Diabetes. 2013; 62 (12): 4270-6. Graca G. et al. J Proteome Res. 2010; 9 (11): 6016-24. Graca G. et al. Mol Biosyst. 2012; 8 (4): 1243-54. Diaz SO. Et al. J Proteome Res. 2011; 10 (8): 3732-42.

  The subject of the present invention is detected in a biological sample that can be collected by a method with a relatively low burden on a pregnant woman who is a subject, and a pregnant woman at a relatively early stage after pregnancy has a high risk of developing gestational diabetes It is an object of the present invention to provide a biomarker for determining the risk of developing gestational diabetes that can be accurately determined.

  In order to solve the above-mentioned problems, the inventors of the present invention have found that pregnant women who have developed gestational diabetes at the time of delivery (preGDM group) and pregnant women who have not developed gestational diabetes (control group) have not developed gestational diabetes. Initially, metabolomic analysis was performed using urine collected in advance, and this metabolite group was found to be a biomarker that can accurately determine the risk of developing gestational diabetes, leading to the completion of the present invention. .

That is, the present invention is as follows.
[1] A method for determining the risk of developing gestational diabetes, comprising the following steps (a) to (c):
(A) a step of measuring the concentration of one or more metabolites selected from the following [Group A] and [Group B] in urine collected from a pregnant woman who has not developed gestational diabetes;
[Group A]
Ethanolamine, methionine [Group B]
1,3-diphosphoglyceric acid, shikimic acid-3-phosphate, N-acetyl-L-alanine (b) The metabolism in the control person who does not develop gestational diabetes is determined according to the concentration of the metabolite measured in step (a). Comparing with the concentration of the substance;
(C) When the metabolite measured in step (a) is a metabolite of [Group A], when the metabolite concentration measured in step (a) is lower than the metabolite concentration in the control, Pregnant women have a high risk of developing gestational diabetes,
When the metabolite measured in step (a) is a metabolite of [Group B], when the metabolite concentration measured in step (a) is higher than the metabolite concentration in the control, the pregnant woman Assessing the high risk of developing gestational diabetes;
[2] The determination method according to [1], wherein the pregnant woman is a pregnant woman of 13 to 22 weeks of pregnancy.
[3] In step (c),
When the ethanolamine concentration measured in step (a) is reduced by at least 44% compared to the ethanolamine concentration in the control, the pregnant woman evaluates that the risk of developing gestational diabetes is high;
When the concentration of methionine measured in step (a) is reduced by at least 51% compared to the concentration of methionine in the control, the pregnant woman evaluates that the risk of developing gestational diabetes is high;
When the concentration of 1,3-diphosphoglyceric acid measured in step (a) is increased by at least 2.5 times compared to the concentration of 1,3-diphosphoglyceric acid in the control, the pregnant woman has gestational diabetes. Assessed that the risk of developing is high,
A pregnant woman develops gestational diabetes when the concentration of shikimic acid-3-phosphate measured in step (a) is increased at least 1.5 times compared to the concentration of shikimic acid-3-phosphate in the control Assess that the risk is high,
A pregnant woman develops gestational diabetes when the concentration of N-acetyl-L-alanine measured in step (a) is increased by at least 1.5 times compared to the concentration of N-acetyl-L-alanine in the control. The determination method according to [1] or [2], wherein the risk is evaluated as high.
[4] The determination method according to any one of [1] to [3], wherein the metabolite is a metabolite containing ethanolamine or 1,3-diphosphoglycerate.
[5] A biomarker for determining the risk of developing gestational diabetes, comprising one or more metabolites selected from the following [Group A] and [Group B],
When the metabolite is a metabolite of [Group A], the concentration of the metabolite in urine collected from a pregnant woman who has a high risk of developing gestational diabetes is the concentration of the metabolite in a control person who does not develop gestational diabetes Lower than
When the metabolite is a [Group B] metabolite, the concentration of the metabolite in urine collected from a pregnant woman who has a high risk of developing gestational diabetes is the concentration of the metabolite in a control person who does not develop gestational diabetes Higher than
The determination biomarker.
[Group A]
Ethanolamine, methionine [Group B]
1,3-diphosphoglyceric acid, shikimic acid-3-phosphate, N-acetyl-L-alanine [6] The concentration of ethanolamine in urine collected from pregnant women at high risk of developing gestational diabetes A 44% decrease compared to the ethanolamine concentration in controls who do not develop diabetes,
The concentration of methionine in urine collected from pregnant women who are at high risk of developing gestational diabetes is reduced by 51% compared to the concentration of methionine in controls who do not develop gestational diabetes;
The concentration of 1,3-diphosphoglyceric acid in urine collected from pregnant women at high risk of developing gestational diabetes is 2 compared to the concentration of 1,3-diphosphoglyceric acid in controls that do not develop gestational diabetes. .5 times increase,
The concentration of shikimate-3-phosphate in urine collected from pregnant women at high risk of developing gestational diabetes is 1.5 compared to the concentration of shikimate-3-phosphate in controls who do not develop gestational diabetes Doubled,
The concentration of N-acetyl-L-alanine in urine collected from pregnant women at high risk of developing gestational diabetes is 1.5 compared to the concentration of N-acetyl-L-alanine in controls who do not develop gestational diabetes. The biomarker for determination according to [5] above, wherein the biomarker is doubled.
[7] The determination biomarker according to [5] or [6] above, wherein the metabolite is a metabolite containing ethanolamine or 1,3-diphosphoglycerate.

As another embodiment of the present invention,
A method of collecting data for diagnosing the risk of developing gestational diabetes, comprising the steps (a) to (c);
A treatment that includes the steps (a) to (c) and further prevents the onset of gestational diabetes for a pregnant woman evaluated (diagnosed) as having a high risk of developing gestational diabetes in the step (c) (for example, , A method for diagnosing the risk of developing gestational diabetes, optionally including a step (p) of giving a diet, exercise, etc .;
One or more metabolites selected from this metabolite group for use as a biomarker in the method for determining (diagnosing) the risk of developing gestational diabetes, wherein the metabolite is the metabolism of the above [Group A] If the substance is a substance, the concentration of the metabolite in urine collected from a pregnant woman who is at high risk of developing gestational diabetes is lower than the concentration of the metabolite in a control person who does not develop gestational diabetes, and the metabolite is When it is a metabolite of [Group B], the concentration of the metabolite in urine collected from a pregnant woman who is at high risk of developing gestational diabetes is compared with the concentration of the metabolite in a control person who does not develop gestational diabetes, High, said metabolite;
Can be mentioned.

  According to the present invention, since pregnant women who have not yet developed gestational diabetes can acquire highly accurate diagnostic data useful for diagnosing whether the risk of developing gestational diabetes is high or low, for example, the risk of developing gestational diabetes Pregnancies with high gestational diabetes can be prevented from undergoing gestational diabetes by applying treatment (for example, diet therapy or exercise) to prevent its onset before the onset of gestational diabetes. In addition, preventing the development of gestational diabetes is complications due to gestational diabetes (eg preeclampsia, hypoglycemia, infertility, pregnancy hypertension syndrome, premature birth, hyperamniotic fluid, etc.); miscarriage, malformation Complications in embryos or fetuses, such as giant infants, hypertrophic cardiomyopathy; complications in neonates such as respiratory distress syndrome, hypoglycemia, hypercalcemia, hypermagnesemia, hyperbilirubinemia) It also helps reduce the risk that pregnant women after childbirth will subsequently suffer from type 2 diabetes.

It is a figure which shows the result of having measured the ethanolamine density | concentration (FIG. 1A) and 1, 3- DPG density | concentration (FIG. 1B) in the urine in a preGDM group and a control group. It is a figure which shows the result of having measured the shikimic acid-3-phosphate density | concentration (FIG. 2A), N-acetyl- L-alanine density | concentration (FIG. 2B), and methionine density | concentration (FIG. 2C) in the preGDM group and a control group. is there.

As a method for determining the risk of developing gestational diabetes of the present invention, the following [Group A] and [A] in urine collected from a pregnant woman who has not developed gestational diabetes (hereinafter sometimes referred to as “subject”): Step (a) of measuring the concentration of one or more metabolites selected from Group B (hereinafter sometimes referred to as “metabolite concentration in a subject”) and quantifying as necessary (Step (a)) (B) comparing the “metabolite concentration in the subject” measured in step (b) with the concentration of the metabolite in the control person who does not develop gestational diabetes (hereinafter sometimes referred to as “metabolite concentration in the control person”); When the metabolite measured in step (a) is a metabolite of the following [Group A], the “metabolite concentration in the subject” measured in step (a) is “metabolite concentration in the control”. , The subject (pregnant woman) evaluates that the risk of developing gestational diabetes is high, and if the metabolite measured in step (a) is a metabolite of [B group] below, When the “metabolite concentration in the subject” measured in a) is higher than the “metabolite concentration in the control”, the step (c) evaluates that the subject (pregnant woman) has a high risk of developing gestational diabetes; If it is a method (henceforth "this determination method" may be called hereafter) including these process (a)-(c), it will not restrict | limit in particular, This determination method assists the diagnosis of the onset risk of gestational diabetes by a doctor. The method does not include a diagnostic action by a doctor.
[Group A]
Ethanolamine, methionine
[Group B]
1,3-diphosphoglycerate (1,3-diphosphoglycerate; 1,3-DPG), shikimate-3-phosphate, N-acetyl-L-alanine (N-Acetyl-L) -alanine)

  In addition, as a biomarker for determining the risk of developing gestational diabetes according to the present invention, determining the risk of developing gestational diabetes comprising one or more metabolites selected from the above [Group A] and [Group B] In the case of a biomarker for use in (diagnosis), where the metabolite is a metabolite of [Group A], the concentration of the metabolite in urine collected from a pregnant woman who has a high risk of developing gestational diabetes is In the urine collected from a pregnant woman who is low in comparison with the concentration of the metabolite in the control who does not develop gestational diabetes, and the metabolite is a metabolite of the above [Group B], the risk of developing gestational diabetes is high. The concentration of the metabolite is higher than the concentration of the metabolite in a control subject who does not develop gestational diabetes. It not particularly limited as long as it is sometimes referred to as marker ").

  The test subject may be any pregnant woman who does not develop gestational diabetes, and is in the early stage of pregnancy (usually 0 to 15 weeks of pregnancy), the second half of pregnancy (usually 16 to 27 weeks of pregnancy), and the second half of pregnancy (usually pregnancy). 28-39 weeks) may be used, but since gestational diabetes often develops in the second trimester, a pregnant woman in the first trimester or the second trimester is preferable. Such an early pregnancy is preferably 4 to 15 weeks of pregnancy, more preferably 8 to 15 weeks of pregnancy, and further preferably 13 to 15 weeks of pregnancy. Moreover, as the said mid pregnancy, the pregnancy 16-25 weeks are preferable, the pregnancy 16-24 weeks are more preferable, and the pregnancy 16-22 weeks are further more preferable. Therefore, as the test subject, a pregnant woman of 4 to 25 weeks of pregnancy is preferable, a pregnant woman of 8 to 24 weeks of pregnancy is more preferable, and a pregnant woman of 13 to 22 weeks of pregnancy is more preferable.

  In the present specification, “gestational diabetes” means a state of sugar metabolism abnormality that has occurred during pregnancy and has not led to diabetes, and more specifically, a fasting blood glucose level during pregnancy is 92 mg / Oral glucose tolerance test (OGTT) using 75 g of glucose (Glucose; Glc) or partial hydrolysate of starch (PHS) during pregnancy. In this case, it means that the blood glucose level is 153 mg / dL or more and less than 200 mg / dL in 2 hours after the implementation. Thus, gestational diabetes does not include diabetes that occurred before or during pregnancy or pregnancy with diabetes. Specifically, this diabetes refers to a state in which the fasting blood glucose level before pregnancy or during pregnancy is 126 mg / dL or more; when the OGTT is performed before or during pregnancy, It means a state in which the value is 200 mg / dL or more; or a state in which the glycated hemoglobin (HbA1c) value is 6.5% or more before or during pregnancy. In addition, the above-mentioned diabetes-complicated pregnancy means a state of having suffered from diabetes before pregnancy and suffering from diabetic retinopathy during pregnancy.

  In the determination method and biomarker for determination in the present case, the concentration of the metabolite group in urine is prepared by preparing a urine sample (sample) from the collected urine, and a known method capable of specifically detecting the metabolite group, For example, it can be measured using mass spectrometry. In such mass spectrometry, a urine sample is converted into gaseous ions using an ion source (ionization), and the urine sample that has been ionized by moving in a vacuum and using electromagnetic force or by a time-of-flight difference is analyzed in the analysis unit. This refers to a measurement method using a mass spectrometer that can be separated and detected according to the charge ratio. The ionization method using an ion source includes an electron ionization (EI) method, a chemical ionization (CI) method, an electric field desorption. A method such as a deionization (FD) method, a fast atom bombardment (FAB) method, a matrix-assisted laser desorption ionization (MALDI) method, an electrospray ionization (ESI) method can be selected as appropriate. Methods for separating ionized urine samples include magnetic field deflection type, quadrupole type, ion trap type, time of flight (TOF) type, Fourier transform ion size. Rotoron method of separating resonance type, etc. can be appropriately selected. Further, tandem mass spectrometry (MS / MS) combining two or more mass spectrometry methods can be used. In addition, the metabolite group can be separated from the contaminants and analyzed by gas chromatography (GC), liquid chromatography (LC), or high performance liquid chromatography (HPLC).

  In the determination method and biomarker for determination, the concentration of the metabolite group in urine may be an absolute value or a relative value. Relative values based on the metabolite group (internal standard) can be mentioned.

  In the determination method and biomarker for determination in this case, the “metabolite concentration in the control person who does not develop gestational diabetes” is the “metabolite concentration in the subject” or “pregnant women who are at high risk of developing gestational diabetes” Corresponding to “the concentration of metabolites in urine collected from”. Therefore, if the “metabolite concentration in the subject” or “the concentration of metabolites in urine collected from pregnant women who are at high risk of developing gestational diabetes” is an absolute value or a relative value, The “metabolite concentration in the control who does not” is an absolute value and a relative value, respectively. The above-mentioned “concentration of metabolite in a control person who does not develop gestational diabetes” may be measured in advance by preparing a urine sample from urine collected from the control person when carrying out the determination method. You may use what you did. The urine sample collected and prepared from the control person is preferably prepared by substantially the same method as the urine sample derived from the subject. In addition, the methods used to measure the “metabolite concentration in controls who do not develop gestational diabetes” were collected from comparative subjects such as “metabolite concentration in subjects” and “pregnant women at high risk of developing gestational diabetes” The method substantially the same as the method used for the measurement of “the concentration of metabolite in urine” is preferable.

  As the above-mentioned control person, it is sufficient if it is clear that it does not develop gestational diabetes, and it is usually a female that is clear that it does not develop gestational diabetes, and a pregnant female that is clear that it does not develop gestational diabetes is preferable. . Moreover, as said control | contrast person, a test subject and the age, BMI (Body Mass Index), and / or the person (Parity) whose birth frequency (Parity) is similar (person with a state without a statistical significant difference) are preferable.

  In step (c) of the determination method, when the metabolite measured in step (a) is the metabolite of [Group A], the “metabolite concentration in the subject” measured in step (a) is “control”. The subject (pregnant woman) evaluates (determines) that the risk of developing gestational diabetes is high, and the “metabolite concentration in the subject” measured in step (a) is “control”. When it is not lower than the “metabolite concentration in the person”, the subject (pregnant woman) evaluates (determines) that the risk of developing gestational diabetes is low. Further, in the step (c) of the determination method, when the metabolite measured in the step (a) is a metabolite of the above [Group B], the “metabolite concentration in the subject” measured in the step (a) is: When higher than the “metabolite concentration in the control”, the subject (pregnant woman) evaluates (determines) that the risk of developing gestational diabetes is high, and the “metabolite concentration in the subject” measured in step (a) is When not higher than the “metabolite concentration in the control person”, the subject (pregnant woman) evaluates (determines) that the risk of developing gestational diabetes is low.

  In order to determine whether the “metabolite concentration in the subject” is lower or higher than the “metabolite concentration in the control person”, an arbitrary threshold (cutoff value) may be set. The threshold value can be, for example, an average value of “metabolite concentration in the control person”, “average value + standard deviation (SD)”, “average value + 2SD”, “average value + 3SD”, median value, “median value” + SD "," median value + 2SD "," median value + 3SD ", and the like. In addition, the threshold is set so that the sensitivity (the rate at which those who subsequently develop gestational diabetes can be correctly determined as positive) and the specificity (the rate at which those who do not subsequently develop gestational diabetes can be correctly determined as negative) are increased. It can also be calculated using ROC (Receiver Operating Characteristic) curve using statistical analysis software based on the data of “metabolite concentration in subject” and the data of “metabolite concentration in control person”.

  In the determination method and biomarker for determination in the present case, the “subject who is not at risk of developing gestational diabetes” or the “pregnant woman who is at high risk of developing gestational diabetes for a control subject who does not develop gestational diabetes” Since the level of decrease in the concentration of metabolites (ethanolamine or methionine) varies depending on the characteristics of the "control subject who does not develop gestational diabetes", "subject", and "pregnant women at high risk of developing gestational diabetes", the measurement method, etc. Although not generally specified, in the case of ethanolamine, for example, a reduction of at least 28%, preferably a reduction of at least 30%, more preferably a reduction of at least 32%, even more preferably a reduction of at least 34%, Even more preferably a reduction of at least 35%, particularly preferably at least 37 Reduction, particularly more preferably at least 39% reduction in, particularly more preferably at least 41% reduction, in particular even more preferably at least 43% reduction, and most preferably at least 44% reduction. In the case of methionine, the reduction level is, for example, a reduction of at least 20%, preferably a reduction of at least 23%, more preferably a reduction of at least 26%, even more preferably a reduction of at least 30%, even more preferably. Is at least 33% reduction, particularly preferably at least 36% reduction, particularly preferably at least 40% reduction, particularly more preferably at least 42% reduction, particularly more preferably at least 44% reduction, particularly still more preferably A reduction of at least 46%, especially even more preferred a reduction of at least 48%, and a particularly preferred reduction of at least 50%, most preferred a reduction of at least 51%.

  In the determination method and biomarker for determination of the present case, in the above [Group B], the “subject for a control person who does not develop gestational diabetes” or the “pregnant woman who has a high risk of developing gestational diabetes for the control person who does not develop gestational diabetes” Increased levels of metabolite (1,3-DPG, shikimic acid-3-phosphate, or N-acetyl-L-alanine) levels are found in “controls who do not develop gestational diabetes”, “subjects”, and “gestational diabetes” However, in the case of 1,3-DPG, for example, the increase is at least 1.1 times, preferably at least 1.1 times. 1.2-fold increase, more preferably at least 1.4-fold increase, even more preferably at least 1.6-fold, even more preferably at least 1. Fold increase, particularly preferably at least 2.0 fold increase, particularly more preferably at least 2.1 fold increase, even more preferably at least 2.2 fold increase, particularly even more preferably at least 2.3 fold increase An increase, particularly preferably an increase of at least 2.4 times, most preferably an increase of at least 2.5 times. In the case of shikimic acid-3-phosphate or N-acetyl-L-alanine, the increase level is, for example, at least a 1.1-fold increase, preferably at least a 1.2-fold increase, more preferably An increase of at least 1.3 times, more preferably an increase of at least 1.4 times, even more preferably an increase of at least 1.5 times, particularly preferably at least an increase of at least 1.6 times.

  In the present determination method and the present determination biomarker, the metabolite may be one or more metabolites selected from the present metabolite group. Specifically, ethanolamine, methionine, 1,3- One selected from the group consisting of DPG, shikimic acid-3-phosphate, and N-acetyl-L-alanine; two combinations of ethanolamine and methionine; 2 of ethanolamine and 1,3-DPG Combination of species; two combinations of ethanolamine and shikimic acid-3-phosphate; two combinations of ethanolamine and N-acetyl-L-alanine; two combinations of methionine and 1,3-DPG Combination; two combinations of methionine and shikimic acid-3-phosphate; two combinations of methionine and N-acetyl-L-alanine; 1,3-DPG and shikimic acid-3- Two combinations with acid; two combinations with 1,3-DPG and N-acetyl-L-alanine; two combinations with shikimic acid-3-phosphate and N-acetyl-L-alanine Three combinations of ethanolamine, methionine and 1,3-DPG; three combinations of ethanolamine, methionine and shikimic acid-3-phosphate; ethanolamine, methionine and N- Three combinations of acetyl-L-alanine; three combinations of ethanolamine, 1,3-DPG, and shikimic acid-3-phosphate; ethanolamine, 1,3-DPG, and N- Three combinations of acetyl-L-alanine; three combinations of ethanolamine, shikimic acid-3-phosphate, and N-acetyl-L-alanine; ethanolamine, methionine, 1,3- DPG and Shi 4 combinations of myic acid-3-phosphate; 4 combinations of ethanolamine, methionine, 1,3-DPG, and N-acetyl-L-alanine; ethanolamine, 1,3- Four combinations of DPG, shikimic acid-3-phosphate, and N-acetyl-L-alanine; ethanolamine, methionine, 1,3-DPG, shikimic acid-3-phosphate, and N -5 types of combinations with acetyl-L-alanine; Among these, those containing ethanolamine or 1,3-DPG are preferable.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

1. Method [Preparation of urine sample]
From the participants of the Chiba Unit Center of the National Survey on Children's Health and Environment (JECS), recruiting women in the first trimester (14-21 weeks) for 3 years (2011-2014) and urine from those who agreed to participate Was collected and stored at -20 ° C. In addition, among pregnant female participants, those who have not developed GDM and who have been diagnosed as having GDM at the time of childbirth in the first pregnancy pregnancy (T1 questionnaire) (PreGDM group). In addition, the age, BMI (Body Mass Index), and the number of births (Parity) of 121 preGDM groups were examined, and those who matched these values, including those who did not develop GDM, including treatment in the early pregnancy A control group was used (see Table 1). This example was carried out in accordance with the declaration of Helsinki, and the protocol was approved by the Chiba University Graduate School of Medicine and the Bioethics Committee of the Graduate School of Medicine. In addition, written informed consent was obtained from pregnant female participants.

[Metabolome analysis by hydrophilic interaction chromatography tandem mass spectrometry (HILIC-MS / MS)]
In order to examine the concentration in urine of 263 kinds of metabolites considered to be stable under urine storage conditions (−20 ° C.), the literature “ Soga T. et al. Anal Chem. 2009; 81 (15): 6165-74. And the literature `` Yuan M. et al. Nat Protoc. 2012; 7 (5): 872-81. '' Metabolomic analysis by HILIC-MS / MS was performed. That is, after centrifuging 60 μL of urine at 14,000 g for 10 minutes, 40 μL of the supernatant and 60 μL of methanol (1 nM lidocaine and positive ions) containing an internal standard for adjusting the sample reduced in the pretreatment procedure were prepared. After placing N, N-diethyl-2-phenylacetamide in the mode and D-camphor-10-sulfonic acid in the negative ion mode on an Amicon Ultra-0.5 3k filter column (Merck Millipore) The filtrate (residue) was used as a urine sample and analyzed using a Prominence UFLC system (manufactured by Shimadzu) and a QTRAP 4500 system (manufactured by AB SCIEX). That is, the filtrate was applied to a mass spectrometer (QTRAP 4500 system) via HILIC chromatography using a 3.5-μm XBridge BEH amide column (Waters) at 4.6 mm (inner diameter) × 100 mm and a flow rate of 350 μL / min. Delivered. In this example, mobile phase A (20 mM ammonium hydroxide / 20 mM ammonium acetate [pH = 9.5] ultrapure water: acetonitrile [95: 5]) and mobile phase B (high performance liquid chromatography-grade acetonitrile ) Was used to analyze the target metabolome. The peak area from the total ion current of each metabolite SRM transition was integrated using MultiQuant v3.0 software (AB SCIEX). The peak integration set values used are as described in the literature “Yuan M. et al. Nat Protoc. 2012; 7 (5): 872-81.”.

[Statistical analysis]
The statistically significant difference between the measured value of the preGDM group and the measured value of the control group was analyzed using unpaired t-test or Mann-Whitney test according to the distribution. Performed orthogonal projections to latent structures Discriminant Analysis (OPLS-DA), Microsoft R open 3.3.0 (R Core Team, 2016, R Core Team R: A Language and Environment for Statistical Computing R Foundation) for Statistical Computing, Vienna 2016) using the package ropls (see literature “Thevenot EA., et al. J Proteome Res. 2015; 14 (8): 3322-35.”) Analyzed. Metabolites having a variable importance of projection (VIP) value exceeding 1.5 were identified as candidate biomarkers for diagnosis of GDM onset risk. Moreover, the sensitivity and specificity of each metabolite were analyzed under the ROC curve (AUC) performed with the ROCR package, using ROC (Receiver Operating Characteristic) curves and corresponding regions.

2. Results When the concentrations of 263 types of metabolites in urine in the preGDM group and the control group were measured, 187 types of metabolites were detected. Among them, it has been shown that there are 20 types of candidate biomarkers for diagnosis of GDM onset risk whose VIP value exceeds 1.5. Furthermore, when the sensitivity and specificity were examined using the ROC curve performed in the ROCR package, five kinds of substances (ethanolamine, 1,3-diphosphoglycerate [1,3-DPG], shikimate-3 -Phosphate, N-acetyl-L-alanine and methionine).

[Ethanolamine]
In the preGDM group, the ethanolamine concentration in urine was 0.039 (median value), which was about 44% lower than the value in the control group (0.070), and a significant difference was observed (P = 0.00000247) (see FIG. 1A). This result shows that the ethanolamine concentration in urine from those who develop GDM (in the future) is lower than those who do not develop GDM.
Moreover, when the ROC curve was created based on the ethanolamine concentration in the urine of the preGDM group and the control group, the AUC value was as high as 0.821. Moreover, when the cutoff value for distinguishing between the preGDM group and the control group was set to 0.056, the sensitivity of the person who develops GDM (the ratio of test positive persons among those who develop GDM) and the specific The degrees (the proportion of test negative among those who did not develop GDM) were 74% and 81%, respectively.
This result shows that when the ethanolamine concentration in urine collected from pregnant women before the onset of GDM is measured and an appropriate cut-off value is set, the risk of developing GDM can be determined using the ethanolamine concentration as an index. .

[1,3-DPG]
In the preGDM group, the urinary 1,3-DPG concentration was 0.037 (median), which was about 2.5 times higher than the value in the control group (0.015), indicating a significant difference. (P = 0.0000000238) (see FIG. 1B). This result shows that the 1,3-DPG concentration in urine from those who develop GDM (in the future) is higher than those who do not develop GDM.
Moreover, when the ROC curve was created based on the 1,3-DPG concentration in the urine of the preGDM group and the control group, the AUC value was as high as 0.848. In addition, when 0.030 was set as a cut-off value for distinguishing between the preGDM group and the control group, the sensitivity of those who developed GDM (the proportion of those who tested positive among those who developed GDM) and uniqueness The degrees (the proportion of test negative among those who did not develop GDM) were 61% and 100%, respectively.
This result shows that when the 1,3-DPG concentration in urine collected from pregnant women before the onset of GDM is measured and an appropriate cut-off value is set, the risk of developing GDM is determined using the 1,3-DPG concentration as an index. It shows that it can be judged.

[Shikimic acid-3-phosphate]
In the preGDM group, the urinary shikimate-3-phosphate concentration was 0.047 (median), which was about 1.5 times higher than the control group value (0.032). Was observed (P = 0.0001113) (see FIG. 2A). This result shows that the shikimate-3-phosphate concentration in urine from those who develop GDM (in the future) is higher than those who do not develop GDM.
Moreover, when the ROC curve was created based on the shikimic acid-3-phosphate concentration in the urine of the preGDM group and the control group, the AUC value was as high as 0.768. In addition, when 0.039 was set as a cut-off value for distinguishing between the preGDM group and the control group, the sensitivity of those who developed GDM (the proportion of those who tested positive among those who developed GDM) and uniqueness The degrees (the proportion of test negative among those who did not develop GDM) were 67% and 74%, respectively.
This result shows that, when the shikimate-3-phosphate concentration in urine collected from a pregnant woman before the onset of GDM is measured and an appropriate cut-off value is set, the shikimate-3-phosphate concentration is used as an index. This indicates that the risk of developing can be determined.

[N-acetyl-L-alanine]
In the preGDM group, the urinary N-acetyl-L-alanine concentration was 0.071 (median), which was about 1.5 times greater than the control group value (0.046). Was observed (P = 0.0008) (see FIG. 2B). This result shows that the concentration of N-acetyl-L-alanine in urine from those who develop GDM (in the future) is higher than those who do not develop GDM.
Moreover, when the ROC curve was created based on the N-acetyl-L-alanine concentration in the urine of the preGDM group and the control group, the AUC value was as high as 0.735. Moreover, when the cut-off value for distinguishing between the preGDM group and the control group was set to 0.05, the sensitivity of those who developed GDM (the proportion of those who tested positive among those who developed GDM) and specific The degrees (the proportion of test negative among those who did not develop GDM) were 78% and 58%, respectively.
This result shows that when N-acetyl-L-alanine concentration in urine collected from a pregnant woman before the onset of GDM is measured and an appropriate cut-off value is set, This indicates that the risk of developing can be determined.

[Methionine]
In the preGDM group, the urinary methionine concentration was 0.0064 (median), a decrease of about 51% compared to the control group value (0.013), and a significant difference was observed (P = 0). .00229) (see FIG. 2C). This result indicates that the urinary methionine concentration from those who develop GDM (in the future) is lower than those who do not develop GDM.
Moreover, when the ROC curve was created based on the urinary methionine concentrations of the preGDM group and the control group, the AUC value was as high as 0.718. In addition, when the cut-off value for distinguishing between the preGDM group and the control group was set to 0.0082, the sensitivity of those who develop GDM (the proportion of those who test positive among those who develop GDM) and peculiarities The degrees (the proportion of test negative among those who did not develop GDM) were 68% and 72%, respectively.
This result indicates that when the methionine concentration in urine collected from a pregnant woman before the onset of GDM is measured and an appropriate cut-off value is set, the risk of developing GDM can be determined using the methionine concentration as an index.

  The present invention relates to diagnosis and treatment of the risk of developing gestational diabetes in pregnant women, fetal malformations due to pregnancy complications of gestational diabetes (eg, cardiac malformations such as cardiac hypertrophy, ventricular septal defect; bone angles such as microcephaly Malformation; renal malformation such as renal dysplasia and ureteral duplication; gastrointestinal malformation such as duodenal obstruction and rectal closure) and the risk of suffering from type 2 diabetes after childbirth.

Claims (7)

The determination method of the onset risk of gestational diabetes characterized by including the following processes (a)-(c).
(A) a step of measuring the concentration of one or more metabolites selected from the following [Group A] and [Group B] in urine collected from a pregnant woman who has not developed gestational diabetes;
[Group A]
Ethanolamine, methionine [Group B]
1,3-diphosphoglyceric acid, shikimic acid-3-phosphate, N-acetyl-L-alanine (b) The metabolism in the control person who does not develop gestational diabetes is determined according to the concentration of the metabolite measured in step (a). Comparing with the concentration of the substance;
(C) When the metabolite measured in step (a) is a metabolite of [Group A], when the metabolite concentration measured in step (a) is lower than the metabolite concentration in the control, Pregnant women have a high risk of developing gestational diabetes,
When the metabolite measured in step (a) is a metabolite of [Group B], when the metabolite concentration measured in step (a) is higher than the metabolite concentration in the control, the pregnant woman Assessing the high risk of developing gestational diabetes;   The method according to claim 1, wherein the pregnant woman is a pregnant woman of 13 to 22 weeks of pregnancy. In step (c),
When the ethanolamine concentration measured in step (a) is reduced by at least 44% compared to the ethanolamine concentration in the control, the pregnant woman evaluates that the risk of developing gestational diabetes is high;
When the concentration of methionine measured in step (a) is reduced by at least 51% compared to the concentration of methionine in the control, the pregnant woman evaluates that the risk of developing gestational diabetes is high;
When the concentration of 1,3-diphosphoglyceric acid measured in step (a) is increased by at least 2.5 times compared to the concentration of 1,3-diphosphoglyceric acid in the control, the pregnant woman has gestational diabetes. Assessed that the risk of developing is high,
A pregnant woman develops gestational diabetes when the concentration of shikimic acid-3-phosphate measured in step (a) is increased at least 1.5 times compared to the concentration of shikimic acid-3-phosphate in the control Assess that the risk is high,
A pregnant woman develops gestational diabetes when the concentration of N-acetyl-L-alanine measured in step (a) is increased by at least 1.5 times compared to the concentration of N-acetyl-L-alanine in the control. The determination method according to claim 1, wherein the risk is evaluated as high.   The determination method according to claim 1, wherein the metabolite is a metabolite containing ethanolamine or 1,3-diphosphoglycerate. A biomarker for determining the risk of developing gestational diabetes, comprising one or more metabolites selected from the following [Group A] and [Group B],
When the metabolite is a metabolite of [Group A], the concentration of the metabolite in urine collected from a pregnant woman who has a high risk of developing gestational diabetes is the concentration of the metabolite in a control person who does not develop gestational diabetes Lower than
When the metabolite is a [Group B] metabolite, the concentration of the metabolite in urine collected from a pregnant woman who has a high risk of developing gestational diabetes is the concentration of the metabolite in a control person who does not develop gestational diabetes Higher than
The determination biomarker.
[Group A]
Ethanolamine, methionine [Group B]
1,3-diphosphoglyceric acid, shikimic acid-3-phosphate, N-acetyl-L-alanine The concentration of ethanolamine in urine collected from pregnant women who are at high risk of developing gestational diabetes is reduced by 44% compared to the concentration of ethanolamine in controls who do not develop gestational diabetes;
The concentration of methionine in urine collected from pregnant women who are at high risk of developing gestational diabetes is reduced by 51% compared to the concentration of methionine in controls who do not develop gestational diabetes;
The concentration of 1,3-diphosphoglyceric acid in urine collected from pregnant women at high risk of developing gestational diabetes is 2 compared to the concentration of 1,3-diphosphoglyceric acid in controls that do not develop gestational diabetes. .5 times increase,
The concentration of shikimate-3-phosphate in urine collected from pregnant women at high risk of developing gestational diabetes is 1.5 compared to the concentration of shikimate-3-phosphate in controls who do not develop gestational diabetes Doubled,
The concentration of N-acetyl-L-alanine in urine collected from pregnant women at high risk of developing gestational diabetes is 1.5 compared to the concentration of N-acetyl-L-alanine in controls who do not develop gestational diabetes. The biomarker for determination according to claim 5, wherein the biomarker is doubled.   The biomarker for determination according to claim 5 or 6, wherein the metabolite is a metabolite containing ethanolamine or 1,3-diphosphoglycerate.

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