Serum / plasma metabolism molecular marker related to screening or early diagnosis of gestational diabetic megathema and application of serum / plasma metabolism molecular marker

Abstract

The invention discloses a serum / plasma metabolism molecular marker related to screening or early diagnosis of gestational diabetic megathema and application of the serum / plasma metabolism molecular marker. The invention relates to a serum / plasma metabolism molecular marker related to GDM huge screening or early diagnosis. The serum / plasma metabolism molecular marker consists of oleic acid, palmitic acid and myristic acid. The reagent for quantitatively detecting the GDM pregnant woman serum / plasma metabolism molecular marker is applied to preparation of a reagent for screening or early diagnosis of a GDM huge high-risk pregnant woman. Laboratory support is provided for screening or early diagnosis of GDM huge, so that a more individualized prevention and treatment scheme is adopted, and the adverse pregnancy outcome is reduced to the maximum extent.

Description

Technical Field

[0001] The present invention belongs to the field of metabolomics and reproductive medicine, and relates to serum / plasma metabolic molecular markers related to the screening or early diagnosis of macrosomia caused by gestational diabetes and applications thereof. Background Art

[0002] Macrosomia refers to infants with a birth weight of 4,000 grams or more, and is the most common adverse pregnancy outcome of gestational diabetes mellitus (GDM). According to the latest statistics from the International Diabetes Federation in 2021, 21.1 million live births worldwide are affected by hyperglycemia during pregnancy, of which 80.3% are GDM. GDM macrosomia is mostly metabolically asymmetric, manifested by a significant increase in body fat weight, while the volume of other tissues / organs is not significantly increased, which not only affects the growth and development of infants, but also greatly increases the risk of obesity or diabetes in children, adolescents and adults. The famous "Developmental Origins of Health and Disease (DOHaD)" theory believes that the nutrition and nurturing environment in the first 1,000 days of life (such as intrauterine high-sugar environment, etc.) are crucial to the health of an individual throughout his or her life. Therefore, early diagnosis, early intervention, and timely and reasonable treatment are of great significance for clinical control of the occurrence of GDM macrosomia, as well as for reducing maternal and infant complications and delaying the development of obesity or diabetes in GDM offspring.

[0003] Metabolomics, as an emerging omics technology, focuses on studying metabolites in organisms. It is known as the omics closest to phenotype and is a comprehensive interpretation of the results of genome and proteome execution. In recent years, metabolomics has shown great potential in the early prediction of diseases and has become an important tool for screening sensitive predictive markers of diseases. It is particularly noteworthy that metabolomics is gaining momentum in the diagnosis and pathogenesis of pregnancy-related diseases, becoming a hot and promising research field. Metabolomics can reveal the mechanism of GDM macrosomia from a new perspective and find sensitive molecular diagnostic markers, which has far-reaching significance for the prevention and treatment of such diseases.

[0004] However, there are relatively few stable serum metabolic molecular markers for GDM macrosomia screening or early diagnosis reported so far. If we can screen out metabolites that are specific or abnormally expressed in GDM macrosomia and use them as molecular markers to further develop corresponding screening or early diagnosis kits, this will greatly improve the current status of GDM macrosomia diagnosis in my country. Summary of the invention

[0005] The primary purpose of the present invention is to address the above technical issues and propose a group of serum / plasma metabolic molecular markers during pregnancy that are related to the screening or early diagnosis of GDM macrosomia.

[0006] Another object of the present invention is to provide applications of the marker.

[0007] Another object of the present invention is to provide a GDM macrosomia screening or early diagnosis kit.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] A serum / plasma metabolic molecular marker associated with the screening or early diagnosis of GDM macrosomia, wherein the group of markers is selected from any one or more of Oleic acid, Palmitic acid and Myristic acid.

[0010] As a preferred embodiment of the present invention, the serum / plasma metabolic molecular markers consist of Oleic acid, Palmitic acid and Myristic acid.

[0011] The serum / plasma metabolic molecular markers are used as detection targets in the preparation of a kit for auxiliary screening or early auxiliary diagnosis of pregnant women at high risk of GDM macrosomia.

[0012] The use of the detection reagent of serum / plasma metabolic molecular markers in the preparation of a kit for auxiliary screening or early auxiliary diagnosis of pregnant women at high risk of GDM macrosomia.

[0013] A kit for auxiliary screening or early auxiliary diagnosis of pregnant women at high risk of GDM macrosomia, comprising reagents and consumables for detecting the serum / plasma metabolic molecular markers of the present invention.

[0014] As a preferred embodiment of the present invention, the kit includes reagents and consumables for detecting the serum / plasma metabolic molecular markers using the UPLC-Q exactive MS method.

[0015] As a further preferred embodiment of the present invention, the diagnostic kit contains oleic acid, palmitic acid and myristic acid standards, carbon 13 labeled oleic acid, palmitic acid and myristic acid stable isotope internal standards, a matching Hypersil GOLD C18 chromatographic column (100 mm×2.1 mm, particle size 1.9 um, ThermoScientific, Germany), reagent A (for protein precipitation, containing 100% methanol), reagent B (for mobile phase, water containing 0.1% formic acid), reagent C (for mobile phase, acetonitrile containing 0.1% formic acid), and reagent D (for reconstitution, ultrapure water).

[0016] The inventive point of the present invention is to find that any one of oleic acid, palmitic acid and myristic acid or a combination thereof can be used as a serum / plasma metabolic molecular marker related to the screening or early diagnosis of GDM macrosomia.

[0017] Specifically, the serum / plasma metabolic molecular markers of the present invention are screened by the following methods: (1) Collecting blood samples that meet the standards and systematically collecting complete demographic and clinical data. (2) Serum metabolomics analysis of pregnant women with GDM macrosomia: Select pregnant women with GDM macrosomia and pregnant women with GDM non-macrosomia (control), use metabolomics methods to detect the content of each metabolite in the serum of pregnant women with GDM macrosomia and the control group, analyze the commonalities and characteristics of metabolites in the serum of pregnant women with GDM macrosomia and the control group, and screen differentially expressed metabolites. (3) Use the UPLC-Q exactive MS method to verify the screened serum / plasma differentially expressed metabolite molecules in an independent sample population.

[0018] Beneficial effects of the present invention:

[0019] The advantages of the serum / plasma metabolic molecular markers provided by the present invention as markers for screening or early diagnosis of GDM macrosomia are:

[0020] The present invention collects blood that meets the standards according to standard operating procedures, systematically collects complete demographic data, clinical data, etc., and adopts metabolomics methods to study the changes in metabolites in the serum of pregnant women with GDM macrosomia and control pregnant women, and finds a group of highly specific and sensitive metabolites that are highly correlated with the occurrence of GDM macrosomia. The results of multivariate logistic regression analysis show that oleic acid, palmitic acid and myristic acid in the serum of pregnant women with GDM are significantly associated with the occurrence of macrosomia. The ROC curves are shown in the attached figure. Figure 1 As shown, it can provide laboratory support for the screening and early auxiliary diagnosis of GDM macrosomia.

[0021] In the initial stage of this invention, the metabolomics research method was used to obtain the molecular expression spectrum of specific and abnormally expressed metabolites in the blood of pregnant women with GDM macrosomia, and the UPLC-Q exactive MS method was simultaneously used to verify it in the serum of independent samples of GDM macrosomia; the application of the above methods and strategies accelerated and guaranteed the application of serum / plasma metabolic molecular markers and diagnostic kits.

[0022] The present invention studies the application prospects of serum / plasma metabolic molecular markers in the screening or early diagnosis of GDM macrosomia by controlling the influencing factors of macrosomia such as age and mid-pregnancy weight gain, expounds the influence of abnormally expressed serum / plasma metabolic molecular markers on the occurrence of GDM macrosomia, and reveals its value for the screening and early diagnosis of GDM macrosomia. Therefore, the present invention obtains a serum / plasma metabolic molecular expression database and specific markers for the occurrence of GDM macrosomia; by determining the development and application of serum / plasma metabolic molecular markers and diagnostic kits, the screening and early diagnosis of GDM macrosomia can be made more convenient and feasible, and support can be provided for clinicians to accurately predict whether GDM pregnant women will give birth to macrosomia, and to promptly adopt more personalized prevention and treatment plans, thereby reducing the incidence of GDM macrosomia to a greater extent and reducing adverse pregnancy outcomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 .The diagnostic value of oleic acid, palmitic acid and myristic acid for GDM macrosomia in the training set

[0024] Figure 2. The diagnostic value of oleic acid, palmitic acid and myristic acid in GDM macrosomia in the validation set DETAILED DESCRIPTION

[0025] The following examples are intended to further illustrate the present invention, but are not intended to limit the present invention.

[0026] Example 1 Collection of samples and organization of sample data

[0027] The applicant collected a large number of peripheral blood samples from pregnant women at 24-28 weeks of gestation from Nanjing Maternal and Child Health Hospital from January to December 2023 (the samples used for research were collected during the same period, with uniform sampling, packaging and storage conditions). By sorting out the sample data, the applicant selected 200 samples that met the following criteria as experimental samples.

[0028] 1. Pregnant women diagnosed with GDM by OGTT when peripheral blood was collected at 24-28 weeks of gestation;

[0029] 2. Pregnant women whose newborns weighed more than 4000g after full-term delivery were defined as the experimental group (100 cases);

[0030] 3. The weight of the newborns born after full term of the above-mentioned research subjects does not exceed 4000g. Pregnant women who match the experimental group in age, BMI, and gestational age are defined as controls (100 cases), and the demographic and clinical data of these samples are systematically collected;

[0031] 4. Use blood coagulation tubes to collect 5 mL of peripheral blood from the individual to be tested. After blood collection, centrifuge at 1000 rpm for 6 minutes, and draw serum into EP tubes and store at -80°C. 40 pairs of GDM macrosomia and controls were used as the screening set (Table 1), and 60 pairs of GDM macrosomia and controls were used as the validation set (Table 2).

[0032] Table 1 Basic clinical information of the screened samples

[0033]

[0034] Example 2 Analysis of differential metabolic profiles in pregnant women with GDM macrosomia

[0035] The above 100 pregnant women with GDM and macrosomia and 100 control pregnant women who met the above conditions were subjected to metabolomics testing to obtain relevant results. The specific experimental methods are as follows:

[0036] 1. Experimental reagents and instruments (Table 3-4)

[0037] Table 3 Experimental reagent list

[0038]

[0039] Table 4 Experimental instruments list

[0040]

[0041] 2. Sample Extraction

[0042] (1) Take 100 μL of sample, add 500 μL of extract containing internal standard (methanol:acetonitrile volume ratio = 1:1, internal standard concentration 20 mg / L), and vortex mix for 30 seconds;

[0043] (2) Ultrasonication for 10 min (ice water bath);

[0044] (3) Stand at -20℃ for one hour;

[0045] (4) Centrifuge the sample at 12000 rpm for 15 min at 4°C;

[0046] (5) Carefully remove 500 μL of supernatant into an EP tube;

[0047] (6) drying the extract in a vacuum concentrator;

[0048] (7) Add 160 μL of extraction solution (acetonitrile-water volume ratio: 1:1) to the dried metabolites for re-dissolution;

[0049] (8) Vortex for 30 seconds and sonicate in an ice water bath for 10 minutes;

[0050] (9) Centrifuge the sample at 12,000 rpm for 15 min at 4°C;

[0051] (10) Carefully remove 120 μL of supernatant into a 2 mL injection vial and take 10 μL of each sample to mix as QC samples for testing.

[0052] 3. On-machine testing

[0053] The LC-MS system used for metabolomics analysis consisted of a Waters Acquity I-Class PLUS ultra-high performance liquid chromatograph connected to a Waters Xevo G2-XS QTOF high-resolution mass spectrometer. The chromatographic column used was an Acquity UPLC HSS T3 column (1.8um2.1*100mm) purchased from Waters. The injection volume was 1ul. The LC mobile phase conditions are shown in Table 5.

[0054] Positive ion mode (POS): Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile

[0055] Negative ion mode (NEG): Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile

[0056] Table 5 Liquid chromatography mobile phase conditions

[0057]

[0058] 4. Qualitative and quantitative analysis of metabolites

[0059] The raw data collected using MassLynxV4.2 were processed by Progenesis QI software for peak extraction, peak alignment and other data processing operations. Identification was performed based on the Progenesis QI software online METLIN database and public database, and theoretical fragment identification was performed at the same time. The deviation of the parent ion mass number was within 100 ppm and the deviation of the fragment ion mass number was within 50 ppm.

[0060] 5. Metabolomics analysis results

[0061] After metabolite qualitative and quantitative analysis, data quality assessment, annotation analysis, differential expression analysis, etc. were performed. Data with at least half of the data in each group as non-null values ​​were selected for significant difference analysis, and the fold change (FC) ≥ 2 or ≤ 0.5 and the P value standard for multiple testing correction (P FDR <0.05) to screen serum metabolites with significant differences between the two groups. We used metabolomics to screen 47 metabolites with significant differences in 40 pregnant women with GDM and 40 controls, among which the expressions of oleic acid, palmitic acid and myristic acid were significantly increased (Table 6). The ROC curve is shown in Figure 1 .

[0062] Table 6 Serum metabolites that differ between GDM macrosomia and controls

[0063]

[0064] Example 3 Verification of the expression of serum / plasma metabolic molecular markers in GDM macrosomia based on UPLC-Q exactive MS method

[0065] Based on the metabolite targeted detection method of UPLC-Q exactive MS, the inventors detected the levels of metabolites oleic acid, palmitic acid and myristic acid in serum samples during pregnancy in an independent population (60 pregnant women with GDM macrosomia and 60 controls), and used this to draw ROC curves. There were significant differences in the levels of metabolic molecular markers oleic acid, palmitic acid and myristic acid in the serum of the GDM macrosomia group and the control group (P<0.05), and the results are shown in Table 7. The results of logistic regression analysis showed that oleic acid, palmitic acid and myristic acid were significantly associated with the occurrence of GDM macrosomia, and the ROC curves were shown in Table 7. Figure 2. The AUC (95% CI) of oleic acid was 0.863 (0.783-0.942), the AUC (95% CI) of palmitic acid was 0.769 (0.662-0.876), the AUC (95% CI) of myristic acid was 0.721 (0.606-0.835), and the AUC (95% CI) of the combination of oleic acid, palmitic acid and myristic acid was 0.932 (0.871-0.993), indicating that oleic acid, palmitic acid and myristic acid are metabolic molecular markers that are highly correlated with the occurrence of GDM macrosomia and can be used to prepare a screening or early diagnosis kit for GDM macrosomia.

[0066] Table 7 Validation results of serum differential metabolites between GDM macrosomia and control groups

[0067]

[0068] Example 4 Kit for early auxiliary diagnosis of GDM

[0069] This kit includes reagents and consumables for the detection of oleic acid, palmitic acid and myristic acid, of which the standards of oleic acid, palmitic acid and myristic acid are used for qualitative and quantitative analysis, and the stable isotope internal standards of oleic acid, palmitic acid and myristic acid labeled with carbon 13 are used for auxiliary quantitative analysis and auxiliary qualitative analysis. Others include a matching reverse chromatographic column for UPLC chromatographic separation (Hypersil GOLD C18 column, 100mm×2.1mm, particle size 1.9μm), a reagent for precipitating plasma proteins (100% methanol), a reagent for mobile phase (water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid), and ultrapure water for reconstitution. The value of this kit is that only 50μl of pregnancy plasma is needed to detect the content of serum oleic acid, palmitic acid and myristic acid, and then through content screening or auxiliary early diagnosis of GDM macrosomia, and dynamic monitoring is easy to carry out.

[0070] The specific kit consists of: oleic acid, palmitic acid and myristic acid standards, carbon 13 labeled oleic acid, palmitic acid and myristic acid stable isotope internal standards, a matching HypersilGOLD C18 chromatographic column (100 mm × 2.1 mm, particle size 1.9 um, Thermo Scientific, Germany), reagent A (100% methanol), reagent B (water containing 0.1% formic acid), reagent C (acetonitrile containing 0.1% formic acid), and reagent D (ultrapure water).

Claims

1. Use of a reagent for detecting serum / plasma metabolic molecular markers related to screening or early diagnosis of gestational diabetes mellitus macrosomia in the preparation of a kit for auxiliary screening or early auxiliary diagnosis of pregnant women at high risk of GDM macrosomia, characterized in that: The serum / plasma metabolic molecular markers are selected from any one or more of Oleic acid, Palmitic acid and Myristic acid.

2. The use according to claim 1, characterized in that: The serum / plasma metabolic molecular markers consist of Oleiric acid, Palmitic acid and Myristic acid.

3. The use according to claim 1, characterized in that: The reagent for detecting serum / plasma metabolic molecular markers related to auxiliary screening or early auxiliary diagnosis of macrosomia caused by gestational diabetes is a reagent for detecting the serum / plasma metabolic molecular markers by UPLC-Q exactive MS.

4. Use of the serum / plasma metabolic molecular markers described in claim 1 as detection targets in the preparation of a kit for auxiliary screening or early auxiliary diagnosis of pregnant women at high risk of GDM macrosomia.

5. A kit for auxiliary screening or early auxiliary diagnosis of pregnant women at high risk of GDM macrosomia, characterized in that A reagent comprising UPLC-Qexactive MS for detecting the serum / plasma metabolic molecular markers related to the screening or early diagnosis of macrosomia caused by gestational diabetes as described in claim 1.

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