Use of a phosphatidylethanolamine

Abstract

The application belongs to the technical field of biology and specifically relates to application of phosphatidylethanolamine. The application takes phosphatidylethanolamine PE (16:0_22:5) as a biomarker, realizes high sensitivity, high specificity and high accuracy in diagnosis of decreased ovarian reserve (DOR) by analyzing metabolites of follicular fluid.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to an application of phosphatidylethanolamine. BACKGROUND

[0002] Diminished ovarian reserve (DOR) refers to that the ovarian reserve function of a female is lower than the normal level of the same age, that is, the number of ovarian follicles decreases, but there is still certain ovarian responsiveness. The patient often shows reproductive dysfunction such as less menstrual flow, irregular menstruation, and infertility in clinic. Medical detection can see the changes of typical biomarkers: the levels of anti-Mullerian hormone (AMH) and antral follicle count (AFC) decrease.

[0003] DOR has a serious impact on the fertility of women of childbearing age, but the pathogenesis of DOR is still unclear. Some scholars believe that it is related to genetic, immune abnormalities, iatrogenic damage (such as chemotherapy) and psychological stress, and its pathogenesis may involve factors such as mitochondrial dysfunction of oocytes, abnormal apoptosis of follicular granulosa cells, local oxidative stress imbalance of ovaries, and abnormal angiogenesis, but the specific action pathway has not been elucidated.

[0004] In the process of assisted reproductive treatment, special treatment for patients with DOR can benefit them, such as giving larger doses of ovulation drugs and applying growth hormone assisted treatment, so it is crucial to accurately identify DOR patients. Clinically, the clinical diagnosis of DOR is mainly made by AMH combined with AFC, but the international diagnostic criteria have not yet reached a consensus. The existing diagnostic indicators of DOR still have some deficiencies in sensitivity, specificity and accuracy. Therefore, it is of great significance to the industry and society to develop a biomarker with high sensitivity, high specificity and high accuracy to improve the accurate diagnosis of DOR. SUMMARY

[0005] In order to overcome the above technical problems, the application provides an application of phosphatidylethanolamine PE (16:0_22:5). The application uses phosphatidylethanolamine PE (16:0_22:5) as a biomarker to realize the diagnosis of DOR with high sensitivity, high specificity and high accuracy by analyzing the metabolites of follicular fluid.

[0006] The application provides a detection method for metabolites in ex vivo follicular fluid, which comprises the following steps: detecting the concentration X of PE (16:0_22:5) in the ex vivo follicular fluid of a to-be-detected object, so as to determine whether the to-be-detected object is a patient with diminished ovarian reserve (DOR), wherein: the X=0.001*R*c*F*V / m.

[0007] R: ratio of peak area of to-be-detected substance to peak area of internal standard.

[0008] c: concentration of internal standard.

[0009] F: internal standard correction factor.

[0010] V: sample extraction solution.

[0011] m: sample amount taken.

[0012] In an embodiment of the present application, when the concentration X of PE (16:0_22:5) in the follicular fluid of the subject to be tested is less than 2.079 x 10 -7 mol / L, it is indicated that the subject to be tested is a patient with decreased ovarian reserve function.

[0013] In an embodiment of the present application, the detection method is a detection method for non-diagnostic and non-therapeutic purposes.

[0014] In an embodiment of the present application, the internal standard is phosphatidylethanolamine PE (17:0-22:4)-d5.

[0015] The present application provides a phosphatidylethanolamine as a biomarker for preparing a product for diagnosing and / or early warning of decreased ovarian reserve function, wherein the phosphatidylethanolamine is PE (16:0_22:5).

[0016] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined in any manner, thereby obtaining various preferred examples of the present application.

[0017] The reagents and raw materials used in the present application are commercially available.

[0018] The positive progress effect of the present application is that the present application uses phosphatidylethanolamine PE (16:0_22:5) as a biomarker, and through analysis of follicular fluid metabolites, high sensitivity, high specificity and high accuracy are achieved in the diagnosis of decreased ovarian reserve function (DOR). BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The red and green dots represent the top ten metabolites that increase and decrease, respectively, in the ranking results of the changes in metabolites in the lipidome of the follicular fluid of DOR patients and the control group.

[0020] Figure 2 The statistical results of the metabolites in the lipidome of the follicular fluid of DOR patients and the control group that change by at least 1.75 times before and after.

[0021] Figure 3 The concentration comparison of different PE metabolites in the follicular fluid of DOR patients and the control group.

[0022] Figure 4To obtain the receiver operating characteristic (ROC) curve of the concentration of PE metabolites in follicular fluid and the DOR patients. DETAILED DESCRIPTION

[0023] The present application is further illustrated by the following examples without thereby limiting the present application to the scope of the examples. The experimental methods in the following examples without specific conditions are selected according to the conventional methods and conditions, or according to the commercial instructions.

[0024] Example 1: The present application detects follicular fluid metabolites by ultra-high performance liquid chromatography-mass spectrometry, and quantifies specific lipids, the specific operation steps are as follows: 1.1 Research subjects and samples: The present application collects follicular fluid samples of patients undergoing assisted reproduction in the affiliated shao yifu hospital of zhejiang university school of medicine, the samples include 100 cases of clinical diagnosis of normal ovarian response (CT, control) control population and 58 cases of DOR patients, among which, the clinical diagnosis of DOR (diminished ovarian reserve) patients includes the following criteria: According to the POR (poor ovarian response) “Bologna Criteria” (Bologna Criteria) published by ESHRE (European Society of Human Reproduction and Embryology) in 2011: AMH (Anti-Mullerian Hormone) <1.1 ng / ml or AFC (Antral Follicle Count) <5. The inclusion criteria of CT (control) patients are as follows: 1.1 ng / ml ≤ AMH <6.0 ng / ml, and 10 ≤ AFC <20, excluding polycystic ovary syndrome, hyperprolactinemia, Cushing's syndrome, congenital adrenal hyperplasia and other abnormal ovarian response diseases. Each patient is informed of the purpose of the sample and signs the informed consent form, and the study is approved by the ethics committee of the affiliated shao yifu hospital of zhejiang university school of medicine.

[0025] 1.2 Collection of follicular fluid: The samples collected in the present application are follicular fluid samples of patients who are about to undergo in vitro fertilization (IVF) treatment in the affiliated shao yifu hospital of zhejiang university school of medicine. The follicular fluid samples are collected by follicular aspiration during the oocyte retrieval procedure of IVF treatment. The follicular fluid samples are collected in 1.5 ml EP tubes, and the samples are stored at -80°C until use. in vitroFollicular fluid of mature follicle (follicle diameter ≥18 mm) of patients treated by in vitro fertilization (IVF) treatment. The patient receives controlled ovarian hyperstimulation treatment, during which the follicle size, serum E2, P, LH, FSH values and other indicators are monitored by transvaginal B-ultrasound to detect ovarian response, and the drug dose is appropriately adjusted according to the individual response of the patient. When at least 1-2 dominant follicles in the dominant follicle group have a diameter of ≥18 mm, and the patient's estrogen level is referred to, HCG 5000-10000 units or HCG + Diphereline is injected at an appropriate time to induce ovulation, and transvaginal ultrasound-guided oocyte retrieval is performed about 36 hours later. The follicle is aspirated with a single lumen tube during the operation, the collected follicular fluid is collected after the constant temperature is picked up, and the remaining follicular fluid is collected, centrifuged at 5000 rpm, aliquoted, and frozen at -80°C for use.

[0026] 1.3 Determination of follicular fluid metabolites: 1.3.1 Sample pretreatment and internal standard addition: after thawing, the sample was vortexed for 10 s and mixed, 50 μL of the sample was taken; 1 mL of internal standard lipid extraction solution (methyl tert-butyl ether: methanol = 3:1, V / V) was added and vortexed for 15 min; 200 μL of water was added, vortexed for 1 min, and centrifuged at 4 ℃ and 12000 r / min for 10 min; after centrifugation, 200 μL of supernatant was taken into the corresponding numbered centrifuge tube, and concentrated to dryness; 200 μL of lipid reconstitution solution (acetonitrile: isopropanol = 1:1, V / V) was added, vortexed for 3 min, centrifuged at 12000 r / min for 3 min, and the supernatant was taken for UPLC-MS analysis.

[0027] 1.3.2 Detection and analysis: The data acquisition instrument system mainly includes ultra-performance liquid chromatography (UltraPerformance Liquid Chromatography, UPLC) and tandem mass spectrometry (Tandem mass spectrometry, MS).

[0028] Among them, the liquid phase conditions mainly include: 1) chromatographic column: Thermo Accucore™ C30 column, i.d. 2.1x100mm, 2.6um.

[0029] 2) Mobile phase: A phase: acetonitrile / water (60 / 40, V / V) (containing 0.1% formic acid, 10 mmol / L ammonium formate); B phase: acetonitrile / isopropanol (10 / 90, V / V) (containing 0.1% formic acid, 10 mmol / L ammonium formate).

[0030] 3) Gradient of mobile phase: 0 min A / B (80:20, V / V), 2 min (70:30, V / V), 4 min (40:60, V / V), 9 min (15:85, V / V), 14 min (10:90, V / V), 15.5 min (5:95, V / V), 17.3 min (5:95, V / V), 17.5 min (80:20, V / V), 20 min (80:20, V / V).

[0031] 4) Flow rate 0.35 ml / min; column temperature 45℃; injection volume 2 μl.

[0032] The mass spectrometry conditions mainly include: electrospray ionization (ESI) temperature 500℃, mass spectrometry voltage 5500V in positive ion mode, mass spectrometry voltage -4500V in negative ion mode, ion source gas 1 (GS1) 45 psi, gas 2 (GS2) 55 psi, curtain gas (CUR) 35 psi. In the triple quadrupole, each ion pair is scanned and detected according to the optimized declustering potential (DP) and collision energy (CE).

[0033] 1.3.3 Lipid quantification: The mass spectrometry data is processed by Analyst 1.6.3 software, and the sample lipids are qualitatively analyzed according to the local lipid database information, and the chromatographic peaks of each substance detected in different samples are corrected to ensure accurate quantification. The integral peak area value of the corresponding substance is extracted and substituted into the formula to calculate the actual concentration X=0.001*R*c*F*V / m, wherein: R: peak area ratio of the substance to be tested to the internal standard peak area.

[0034] c: internal standard concentration (μmol / L).

[0035] F: internal standard correction factor (F=1+0.02*(total carbon number-36)+0.05*total double bond number).

[0036] V: sample extraction solution (μL).

[0037] m: sample amount taken (μL).

[0038] According to the above method, the concentration of specific lipids in the follicular fluid of the subject can be detected and calculated, and the obtained X value is related to the risk value of the subject being a DOR patient. According to the specific application scenario, a suitable X value can be selected as the threshold for the subject to have DOR.

[0039] Specifically, ROC analysis was performed with single lipid concentration as the independent variable, and the area under the ROC curve (AUC) was obtained to determine the prediction efficiency of the model. The optimal cutoff value X (concentration of a specific lipid) of the metabolite was obtained by calculating the Youden index, and the threshold for subjects with DOR was obtained.

[0040] 1.4 Result analysis and statistics: follicular fluid metabolome data Figure 1 、 Figure 2 and Figure 4 Graphing and statistical analysis were performed using the Mavi cloud platform (https: / / cloud.metware.cn / # / tools / tool-list); Figure 3 Graphing and statistical analysis were performed using GraphPad Prism 8 software.

[0041] 1.5 Discussion of results: 1.5.1 Lipidome detection of follicular fluid: First, the top ten metabolites in the lipidome that changed in DOR patients and controls were selected, and the results are shown in Figure 1 The target metabolites are green dots with black text, and the metabolites that did not change significantly between DOR patients and controls are gray dots with gray text.

[0042] According to Figure 1 , PE(16:0_22:5) ranks among the top ten changes between DOR patients and controls, and the change (decrease) is relatively significant. Therefore, PE(16:0_22:5) is likely to become a biomarker for diagnosing DOR.

[0043] Further analysis found that the follicular fluid lipids ranked 600-1200 in the change ranking between DOR patients and controls were relatively weak, such as PE(13:0_20:5), PE(20:1_20:5), FFA(28:0), and Cer(d18:1 / 40:2(2OH)). Since these substances do not change significantly between DOR patients and controls, they obviously do not have an indication and cannot be used as biomarkers for diagnosing DOR.

[0044] To obtain higher quality signals, further select metabolites in the lipidome that change at least 1.75 times between DOR patients and controls, and the experimental results are shown in Figure 2 .

[0045] According to Figure 2 , there are 16 metabolites that change significantly by 1.75 times between DOR patients and controls, all of which are down-regulated, and the decrease in PE(16:0_22:5) is significantly higher than other lipid metabolites, such as FFA(28:0) and Cer(d18:1 / 40:2(2OH)).

[0046] In summary Figure 1 and Figure 2 The results show that PE(16:0_22:5) has more potential to be used as a biomarker to diagnose DOR than other lipid species, such as FFA(28:0) and Cer(d18:1 / 40:2(2OH)).

[0047] 1.5.2 Evaluation of the concentrations of specific PE metabolites in the lipidome of follicular fluid of DOR patients and controls: According to the method for quantifying lipids described above, specific PE metabolites in follicular fluid were selected as evaluation objects, and the concentrations of specific PE metabolites were determined, wherein the specific PE metabolites included PE(13:0_20:5), PE(20:1_20:5) or PE(16:0_22:5), and the results are shown in Figure 3 .

[0048] According to Figure 3 , the average relative intensity of PE(16:0_22:5) in the PE metabolites of follicular fluid in the control group was 3.00x10 5 , and in the DOR group was 1.51x10 5 , P<0.001; the average relative intensity of PE(13:0_20:5) in the control group was 2.20x10 7 , and in the DOR group was 2.20x10 7 , P>0.05; the average relative intensity of PE(20:1_20:5) in the control group was 8.37x10 4 , and in the DOR group was 9.04x10 4 , P>0.05. Therefore, compared with PE(13:0_20:5) and PE(20:1_20:5), the metabolite concentration of PE(16:0_22:5) in DOR patients was significantly down-regulated.

[0049] Therefore, PE(16:0_22:5) can be used as a biomarker to diagnose DOR.

[0050] 1.5.3 ROC curve evaluation of the model performance of specific PE metabolites for classifying CT and DOR: Further, the ROC curve was drawn with the relative intensity of specific PE as the test variable and whether DOR as the state variable, the vertical coordinate was the sensitivity, the horizontal coordinate was 1-specificity, the AUC was the area under the curve, and the ROC curve was used to evaluate the model performance of using metabolites to classify CT and DOR. Specifically, the ROC analysis was performed with the concentration of specific PE metabolites as the independent variable, and the area under the ROC curve (AUC) was obtained, and the results are shown in Figure 4 .

[0051] According to Figure 4It can be seen that the AUC of PE (16:0_22:5) is 0.830 (0.765-0.894, P<0.01); the AUC of PE (13:0_20:5) is 0.528 (0.433-0.623, P>0.05), and the AUC of PE (20:1_20:5) is 0.535 (0.437-0.634, P>0.05).

[0052] The above experimental results show that not all PEs can be used as biomarkers to diagnose and warn DOR patients, but PE (16:0_22:5) can be used to diagnose and warn whether a DOR patient.

[0053] 1.5.4 Establishing a model for diagnosing DOR with PE (16:0_22:5): PE (16:0_22:5), PE (13:0_20:5), and PE (20:1_20:5) are respectively as shown in Figure 4 It can be seen that the AUC of any single lipid is lower than that of PE (16:0_22:5), so the diagnostic or warning potential of PE (16:0_22:5) is significantly better than that of other lipid molecules.

[0054] Therefore, when PE (16:0_22:5) is selected as a biomarker for diagnosing or warning DOR, the internal standard substance is phosphatidylethanolamine PE (17:0-22:4)-d5, and the F value is calculated to be 1.26. The best cutoff value of PE (16:0_22:5) is 2.079x10 -7 mol / L, that is, if the concentration X of PE (16:0_22:5) in the sample is less than 2.079x10 -7 mol / L, it can be suggested that the subject has DOR.

[0055] Example 2: Evaluation of the detection effect of phosphatidylethanolamine PE (16:0_22:5).

[0056] In this example, 68 women who underwent assisted reproductive treatment in the Shao Yifu Hospital Affiliated to Zhejiang University School of Medicine were diagnosed as 24 DOR positive patients and 44 negative patients according to the "Bologna criteria", and then the AMH diagnosis method, the AFC diagnosis method and the diagnosis method of Example 1 (PE diagnosis method) were used to diagnose whether they had DOR.

[0057] The diagnostic criteria of the diagnosis method of Example 1 (PE diagnosis method) are as follows: the best cutoff value of PE (16:0_22:5) is 2.079x10 -7mol / L, AUC is 0.830, that is, when the material concentration of PE (16:0_22:5) in the sample is less than 2.079 x 10 -7 mol / L, the subject is prompted to have DOR, counted as positive, otherwise counted as negative.

[0058] The diagnosis results of DOR by AMH are shown in Table 1 as follows:

[0059] Table 1:

[0060] The diagnosis results of DOR by AFC are shown in Table 2 as follows:

[0061] Table 2:

[0062] The diagnosis results of DOR by PE (16:0_22:5) are shown in Table 3 as follows:

[0063] Table 3:

[0064] Based on the data in Tables 1-3, the consistency rate of PE diagnosis method and clinical diagnosis results is calculated by sensitivity, specificity and accuracy.

[0065] Sensitivity: the ability to diagnose DOR positive patients, calculated as: Sensitivity = True Positive Number / (True Positive Number + False Negative Number), the larger the value, the more effective the experimental results of detecting DOR.

[0066] Specificity: measures the ability of the diagnostic model to correctly identify DOR negative patients, the higher the value, the lower the probability of misdiagnosing negative patients, calculated as: Specificity = True Negative Number / (True Negative Number + False Positive Number).

[0067] Accuracy: measures the proportion of samples correctly classified by the diagnostic model as a whole, calculated as: Accuracy = (True Positive Number + True Negative Number) / Sample Number.

[0068]

[0069] As can be seen, compared with AMH diagnosis method and AFC diagnosis method, PE diagnosis method has higher sensitivity and accuracy for the diagnosis of DOR patients, and its specificity effect is also better.

Claims

1. The application of phosphatidylethanolamine as a biomarker in the preparation of products for diagnosing impaired ovarian reserve, wherein the phosphatidylethanolamine is PE (16:0-22:5).

2. The application as described in claim 1, characterized in that, The diagnosis of diminished ovarian reserve is achieved by detecting metabolites in isolated follicular fluid. The detection method includes the following steps: detecting the concentration X of PE (16:0-22:5) in the follicular fluid of the test subject to determine whether the test subject is a patient with diminished ovarian reserve (DOR), wherein: The value of X is 0.001 * R * c * F * V / m. R: The ratio of the peak area of ​​the analyte to the peak area of ​​the internal standard; c: Internal standard concentration; F: Internal standard correction factor; V: Sample extraction solution; m: The sample size.

3. The application as described in claim 2, characterized in that, When the concentration X of PE (16:0_22:5) in the follicular fluid of the test subject is less than 2.079×10 -7 A concentration of mol / L indicates that the subject of the test is a patient with diminished ovarian reserve.

4. The application as described in claim 2, characterized in that, The internal standard mentioned is phosphatidylethanolamine PE(17:0-22:4)-d5.

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