Application of phosphatidyl ethanolamine

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of phosphatidyl ethanolamine. According to the invention, phosphatidyl ethanolamine PE (16: 022: 5) is taken as a biomarker, follicular fluid metabolites are analyzed, and high-sensitivity, high-specificity and high-accuracy diagnosis of the hypoovarian reserve function (DOR) is realized.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically relates to an application of phosphatidylethanolamine. Background Art

[0002] Diminished ovarian reserve (DOR) refers to a condition in which a woman's ovarian reserve is lower than normal for her age. This refers to a decrease in the number of ovarian follicles, despite a certain degree of ovarian responsiveness. Clinically, patients often present with reproductive dysfunction, including scanty menstruation, irregular menstruation, and infertility. Medical testing can reveal changes in typical biomarkers, including decreased anti-Mullerian hormone (AMH) levels and antral follicle count (AFC).

[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 factors such as genetics, immune abnormalities, iatrogenic damage (such as radiotherapy and chemotherapy) and psychological stress. Its pathogenesis may involve factors such as oocyte mitochondrial dysfunction, abnormal apoptosis of granulosa cells, local ovarian oxidative stress imbalance and abnormal angiogenesis, but the specific action pathway has not yet been elucidated.

[0004] During assisted reproductive treatment, patients with DOR can benefit from specialized treatments, such as higher doses of ovulation-inducing drugs and growth hormone therapy. Therefore, accurate identification of patients with DOR is crucial. Clinically, DOR is primarily diagnosed using a combination of AMH and AFC, but international consensus on diagnostic criteria remains. Existing diagnostic indicators for DOR have some limitations in terms of sensitivity, specificity, and accuracy. Therefore, developing a highly sensitive, specific, and accurate biomarker to improve the precise diagnosis of DOR is of great significance to both the industry and society. Summary of the Invention

[0005] To overcome the above technical problems, the present invention provides a use of phosphatidylethanolamine (PE) (16:0-22:5). The present invention uses PE (16:0-22:5) as a biomarker to diagnose DOR with high sensitivity, specificity, and accuracy by analyzing follicular fluid metabolites.

[0006] The present invention provides a method for detecting metabolites in isolated follicular fluid, comprising the following steps: detecting the concentration X of PE (16:0_22:5) in the isolated follicular fluid of a test subject, thereby determining whether the test subject is a patient with diminished ovarian reserve (DOR), wherein: X = 0.001*R*c*F*V / m.

[0007] R: Ratio of the peak area of ​​the analyte to the peak area of ​​the internal standard.

[0008] c: internal standard concentration.

[0009] F: internal standard correction factor.

[0010] V: Sample extract.

[0011] m: the sample size.

[0012] In one embodiment of the present invention, when the concentration X of PE (16:0_22:5) in the isolated follicular fluid of the test subject is less than 2.079×10 -7 mol / L, it indicates that the subject to be tested is a patient with diminished ovarian reserve function.

[0013] In one embodiment of the present invention, the detection method is a non-diagnostic and non-therapeutic detection method.

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

[0015] The present invention provides a use of phosphatidylethanolamine as a biomarker in the preparation of a product for diagnosing and / or warning of diminished ovarian reserve function, wherein the phosphatidylethanolamine is PE (16:0_22:5).

[0016] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

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

[0018] The positive progress of the present invention is that the present invention uses phosphatidylethanolamine PE (16:0_22:5) as a biomarker and analyzes follicular fluid metabolites, thereby achieving high sensitivity, high specificity and high accuracy diagnosis of diminished ovarian reserve (DOR). BRIEF DESCRIPTION OF THE DRAWINGS

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

[0020] Figure 2 Statistical results for metabolites that changed at least 1.75-fold in the follicular fluid lipid profiles of DOR patients and controls.

[0021] Figure 3 Comparison of the concentrations of different PE metabolites in the follicular fluid of DOR patients and controls.

[0022] Figure 4Figure 3 is the receiver operating characteristic curve (ROC curve) of the PE metabolite concentration in follicular fluid and DOR patients. DETAILED DESCRIPTION

[0023] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0024] Example 1: The present invention uses ultra-high performance liquid chromatography-mass spectrometry to detect follicular fluid metabolites and quantify specific lipids. The specific operating steps are as follows: 1.1 Research subjects and samples: In this example, samples of follicular fluid from patients undergoing assisted reproduction at the Sir Run Run Shaw Hospital affiliated to Zhejiang University School of Medicine were collected. The samples included 100 clinically diagnosed normal ovarian response (CT, control) control groups and follicular fluid from 58 DOR patients. Among them, the inclusion criteria for the clinical diagnosis of DOR (diminished ovarian reserve) patients were: referring to the POR (poor ovarian response) "Bologna Criteria" published by ESHRE (European Society of Human Reproduction and Embryology) in 2011: AMH (Anti-Müllerian Hormone) <1.1 ng / ml or AFC (Antral Follicle Count) <5. Inclusion criteria for CT (control) patients were: AMH 1.1 ng / ml ≤ < 6.0 ng / ml, and AFC 10 ≤ < 20. Patients with abnormal ovarian response, such as polycystic ovary syndrome, hyperprolactinemia, Cushing's syndrome, and congenital adrenal hyperplasia, were excluded. Each patient was informed of sample use and provided written informed consent. This study was approved by the Ethics Committee of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine.

[0025] 1.2 Collection of follicular fluid: The samples collected in this invention are all intended for in vitro fertilization ( in vitroFollicular fluid from mature follicles (follicle diameter ≥18 mm) from patients undergoing IVF treatment. Patients undergo controlled ovarian hyperstimulation (CHH). During this period, ovarian response is monitored by vaginal ultrasound, including follicle size, serum E2, P, LH, and FSH levels. Medication dosages are adjusted based on individual patient responses. When at least one or two dominant follicles in the dominant follicle cluster are ≥18 mm in diameter, and based on the patient's estrogen level, ovulation is induced with a single injection of 5,000–10,000 units of hCG or hCG combined with Dabiq, as appropriate. Oocyte retrieval is performed approximately 36 hours later under transvaginal ultrasound guidance. During the procedure, follicles are aspirated using a single-lumen cannula. The collected follicular fluid is then collected at a constant temperature to remove the cumulus-ovarian complex. The remaining follicular fluid is then collected, centrifuged at 5,000 rpm, aliquoted, and stored frozen at -80°C until use.

[0026] 1.3 Determination of follicular fluid metabolites: 1.3.1 Sample pretreatment and internal standard addition: After thawing, the samples were vortexed for 10 s to mix, and 50 μL of each sample was transferred; 1 mL of lipid extract containing internal standard (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 12,000 r / min for 10 min at 4°C; after centrifugation, 200 μL of the supernatant was transferred to the corresponding numbered centrifuge tube and concentrated to complete dryness; 200 μL of lipid reconstitution solution (acetonitrile: isopropanol = 1:1, v / v) was added, vortexed for 3 min, and centrifuged at 12,000 r / min for 3 min. The supernatant was transferred for UPLC-MS analysis.

[0027] 1.3.2 Detection and Analysis: The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (UPLC) and tandem mass spectrometry (MS).

[0028] The liquid phase conditions mainly include: 1) Chromatographic column: Thermo Accucore™ C30 column, ID2.1x100mm, 2.6um.

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

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

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

[0032] Mass spectrometry conditions included an electrospray ionization (ESI) source temperature of 500°C, a mass spectrometer voltage of 5500 V in positive ion mode, a mass spectrometer voltage of -4500 V in negative ion mode, ion source gas 1 (GS1) of 45 psi, gas 2 (GS2) of 55 psi, and a curtain gas (CUR) of 35 psi. In a triple quadrupole, each ion transition was scanned and detected based on optimized declustering potential (DP) and collision energy (CE).

[0033] 1.3.3 Lipid Quantification: Mass spectrometry data were processed using Analyst 1.6.3 software, and qualitative analysis of sample lipids was performed based on the local lipid database. Chromatographic peaks detected for each substance in different samples were then corrected to ensure accurate quantification. The integrated peak area values ​​of the corresponding substances were extracted and substituted into the formula to calculate the actual concentration: X = 0.001 * R * c * F * V / m, where: R is the ratio of the peak area of ​​the analyte to the peak area of ​​the internal standard.

[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 bonds number).

[0036] V: sample extract (μL).

[0037] m: sample volume taken (μL).

[0038] According to the above method, the concentration of specific lipids in the subject's follicular fluid can be detected and calculated, and the resulting X value can be correlated with the subject's risk of DOR. Based on the specific application scenario, an appropriate X value can be selected as the threshold for the subject to have DOR.

[0039] Specifically, receiver operating characteristic (ROC) analysis was performed using individual lipid concentrations as independent variables to obtain the area under the ROC curve (AUC) to assess the model's predictive efficiency. The Youden index was calculated to determine the optimal cutoff value X (specific lipid concentration) for the metabolite, providing the threshold for determining if a subject has DOR.

[0040] 1.4 Results Analysis and Statistics: Follicular Fluid Metabolome Data Figure 1 、 Figure 2 and Figure 4 The Maiwei Cloud Platform (https: / / cloud.metware.cn / # / tools / tool-list) was used for graphing and statistical analysis; Figure 3 GraphPad Prism 8 software was used for graphing and statistical analysis.

[0041] 1.5 Results and Discussion: 1.5.1 Lipidome analysis of follicular fluid: First, the top ten metabolites with the highest changes between DOR patients and the control group were selected. The results are as follows: Figure 1 As shown, the target metabolites are green dots and black characters, and the metabolites with no significant difference between DOR patients and the control group are gray dots and gray characters.

[0042] according to Figure 1 As can be seen, PE (16:0_22:5) ranked in the top ten changes in the lipid profile between DOR patients and controls, with a significant decrease. This suggests that PE (16:0_22:5) may be a biomarker for diagnosing DOR.

[0043] Further analysis revealed that, among the lipid profiles ranked 600-1200, follicular fluid lipids with minimal changes between DOR patients and controls, 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 showed minimal changes between DOR patients and controls, they clearly lack indicative value and cannot be used as biomarkers for diagnosing DOR.

[0044] In order to obtain higher quality signals, we further selected metabolites in the lipid group that had a change of at least 1.75 times between DOR patients and the control group. The experimental results are as follows: Figure 2 shown.

[0045] according to Figure 2 It can be seen that in the lipid group, there were 16 metabolites that showed a significant 1.75-fold change in DOR patients compared with the control group, all of which showed downregulation, among which the decrease level of PE (16:0_22:5) was significantly higher than that of other lipid metabolites, such as FFA (28:0) and Cer (d18:1 / 40:2(2OH)).

[0046] comprehensive Figure 1 and Figure 2 The results showed that compared with other lipid species, such as FFA (28:0) and Cer (d18:1 / 40:2(2OH)), PE (16:0_22:5) has greater potential as a biomarker for the diagnosis of DOR.

[0047] 1.5.2 Evaluation of the concentration of specific PE metabolites in the follicular fluid of DOR patients and the control group in the lipid group: According to the above-mentioned lipid quantification method, specific PE metabolites in the follicular fluid were selected as the evaluation objects, and the concentration of specific PE metabolites was measured, wherein the specific PE metabolites included PE (13:0_20:5), PE (20:1_20:5) or PE (16:0_22:5). The results are as follows: Figure 3 shown.

[0048] according to Figure 3 It can be seen that the average relative intensity of PE (16:0_22:5) in the follicular fluid PE metabolites in the control group was 3.00×10 5 , and 1.51×10 in the DOR group 5 , P<0.001; the average relative intensity of PE(13:0_20:5) in the control group was 2.20×10 7 , and 2.20×10 in the DOR group 7 , P>0.05; the average relative intensity of PE (20:1_20:5) in the control group was 8.37×10 4 , and 9.04×10 in the DOR group 4 , P>0.05. Compared with PE(13:0_20:5) and PE(20:1_20:5), the metabolite concentrations of PE(16:0_22:5) in DOR patients were significantly downregulated.

[0049] These results indicate that 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 in classifying CT and DOR: Further, the ROC curve was drawn with the relative intensity of specific PE as the test variable and whether DOR was the state variable. The vertical axis was sensitivity, the horizontal axis was 1-specificity, and the AUC was the area under the curve. The ROC curve was used to evaluate the performance of the model for classifying CT and DOR using metabolites. 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. The results are shown as follows: Figure 4 shown.

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

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

[0053] 1.5.4 Establish the PE(16:0_22:5) model for diagnosing DOR: PE(16:0_22:5), PE(13:0_20:5), and PE(20:1_20:5) are as follows: Figure 4 As shown in the figure, it can be seen that the AUC of any single lipid is lower than that of PE(16:0_22:5). Therefore, the diagnostic or early 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 diagnosis or early warning of DOR, the internal standard substance is phosphatidylethanolamine PE (17:0-22:4)-d5, and its F value is calculated to be 1.26. The Youden index calculated by the ROC curve shows that the optimal cutoff value of PE (16:0_22:5) is 2.079×10 -7 mol / L, that is, if the concentration X of PE (16:0_22:5) in the sample is less than 2.079×10 -7 mol / L, it may indicate that the subject suffers from 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 at the Sir Run Run Shaw Hospital affiliated to Zhejiang University School of Medicine were diagnosed as 24 DOR-positive patients and 44 DOR-negative patients according to the "Bologna criteria". They were then diagnosed for DOR using the AMH diagnostic method, the AFC diagnostic method, and the diagnostic method of Example 1 (PE diagnostic method), respectively.

[0057] The diagnostic criteria of the diagnostic method (PE diagnostic method) of Example 1 are as follows: The optimal cutoff value of PE (16:0_22:5) was obtained by calculating the Youden index to be 2.079×10 -7mol / L, AUC was 0.830, that is, when the concentration of PE (16:0_22:5) in the sample was less than 2.079×10 -7 mol / L, it may indicate that the subject suffers from DOR and is counted as positive, otherwise it is counted as negative.

[0058] Among them, the diagnostic results of DOR by AMH are shown in Table 1 below: Table 1:

[0059] The diagnostic results of DOR by AFC are shown in Table 2 below: Table 2:

[0060] The diagnostic results of DOR by PE (16:0_22:5) are shown in Table 3 below: Table 3:

[0061] Based on the data in Tables 1-3 above, the consistency rate between the PE diagnostic method and clinical diagnosis results was calculated using sensitivity, specificity, and accuracy.

[0062] Sensitivity: The ability to diagnose DOR-positive patients. It is calculated as: Sensitivity = number of true positives / (number of true positives + number of false negatives). The larger the value, the more effective the experimental results for detecting DOR.

[0063] Specificity: This measures the ability of the diagnostic model to correctly identify DOR-negative patients. A higher value indicates a lower probability of the diagnostic model misdiagnosing negative patients. Specificity is calculated as: Specificity = number of true negatives / (number of true negatives + number of false positives).

[0064] Accuracy: measures the proportion of samples correctly classified by the diagnostic model as a whole, calculated as: Accuracy = (number of true positives + number of true negatives) / number of samples.

[0065]

[0066] It can be seen that compared with the AMH diagnosis method and the AFC diagnosis method, the PE diagnosis method has higher sensitivity and accuracy for the diagnosis of DOR patients, and its specificity is also better.

Claims

1. A method for detecting metabolites in isolated follicular fluid, comprising the following steps: detecting the concentration X of PE (16:0-22:5) in the isolated follicular fluid of a subject to be tested, thereby determining whether the subject to be tested is a patient with diminished ovarian reserve (DOR), wherein: X=0.001*R*c*F*V / m; R: 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 extract; m: sample volume.

2. The detection method according to claim 1, wherein When the concentration of PE (16:0_22:5) in the isolated follicular fluid of the test subject is X<2.079×10 -7 mol / L, it indicates that the subject to be tested is a patient with diminished ovarian reserve function.

3. The detection method according to claim 1, wherein The internal standard is phosphatidylethanolamine PE (17:0-22:4)-d5.

4. A use of phosphatidylethanolamine as a biomarker in the preparation of a product for diagnosing and / or warning of diminished ovarian reserve function, wherein the phosphatidylethanolamine is PE (16:0_22:5).

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