Application of pyrophosphatase in judging maturity of oocyte

Abstract

The present application relates to the application of pyrophosphatase in judging the maturity of oocytes, and the correlation between serum ENPP2 level and follicular fluid ENPP2 level is determined through enzyme-linked immunoassay of follicular fluid and serum protein components. Then, the serum ENPP2 level during follicular maturation is detected, and it is found that serum ENPP2 can be a protein for indicating human oocyte maturation. Through research, a model is obtained, and after the serum ENPP2 concentration is detected and brought into the model, the P1 value is calculated. When the model P1 value is greater than or equal to 0.50, the current oocyte is determined to be mature. When the model P1 value is less than 0.50, the current oocyte is determined to be immature. At the same time, AMH and AFC are used as auxiliary diagnostic parameters to establish two other models P2 and P3. When the P2 value is greater than or equal to 0.99 or the P3 value is greater than or equal to 0.99, the current oocyte is determined to be mature. When the P2 value is less than 0.99 or the P3 value is less than 0.99, the current oocyte is determined to be immature.

Description

[Technical Field]

[0001] This invention relates to the field of reproductive medicine technology, specifically to the application of pyrophosphatase in determining oocyte maturity. [Background Technology]

[0002] The oocyte is one of the few cells visible to the naked eye, and its maturation process is a prerequisite physiological event determining successful fertilization, zygote formation, and normal embryonic development. Human oocytes enter the early stages of meiosis during fetal development and arrest in prophase I of meiosis until they undergo hormonal regulation or physical stimulation, completing both meiosis I and II to reach maturity. Human oocyte maturation is divided into three stages: the genital vesicle (GV) stage, metaphase I (MI) stage, and metaphase II (MII) stage. In the GV stage, the oocyte has not resumed meiosis; after removing the granulosa cells, a distinct nucleus can be seen. In the MI stage, the first polar body has not been expelled, and the granulosa cells have not diffused well. In the MII stage, the oocyte has expelled the first polar body and is clinically considered a "mature" oocyte, capable of fertilizing with sperm and expelling the second polar body to become a zygote.

[0003] Clinically, during ovulation induction, because oocytes are located within the body, specifically within follicles in the ovarian tissue, oocyte maturation can only be assessed indirectly. Ultrasound is a major monitoring standard for oocyte maturation, particularly in determining the number and size of follicles. Due to differences in population, medications used, and ultrasound monitoring techniques, different reproductive centers may have slightly different criteria for determining oocyte maturity. Generally, at least 4-5 follicles with a diameter of 16-18 mm are considered the optimal main follicle group. Peripheral serum estradiol (E2) and progesterone (P) levels are also commonly used to assist in determining oocyte maturity. Generally, a serum E2 level of 200-350 pg / ml per follicle (follicle with a diameter ≥14 mm) is established as the oocyte maturation point. In addition to the commonly used E2, some centers also use serum P and the P / E2 ratio to assist in the determination. Determining oocyte maturity during ovulation induction requires a comprehensive analysis of multiple data points and years of clinical experience; currently, there is a lack of a unified and objective standard.

[0004] Inaccurate determination of oocyte maturity can affect the outcome of in vitro fertilization and embryo transfer (IVF-ET). Immature oocytes can lead to failure to fertilize and obtain effective embryos, while overmature oocytes can lead to decreased oocyte quality and adverse outcomes such as polyspermia. Therefore, there is an urgent need to find a new method that can intuitively and qualitatively and quantitatively determine oocyte maturity.

[0005] Pyrophosphatase / phosphodiesterase 2 (ENPP2), also known as autotaxin, is a secretase with lysophospholipase D activity. It hydrolyzes lysophosphatidylcholine to produce lysophosphatidic acid (LPA). LPA is the smallest and simplest phospholipid discovered to date (430–480 Da), and it is a key precursor in the early stages of eukaryotic phospholipid biosynthesis. Previous research has shown that LPA is significantly elevated in the follicular fluid of mature follicles and is an important factor in the in vitro maturation of oocytes. Prostatic acid phosphatase (ACPP) can inactivate LPA. Anti-Müllerian hormone (AMH) is an important hormone in gonadal development. In adult women, AMH inhibits the recruitment of primordial follicles and the development of antral follicles, preventing premature follicular depletion. Antral follicles typically range in diameter from 2 to 9 mm. The antral follicle count (AFC) is usually checked on days 2-5 of the menstrual cycle, with a gynecological transvaginal ultrasound providing the most accurate assessment. AMH and AFC are important indicators for evaluating ovarian reserve function and are closely related to the dosage of medications used in ovulation induction treatment and the final number of eggs retrieved. [Summary of the Invention]

[0006] The purpose of this invention is to address the aforementioned problems by providing the application of pyrophosphatase in determining oocyte maturity, which provides a more accurate reflection of the maturation status of oocytes in follicles.

[0007] One of the objectives of this invention is to provide the application of pyrophosphatase in determining oocyte maturity.

[0008] The second objective of this invention is to provide a method for evaluating oocyte maturity, the specific steps of which are as follows:

[0009] (1) Detect the concentration of serum pyrophosphatase, the concentration of anti-Müllerian hormone, and the number of antral follicles;

[0010] (2) Input the detected data into the calculation according to the model;

[0011] The model is:

[0012] Model 1: P1 = exp[-4.8960 + 0.03794 × (serum pyrophosphatase concentration)] / (1 + exp[-4.8960 + 0.03794 × (serum pyrophosphatase concentration)]) determines the value of P1;

[0013] Model 2: P2 = exp[-5.9364 + 0.1518 × (serum pyrophosphatase concentration / anti-Müllerian hormone concentration)] / (1 + exp[-5.9364 + 0.1518 × (serum pyrophosphatase concentration / anti-Müllerian hormone concentration)]) to determine the P2 value;

[0014] Model 3: P3 = exp[-6.1438 + 0.6012 × (serum pyrophosphatase concentration / antral follicle count)] / (1 + exp[-6.1438 + 0.6012 × (serum pyrophosphatase concentration / antral follicle count)]) determines the P3 value;

[0015] (3) Determine the maturity of the oocytes.

[0016] When the P1 value of Model 1 is ≥0.50 and / or the P2 value of Model 2 is ≥0.99 and / or the P3 value of Model 3 is ≥0.99, the current oocyte is considered mature; when the P1 value of Model 1 is <0.50 and / or the P2 value of Model 2 is <0.99 and / or the P3 value of Model 3 is <0.99, the current oocyte is considered immature.

[0017] Further explanation: The specific steps for detecting serum pyrophosphatase concentration are as follows: After equilibration at room temperature for 30 min, add 50 μL of standard curve solution to each well of the ELISA plate coated with pyrophosphatase antibody, and add 50 μL of serum to be tested to each well; then add 50 μL of freshly diluted 1:100 biotin-labeled antibody mixture to each reaction well; incubate at 37℃ for 1 h, wash 3 times with washing buffer, and spin dry; add 50 μL of hydrogen peroxide and 50 μL of tetramethylbenzidine (TMB) chromogenic solution to each well; incubate at room temperature in the dark for about 10-20 min; finally, add 50 μL of stop solution, measure at a wavelength of 450 nm, and record the OD value; calculate the protein concentration of each sample based on the standard curve.

[0018] Further explanation: The specific steps for detecting serum AMH concentration are as follows: The detection equipment is a Roche Cobas e411 electrochemiluminescence immunoassay analyzer, and the reagent is a Roche AMH detection kit. Calibration was performed using Roche Calibrators before detection; after equilibration at room temperature for 30 min, 50 μL of serum sample, biotinylated anti-AMH monoclonal antibody, and ruthenium complex-labeled anti-AMH monoclonal antibody were added to form an antigen-antibody sandwich complex; streptavidin-coated magnetic beads were added; the reaction solution was drawn into the measuring cell, a certain voltage was applied to the electrode to cause the complex to luminesce, and the result was measured; the calibration curve was obtained using the quality control material PerciControl AMH.

[0019] To further explain, the specific steps for detecting AFC are as follows: On days 2-5 of the menstrual cycle, a gynecological vaginal ultrasound examination is performed to record the number of recruitable antral follicles with a diameter of 2-9 mm, and records are made on both sides of the ovary.

[0020] A third objective of this invention is to provide a serum protein that indicates oocyte maturation, wherein the serum protein is a pyrophosphatase.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] Serum biomarker detection is a novel, non-subjective, efficient, and quantitative method for determining oocyte maturity. Based on serum sex hormone detection, it simultaneously detects proteins related to oocyte development and maturation, aiming to identify specific biomarkers reflecting oocyte maturation from peripheral blood. Combined with current clinical monitoring data, this provides a more accurate reflection of the maturation status of oocytes within follicles. [Attached Image Description]

[0023] Figure 1 This is a correlation diagram of ENPP2 expression in follicular fluid and serum in the experimental examples of this invention.

[0024] Figure 2 This is a correlation diagram of LPA expression in follicular fluid and serum in the experimental examples of this invention.

[0025] Figure 3 This is a correlation diagram of ACPP expression in follicular fluid and serum in the experimental examples of this invention.

[0026] Figure 4 This is a graph showing the differential expression of serum ENPP2 on the day of basal sex hormones, during follicular development, and at follicular maturation in the experimental examples of this invention.

[0027] Figure 5 This is a graph showing the differential expression of serum LPA on the day of basal hormone, during follicular development, and at follicular maturation in the experimental examples of this invention.

[0028] Figure 6 This is a graph showing the differential expression of serum ACPP in the experimental examples of this invention at the time of basal hormone release, during follicular development, and at follicular maturation.

[0029] Figure 7 This is a schematic diagram of the ROC curves of serum ENPP2, LPA, and ACPP in the experimental examples of this invention.

Detailed Implementation Methods

[0030] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0031] Experimental example:

[0032] 1. Objects and Methods

[0033] 1.1 Inclusion of Study Subjects

[0034] This study collected cases aged 20-44 years with regular menstrual cycles of 28-32 days, normal basal endocrine levels, no endocrine / immune and metabolic diseases, no tuberculosis / liver and kidney diseases, no history of smoking or alcohol abuse, and who had not received hormone therapy within 3 months prior to the procedure. These patients underwent assisted reproductive technologies (such as in vitro fertilization, IVF; intracytoplasmic sperm injection, ICSI) due to male factors.

[0035] From November 2021 to May 2022, follicular fluid and serum were collected from the same IVF patient (65 cases, mean age 32.60 ± 3.98 years). Serum was also collected from patients undergoing IVF treatment during follicular monitoring (training group, 35 cases, mean age 31.91 ± 3.53 years; validation group, 57 cases, mean age 34.31 ± 4.83 years), and divided into basal hormone day group, follicular development group, and follicular maturation group.

[0036] Follicular fluid was collected as follows: During oocyte retrieval for IVF / ICSI treatment, the first tube of pale yellow, bloodless, nearly transparent follicular aspiration fluid (follicle diameter >16mm) was collected, and mature oocytes were confirmed under a microscope. The fluid was centrifuged at 3000 rpm for 15 min, and the supernatant was separated, aliquoted, and labeled as oocyte follicular fluid. Immediately after collection, the follicular fluid was centrifuged at 3000 g for 15 min, and the supernatant was collected. 1 mL of the supernatant was aliquoted and stored at -80°C for later use. Serum was collected as follows: Peripheral blood samples (3.0 mL) from all participants were collected using disposable vacuum non-anticoagulating blood collection tubes. The blood was centrifuged over 4 hours (3000 rpm, 10 min, 4°C), and the supernatant was aliquoted into 500 μL portions and stored at -80°C.

[0037] Simultaneously, patient clinical data and test indicators are collected. After case adoption, cases are grouped and numbered to establish a complete and systematic case database. Cases are also followed up regularly, and data on patient clinical pregnancy, miscarriage, and multiple pregnancies are recorded.

[0038] 1.2 Enzyme-linked immunosorbent assay

[0039] The experiments used the human ENPP2 (ATX) ELISA kit (CSB-EL007680HU; Wuhan Huamei), the human LPA kit (CSB-EQ028005HU; Wuhan Huamei), and the human PAP (ACPP) ELISA kit (ab267802; Abcam, Cambridge, MA, USA). No dilution was performed when detecting ENPP2; follicular fluid and serum were diluted 1:400 when detecting LPA; and follicular fluid and serum were diluted 1:100 and 1:2 respectively when detecting ACPP protein.

[0040] After equilibration at room temperature for 30 minutes, a standard curve was prepared. ENPP2 standards were provided by the kit at six concentrations: 2000 ng / mL, 750 ng / mL, 375 ng / mL, 125 ng / mL, 25 ng / mL, and 0 ng / mL. LPA and ACPP protein standards were prepared at concentrations of 250 ng / mL and 2400 pg / mL, respectively. When using the diluent, serially dilute the LPA standard to establish standard curves at eight concentrations: 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, 15.6 ng / mL, 8.8 ng / mL, 3.9 ng / mL, and 0 ng / mL. Serially dilute the ACPP standard to establish standard curves at eight concentrations: 2400 pg / mL, 1200 pg / mL, 600 pg / mL, 300 pg / mL, 150 pg / mL, 75 pg / mL, 37.5 pg / mL, and 0 pg / mL.

[0041] Add 50 μL of diluted follicular fluid and serum to ELISA plates coated with anti-ENPP2 antibody, anti-LPA antibody, and anti-ACPP antibody, respectively. Add 50 μL of freshly diluted biotin-labeled antibody solution (1:100) to the anti-ENPP2 antibody and anti-LPA antibody ELISA plates; add 50 μL of freshly diluted antibody solution (1:10) to the anti-ACPP antibody ELISA plates. Apply the ELISA plate sticker and incubate at 37°C for 1 hour.

[0042] Wash each well three times with 300 μL of washing buffer for 2 min each time, then spin dry. Add 50 μL of hydrogen peroxide and 50 μL of tetramethylbenzidine (TMB) chromogenic solution to the anti-ENPP2 antibody ELISA plate; add 100 μL of TMB chromogenic solution to the anti-LPA antibody ELISA plate and the anti-ACPP antibody ELISA plate, and incubate at room temperature in the dark for about 10-20 min. Finally, add 50 μL of stop solution (sulfuric acid), and measure the OD value at 450 nm. Calculate the protein concentration of each sample according to the standard curve.

[0043] 1.3 Anti-Müllerian Hormone (AMH) Detection

[0044] The detection equipment was a Roche Cobas e411 electrochemiluminescence immunoassay analyzer, and the reagent was a Roche AMH detection kit. Calibration was performed using Roche Calibrators before detection. After equilibration at room temperature for 30 min, 50 μL of serum sample, biotinylated anti-AMH monoclonal antibody, and ruthenium complex-labeled anti-AMH monoclonal antibody were added to form an antigen-antibody sandwich complex; streptavidin-coated magnetic beads were then added. The reaction solution was drawn into the measurement cell, and a certain voltage was applied to the electrodes to cause the complex to luminesce, which was then measured. The calibration curve was obtained using the quality control material Perci Control AMH.

[0045] 1.4 Antral Follicle Count (AFC) Detection

[0046] On days 2-5 of the menstrual cycle, a gynecological vaginal ultrasound is performed to record the number of recruitable antral follicles with a diameter of 2-9 mm, and the number of follicles is recorded on both sides of the ovary.

[0047] 1.5 Data Processing and Analysis

[0048] Based on the OD values ​​of each well in the ELISA plate, and after converting them to the protein concentrations of each sample according to the standard curves, Pearson correlation analysis was used to examine the correlation between ENPP2, LPA, and ACPP in serum and follicular fluid (see [link to relevant documentation]). Figures 1-3 Statistical analysis was performed using SPSS 16.0 software. The t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among three groups. GraphPad Prism 5 software was used to plot scatter plots including error bars (see [link to statistic]). Figures 4-6 The study aimed to clarify the correlation between serum ENPP2, LPA, and ACPP levels and follicular oocyte maturation. ROC curves, including sensitivity, specificity, and area under the curve (AUC), were calculated using MedCalc software (Version 12.4.2.0, Belgium).

[0049] 2. Results:

[0050] 2.1 Correlation between ENPP2, LPA, and ACPP in serum and follicular fluid

[0051] We collected serum samples before oocyte retrieval and follicular fluid samples on the day of oocyte retrieval from 65 IVF patients, and measured ENPP2, LPA, and ACPP levels using enzyme-linked immunosorbent assay (ELISA). The results showed a positive correlation between serum ENPP2 levels and follicular fluid ENPP2 levels (r = 0.29, P = 0.031). Figure 1 Serum LPA and ACPP levels were not correlated with follicular fluid levels (r = 0.11, P = 0.434). Figure 2 r = -0.06, P = 0.769 Figure 3This indicates that the level of ENPP2 in peripheral blood can reflect the maturation status of oocytes in follicles by indicating the level of ENPP2 in follicular fluid.

[0052] 2.2 Correlation between serum ENPP2, LPA, and ACPP levels and follicular oocyte maturation

[0053] We collected serum samples from 35 IVF patients in the training group on the day of basal hormone administration (immature), during follicular development, and at the time of follicular maturation. The levels of ENPP2, LPA, and ACPP were measured using enzyme-linked immunosorbent assay (ELISA). The results showed that serum ENPP2, LPA, and ACPP levels on the day of maturation were correlated with those on the day of basal hormone administration (r = 0.57, P < 0.0001; r = 0.34, P = 0.011; r = 0.50, P = 0.0003), and there were significant differences in ENPP2 levels on the day of maturation compared to those during follicular development and on the day of basal hormone administration. Figures 4-6 ***When P < 0.0001, the difference is highly significant.

[0054] 2.3 Establishing a Logistic Regression Model

[0055] The ROC curves of single serum ENPP2, LPA, and ACPP indicative of follicular oocyte maturation were calculated using MedCalc software. Figure 7 The results showed that the AUCs of serum ENPP2, LPA, and ACPP were 0.998, 0.522, and 0.603, respectively, with sensitivities of 100.00%, 70.59%, and 29.03%, and specificities of 97.14%, 44.12%, and 96.43%, respectively.

[0056] The ROC curve of serum ENPP2 indicating follicular oocyte maturation was calculated using a logistic stepwise regression model. AMH and AFC were used as auxiliary diagnostic parameters. Through data calculation and analysis, a model indicating follicular oocyte maturation was constructed.

[0057] Model 1: Logit(p1) = -4.8960 + 0.03794 × (serum ENPP2 concentration)

[0058] Obtain the Logit(p1) value, and convert it to P1 value by P1 = exp[Logit(p1)] / (1+exp[Logit(p1)]). When the P1 value of Model 1 is ≥ 0.50, the current oocyte is determined to be mature; when the P1 value is < 0.50, the current oocyte is determined to be immature.

[0059] Model 2: Logit(p2) = -5.9364 + 0.1518 × (serum ENPP2 concentration / AMH),

[0060] Obtain the Logit(p2) value, and convert it to P2 value using P2 = exp[Logit(p2)] / (1 + exp[Logit(p2)]). If the model P2 value is ≥ 0.99, the current oocyte is determined to be mature; if the model P2 value is < 0.99, the current oocyte is determined to be immature.

[0061] Model 3: Logit(p3) = -6.1438 + 0.6012 × (serum ENPP2 concentration / AFC),

[0062] Obtain the Logit(p3) value, and convert it to P3 value using P3 = exp[Logit(p3)] / (1 + exp[Logit(p3)]). If the model P3 value is ≥ 0.99, the current oocyte is determined to be mature; if the model P3 value is < 0.99, the current oocyte is determined to be immature.

[0063] The ROC curves for Model 1 showed a sensitivity of 97.14%, a specificity of 97.14%, and an AUC of 0.998 (P < 0.0001, 95% Confidence Interval = 0.945–1.000); Model 2 showed a sensitivity of 97.14%, a specificity of 97.14%, and an AUC of 0.998 (P < 0.0001, 95% Confidence Interval = 0.945–1.000); and Model 3 showed a sensitivity of 97.14%, a specificity of 97.14%, and an AUC of 0.999 (P < 0.0001, 95% Confidence Interval = 0.947–1.000). The models demonstrated high accuracy, sensitivity, and specificity. This indicates that the combination of serum ENPP2, AMH, and AFC can be used to determine oocyte maturity.

[0064] 2.4 Application of Logistic Regression Model

[0065] In addition, a blinded validation was conducted using serum clinical samples from 57 IVF patients in the clinical follicle maturation verification group. The detected ENPP2 protein levels, clinically measured AMH levels, and AFC were substituted into the regression equation of a pre-constructed binary logistic stepwise model for follicle-oocyte maturation to obtain P1, P2, and P3 values. The clinical concordance rate of Model 1 was 85.96% (49 / 57), the clinical concordance rate of Model 2 was 96.49% (55 / 57), and the clinical concordance rate of Model 3 was 98.25% (56 / 57). This indicates that this specific serum ENPP2 protein method has high accuracy in indicating follicle-oocyte maturation, and each model can be flexibly selected based on the center's detection data for clinical application.

[0066] Table 1 shows the serum ENPP2, AMH, AFC, and model determination values ​​of the experimental verification group of this invention.

[0067]

[0068]

[0069] The above description is a detailed description of the preferred embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.

Claims

1. Use of reagents for detecting serum ENPP2 protein concentration in the preparation of kits for assessing oocyte maturity.

2. The use according to claim 1, characterized in that, The kit also includes reagents for detecting anti-Müllerian hormone (AMH) concentrations and / or tools for assessing antral follicle count (AFC).

3. The use according to claim 1, characterized in that, Based on the detected serum ENPP2 protein concentration, the P1 value is calculated using the following formula, and a signal indicating the oocyte maturity evaluation result is output based on the comparison between the P1 value and the threshold: The formula P1 = exp[-4.8960 + 0.03794 × (serum ENPP2 protein concentration)] / (1 + exp[-4.8960 + 0.03794 × (serum ENPP2 protein concentration)]); When the P1 value is ≥ 0.50, a signal indicating oocyte maturity is output; when the P1 value is < 0.50, a signal indicating oocyte immaturity is output.

4. The use according to claim 1, characterized in that, The kit also includes reagents for detecting AMH concentration; based on the detected serum ENPP2 protein concentration and AMH concentration, the P2 value is calculated using the following formula, and a signal indicating the oocyte maturity evaluation result is output based on the comparison result of the P2 value and the threshold: The formula P2 = exp[-5.9364 + 0.1518 × (serum ENPP2 protein concentration / AMH concentration)] / (1 + exp[-5.9364 + 0.1518 × (serum ENPP2 protein concentration / AMH concentration)]); When the P2 value is ≥0.99, a signal indicating oocyte maturity is output; when the P2 value is <0.99, a signal indicating oocyte immaturity is output.

5. The use according to claim 1, characterized in that, The kit also includes tools for assessing AFC; based on the detected serum ENPP2 protein concentration and the assessed AFC, the P3 value is calculated using the following formula, and a signal indicating the oocyte maturity assessment result is output based on the comparison result of the P3 value with a threshold: The formula P3 = exp[-6.1438 + 0.6012 × (serum ENPP2 protein concentration / AFC)] / (1 + exp[-6.1438 + 0.6012 × (serum ENPP2 protein concentration / AFC)]); When the P3 value is ≥0.99, a signal indicating oocyte maturity is output; when the P3 value is <0.99, a signal indicating oocyte immaturity is output.

Images