Non-invasive joint evaluation method and system for embryo planting potential, medium and terminal

Abstract

The invention relates to a non-invasive combined evaluation method and system for embryo planting potential, a medium and a terminal, and relates to the technical field of reproductive medicines.The combined evaluation method comprises the following steps that 1, a single embryo cultured to the cleavage period of the third day is cultured to the fifth day or the sixth day, and a blastocyst is formed; obtaining a cell-free cultured single blastocyst condition culture solution; (2) collecting morphological scoring data of the formed blastocyst and content detection data of the single blastocyst condition culture solution by a Gardner scoring system; (3) constructing a logistic regression prediction model containing the blastocyst morphological scoring data and content detection data of IL-8, hCG beta and RANTES by using a logistic regression equation; and (4) using the logistic regression prediction model to carry out non-invasive planting potential evaluation or value prediction on a to-be-evaluated blastocyst. According to the method, the system, the medium and the terminal, IL-8, hCG beta and RANTES are used as biomarkers, and embryos with higher development potential can be screened out through the prediction model.

Description

Technical Field

[0001] The present invention relates to the field of reproductive medicine technology, and in particular to a non-invasive combined evaluation method, system, medium and terminal for embryo implantation potential. Background Art

[0002] Assisted reproductive technology (ART) is an advanced technology for treating infertile patients. With the rapid development of assisted reproductive technology, more and more infertile patients have achieved their fertility needs, but the current success rate of embryo transplantation still cannot meet the expectations of doctors and patients. Selecting high-quality embryo transplantation is one of the key factors that determine the success rate of assisted reproductive technology. In order to improve the implantation rate and live birth rate of transplanted embryos and reduce the rate of multiple pregnancy, selecting single embryos with higher developmental potential for transplantation has gradually become a trend. Traditional embryo selection is mainly based on the morphological score of embryos, which is highly subjective and has limited ability to reflect the quality and implantation of embryos. The time-lapse embryo culture monitoring and analysis system (Time-Lapse) combines AI technology to predict embryos, but there is still some controversy about the evaluation effect of this method on embryos. In addition, selecting euploid embryos for transplantation may improve the implantation rate of embryos, but this invasive technology (such as PGT-A) may cause damage to embryos. Therefore, an objective and non-invasive evaluation method is needed to predict the embryo implantation potential in order to screen higher-quality embryos for transplantation and improve the success rate of transplanted embryos.

[0003] Numerous research evidences show that embryos can secrete various proteins, such as cytokines, chemokines, growth factors, etc., and these secretory proteins are closely related to functions such as embryonic development or implantation. Secretory proteins may act on themselves in the form of autocrine to regulate, or they may act on the endometrium in the form of paracrine to achieve maternal-fetal dialogue. Existing research reports have shown that hCGβ is expressed from the embryonic 2 cell to the blastocyst period, and then synthesized and released in large quantities during the syncytial trophoblast period. hCGβ secreted by the embryo can predict embryo quality and may be related to embryo implantation potential. In addition, studies have shown that IL-8 secreted by cleavage-stage embryos is related to clinical pregnancy outcomes. The previous study of the applicant found that the RANTES concentration in the single blastocyst conditioned medium was correlated with embryo quality and embryo implantation potential. In general, embryonic secretory proteins may act on themselves or the endometrium in the form of autocrine or paracrine to regulate processes such as their own development or embryo implantation.

[0004] In view of the above analysis, it is possible to non-invasively predict embryo implantation potential by detecting secretory proteins in embryo conditioned medium. Therefore, the present invention provides a non-invasive combined assessment method, system, medium and terminal for embryo implantation potential. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a non-invasive combined evaluation method, system, medium and terminal for embryo implantation potential. The purpose is to select embryos with higher developmental potential for transplantation by detecting the levels of IL-8, hCGβ and RANTES in the single blastocyst conditioned medium and combining embryo morphology scoring evaluation, thus having a higher predictive value for clinical pregnancy outcomes.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] In a first aspect, a non-invasive combined evaluation method for embryo implantation potential, wherein the non-invasive combined evaluation method for embryo implantation potential does not belong to the treatment and diagnosis of a disease, comprises the following steps:

[0008] (1) transferring a single embryo cultured to the cleavage stage on the third day to a culture system containing a blastocyst culture medium, and culturing the embryo to the fifth or sixth day to form a blastocyst; collecting the blastocyst culture medium from the culture system to obtain a cell-free cultured single blastocyst conditioned culture medium;

[0009] (2) collecting the morphological scoring data of the formed blastocyst using the Gardner scoring system and the content detection data of IL-8, hCGβ and RANTES in the conditioned culture medium of the single blastocyst;

[0010] (3) constructing a logistic regression prediction model containing the blastocyst morphology scoring data and the content detection data of IL-8, hCGβ and RANTES using a logistic regression equation;

[0011] (4) Using the logistic regression prediction model to non-invasively evaluate the implantation potential or predictive value of the blastocyst to be evaluated.

[0012] Based on the above technical solution, the present invention can also be improved as follows.

[0013] Furthermore, the blastocyst culture medium of the culture system in step (1) contains 10% human serum albumin, and the blastocyst culture medium of the culture system is 25 μL.

[0014] Furthermore, the Gardner scoring system in step (2) includes grade 1, grade 2 and grade 3; grade 1 is any one of AA, AB, BA, BB, grade 2 is any one of AC, BC, CB, CA, and grade 3 is CC; grade 1 is defined as high-quality blastocyst, and grades 1 and 2 are defined as usable blastocyst.

[0015] Furthermore, in step (2), the detection data of the contents of IL-8, hCGβ and RANTES in the single blastocyst conditioned medium are collected by the following specific detection method: the single blastocyst conditioned medium is diluted, and the contents of IL-8, hCGβ and RANTES in the diluted single blastocyst conditioned medium are detected by single molecule immunoassay technology.

[0016] Furthermore, the reagents used in the single-molecule immunoassay technology include capture antibody-coated magnetic beads, biotinylated detection antibodies, streptavidin-β-galactosidase solution and fluorescent substrate solution; the capture antibody-coated magnetic beads include IL-8 capture antibody-coated magnetic beads, hCGβ capture antibody-coated magnetic beads and RANTES capture antibody-coated magnetic beads; the magnetic beads in the IL-8 capture antibody-coated magnetic beads, the hCGβ capture antibody-coated magnetic beads and the RANTES capture antibody-coated magnetic beads are magnetic beads with different fluorescent codes; the biotinylated detection antibodies include biotinylated IL-8 detection antibodies, biotinylated hCGβ detection antibodies and biotinylated RANTES detection antibodies.

[0017] Furthermore, the formula of the logistic regression prediction model in step (3) is:

[0018] Logit (p = clinical pregnancy) = 0.570 + 0.987 × IL-8 high concentration group + 1.163 × hCGβ high concentration group - 1.378 × RANTES high concentration group - 0.184 × blastocyst score 2 + 0.142 × blastocyst score 3 - 0.221 × blastocyst score 4 - 0.669 × blastocyst score 5 ； Among them, the IL-8 high concentration group has a concentration greater than 0.341pg / mL, the hCGβ high concentration group has a concentration greater than 0.208mIU / mL; the RANTES high concentration group has a concentration greater than 9.87pg / mL. Explanation of the part of the formula "0.184×blastocyst score 2+0.142×blastocyst score 3-0.221×blastocyst score 4-0.669×blastocyst score 5": Blastocyst score is a categorical variable: AA is defined as 1, AB is defined as 2, BA is defined as 3, BB is defined as 4, and AC, BC, CA or CB are defined as 5.

[0019] Furthermore, step (4) is specifically as follows: using the formula of the logistic regression prediction model to non-invasively evaluate the implantation potential or predictive value of the blastocyst to be evaluated.

[0020] In a second aspect, a non-invasive combined evaluation system for embryo implantation potential is provided, wherein the system based on the combined evaluation method comprises:

[0021] Culture module: transferring a single embryo cultured to the cleavage stage on the third day to a culture system of blastocyst culture medium, and culturing to the fifth or sixth day to form a blastocyst; collecting the blastocyst culture medium from the culture system to obtain a cell-free cultured single blastocyst conditioned culture medium;

[0022] Data collection module: collecting the morphological scoring data of the formed blastocyst using the Gardner scoring system and the content detection data of IL-8, hCGβ and RANTES in the single blastocyst conditioned medium;

[0023] Non-invasive evaluation prediction module: Use the constructed logistic regression prediction model to perform non-invasive evaluation of the implantation potential or predictive value of the blastocyst to be evaluated.

[0024] In a third aspect, a computer-readable storage medium stores a non-invasive joint assessment program for embryo implantation potential, wherein when the non-invasive joint assessment program for embryo implantation potential is executed by a processor, step (4) of the non-invasive joint assessment method for embryo implantation potential is implemented, and the implantation potential or predictive value of the blastocyst to be assessed is non-invasively assessed.

[0025] In a fourth aspect, a terminal comprises: a memory for storing a non-invasive joint assessment program for embryo implantation potential; and a processor for implementing step (4) of a non-invasive joint assessment method for embryo implantation potential when executing the non-invasive joint assessment program for embryo implantation potential, and performing a non-invasive assessment of the implantation potential or predictive value of the blastocyst to be assessed.

[0026] The beneficial effects of the present invention are:

[0027] (1) The present invention provides a method, system, medium and terminal for non-invasively evaluating and predicting embryo quality and embryo implantation potential in assisted reproduction, thereby improving the success rate after embryo transplantation.

[0028] (2) The present invention proposes through research that the levels of IL-8, hCGβ and RANTES in the conditioned medium of single blastocysts can jointly predict the implantation potential of embryos.

[0029] (3) The present invention provides a method, system, medium and terminal for screening embryos for assisted reproductive transplantation, using IL-8, hCGβ and RANTES as biomarkers, and using a prediction model to screen out embryos with higher developmental potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A comparative bar graph of clinical pregnancy rates grouped by different concentrations of various factors in the single blastocyst conditioned medium of the present invention;

[0031] Figure 2The present invention compares ROC curves for predicting clinical pregnancy outcomes using IL-8, hCGβ, and RANTES concentration grouping in single blastocyst conditioned medium combined with embryo morphology scoring and embryo morphology scoring alone. DETAILED DESCRIPTION

[0032] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0033] Example

[0034] 1. A non-invasive combined assessment method for embryo implantation potential.

[0035] 1.1 Research methods

[0036] A non-invasive combined evaluation method for embryo implantation potential, which does not belong to the treatment and diagnosis of a disease, comprises the following steps:

[0037] (1) transferring a single embryo cultured to the cleavage stage on the third day to a culture system containing a blastocyst culture medium, and culturing the embryo to the fifth or sixth day to form a blastocyst; collecting the blastocyst culture medium from the culture system to obtain a cell-free cultured single blastocyst conditioned culture medium;

[0038] (2) collecting the morphological scoring data of the formed blastocyst using the Gardner scoring system and the content detection data of IL-8, hCGβ and RANTES in the conditioned culture medium of the single blastocyst;

[0039] (3) constructing a logistic regression prediction model containing the blastocyst morphology score data and the IL-8, hCGβ and RANTES content data using a logistic regression equation;

[0040] (4) Using the logistic regression prediction model to non-invasively evaluate the implantation potential or predictive value of the blastocyst to be evaluated.

[0041] This embodiment specifically includes the following steps:

[0042] 1) Ovulation induction program: According to the ovarian reserve and response of infertile patients who come to the hospital for assisted reproductive technology treatment, an appropriate ovulation induction program is adopted. When there are two or more dominant follicles with a diameter of ≥18mm, 10,000U of hCGβ is injected intramuscularly, and B-ultrasound puncture and negative pressure extraction of eggs are performed 34h-36h later. The insemination method is determined by the routine condition of semen on the day, which is divided into IVF and ICSI.

[0043] 2) Embryo culture and single blastocyst conditioned medium sample collection: Observe and score the cleavage-stage embryos 72h after fertilization (Day 3). After embryo scoring on Day 3, transfer the cleavage-stage embryos individually to 25ul droplets of blastocyst culture medium containing 10% human serum albumin. One embryo is cultured in one droplet until Day 5 or Day 6 to form a blastocyst. The formed blastocysts are then scored according to the Gardner scoring system and divided into grade 1 (AA, AB, BA, BB), grade 2 (AC, BC, CB, CA) and grade 3 (CC). Grade 1 is defined as high-quality blastocysts, and grades 1 and 2 are defined as usable blastocysts. The usable blastocysts are vitrified and frozen; after the embryos are frozen, the blastocyst culture medium is collected to obtain the cell-free cultured single blastocyst conditioned medium, which is frozen at -80°C as samples to be tested in the study;

[0044] 3) Endometrial preparation and embryo transfer: Choose the appropriate endometrial preparation plan according to the patient's own situation. When the thickness of the endometrium reaches 7mm to 8mm monitored by B-ultrasound, progesterone is injected intramuscularly until the day of embryo transfer. On the day of embryo transfer, the thawed blastocyst is transferred under B-ultrasound guidance, and luteal support is given after the operation.

[0045] 4) Tracking of clinical pregnancy outcomes: If a gestational sac is seen under ultrasound 30 days after embryo transfer, it is considered a clinical pregnancy.

[0046] 5) Selection and pretreatment of test samples: Select the single blastocyst conditioned medium from transplanted frozen-thawed embryos, thaw the sample at room temperature for 30 minutes, measure the sample volume, and perform pretreatment to dilute the sample to a total volume of 130 μL;

[0047] 6) Sample testing: The Simoa triple detection kit for biomarkers IL-8, hCGβ and RANTES was used to detect the concentrations of IL-8, hCGβ and RANTES in the conditioned medium of single blastocysts from transferred embryos;

[0048] Regarding the above-mentioned Simoa triple detection reagent kit for biomarkers IL-8, hCGβ and RANTES, the content of IL-8, hCGβ and RANTES in the conditioned medium of single blastocysts from transplanted embryos is detected. The specific method is operated according to the public content of the kit, which can be simply summarized as the following steps:

[0049] ①Simoa triple detection kit for IL-8, hCGβ and RANTES:

[0050] The Simoa triple detection kit for biomarkers IL-8, hCGβ and RANTES includes capture antibody-coated magnetic beads, biotinylated detection antibodies, streptavidin-β-galactosidase solution (SBG), fluorescent substrate solution (RGP, resorufin-β-D-galactoside), IL-8 standard, hCGβ standard, RANTES standard, sample diluent, magnetic bead diluent, and SBG diluent; the capture antibody-coated magnetic beads include IL-8 capture antibody-coated magnetic beads, hCGβ capture antibody-coated magnetic beads and RANTES capture antibody-coated magnetic beads; the magnetic beads in the IL-8 capture antibody-coated magnetic beads, the hCGβ capture antibody-coated magnetic beads and the RANTES capture antibody-coated magnetic beads are magnetic beads with different fluorescence codes; the biotinylated detection antibodies include biotinylated IL-8 detection antibodies, biotinylated hCGβ detection antibodies and biotinylated RANTES detection antibodies.

[0051] The magnetic beads with different fluorescence codes are 488nm, 700nm and 750nm magnetic beads.

[0052] (A) Preparation of dilution solutions of the IL-8, hCGβ and RANTES standards:

[0053] a. Prepare IL-8 standard gradient solution, dilute IL-8 standard of known concentration with sample diluent (120pg / mL, 40pg / mL, 13.3pg / mL, 4.4pg / mL, 1.48pg / mL, 0.49pg / mL, 0.17pg / mL, 0pg / mL);

[0054] b. Prepare a gradient solution of hCGβ standard, dilute the known concentration of hCGβ standard with sample diluent (135mIU / mL, 43.55mIU / mL, 14.05mIU / mL, 4.53mIU / mL, 1.46mIU / mL, 0.47mIU / mL, 0.15mIU / mL, 0mIU / mL);

[0055] c. Prepare a gradient solution of RANTES standard and dilute RANTES standard of known concentration with sample diluent (48 pg / mL, 19.2 pg / mL, 7.68 pg / mL, 3.07 pg / mL, 1.23 pg / mL, 0.49 pg / mL, 0.20 pg / mL, 0 pg / mL).

[0056] The concentration points of the three standards were mixed at a ratio of 1:1:1, that is, the final three-factor standard curve was the IL-8 standard curve (40pg / mL, 13.3pg / mL, 4.4pg / mL, 1.48pg / mL, 0.49pg / mL, 0.17pg / mL, 0.055pg / mL, 0pg / mL); the hCGβ standard curve (45mIU / mL, 14.52, 4.68mIU / mL) / mL, 1.51mIU / mL, 0.49mIU / mL, 0.16mIU / mL, 0.051mIU / mL, 0mIU / mL); RANTES standard curve (16pg / mL, 6.4pg / mL, 2.56pg / mL, 1.02pg / mL, 0.41pg / mL, 0.164pg / mL, 0.066pg / mL, 0pg / mL).

[0057] (B) Preparation of the diluted SBG reagent:

[0058] Dilute the SBG reagent and dilute the SBG stock solution to 150PM using SBG diluent as the working solution.

[0059] (C) Pre-treatment of the single blastocyst conditioned medium sample:

[0060] Measure the volume of the single blastocyst conditioned medium sample and add sample diluent to a total volume of 130ul. Record the data and calculate the dilution multiple.

[0061] Note: The above three fluorescent coded magnetic beads, BCB, BWB, BBB, BRB, NHS-Biotin, EDC, SBG, RGP, sample diluent, magnetic bead diluent, and SBG diluent are all supporting reagents provided by Simoa's homemade kits.

[0062] ③How to use the kit

[0063] The method for using the Simoa triple assay for biomarkers IL-8, hCGβ and RANTES comprises the following steps:

[0064] (A) First, import the electronic file of the multiple detection test method produced by Quanterix on the Quanterix HD-X instrument; set the experimental conditions: in the first step, mix the capture antibody-coated magnetic beads (25 μL / sample), standard / sample (100 μL / sample), and biotinylated detection antibody (20 μL / sample) and incubate for 35 minutes; in the second step, add SBG working solution (100 μL / sample) and incubate for 5 minutes; set the concentration points of the standard curve of each factor as described above; select the fluorescence channel for each factor detection according to the actual situation of the fluorescence-encoded magnetic beads coupled to the capture antibody.

[0065] (B) Then prepare a sufficient amount of mixed solution of magnetic beads coated with different capture antibodies according to the total number of standards and samples to be tested (total magnetic bead concentration 2×10 7 All reagents were loaded into the instrument together: 1 μg / mL, 1 μg / mL biotinylated detection antibody mixed solution (IL-8: 1 μg / mL, hCGβ: 10 μg / mL, RANTES: 1 μg / mL), SBG working solution (150 PM), RGP solution (activated by vibration at 30 °C for 30 min), and a 96-well plate with standards and samples.

[0066] (C) Finally, check whether the consumables and reagents are sufficient. If they are sufficient, run the test and export the fitting curve and sample test results after completion.

[0067] 7) Data statistical analysis: The detection data of IL-8, hCGβ, and RANTES in the conditioned medium of single blastocysts of transplanted embryos from different patients were statistically analyzed to determine the predictive value of IL-8, hCGβ, and RANTES for embryo implantation potential. SPSS22.0 software was used for statistical analysis, and the comparison of count data between groups was performed using the χ2 test. 2 Test; measurement data were expressed as mean ± standard deviation. If the sample was normally distributed, the independent sample t test was used for inter-group comparison. If it was not normally distributed, the non-parametric Mann-Whitney U test was used. P < 0.05 was considered statistically significant. Multivariate Logistic regression was used to analyze the effects of different factors on clinical pregnancy; the Logit model and receiver operating characteristic curve (ROC curve) were used to evaluate the predictive value of the three factors combined with embryo morphology score for clinical pregnancy after blastocyst transfer.

[0068] 1.2 Research results

[0069] After the patient's embryos formed blastocysts on Day 5 or Day 6, they were vitrified and the blastocyst conditioned medium was collected and stored at -80°C. A total of 240 samples of frozen-thawed blastocyst transfer patients were tested, of which 162 were clinically positive and 78 were negative after embryo transfer. The above-mentioned IL-8, hCGβ and RANTES Simoa triple kits were used for testing. The test results and patient information were statistically analyzed.

[0070] There were no significant differences in the female age, BMI, endometrial thickness on the day of luteal transformation, blastocyst score (grade 1 and grade 2), IL-8 concentration and RANTES concentration between the two groups with positive clinical pregnancy and negative clinical pregnancy (P>0.05). There were significant differences in the years of infertility and hCGβ concentration between the two groups (P<0.05). With clinical pregnancy outcome as the dependent variable, multivariate logistic regression analysis was performed with female age, years of infertility, BMI, endometrial thickness on the day of luteal transformation, blastocyst score, IL-8 concentration, hCGβ concentration and RANTES concentration included. The results showed that the effect of hCGβ concentration on clinical pregnancy was statistically significant (P<0.05) (Table 1).

[0071] The IL-8 concentration, hCGβ concentration, and RANTES concentration were divided into concentration groups. When the IL-8 concentration was ≤0.341pg / mL, it was the low concentration group (102 cases), and vice versa, it was the high concentration group (n=138 cases). When the hCGβ concentration was ≤0.208mIU / mL, it was the low concentration group (114 cases), and vice versa, it was the high concentration group (126 cases). When the RANTES concentration was ≤9.87pg / mL, it was the low concentration group (116 cases), and vice versa, it was the high concentration group (124 cases). According to the above grouping, the clinical pregnancy rate after embryo transfer in each group was calculated, and the pregnancy outcomes of different concentration groups were compared. The results showed that the clinical pregnancy rate in the high-concentration IL-8 group was 75.4%, significantly higher than that in the low-concentration group (56.9%) (P < 0.01); the clinical pregnancy rate in the high-concentration hCGβ group was as high as 77.0%, significantly higher than that in the low-concentration group (57.0%) (P < 0.01); the clinical pregnancy rate in the low-concentration RANTES group was 77.6%, significantly higher than that in the high-concentration group (58.1%) (P < 0.01) (Table 2, Figure 1 ). Figure 1 **P<0.01. In multivariate logistic regression analysis, the dependent variable was clinical pregnancy outcome, with positive defined as 1 and negative defined as 0; the independent variables were IL-8 concentration grouping, hCGβ concentration grouping and RANTES concentration grouping, with the low concentration group of each factor defined as 1 and the high concentration group defined as 2. The results showed that the effects of IL-8 concentration grouping (OR=2.694, P<0.01), hCGβ concentration grouping (OR=3.301, P<0.001) and RANTES concentration grouping (OR=0.238, P<0.001) on clinical pregnancy were statistically significant (Table 2).

[0072] Table 1 is a comparison of the basic data of patients and sample test results in different clinical pregnancy outcome groups

[0073]

[0074] Table 2 shows the comparison of clinical pregnancy rates in different concentration groups of each test factor and the effects of the three factors on clinical pregnancy

[0075]

[0076] The present invention adopts a logistic regression prediction model for predicting clinical pregnancy outcome by combining IL-8, hCGβ, and RANTES concentration grouping in a single blastocyst conditioned medium with embryo morphology scoring, and uses ROC curve analysis to analyze the prediction value. The IL-8, hCGβ, and RANTES concentration grouping combined with embryo morphology scoring model includes dependent variables and independent variables. The dependent variable is the clinical pregnancy outcome (clinical pregnancy positive is defined as 1, and negative is defined as 0). The independent variables are IL-8, hCGβ, and RANTES concentration grouping and embryo morphology scoring, IL-8 concentration grouping, hCGβ concentration grouping, and RANTES concentration grouping, and the low concentration group of each factor is defined as 1, and the high concentration group is defined as 2; the embryo morphology scoring is the Gardner scoring rule, and according to the embryo scoring result, AA is defined as 1, AB is defined as 2, BA is defined as 3, BB is defined as 4, and AC / BC / CA / CB are all defined as 5.

[0077] The logistic regression prediction model formula for predicting clinical pregnancy outcome by combining IL-8, hCGβ, and RANTES concentration groups with embryo morphology score is as follows: Logit (p = clinical pregnancy) = 0.570 + 0.987 × IL-8 high concentration group + 1.163 × hCGβ high concentration group - 1.378 × RANTES high concentration group - 0.184 × blastocyst score 2 + 0.142 × blastocyst score 3 - 0.221 × blastocyst score 4 - 0.669 × blastocyst score 5. The logistic regression prediction model formula for predicting clinical pregnancy outcome by embryo morphology score is as follows: Logit (p = clinical pregnancy) = 0.944 - 0.123 × blastocyst score 2 + 0.245 × blastocyst score 3 - 0.639 × blastocyst score 4 - 0.881 × blastocyst score 5. The ROC curves of the logistic distribution formulas of the above two logistic regression models were plotted, and the area under the curve (AUC) was used as an indicator to evaluate the predictive value of each model. The AUC of the ROC curve of the prediction model for clinical pregnancy outcome using IL-8, hCGβ, and RANTES concentration groups combined with embryo morphology score was 0.727 (95% CI: 0.658-0.797) (P < 0.001). The AUC of the ROC curve of the prediction model for clinical pregnancy outcome using embryo morphology score was 0.589 (95% CI: 0.511-0.667) (P < 0.05). The difference in AUC of the ROC curves of the two prediction models was statistically significant (P < 0.001) (see Figure 2 ). Therefore, the prediction model of clinical pregnancy outcome based on IL-8, hCGβ, and RANTES concentration grouping combined with embryo morphology score has better predictive value.

[0078] It can be seen that the present invention can predict the success rate of embryo transplantation by quantitatively detecting the concentrations of IL-8, hCGβ, and RANTES in the single blastocyst conditioned medium of the blastocyst, and applying the IL-8, hCGβ, and RANTES concentration grouping combined with the embryo's morphological score. ROC curve analysis and evaluation show that compared with the traditional prediction method of embryo morphological score, IL-8, hCGβ, and RANTES combined with embryo morphological score have higher prediction accuracy. Therefore, the prediction method of combining IL-8, hCGβ, and RANTES levels in the single blastocyst conditioned medium with embryo morphological score can more effectively predict clinical pregnancy outcomes, and the results are more objective and accurate.

[0079] 2. A non-invasive combined evaluation system, medium and terminal for embryo implantation potential.

[0080] 2.1 A non-invasive combined assessment system for embryo implantation potential.

[0081] This embodiment involves a non-invasive joint evaluation system for embryo implantation potential. The system based on the joint evaluation method includes:

[0082] Culture module: transferring a single embryo cultured to the cleavage stage on the third day to a culture system of blastocyst culture medium, and culturing to the fifth or sixth day to form a blastocyst; collecting the blastocyst culture medium from the culture system to obtain a cell-free cultured single blastocyst conditioned culture medium;

[0083] Data collection module: collecting the morphological scoring data of the formed blastocyst using the Gardner scoring system and the content detection data of IL-8, hCGβ and RANTES in the single blastocyst conditioned medium;

[0084] Non-invasive evaluation prediction module: Use the logistic regression prediction model to perform non-invasive evaluation of the implantation potential or predictive value of the blastocyst to be evaluated.

[0085] 2.2 Computer-readable storage medium.

[0086] The present embodiment relates to a computer-readable storage medium, on which is stored a non-invasive joint assessment program for embryo implantation potential. When the non-invasive joint assessment program for embryo implantation potential is executed by a processor, step (4) of the non-invasive joint assessment method for embryo implantation potential is implemented, and the implantation potential or predictive value of the blastocyst to be assessed is non-invasively assessed.

[0087] 2.3 A terminal.

[0088] A terminal involved in this embodiment includes: a memory for storing a non-invasive joint assessment program for embryo implantation potential; a processor for implementing step (4) of the non-invasive joint assessment method for embryo implantation potential when executing the non-invasive joint assessment program for embryo implantation potential, and performing non-invasive assessment of the implantation potential or predictive value of the blastocyst to be assessed.

[0089] In summary, the present invention provides a method, system, medium and terminal for non-invasive evaluation and prediction of embryo quality and embryo implantation potential of assisted reproduction, thereby improving the success rate after embryo transplantation. The present invention proposes that the levels of IL-8, hCGβ and RANTES in the conditioned medium of a single blastocyst can jointly predict the implantation potential of the embryo through research. The present invention provides a method, system, medium and terminal for screening embryos for assisted reproductive transplantation, using IL-8, hCGβ and RANTES as biomarkers, and a prediction model of IL-8, hCGβ, RANTES concentration grouping combined with embryo morphological scoring can screen out embryos with higher developmental potential.

[0090] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A non-invasive combined evaluation method for embryo implantation potential, wherein the non-invasive combined evaluation method for embryo implantation potential does not belong to the treatment and diagnosis of a disease, and is characterized in that: The steps include: (1) transferring a single embryo cultured to the cleavage stage on the third day to a culture system containing a blastocyst culture medium, and culturing the embryo to the fifth or sixth day to form a blastocyst; collecting the blastocyst culture medium from the culture system to obtain a cell-free cultured single blastocyst conditioned culture medium; (2) collecting the morphological scoring data of the formed blastocyst using the Gardner scoring system and the content detection data of IL-8, hCGβ and RANTES in the conditioned culture medium of the single blastocyst; (3) constructing a logistic regression prediction model containing the blastocyst morphology scoring data and the content detection data of IL-8, hCGβ and RANTES using a logistic regression equation; (4) Using the logistic regression prediction model to non-invasively evaluate the implantation potential or predictive value of the blastocyst to be evaluated.

2. A non-invasive combined assessment method for embryo implantation potential according to claim 1, characterized in that: The blastocyst culture medium of the culture system in step (1) contains 10% human serum albumin, and the blastocyst culture medium of the culture system is 25 μL.

3. A non-invasive combined assessment method for embryo implantation potential according to claim 1, characterized in that: The Gardner scoring system in step (2) includes level 1, level 2 and level 3; level 1 is any one of AA, AB, BA, BB, level 2 is any one of AC, BC, CB, CA, and level 3 is CC.

4. A non-invasive combined assessment method for embryo implantation potential according to claim 1, characterized in that: In step (2), the content detection data of IL-8, hCGβ and RANTES in the single blastocyst conditioned culture medium is collected by the following specific method: the single blastocyst conditioned culture medium is diluted, and the single molecule immunoassay technology is used to detect the content of IL-8, hCGβ and RANTES in the diluted single blastocyst conditioned culture medium.

5. A non-invasive combined assessment method for embryo implantation potential according to claim 4, characterized in that: The reagents used in the single-molecule immunoassay technology include capture antibody-coated magnetic beads, biotinylated detection antibodies, streptavidin-β-galactosidase solution and fluorescent substrate solution; the capture antibody-coated magnetic beads include IL-8 capture antibody-coated magnetic beads, hCGβ capture antibody-coated magnetic beads and RANTES capture antibody-coated magnetic beads; the magnetic beads in the IL-8 capture antibody-coated magnetic beads, the hCGβ capture antibody-coated magnetic beads and the RANTES capture antibody-coated magnetic beads are magnetic beads with different fluorescence codes; the biotinylated detection antibodies include biotinylated IL-8 detection antibodies, biotinylated hCGβ detection antibodies and biotinylated RANTES detection antibodies.

6. The non-invasive combined assessment method for embryo implantation potential according to claim 1, characterized in that: The formula of the logistic regression prediction model in step (3) is: Logit (p = clinical pregnancy) = 0.570 + 0.987 × IL-8 high concentration group + 1.163 × hCGβ high concentration group - 1.378 × RANTES high concentration group - 0.184 × blastocyst score 2 + 0.142 × blastocyst score 3 - 0.221 × blastocyst score 4 - 0.669 × blastocyst score 5 ； Among them, the IL-8 high concentration group had a concentration greater than 0.341 pg / mL, the hCGβ high concentration group had a concentration greater than 0.208 mIU / mL; and the RANTES high concentration group had a concentration greater than 9.87 pg / mL.

7. A non-invasive combined assessment method for embryo implantation potential according to claim 6, characterized in that: Step (4) specifically includes: using the formula of the logistic regression prediction model to non-invasively evaluate the implantation potential or predictive value of the blastocyst to be evaluated.

8. A non-invasive combined assessment system for embryo implantation potential, characterized in that: The system used in the joint evaluation method according to any one of claims 1 to 7 comprises: Culture module: transferring a single embryo cultured to the cleavage stage on the third day to a culture system of blastocyst culture medium, and culturing to the fifth or sixth day to form a blastocyst; collecting the blastocyst culture medium from the culture system to obtain a cell-free cultured single blastocyst conditioned culture medium; Data collection module: collecting the morphological scoring data of the formed blastocyst using the Gardner scoring system and the content detection data of IL-8, hCGβ and RANTES in the single blastocyst conditioned medium; Non-invasive evaluation prediction module: Use the constructed logistic regression prediction model to perform non-invasive evaluation of the implantation potential or predictive value of the blastocyst to be evaluated.

9. A computer-readable storage medium, characterized in that: The readable storage medium stores a non-invasive joint assessment program for embryo implantation potential. When the non-invasive joint assessment program for embryo implantation potential is executed by a processor, step (4) of a non-invasive joint assessment method for embryo implantation potential as described in any one of claims 1 to 7 is implemented, and the implantation potential or predictive value of the blastocyst to be assessed is non-invasively assessed.

10. A terminal, characterized in that: include: A memory for storing a non-invasive combined assessment procedure for embryo implantation potential; A processor is used to implement step (4) of the method for non-invasively combining the implantation potential of an embryo as described in any one of claims 1 to 7 when executing the non-invasively combined evaluation procedure for the implantation potential of an embryo, and to perform a non-invasive evaluation of the implantation potential or the predictive value of the blastocyst to be evaluated.

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