Method and system for non-invasive evaluation of embryo planting potential

Abstract

The invention relates to a method and a system for non-invasive evaluation of embryo planting potential, and relates to the field of assisted reproduction. The method for non-invasive evaluation of embryo planting potential does not belong to disease treatment and diagnosis, and comprises the following steps: (1) transferring a single blastocyst cultured to a cleavage period of the third day into a culture system containing a blastocyst culture solution, and culturing to a fifth day or a sixth day; (2) after the blastocyst is formed, carrying out vitrification treatment on the blastocyst, and collecting a blastocyst culture solution from the culture system to obtain a cell-free cultured blastocyst culture solution as a sample to be detected; and (3) detecting the content of RANTES in the sample to be detected by adopting a single molecule immunodetection technology so as to noninvasively evaluate the planting potential or prediction value of the embryo. According to the method, the embryo with higher development potential is selected for embryo transplantation by detecting the level of RANTES in the single blastocyst condition culture solution, so that the success rate of embryo planting can be increased.

Description

Technical Field

[0001] The present invention relates to the field of assisted reproduction, and in particular to a method and system for non-invasively evaluating embryo implantation potential. Background Art

[0002] With the development of assisted reproductive technology, in order to improve embryo implantation rate, live birth rate and reduce multiple birth rate, the selection of single embryo transplantation has gradually become a future trend. At present, the traditional method of embryo selection is morphological scoring, but it is highly subjective and has certain limitations, and cannot truly reflect the quality and implantation ability of the embryo. In addition, the selection of euploid embryos for transplantation may increase the embryo implantation rate, but this invasive technology (such as PGT-A) may cause damage to the embryo. Therefore, it is hoped that a non-invasive method and strategy for evaluating the potential of embryo development can be selected. At present, some studies have performed non-invasive evaluations by detecting some components of embryo conditioned medium, such as embryo metabolomics, proteomics, and embryo development dynamics.

[0003] Numerous research evidences show that embryos can secrete various proteins, such as cytokines, chemokines, growth factors, etc., and these secreted proteins may play an important role in embryo implantation. Therefore, it is possible to non-invasively predict embryo implantation potential by detecting secreted proteins in embryo conditioned medium. In view of this, the present invention provides a method and system for non-invasively evaluating embryo implantation potential. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method and system for non-invasively evaluating embryo implantation potential, with the purpose of non-invasively evaluating embryo implantation potential by detecting the level of RANTES in the conditioned culture medium of a single blastocyst.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] In a first aspect, a method for non-invasively assessing embryo implantation potential, wherein the method does not belong to the treatment and diagnosis of a disease, comprises the following steps:

[0007] (1) transferring a single blastocyst cultured to the cleavage stage on the third day into a culture system containing blastocyst culture medium and culturing the system until the fifth or sixth day;

[0008] (2) After the blastocyst is formed, the blastocyst is vitrified and the blastocyst culture fluid is collected from the culture system to obtain a cell-free cultured blastocyst culture fluid as a sample to be tested, which is frozen at -80°C for future use;

[0009] (3) Single-molecule immunoassay technology is used to detect the content of RANTES in the test sample to non-invasively evaluate the embryo's implantation potential or predictive value.

[0010] Among them, RANTES (CCL5) is a member of the CC family of chemokines, which can chemotactic T cells, eosinophils and basophils, and plays an important role in the activation of killer T cells in immune response. In addition, it is related to the activation of proliferation, apoptosis, cytokine release, etc. At present, no research has found the predictive potential of RANTES content in embryo conditioned medium for embryo quality and implantation. The present invention non-invasively evaluates the implantation potential of embryos by detecting the level of RANTES in the conditioned medium of single blastocysts and analyzing the relationship between the concentration of RANTES and embryo quality and embryo implantation potential.

[0011] The beneficial effects of the present invention are as follows: the present invention unexpectedly discovered through research that the level of RANTES in the single blastocyst conditioned medium is correlated with the embryo development potential. Therefore, by detecting the level of RANTES in the single blastocyst conditioned medium, embryos with higher development potential can be selected for embryo transplantation, thereby improving the success rate of embryo implantation; and providing a method for non-invasively evaluating and predicting embryo quality and embryo implantation potential, thereby improving the implantation rate after embryo transplantation.

[0012] 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.

[0013] Furthermore, step (3) includes the following specific steps: thawing the sample to be tested at room temperature for 30 minutes to 60 minutes, measuring the volume of the sample to be tested, and diluting the sample to be tested; using single molecule immunoassay technology to detect the content of RANTES in the diluted sample to non-invasively evaluate the implantation potential or predictive value of the embryo.

[0014] Furthermore, the sample to be tested is diluted to a total volume of at least 130 μL.

[0015] Furthermore, in the method for non-invasively evaluating embryo implantation potential described in step (3), the reagents used in the single-molecule immunoassay technology include capture antibody-coated magnetic beads, biotinylated detection antibodies, streptavidin-β-galactosidase solution, fluorescent substrate solution and RANTES standard; the capture antibody-coated magnetic beads include RANTES capture antibodies coupled to the magnetic beads, and the biotinylated detection antibodies are prepared by connecting the RANTES capture antibodies to biotin.

[0016] In a second aspect, a system for non-invasively assessing embryo implantation potential comprises:

[0017] A culture module, wherein the culture module is used to transfer the embryos cultured to the cleavage stage on the third day into a culture system containing 10% human serum albumin blastocyst culture solution, and culture them to the fifth or sixth day;

[0018] A sampling module, wherein the sampling module is used to collect blastocyst culture fluid from the culture system, obtain cell-free cultured blastocyst culture fluid, and freeze it at -80°C as a sample to be tested;

[0019] A detection module, wherein the detection module is used for single molecule immunoassay to detect the content of RANTES in the sample to be tested;

[0020] A discrimination processing module, based on the content of RANTES obtained by the detection module, screens blastocysts with greater embryo implantation potential, thereby achieving non-invasive evaluation of the implantation potential of the blastocysts;

[0021] An output module is used to output the evaluation result of the embryo implantation potential and complete the work of selecting the optimal embryo. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A comparison chart of the RANTES concentration of the high-quality blastocyst group and the non-high-quality blastocyst group of the present invention;

[0023] Figure 2 This is a comparison chart of the RANTES concentration of the clinical pregnancy positive group and the RANTES concentration of the clinical pregnancy negative group of the present invention;

[0024] Figure 3 The figure is a comparison chart of the clinical pregnancy rate of the low-concentration group and the high-concentration group of RANTES of the present invention. DETAILED DESCRIPTION

[0025] 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.

[0026] Terminology Note:

[0027] As used herein, the terms "blastocyst" and "embryo" are used interchangeably and refer to the embryonic in vitro culture stage, which is usually formed on the 5th to 7th day after the fertilization of the egg.

[0028] Example

[0029] 1. A non-invasive method for assessing embryo implantation potential

[0030] 1.1 Research Methods

[0031] A research method for non-invasively evaluating embryo implantation potential mainly includes the following steps:

[0032] (1) Embryo culture: After patients undergoing assisted reproductive technology treatment adopt appropriate ovulation induction programs, eggs are obtained on the day of egg retrieval. 72 hours after fertilization (D3), cleavage-stage embryos are individually transferred to 25 μL droplets of blastocyst culture medium containing 10% human serum albumin and cultured until the fifth day (D5) or the sixth day (D6).

[0033] First, the blastocysts formed on D5 or D6 are scored according to the Gardner scoring system. The blastocysts are divided into grade 1 (AA, AB, BA, BB), grade 2 (AC, CA, BC, CB), and grade 3 (CC) blastocysts. Among them, grade 1 is defined as high-quality blastocysts, grade 2 is non-high-quality embryos, and grades 1 and 2 are defined as usable blastocysts.

[0034] (2) Collection of single blastocyst conditioned medium samples: The vitrified blastocysts formed on D5 and D6 were vitrified on the day of formation. After embryo freezing, the blastocyst conditioned medium was collected and stored at -80°C as samples for research.

[0035] (3) Embryo transfer and pregnancy outcome tracking: Select the appropriate endometrial preparation plan based on the patient's own situation, thaw the blastocyst and perform embryo transfer under the guidance of B-ultrasound; 30 days after blastocyst transfer, if the gestational sac is seen on B-ultrasound, it is considered clinical pregnancy; during this period, collect relevant information about the patient;

[0036] (4) Selection and pretreatment of samples to be tested: Select single blastocyst conditioned medium samples from frozen-thawed embryo transfer; thaw the samples at room temperature for 30-60 min, measure the volume of the samples to be tested, and dilute the samples to be tested until the total volume reaches at least 130 μL; place at 4°C for testing;

[0037] (5) Sample detection: using single molecule immunoassay technology to detect the content of RANTES in the conditioned medium of a single blastocyst derived from a transplanted embryo (the sample to be tested);

[0038] (6) Statistical analysis: The detection data of RANTES in the conditioned medium of single blastocysts were statistically analyzed to determine the predictive value of RANTES content for embryo implantation potential.

[0039] SPSS 24.0 software was used for statistical analysis. The Mann-Whitney U test was used for comparison of intergroup measurement data, and the chi-square test was used for comparison of intergroup count data. Binary logistic regression analysis was performed for blastocyst quality, clinical pregnancy outcome, or RANTES group outcome. P < 0.05 was considered statistically significant.

[0040] Among them, step (4) uses the RANTES SIMOA single molecule immunoassay kit to detect the content of RANTES in the conditioned medium of a single blastocyst from a transplanted embryo. The specific method is performed according to the disclosure of the kit and can be simply summarized as follows:

[0041] 1) RANTES SIMOA kit

[0042] A SIMOA kit for detecting the biomarker RANTES, comprising magnetic beads coated with a capture antibody, a biotinylated detection antibody, a RANTES standard, a streptavidin-β-galactosidase solution (SBG solution), a resorufin-β-D-galactoside solution (RGP), a sample diluent, a magnetic bead diluent, and a SBG diluent.

[0043] (A) Standard solution dilution method: dilute the RANTES standard with sample diluent to 16 pg / mL, 6.4 pg / mL, 2.56 pg / mL, 1.024 pg / mL, 0.41 pg / mL, 0.164 pg / mL, 0.066 pg / mL, and 0 pg / mL, and make a standard curve with a total of 8 concentration points;

[0044] (B) The SBG solution dilution method: dilute the SBG stock solution to 150PM using SBG diluent;

[0045] (C) Dilution method of the single blastocyst conditioned medium sample: the single blastocyst conditioned medium sample is added with sample diluent to a total volume of 130 μL.

[0046] 2) How to use the RANTES SIMOA test kit

[0047] A method for using a SIMOA kit for the biomarker RANTES comprises the following steps:

[0048] ①First, import the HD-X Homebrew Assay Definition 1 file (Quanterix official website) on the Quanterix HD-X instrument and set the experimental conditions: two-step method, i.e. the reaction time of capture antibody-coated magnetic beads, samples, and biotinylated detection antibodies is 35 min, the SBG reaction time is 5 min, and the standard curve concentration points are set (16 pg / mL, 6.4 pg / mL, 2.56 pg / mL, 1.024 pg / mL, 0.41 pg / mL, 0.164 pg / mL, 0.066 pg / mL, 0 pg / mL).

[0049] ②Then the magnetic bead solution coated with RANTES capture antibody (2.0×10 7 / mL), biotinylated RANTES detection antibody solution (1 μg / mL), SBG working solution (150PM), RGP solution, and a 96-well plate with standards and samples were loaded into the instrument.

[0050] ③Finally, run the test, export the sample test results and perform statistical analysis (Table 1-2).

[0051] 1.2 Research Results

[0052] Table 1 RANTES concentration in the conditioned medium of single blastocysts of human preimplantation embryos

[0053]

[0054] Table 2 Comparison of clinical pregnancy rates among groups of RANTES concentrations in the conditioned medium of human preimplantation single blastocyst embryos

[0055]

[0056] As shown in Tables 1 and 2 above, RANTES content was detected in all 67 samples of single blastocyst conditioned culture medium, and the detection rate reached 100% (67 / 67).

[0057] According to the Gardner scoring system, embryos were divided into a high-quality blastocyst group (n = 51) and a non-high-quality blastocyst group (n = 16). The difference in RANTES concentration between the two groups was statistically significant (P < 0.05). Binary logistic regression analysis was performed, with blastocyst quality as the dependent variable and RANTES concentration as the independent variable, while correcting for the woman's age, infertility years, BMI, and endometrial thickness. The results showed that the RANTES concentration in the conditioned medium of high-quality blastocysts was significantly lower than that in non-high-quality blastocysts (P < 0.05) (Table 1 and Figure 1 ).

[0058] According to the clinical pregnancy outcome, the women were divided into a clinical pregnancy positive group (n=34) and a clinical pregnancy negative group (n=33). The difference in RANTES concentration between the two groups was statistically significant (P<0.05). Binary logistic regression analysis was performed, with clinical pregnancy outcome as the dependent variable and RANTES concentration as the independent variable. The women's age, infertility years, BMI, endometrial thickness, and embryo score were corrected. The results showed that the RANTES concentration in the conditioned medium of the clinical pregnancy positive group was significantly lower than that of the clinical pregnancy negative group (P<0.05) (Table 1 and Figure 2 ).

[0059] With RANTES concentration of 8.21pg / mL as the cut-off value, the samples were divided into a low-concentration group (≤8.21pg / mL) and a high-concentration group (>8.21pg / mL). The clinical pregnancy positive rates of the two groups were 69.6% and 40.9%, respectively. The clinical pregnancy positive rate of the low-concentration group was significantly higher than that of the high-concentration group (P<0.05). Through binary logistic regression analysis, the RANTES concentration group was used as the dependent variable, the clinical pregnancy outcome was the independent variable, and the female age, infertility years, BMI, endometrial thickness, and embryo score were corrected. The results showed that the clinical pregnancy rate of the low-concentration group was significantly different from that of the high-concentration group (P<0.05) (Table 2 and Figure 3 ).

[0060] 2. A system for non-invasive assessment of embryo implantation potential

[0061] This embodiment relates to a system for non-invasively assessing embryo implantation potential, comprising:

[0062] A culture module, wherein the culture module is used to transfer the embryos cultured to the cleavage stage on the third day into a culture system containing 10% human serum albumin blastocyst culture solution, and culture them to the fifth or sixth day;

[0063] A sampling module, wherein the sampling module is used to collect blastocyst culture fluid from the culture system, obtain cell-free cultured blastocyst culture fluid, and freeze it at -80°C as a sample to be tested;

[0064] A detection module, wherein the detection module is used to detect the content of RANTES in the sample to be tested using a self-developed RANTES SIMOA single molecule immunoassay kit;

[0065] The discrimination processing module selects blastocysts with greater embryo implantation potential based on the RANTES content obtained by the detection module, such as selecting blastocysts derived from samples with low RANTES concentration, to achieve non-invasive assessment of the implantation potential of blastocysts;

[0066] An output module is used to output the evaluation result of the embryo implantation potential and select a blastocyst with greater embryo implantation potential for transplantation.

[0067] In summary, the present invention unexpectedly discovered through research that the level of RANTES in the conditioned culture medium of single blastocysts is correlated with the embryo developmental potential. Therefore, by detecting the level of RANTES in the conditioned culture medium of single blastocysts, embryos with higher developmental potential can be selected for embryo transplantation, thereby improving the success rate of embryo implantation; and providing a method for non-invasively evaluating and predicting embryo quality and embryo implantation potential, thereby improving the implantation rate after embryo transplantation.

[0068] 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 method for non-invasively assessing embryo implantation potential, wherein the method does not belong to the treatment and diagnosis of a disease, and is characterized in that: The steps include: (1) transferring a single blastocyst cultured to the cleavage stage on the third day into a culture system containing blastocyst culture medium and culturing the system until the fifth or sixth day; (2) After the blastocyst is formed, the blastocyst is vitrified and the blastocyst culture fluid is collected from the culture system to obtain a cell-free cultured blastocyst culture fluid as a sample to be tested, which is frozen at -80°C for future use; (3) Using single molecule immunoassay technology to detect the content of RANTES in the test sample to non-invasively evaluate the implantation potential or predictive value of the embryo.

2. A method for non-invasively assessing 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. The method for non-invasively assessing embryo implantation potential according to claim 1, characterized in that: Step (3) includes the following specific steps: thawing the sample to be tested at room temperature for 30 minutes to 60 minutes, measuring the volume of the sample to be tested, and diluting the sample to be tested; using single molecule immunoassay technology to detect the content of RANTES in the diluted sample to non-invasively evaluate the implantation potential or predictive value of the embryo.

4. A method for non-invasively assessing embryo implantation potential according to claim 3, characterized in that: The dilution process of the sample to be tested is specifically: diluting the sample to be tested to a total volume of at least 130 μL.

5. The method for non-invasively assessing embryo implantation potential according to claim 3, 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, fluorescent substrate solution and RANTES standard; the capture antibody-coated magnetic beads include RANTES capture antibodies coupled to the magnetic beads, and the biotinylated detection antibodies are prepared by connecting the RANTES capture antibodies with biotin.

6. A system for non-invasively assessing embryo implantation potential based on the method according to any one of claims 1 to 5, characterized in that: include: A culture module, wherein the culture module is used to transfer the embryos cultured to the cleavage stage on the third day into a culture system containing 10% human serum albumin blastocyst culture solution, and culture them to the fifth or sixth day; A sampling module, wherein the sampling module is used to collect blastocyst culture fluid from the culture system, obtain cell-free cultured blastocyst culture fluid, and freeze it at -80°C as a sample to be tested; A detection module, wherein the detection module is used to detect the content of RANTES in the sample to be tested by using single molecule immunoassay; A discrimination processing module, based on the content of RANTES obtained by the detection module, screens blastocysts with greater embryo implantation potential, thereby achieving non-invasive evaluation of the implantation potential of the blastocysts; An output module is used to output the evaluation result of the embryo implantation potential and complete the work of selecting the optimal embryo.

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