A method for detecting embryonic chromosomal abnormalities using blastocyst culture medium without zona pellucida

Abstract

The present invention provides a method for detecting chromosomal abnormalities of embryos by using blastocyst culture medium without zona pellucida. Specifically, the present invention provides an in vitro non-therapeutic method for detecting chromosomal abnormalities of embryos by using blastocyst culture medium, comprising the steps of: (a) providing a culture medium from a blastocyst culture system, wherein the embryos in the blastocyst culture system have been stripped of zona pellucida before culture, and the embryos are cultured in the blastocyst culture system for 3-6 days, preferably 4 days, and then the culture medium is separated from the culture system; (b) performing genetic testing on the culture medium to identify whether the chromosomes of the embryos are abnormal. The method provided by the present invention can eliminate the risk of contamination and interference by excess sperm and maternal granulosa cells during the IVF and ICSI techniques, and can accurately identify whether the chromosomes of the embryos are abnormal.

Description

Technical Field

[0001] The present invention relates to the fields of biomedicine and molecular cell biology. Specifically, it relates to a method for detecting chromosomal abnormalities in embryos using blastocyst culture medium without zona pellucida. Background Art

[0002] Assisted reproductive technology has become an important means for treating infertility. With the continuous improvement of technical levels, the clinical pregnancy rate of assisted reproduction has also been continuously increasing. However, the embryo implantation rate has not increased significantly. The quality of embryos is the main factor affecting embryo implantation. Currently, the selection of high-quality embryos mainly relies on embryo developmental morphological scoring, which is highly subjective and cannot fundamentally distinguish good-quality embryos from poor-quality ones. Some studies have shown that even some morphologically good-quality blastocysts are aneuploid, or the morphological scores of chromosomally euploid embryos are not high. Therefore, relying solely on embryo morphological evaluation cannot guarantee that the chromosomes of transplanted embryos are normal. A large number of studies have shown that a considerable proportion of embryos derived from in vitro fertilization are chromosomally aneuploid, especially embryos from elderly infertile women. The transplantation of aneuploid embryos leads to implantation failure or miscarriage after transplantation, which is also the main reason for the low success rate of in vitro fertilization currently.

[0003] Applying pre-implantation genetic screening (PGS) to select embryos with normal chromosomes can effectively improve the embryo implantation success rate of in vitro fertilization and reduce the pregnancy miscarriage rate at the same time. However, PGS detection requires biopsy of embryos, and 1 to several cells are taken from the embryos for detection, which poses a risk of embryo damage. Moreover, the current method of using blastocyst culture medium to check embryo chromosomes also has the risk of contamination and interference by excess sperm and maternal granulosa cells during the IVF and ICSI technical processes.

[0004] During the IVF technical process, hundreds of millions of sperm are mixed with an egg in an in vitro artificial culture environment. As a result, multiple sperm bind to the egg simultaneously. Although only one sperm completes normal fertilization with the egg, the other excess sperm that cannot complete fertilization still remain in the zona pellucida around the egg cell. In addition, a large number of maternal-derived granulosa cells will adhere to the zona pellucida around the egg at this time. These excess sperm remaining in the zona pellucida and the granulosa cells adhering to the surface of the zona pellucida may release DNA into the culture medium during the in vitro culture of the fertilized egg and its development into a blastocyst. Therefore, the DNA in the blastocyst culture medium is easily contaminated by sperm and granulosa cell DNA.

[0005] During the ICSI technical process, the technical method of injecting a single sperm into the egg avoids the contamination of blastocyst culture medium by excess sperm. However, the maternal-derived granulosa cells adhering to the zona pellucida of the egg still cause contamination to blastocyst culture and interfere with subsequent detection.

[0006] Therefore, there is an urgent need in the art to develop a method that can not only remove the risks of contamination and interference from excess sperm and maternal granulosa cells during the IVF and ICSI procedures, but also accurately identify whether the embryo chromosomes are abnormal. Summary of the Invention

[0007] The object of the present invention is to provide a method that can not only remove the risks of contamination and interference from excess sperm and maternal granulosa cells during the IVF and ICSI procedures, but also accurately identify whether the embryo chromosomes are abnormal.

[0008] The first aspect of the present invention provides a method for detecting embryo chromosomal abnormalities using blastocyst culture medium in vitro, comprising the steps of:

[0009] (a) Providing a culture medium from a blastocyst culture system, wherein the embryo in the blastocyst culture system has had its zona pellucida removed before culturing, and the embryo is cultured in the blastocyst culture system for 3 - 6 days, preferably, after 4 days, the culture medium is separated from the culture system;

[0010] (b) Performing genetic testing on the culture medium to identify whether the embryo chromosomes are abnormal.

[0011] In another preferred example, only one embryo is contained in the blastocyst culture system.

[0012] In another preferred example, the blastocyst culture system is a single - embryo culture system, and there is no zona pellucida in the single - embryo culture system, containing 10 - 100 μl, preferably, 15 - 50 μl, more preferably, 20 - 35 μl of culture medium.

[0013] In another preferred example, the volume of the culture medium separated in step (a) is 30 - 100% of the volume of the culture medium in the single - embryo culture system, preferably, 40 - 100%, more preferably, 50 - 100%, most preferably, 80 - 100%.

[0014] In another preferred example, step (c) is further included in step (b):

[0015] (i) Mixing the culture medium with a lysis solution to obtain a first mixture containing the culture medium and the lysis solution;

[0016] (ii) Mixing the first mixture with a lytic enzyme, incubating, and inactivating the lytic enzyme to obtain a lysate; and

[0017] (iii) Performing genomic analysis on the lysate to identify whether the embryo chromosomes are abnormal.

[0018] In another preferred embodiment, the method for genomic analysis is selected from the group consisting of: next-generation sequencing, nucleic acid microarray, immunofluorescence detection, fluorescent PCR detection, first-generation sequencing, third-generation sequencing, mass spectrometry detection, or a combination thereof.

[0019] In another preferred embodiment, the lysis buffer is selected from the group consisting of: Tris buffer, chelating agent, hydrochloride, nonionic surfactant, or a combination thereof.

[0020] In another preferred embodiment, the Tris buffer includes Tris-Cl.

[0021] In another preferred embodiment, the concentration of the Tris buffer is 10 - 60 mM, preferably 15 - 50 mM, more preferably 20 - 40 mM.

[0022] In another preferred embodiment, the pH of the Tris buffer is 5 - 10, preferably 6 - 9, more preferably 7 - 8.

[0023] In another preferred embodiment, the chelating agent includes EDTA.

[0024] In another preferred embodiment, the concentration of the chelating agent is 0.2 - 8 mM, preferably 0.5 - 6 mM, more preferably 1 - 4 mM.

[0025] In another preferred embodiment, the hydrochloride is selected from the group consisting of: KCl, NaCl, or a combination thereof.

[0026] In another preferred embodiment, the concentration of the hydrochloride is 5 - 60 mM, preferably 8 - 40 nM, more preferably 10 - 40 mM.

[0027] In another preferred embodiment, the nonionic surfactant is selected from the group consisting of: Triton X-100, Triton X-114, Tween 20, NP40, SDS, or a combination thereof.

[0028] In another preferred embodiment, the concentration of the nonionic surfactant is 0.02 - 10%, preferably 0.05 - 5%, more preferably 0.1 - 3%, based on the total weight of the lysis buffer.

[0029] In another preferred embodiment, in the step (i), the volume ratio of the culture medium to the lysis buffer is 1:10 - 10:1, preferably 1:5 - 5:1, more preferably 1:2 - 2:1.

[0030] In another preferred embodiment, the lytic enzyme is selected from the group consisting of: Proteinase K, Qiagen Protease, Pepsin, Papain, Trypsin, Lysozyme, or a combination thereof.

[0031] In another preferred embodiment, the concentration of the lyase is 1 - 25 μg / ml, preferably 5 - 20 μg / ml, more preferably 10 - 15 μg / ml.

[0032] In another preferred embodiment, the addition amount of the lyase is 0.1 - 10 μl, preferably 0.5 - 6 μl, more preferably 0.8 - 3 μl.

[0033] In another preferred embodiment, the step (c) includes one or more features selected from the following group:

[0034] (i) The incubation temperature is 20 - 70°C, preferably 30 - 60°C;

[0035] (ii) The incubation time is 1 min - 12 h, preferably 10 min - 6 h, more preferably 30 min - 2 h;

[0036] (iii) The inactivation temperature is 60 - 100°C, preferably 75 - 95°C;

[0037] (iv) The inactivation time is 0.5 - 20 min, preferably 10 - 15 min;

[0038] In another preferred embodiment, the culture medium is obtained by the following method:

[0039] (i) After culturing the fertilized embryo for 3 - 6 days (preferably 4 days), removing the zona pellucida of the embryo to obtain an embryo without zona pellucida;

[0040] (ii) Transferring the zona - pellucida - free embryo obtained in step (i) to a blastocyst culture droplet and culturing for 0.5 - 3 days (preferably 1 - 2 days) to obtain a culture medium containing blastocysts; and

[0041] (iii) Separating the culture medium obtained in step (ii), which is the culture medium for identifying whether the chromosomes of the embryo are abnormal.

[0042] In another preferred embodiment, the volume of the blastocyst culture droplet is 10 - 100 μl, preferably 15 - 50 μl, more preferably 20 - 35 μl.

[0043] In another preferred embodiment, the method is non - therapeutic and non - diagnostic.

[0044] The second aspect of the present invention provides a method for preparing a gene detection sample or a chromosome detection sample, including the steps:

[0045] (i) Providing a culture system containing an embryo without zona pellucida, culturing for 3 - 6 days, preferably 4 days, and separating the liquid from the culture system, which is the detection sample.

[0046] In another preferred example, the method further includes step (ii): performing a genetic test on the obtained test sample to identify whether the chromosomes of the embryo are abnormal.

[0047] In another preferred example, only one embryo is contained in the culture system.

[0048] In another preferred example, the culture system is a single-embryo culture system, containing 10 - 100 μL, preferably 15 - 50 μL, more preferably 20 - 35 μL of culture medium.

[0049] In another preferred example, in step (ii), the volume of the test sample is 30 - 100% of the volume of the culture medium in the culture system, preferably 40 - 100%, more preferably 50 - 100%, and most preferably 80 - 100%.

[0050] In another preferred example, in step (ii), the test sample is centrifuged, the supernatant is taken, and a genetic test is performed to identify whether the chromosomes of the embryo are abnormal.

[0051] It should be understood that within the scope of the present invention, the above-mentioned various technical features of the present invention and the various technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG. 1 shows that when detecting a conventional ICSI embryo, due to the interference of granulosa cell contamination, the NICS test result (1a) is deviated and inconsistent with the conventional PGS (embryo invasive biopsy) test result (1b).

[0053] FIG. 2 shows that when detecting a conventional IVF embryo, due to the interference of excess sperm contamination, the NICS test (2a) result is deviated and inconsistent with the conventional PGS (embryo invasive biopsy) test result (2b).

[0054] FIG. 3 shows that when using the technical solution of the present invention to detect a normal IVF embryo, due to the elimination of interference, the NICS test (3a) result is accurate and consistent with the conventional PGS (embryo invasive biopsy) test result (3b).

[0055] FIG. 4 shows that when using the technical solution of the present invention to detect an abnormal IVF embryo, due to the elimination of interference, the NICS test (4a) result is accurate and consistent with the conventional PGS (embryo invasive biopsy) test result (4b). DETAILED DESCRIPTION OF THE INVENTION

[0056] After long-term extensive and in-depth research, through a large number of screenings and tests, the present inventor unexpectedly discovered for the first time that after removing the zona pellucida and adopting a single embryo culture system, when culturing embryos in a culture solution of 20-30 μL, and taking out a small amount of the culture solution for detection, it was found that the detection results of chromosomal abnormalities obtained had extremely high accuracy, and could greatly eliminate the risk of interference from excess sperm and maternal granulosa cell contamination. On this basis, the present inventor completed the present invention.

[0057] IVF embryo

[0058] IVF refers to in vitro fertilization (test-tube baby, in vitro fertilization), specifically referring to the technique of in vitro fertilization combined with embryo transfer (IVF), also known as test-tube baby, which means that after removing eggs and sperm respectively, they are placed in a test tube for fertilization, and then the embryo precursor - fertilized egg is transplanted back into the mother's uterus to develop into a fetus.

[0059] ICSI

[0060] ICSI (Intracytoplasmic sperm injection) is the technique of intracytoplasmic sperm microinjection, that is, the second-generation "test-tube baby". This technique is to inject a single sperm into an egg with the aid of a micromanipulation system to make it fertilized.

[0061] Zona pellucida

[0062] The outside of the egg has an outer coat, the main component of which is glycoprotein and is secreted by the egg cell or other cells. In mammals, this outer coat is called the zona pellucida, and its function is to protect the egg and prevent foreign sperm from entering.

[0063] Detection method

[0064] The present invention provides a method for gene detection of the depleted medium (i.e., the culture solution separated from the culture system) after culturing blastocysts, so as to identify whether the chromosomes of the embryos are abnormal.

[0065] In the present invention, the method for gene detection of the "depleted" culture solution (i.e., the culture solution separated from the culture system) after culturing blastocysts is not particularly limited, and can be detected by conventional methods, such as next-generation sequencing, nucleic acid chip, immunofluorescence detection, fluorescence PCR detection, first-generation sequencing, third-generation sequencing, mass spectrometry detection, or a combination thereof.

[0066] In one embodiment, the detection method includes the following steps:

[0067] (a) Provide a culture medium from a blastocyst culture system, wherein the embryos in the blastocyst culture system have had their zona pellucida removed before culturing, the embryos are cultured in the blastocyst culture system for 3 - 6 days, preferably, after 4 days, the culture medium is separated from the culture system;

[0068] (b) Perform genetic testing on the culture medium to identify whether the chromosomes of the embryo are abnormal.

[0069] In a preferred embodiment, step (c) is further included in step (b):

[0070] (i) Mix the culture medium with a lysis solution to obtain a first mixture containing the blastocyst culture medium and the lysis solution;

[0071] (ii) Mix the first mixture with a lysing enzyme, incubate, and inactivate the lysing enzyme to obtain a lysate; and

[0072] (iii) Perform genomic analysis on the lysate to identify whether the chromosomes of the embryo are abnormal.

[0073] Sample preparation and its detection

[0074] The present invention also provides a method for preparing a genetic testing sample or a chromosome testing sample, including the steps:

[0075] (i) Provide a culture system containing embryos with their zona pellucida removed, culture for 3 - 6 days, preferably, after 4 days, separate the liquid from the culture system, which is the testing sample.

[0076] In a preferred embodiment, the method further includes step (ii): Perform genetic testing on the testing sample to identify whether the chromosomes of the embryo are abnormal.

[0077] Removal of zona pellucida

[0078] Methods for removing the zona pellucida include (but are not limited to): mechanical removal, laser removal, Tyrode solution digestion, etc. For specific methods of removing the zona pellucida, refer to reference 1 (A comparison of four different techniques of assisted hatching, 2002).

[0079] In the present invention, since the mechanical removal method has a better effect on removing the zona pellucida, and the obtained embryos are more complete, which can exclude the interference of various substances, therefore, the present invention selects the mechanical removal method to remove the zona pellucida. Specifically, in the present invention, the zona pellucida is removed by a conventional mechanical removal method and commercially available equipment.

[0080] The main advantages of the present invention include:

[0081] (1) In the present invention, after completely removing the zona pellucida, the embryo is cultured in 20 - 30 μL of culture medium, and a small amount of the culture medium is detected. The obtained detection result of chromosomal abnormalities has extremely high accuracy, and can greatly exclude the risk of interference from excess sperm and maternal granulosa cell contamination.

[0082] (2) The present invention adopts a single - embryo culture system, that is, only one embryo is cultured in one culture medium micro - drop. The detection result of chromosomal abnormalities obtained by this system is more accurate.

[0083] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0084] The materials used in the present invention are all commercially available products unless otherwise specified.

[0085] Qiagen Protease: crude protease, purchased from Qiagen.

[0086] General method

[0087] Obtaining of the culture medium

[0088] (1) The fertilized eggs (in volunteer couples, eggs are obtained from female volunteers and sperm are obtained from male volunteers, and in vitro fertilization is performed to obtain fertilized eggs) are cultured in vitro from the 3rd day to the 6th day. Preferably, on the 4th day, that is, when the embryo develops to the morula stage and the blastomeres are completely fused, there is a large gap between the embryo and the zona pellucida at this time.

[0089] (2) Use a mechanical stripping method (such as mechanical methods like PIZO) to strip the zona pellucida around the embryo, and gently aspirate and blow the embryo with a 135 - micron egg - stripping needle to completely separate the zona pellucida from the embryo.

[0090] (3) Transfer at least 3 zona - pellucida - removed embryos to at least 3 30 - μL blastocyst culture medium micro - drops in sequence (ensuring that there is only one embryo in one micro - drop). Each micro - drop is gently aspirated and rinsed 2 - 3 times, and then the embryo is transferred to a micro - drop (30 μL) under the conditions of 37 °C, 5% CO2, and 5% O2 for overnight equilibration and continued culture to the blastocyst stage.

[0091] (4) Transfer the culture medium of the blastocyst obtained in step (3) (i.e., the exhausted culture medium, with a volume of about 24 - 30 μL) into 30 μL of lysis buffer (30 mM Tris-Cl, 2 mM EDTA, 20 mM KCl, 0.2% Triton X-100 at pH 7.8), and mark the sample name on the collection tube with a marker pen. Centrifuge in a microcentrifuge for 30 seconds. The sample can immediately enter the next whole genome amplification step or be stored frozen at -20 °C or -80 °C.

[0092] Whole Genome Amplification of Trace DNA in Blastocyst Culture Medium

[0093] (1) Thaw the mixture of blastocyst culture medium and lysis buffer at room temperature.

[0094] (2) Add 1 μL of lysing enzyme (12.5 μg / ml Qiagen Protease) to the tube and pipette up and down to mix well.

[0095] (3) Incubate the tube at 50 °C for 90 minutes.

[0096] (4) Incubate the tube at 80 °C for 10 minutes to inactivate the lysing enzyme.

[0097] (5) Take out 10 μL of the lysate from the tube and add it to a PCR reaction tube.

[0098] (6) Add 60 μL of pre-amplification mixture to the PCR reaction tube.

[0099] (7) Place the PCR reaction tube in a PCR instrument for pre-amplification, and the thermal cycling program is:

[0100]

[0101] (8) Add 60 μL of amplification mixture to the PCR reaction tube (the composition of the amplification mixture is 10 - 25 mM Tris-HCl, 5 - 25 mM (NH4)2SO4, 5 - 30 mM KCl, 0.5 - 5 mM MgSO4, 0.1% - 20% DMSO, and 0.05% - 5% Triton X-100. Preferably, the composition of the amplification mixture is 15 mM Tris-HCl, 15 mM (NH4)2SO4, 20 mM KCl, 1 mM MgSO4, 5% DMSO, and 2% Triton X-100).

[0102] (9) Place the PCR reaction tube in a PCR instrument for exponential amplification, and the thermal cycling program is:

[0103]

[0104] NICS Method

[0105] (1) Thaw the mixture of blastocyst culture medium and lysis solution at room temperature.

[0106] (2) Add 1 μl of lytic enzyme (12.5 μg / ml Qiagen Protease) to the tube and pipette up and down to mix evenly.

[0107] (3) Incubate the tube at 50 °C for 90 minutes.

[0108] (4) Place the tube at 80 °C for 10 minutes to inactivate the lytic enzyme.

[0109] (5) Take out 10 μl of the lysate from the tube and add it to a PCR reaction tube.

[0110] (6) Add 60 μl of pre-amplification mixture to the PCR reaction tube.

[0111] (7) Place the PCR reaction tube in a PCR instrument for pre-amplification. The thermal cycling program is:

[0112]

[0113]

[0114] (8) Add 60 μl of amplification mixture to the PCR reaction tube (the components of the amplification mixture are 10 - 25 mM Tris-HCl, 5 - 25 mM (NH4)2SO4, 5 - 30 mM KCl, 0.5 - 5 mM MgSO4, 0.1% - 20% DMSO, and 0.05% - 5% Triton X-100. Preferably, the components of the amplification mixture are 15 mM Tris-HCl, 15 mM (NH4)2SO4, 20 mM KCl, 1 mM MgSO4, 5% DMSO, and 2% Triton X-100).

[0115] (9) Place the PCR reaction tube in a PCR instrument for exponential amplification. The thermal cycling program is:

[0116]

[0117] (10) Sequence the amplified DNA product by next-generation sequencing according to the conventional method to identify whether the chromosomal status of the embryo is normal.

[0118] Treatment of samples and acquisition of samples

[0119] Samples: Take 5 fertilized eggs from 5 pairs of volunteer couples respectively (wherein, take eggs from female volunteers and sperm from male volunteers, and perform in vitro fertilization to obtain fertilized eggs).

[0120] Sample treatment and sample acquisition:

[0121] (1) The fertilized eggs are cultured in vitro from the 3rd day to the 6th day. Preferably, on the 4th day, when the embryo develops to the morula stage and the blastomeres are completely fused, a large gap is generated between the embryo and the zona pellucida at this time.

[0122] (2) The zona pellucida around the embryo is removed by mechanical stripping methods (such as mechanical methods like PIZO). The embryo is gently aspirated with a 135-micron egg stripper needle to completely separate the zona pellucida from the embryo.

[0123] (3) At least 3 zona pellucida-removed embryos are sequentially transferred to at least 3 30-μL blastocyst culture medium microdrops (ensuring that there is only one embryo in one microdrop). Each microdrop is gently aspirated and rinsed 2 - 3 times, and then the embryos are transferred to a microdrop (30 μL) for overnight equilibration under the conditions of 37 °C, 5% CO2, and 5% O2 and continue to be cultured to the blastocyst stage.

[0124] (4) 30 μL (volume) (supplemented) of the culture medium (i.e., spent culture medium) (volume is about 24 - 30 μL) is taken and transferred into 30 μL of lysis buffer (30 mM Tris-Cl with pH 7.8, 2 mM EDTA, 20 mM KCl, 0.2% Triton X-100), and thus a culture medium sample without zona pellucida is obtained. It is centrifuged in a microcentrifuge for 30 seconds, and then enters the next whole-genome amplification step or is stored frozen at -20 °C or -80 °C.

[0125] Example 1

[0126] Using the detection method of the present invention (i.e., stripping the zona pellucida of the embryo at the morula stage and then changing the culture medium for culture), the chromosomal status of two IVF embryos (Samples C and D) is evaluated by the methods of blastocyst cell biopsy detection and blastocyst culture medium detection respectively. The blastocyst culture medium detection method obtains the same results as the blastocyst cell detection method.

[0127] The results of next-generation sequencing data show that in Sample C, both the blastocyst culture medium detection method ( Figure 3a ) and the blastocyst cell detection method ( Figure 3b ) detect normal chromosomes. In Sample D, both the blastocyst culture medium detection method ( Figure 4a ) and the blastocyst cell detection method ( Figure 4b ) detect deletions in some segments of chromosome 2 and amplifications in some segments of chromosome 4.

[0128] The fertilized eggs of Sample C are implanted into the uterus of the couple's biological mother, and embryos with normal chromosomal status and capable of normal development can be obtained.

[0129] Comparative Example 1

[0130] The blastocyst culture medium and blastocyst biopsy cells of an ICSI embryo were respectively detected using the conventional NICS method (i.e., only changing the culture medium and culturing without removing the zona pellucida) to evaluate its chromosomal status. The detection result of the blastocyst biopsy cells was used as the standard to evaluate the detection result of the blastocyst culture medium.

[0131] The results of next-generation sequencing showed that, compared with the blastocyst biopsy cell detection method ( Figure 1b ), the detection result of the blastocyst culture medium detection method ( Figure 1a ) had granulosa cell contamination, resulting in false-positive amplification of the X chromosome, and at the same time, the amplification variations of chromosome 6 and chromosome 16 could not be detected.

[0132] Comparative Example 2

[0133] The blastocyst culture medium and blastocyst biopsy cells of an IVF embryo were respectively detected using the conventional NICS method (i.e., only changing the culture medium and culturing without removing the zona pellucida) to evaluate its chromosomal status. The detection result of the blastocyst biopsy cells was used as the standard to evaluate the detection result of the blastocyst culture medium.

[0134] The results of next-generation sequencing showed that, compared with the blastocyst biopsy cell detection method ( Figure 2b ), the result of the blastocyst culture medium detection method ( Figure 2a ) had sperm contamination, resulting in false-positive deletion of the X chromosome copy number, and the deletion of chromosome 1 could not be detected, and the detection results of the two methods were inconsistent.

[0135] All the documents mentioned in the present invention are cited in this application as references, just as if each document was cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A method for detecting embryonic chromosomal abnormalities in vitro using blastocyst culture medium, characterized in that, Comprising the steps: (a) Providing a culture medium from a blastocyst culture system, wherein the culture medium is obtained by the following method: (i) After culturing fertilized embryos for 3 - 4 days, removing the zona pellucida of the embryos to obtain zona pellucida-free embryos; (ii) Transferring the zona pellucida-free embryos obtained in step (i) into a blastocyst culture droplet and culturing for 0.5 - 3 days to obtain a culture medium containing blastocysts; and (iii) Separating the culture medium obtained in step (ii), which is the culture medium for identifying whether the chromosomes of the embryos are abnormal; (b) Performing gene detection on the culture medium to identify whether the chromosomes of the embryos are abnormal, wherein the method is non-therapeutic and non-diagnostic.

2. The method according to claim 1, characterized in that, The blastocyst culture system is a single-embryo culture system, and there is no zona pellucida in the single-embryo culture system, containing 10 - 100 μl of culture medium.

3. The method according to claim 1, characterized in that, The volume of the culture medium separated in step (a) is 30 - 100% of the volume of the culture medium in the single-embryo culture system.

4. The method according to claim 1, characterized in that, Step (c) is further included in step (b): (i) Mixing the culture medium with a lysis solution to obtain a first mixture containing the culture medium and the lysis solution; (ii) Mixing the first mixture with a lyase, incubating, and inactivating the lyase to obtain a lysis product; and (iii) Performing genomic analysis on the lysis product to identify whether the chromosomes of the embryos are abnormal.

5. The method according to claim 4, characterized in that, The method of genomic analysis is selected from the group consisting of: next-generation sequencing, nucleic acid microarray, immunofluorescence detection, fluorescence PCR detection, first-generation sequencing, third-generation sequencing, mass spectrometry detection, or a combination thereof.

6. The method according to claim 4, characterized in that, The lysis solution is selected from the group consisting of: Tris buffer, chelating agent, hydrochloride, non-ionic surfactant, or a combination thereof.

7. The method according to claim 4, characterized in that, In step (i), the volume ratio of the culture medium to the lysis solution is 1:10 - 10:

1.

8. The method according to claim 4, characterized in that, The concentration of the lyase is 1 - 25 μg / ml.

9. A method for preparing a gene detection sample or a chromosome detection sample, characterized in that, The test sample is obtained by the following method: (a) After culturing fertilized embryos for 3 - 4 days, removing the zona pellucida of the embryos to obtain zona pellucida-free embryos; (b) Transferring the zona pellucida-free embryos obtained in step (a) into a blastocyst culture droplet and culturing for 0.5 - 3 days to obtain a culture medium containing blastocysts; (c) Separating the culture medium obtained in step (b), which is the culture medium for identifying whether the chromosomes of the embryos are abnormal and is the test sample.

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