METHOD AND PRODUCT FOR DETECTING EMBRYO HEALTH CONDITION BY USING BLASTOCYST CULTURE SOLUTION

Abstract

The invention provides a method and a product for detecting the health status of an embryo by using a blastocyst culture medium. Specifically, the present invention provides an in vitro method for detecting the health status of embryos using blastocyst culture medium, characterized in that it comprises the steps of: (a) providing a first blastocyst culture system containing a blastocyst of in vitro culture on Day 5 (D5) to Day 6 (D6); (b) transferring the blastocysts into a second blastocyst culture system containing a fresh blastocyst culture medium, and culturing in the exchanged medium for a time of T1, thereby obtaining a spent blastocyst culture medium; (c) collecting the spent blastocyst culture medium, thereby obtaining a cell-free spent blastocyst culture medium, i.e. a test sample; and (d) performing a genetic assay to identify the health status of the blastocyst. The method can reduce the risk of being Interfered by contaminants from extra sperms and maternal-derived granulosa cells during IVF and ICSI treatment, and can accurately identify the health status of the embryo.

Claims

1. An in vitro method for detecting the health status of blastocysts using blastocyst culture medium, characterized in that it comprises the steps of: (a) providing a first blastocyst culture system, which comprises a blastocyst on Day 5 (D5) to Day 6 (D6) of in vitro culture; (b) transferring the blastocysts into a second blastocyst culture system containing a fresh blastocyst culture medium, and culturing in the exchanged medium for a time of T1, thereby obtaining a spent blastocyst culture medium; (c) collecting the spent blastocyst culture medium, thereby obtaining a cell-free spent blastocyst culture medium, i.e., a test sample; and (d) performing a genetic assay to identify the health status of the blastocyst.

2. The method of claim 1, wherein in step (a), the in vitro culture of the blastocyst is a de novo culture or a staged culture.

3. The method of claim 1, wherein in step (a), the in vitro culture of the blastocyst includes a first-stage blastocyst culture and a second-stage blastocyst culture.

4. The method of claim 3, wherein the first-stage blastocyst culture includes culturing on Day 1 to Day 3 in a cleavage-stage culture medium.

5. The method of claim 3, wherein the second-stage blastocyst culture includes culturing on Day 3 to Day 5 in a blastocyst culture medium.

6. The method of claim 1, wherein the T1 time is 2 to 8 hours, for example, 3 to 6 hours, preferably 3 to 5 hours.

7. The method of claim 1, wherein the method has one or more of the following characteristics: (i) a high signal-to-noise ratio, wherein the signal-to-noise ratio S.sub.1/S.sub.0>2, preferably, >5, more preferably, >10, where S.sub.1 is the signal from the embryo and S.sub.0 is the signal from the background; (ii) low false-negative rate, wherein the false-negative rate is <8%, preferably, <5%, more preferably, <3%; (iii) high accuracy, wherein the accuracy rate is ≥80%, preferably, 85%≥, more preferably, ≥90%.

8. A method for preparing a sample for a gene detection or for a chromosome detection, characterized in that it comprises the steps: (a) providing a first blastocyst culture system, which comprises a blastocyst on Day 5 (D5) to Day 6 (D6) of in vitro culture; (b) transferring the blastocysts into a second blastocyst culture system containing a fresh blastocyst culture medium, and culturing in the exchanged medium for a time of T1, thereby obtaining a spent blastocyst culture medium; (c) collecting the spent blastocyst culture medium, thereby obtaining a cell-free spent blastocyst culture medium, i.e., a test sample.

9. The method of claim 1, further including using a culture module that includes a culture module for a later stage blastocyst, functioning to exchange the medium of the cultured blastocyst on Day 5 to Day 6, and to perform the later-stage culture of the blastocyst for a period of time (T1).

10. A device for assisting in diagnosing the health status of a blastocyst, characterized in that it comprises: (a) a culture module, which includes a culture module for a later stage blastocyst, functioning to exchange the medium of the cultured blastocyst on Day 5 to Day 6, and to perform the later-stage culture of the blastocyst for a period of time (T1); (b) a sampling module, functioning to collect from the later-stage blastocyst culture module, the spent culture medium of the blastocyst as a test sample; (c) a detection module, functioning to perform a gene detection on the test sample collected from the sampling module, thereby obtaining a detection result; (d) an evaluation module, functioning to determine based on the detection result the health status of the blastocyst, thereby providing the evaluation result of the blastocyst health status; (e) an output module, functioning to output the evaluation result of the health status of the blastocyst.

11. The method of claim 1, wherein the method further comprises, after exchanging the medium and before collecting the test sample, perforating the zona pellucida of the blastocyst.

12. The method of claim 9, wherein the culture module further comprises a device for perforating the zona pellucida of the blastocyst after the medium is exchanged.

13. The device of claim 10, wherein the culture module further comprises a device for perforating the zona pellucida of the blastocyst after the medium is exchanged.

14. The method of claim 2, wherein the method further comprises, after exchanging the medium and before collecting the test sample, perforating the zona pellucida of the blastocyst.

15. The method of claim 3, wherein the method further comprises, after exchanging the medium and before collecting the test sample, perforating the zona pellucida of the blastocyst.

16. The method of claim 4, wherein the method further comprises, after exchanging the medium and before collecting the test sample, perforating the zona pellucida of the blastocyst.

17. The method of claim 5, wherein the method further comprises, after exchanging the medium and before collecting the test sample, perforating the zona pellucida of the blastocyst.

18. The method of claim 6, wherein the method further comprises, after exchanging the medium and before collecting the test sample, perforating the zona pellucida of the blastocyst.

19. The method of claim 7, wherein the method further comprises, after exchanging the medium and before collecting the test sample, perforating the zona pellucida of the blastocyst.

20. The method of claim 8, wherein the method further comprises, after exchanging the medium and before collecting the test sample, perforating the zona pellucida of the blastocyst.

Description

DESCRIPTION OF THE DRAWINGS

[0116] FIG. 1 shows a schematic diagram of the difference between the conventional sampling method and the sampling method of the present invention in the exposure time of a test sample.

[0117] FIG. 2 shows the CNV analysis results of the culture medium collected using the conventional D3-D5 sampling method.

[0118] FIG. 3 shows the CNV analysis results of the culture medium collected using the D5 sampling method of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0119] After a long-term, extensive and in-depth research, through a large number of screenings and tests, the inventors unexpectedly discovered for the first time that: by using the 10-20 microliter blastocyst culture system of the present invention to culture an embryo (blastocyst) in a blastocyst culture medium to Day, D5 to D6, and then transferring the embryo to a fresh blastocyst culture medium for culture, and collecting a small amount of culture medium from the fresh blastocyst culture medium for testing, the embryo health status (such as chromosome aneuploidy, mitochondrial copy number, DNA content is normal, and pathogenic gene detection) can be accurately detected with extremely low false-negative rate (<8%, preferably, <5%). The method can greatly eliminate the risk of interference by contaminants from extra sperm and maternal-derived granular cells. On this basis, the inventor completed the present invention.

[0120] As used herein, the terms “blastocyst” and “embryo” are used interchangeably and refer to embryo at the final stage of in vitro culture, which is usually formed on the 5.sup.th to the 7.sup.th day after fertilization of the egg.

[0121] As used herein, a depleted medium refers to a spent medium, for example, a medium separated from a culture system which has been used to culture embryos.

[0122] As used herein, a de novo culture refers to an embryo culture process in which the culture medium is not exchanged from the start of the culture process to the end of the culture process. For example, a de novo culture on D1 to D5 means that the embryo culture process starts from D1 and continues to D5, during which the culture medium is not replaced.

[0123] As used herein, a staged culture refers to an embryo culture process in which the embryo undergoes one or more medium exchanges. For example, a staged culture on D1 to D5 means that the embryo culture process starts from D1 and continues to D5, during which the embryo undergoes one or more medium exchanges, for example, on D1 to D3, embryos are cultured in a cleavage-stage medium; and on D3 to D5, embryos are cultured in a blastocyst culture medium.

[0124] As used herein, “Dx” (where x=a positive integer of 1, 2, 3 . . . ) means that, with the start of the in vitro culture of a fertilized oocyte set to day 1 (D1), Dx is the X day of the in vitro culture.

[0125] As used herein, “D3-D5 sampling” means that a culture medium is used to culture embryos from D3 to D5, and a sample is collected from the spent culture medium (i.e., the depleted culture medium) on D5. Therefore, the culture medium sample obtained by this sampling method will be in contact with the cultured embryo for a period of time (i.e., the exposure time of the test medium sample), which is at least about 48 hours or more.

[0126] As used herein, “on-the-day sampling” refers to collecting a sample from a spent culture medium (i.e., depleted culture medium) on the day of culture medium exchange and after embryo is cultured in the exchanged medium for a short period. Therefore, the time for the culture medium sample obtained by this sampling method to contact the cultured embryo (i.e., the exposure time of the test sample) will be short. In the present invention, it is preferable that the exposure time does not exceed 8 hours, such as 3-6 hours, more preferably 3-5 hours.

[0127] As used herein, “lysis solution” refers to a lysis buffer used to decompose proteins and cells in a culture medium sample. In some embodiments, the lysis solution may or may not include a lytic enzyme. In some embodiments, the lysis solution and the lytic enzyme can be added to the culture medium sample at the same time. In other embodiments, the lytic enzyme is supplemented after mixing the lysis solution with the culture medium sample.

[0128] As used herein, “in vitro incubation time of test sample”, “culture time of test sample” and “exposure time of test sample” are used interchangeably, which means the length of time for which the culture medium used for sampling has been in vitro in contact with embryos, or has been incubated with embryo.

[0129] As used herein, “CNV” refers to chromosome copy number variants. CNV nomenclature complies with the International System for Human Cytogenetic Nomenclature (ISCN). See, Shaffer LG, Slovak ML, Campbell LJ (2009): ISCN 2009 an international system for human cytogenetic nomenclature, Human Genetics volume 126, Article number: 603 (2009).

[0130] IVF and IVF Embryos

[0131] IVF refers to in vitro fertilization, specifically refers to in vitro fertilization combined embryo transfer technology, also known as test tube baby, by which an oocyte and a sperm are taken, placed in a test tube to fertilize, and then the fertilized oocyte (i.e., precursor of embryo) is transplanted back into the mother's uterus to develop into a fetus.

[0132] “Polysperm-fertilized blastocyst” or “IVF blastocysts” refer to a blastocyst obtained by in vitro co-culturing more than one sperm with an oocyte to fertilize using IVF technology.

[0133] ICSI and ICSI Embryos

[0134] ICSI (Intracytoplasmic sperm injection) is a technology by which a single sperm is microinjected into the cytoplasm of an egg, which is the second generation of “test tube baby”. This technology uses a microscopic operating system to inject a single sperm into an egg to fertilize it. “monosperm-fertilized blastocyst” or “ICSI blastocyst” refers to a blastocyst obtained by using ICSI, the technique of intracytoplasmic sperm microinjection.

[0135] Detection Method

[0136] The present invention provides a detection method that performs a genetic detection on a depleted medium used for a blastocyst culture (i.e., the culture medium separated from a culture system which has been used to culture a blastocyst for a period of time), thereby identifying the health status of the embryo.

[0137] In the present invention, the method for genetic detection on a depleted culture medium used for blastocyst culture is not particularly limited, and conventional methods can be used for detection, such as second-generation sequencing, nucleic acid chip, immunofluorescence detection, fluorescent PCR detection, first-generation sequencing, third-generation sequencing, mass spectrometry detection, or a combination thereof.

[0138] In one embodiment, the detection method includes the following steps:

[0139] (a) providing a first blastocyst culture system, which comprises a blastocyst on Day 5 (D5) to Day 6 (D6) of in vitro culture;

[0140] (b) transferring the blastocyst into a second blastocyst culture system containing a fresh blastocyst culture medium, and culturing in the exchanged medium for a time of T1, thereby obtaining a spent blastocyst culture medium;

[0141] (c) collecting the spent blastocyst culture medium, thereby obtaining a cell-free spent blastocyst culture medium, which is the test sample; and

[0142] (d) performing a genetic assay to identify the health status of the blastocyst.

[0143] In a preferred embodiment, the step (d) further includes step (e):

[0144] (i) mixing the culture medium with a lysis solution to obtain a first mixture containing the culture medium and the lysis solution;

[0145] (ii) mixing the first mixture with a lytic enzyme, incubating, and then inactivating the lytic enzyme, thereby obtaining a lysate; and

[0146] (iii) Perform genomic analysis on the lysate to identify the health status of the blastocyst (embryo).

[0147] In a specific embodiment, the method according to the present invention by using blastocyst culture medium as a test sample to reflect the health status of embryos include the following main steps:

[0148] (A) an oocyte is fertilized by IVF or ICSI, and after fertilization, the fertilized oocyte is transferred to a culture medium and cultured to the D3 cleavage stage, and then the embryo is transferred to a blastocyst culture medium on D3 and cultured to D5-D6;

[0149] (b) blastocyst grading is performed, and the well-developed blastocyst is transferred to another fresh blastocyst culture medium on D5±0.5 days, and a laser puncture instrument is used to perforate the zona pellucida under a high-power microscope to make the embryo shrink, the blastocyst is removed out 3-5 hours later and subjected to vitrification, and the remaining spent blastocyst culture medium is the test sample to be collected;

[0150] (c) the spent blastocyst culture medium obtained in step (b) is transferred into a lysis solution, and after centrifugation, the sample is subjected to a whole genome amplification, including but not limited to, the amplification of NICS-INST, and an analysis, thereby obtaining the test result reflecting the embryo's health status.

[0151] The present invention performs a culture medium exchange on D5±0.5 days and a 3-5 hours short-time culture before the blastocyst culture medium is collected as a test sample. By this, the accuracy of the test result is significantly improved, the false negative rate is reduced to less than 5%, and the interference from external substances, especially maternal-derived interference, is avoided to a great extent. In addition, by this, the method according to the present invention is applicable to both ICSI and IVF fertilization.

[0152] In the present invention, it is believed that the following contributes to the improved accuracy of the detection result: First, compared with the exposure time of 2-3 days in the conventional operation method (as shown in FIG. 1), the exposure time of the test sample according to the present invention is shorten to a very short period, by which the interference from external substances, especially the maternal-derived interference, is avoided to the maximum extent possible, and irrespective of the fertilization process, the accuracy of the detection results is greatly improved. Second, embryo has the ability to repair itself, and abnormal fragments are released into the culture medium, which may affect the accuracy in the conventional culture method. In the present invention, sampling is made at the time of 3-5 hours incubation after D5 culture medium exchange, so that the culture time for embryo in the exchanged medium is short. The self-repairing of embryo occurs during the D3-D5 culture period and before the D5 culture medium exchange, with most of abnormal fragments released during the D3-D5 culture period. This causes a low release rate of abnormal fragments in the test sample collected according to the present invention, and consequently an improved accuracy of the detection result. Before the present invention is filed, generally the culture medium of ICSI fertilized embryo is collected, but no one has tried to collect the culture medium of IVF embryo. The most important factor for external interference is the excessively long in vitro incubation time of the test sample. The present invention adopts a short-time incubation method for collecting culture medium sample. The method avoids external interference from such as sperm and granular cells due to excessively long incubation time, which will reduce the ratio of embryo-derived DNA in the test sample and affect the accuracy of the results.

[0153] The amount of the test sample in the step (c) is 8-15 ul, preferably 10 ul. The amount of the fresh blastocyst culture medium (i.e., the second blastocyst culture medium) is preferably 10-15 ul.

[0154] Preferably, the blastocyst grading includes selecting a well-developed embryo that has developed to stage 4 or higher.

[0155] The perforation in step (b) increases the release of blastocoel fluid, that is, the release of free nucleic acid, so that the initial amount of the nucleic acid substance is increased in the test sample under the premise of short-term incubation for 3-5 hours, to a level sufficient for subsequent amplification and detection, thereby ensuring the success detection rate can reach more than 97%. Embryos are usually observed, graded and frozen in the blastocyst stage. It is believed that operations at the blastocyst stage will not cause damage to the embryo. The present invention also chooses the D5 blastocyst stage to perform laser drilling and in vitro incubation, so those operations are performed without affecting embryonic development.

[0156] In the present invention, step (b) may also include a step of washing embryos before changing the culture medium on D5, which is helpful to further improve the accuracy of the detection results.

[0157] In the present invention, in the step (a), after the exchange of a blastocyst culture medium on D3, multiple embryos can be mixed in culture, or a single embryo culture can be performed.

[0158] Sample Preparation and Testing

[0159] The present invention also provides a method for preparing a genetic test sample or a chromosome test sample, including the steps:

[0160] (a) providing a first blastocyst culture system, which comprises a blastocyst on Day 5 (D5) to Day 6 (D6) of in vitro culture;

[0161] (b) transferring the blastocysts into a second blastocyst culture system containing a fresh blastocyst culture medium, and culturing in the exchanged medium for a time of T1, thereby obtaining a spent blastocyst culture medium;

[0162] (c) collecting the spent blastocyst culture medium, thereby obtaining a cell-free spent blastocyst culture medium, i.e., a test sample.

[0163] In another preferred embodiment, the test sample obtained by the method can be used for genetic testing to identify the health status of the embryo.

[0164] Zona Pellucida Perforation

[0165] An embryo grows to the blastocyst stage, and the blastocyst is positioned on the operating table. A site far from the inner cell mass and with thin trophectoderm is chosen, a laser beam is used to punch the blastocyst cavity, and create a small hole in the zona pellucida to allow the Blastocoel fluid release into the blastocyst culture medium. The opening size of the zona pellucida is not particularly limited. A preferred opening size of the zona pellucida is 10-40 μm, preferably, 10-30 μm, and more preferably, 10-20 μm.

[0166] In the present invention, the perforation of the zona pellucida increases the release of blastocoel fluid, that is, the release of free nucleic acid, which leads to an increase in the initial amount of the nucleic acid substance in the test sample under the premise of a short incubation of 3-5 hours. As a consequence, the test sample has enough nucleic acid substance for subsequent amplification and detection, thereby ensuring the success detection rate can reach more than 97%.

[0167] If the laser drilling operation is not performed, the detection success rate is also high, up to about 70%.

[0168] Detection Kit

[0169] In the present invention, a kit for detecting the health status of embryos using blastocyst culture medium is provided, which includes:

[0170] (i) a first container, and a cleavage-stage culture medium contained in the first container;

[0171] (ii) a second container, and a first blastocyst culture medium contained in the second container;

[0172] (iii) a third container, and a second blastocyst culture medium contained in the third container; and

[0173] (iv) labels or instructions.

[0174] Device for Assisting in the Diagnosis of Embryo Health

[0175] In the present invention, a device for assisting in diagnosing the health status of embryos is provided, including:

[0176] (a) a culture module, which includes a culture module for a later-stage blastocyst, functioning to exchange the medium of the cultured blastocyst on Day 5 to Day 6, and to perform the later-stage culture of the blastocyst for a period of time (T1);

[0177] (b) a sampling module, functioning to collect from the later-stage blastocyst culture module, the spent culture medium of the blastocyst as a test sample;

[0178] (c) a detection module, functioning to perform a gene detection on the test sample collected from the sampling module, thereby obtaining a detection result;

[0179] (d) an evaluation module, functioning to determine based on the detection result the health status of the blastocyst, thereby providing the evaluation result of the blastocyst health status;

[0180] (e) an output module, functioning to output the evaluation result of the health status of the blastocyst.

[0181] The present invention also relates to the use of said module (a), optionally in combination with modules (b)-(e), in the preparation of a device used in the method for evaluating the health status of blastocysts according to the present invention.

[0182] The main advantages of the present invention include:

[0183] (1) In the present invention, it was discovered for the first time that by transferring a blastocyst on D5-D6 of in vitro culture to a fresh blastocyst culture medium for a short-term culture and then separating the spent culture medium therefrom and taking a small amount of the culture medium for genetic testing, the embryo's health status (such as whether chromosomes are abnormal, chromosome aneuploidy, mitochondrial copy number, whether the DNA content is normal, pathogenic gene testing, etc.) can be detected in an extremely high accuracy and with a very low false-negative rate (less than 8%, preferably, less than 5%), and the risk of the interference by contaminants from extra sperm and maternal-derived granular cells can be greatly reduced.

[0184] (2) The present invention is applicable to a single embryo culture system, that is, only one embryo is cultured in one culture medium, and the detection result of the embryo's health status obtained by the system is more accurate.

[0185] (3) The method according to the present invention is a universal method, which can be used for monosperm-fertilized embryos, and can also be used for polysperm-fertilized embryos, and polysperm-fertilized embryos are preferred.

[0186] (4) The method of the present invention may include a step of zona pellucida drilling, by which the detection result can be further improved.

[0187] (5) The method of the present invention has a high signal-to-noise ratio.

[0188] (6) In the method of the present invention, the collection operation of embryo culture medium is performed after the embryo has been cultured to Day 5 (D5). The operation time is extremely short, and the incubation time of the test sample is controllable, and the risk of external interference is low. In addition, the sample collection method is not limited by fertilization process, and is a culture medium sampling method applicable to both IVF and ICSI embryos.

[0189] (7) The process according to the present invention using D5 medium exchange and a short-term culture has the advantage of controlling the in vitro incubation time of the test sample, thereby controlling external interferences such as maternal-derived interference and sperm-derived interference to the greatest extent, so that the application of the present invention is not limited to ICSI embryos, but also can be applied to IVF embryos.

[0190] The present invention will be further explained below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the examples usually follow the conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0191] Unless otherwise specified, the materials used in the present invention are all commercially available products.

[0192] General Method

[0193] Obtaining blastocyst culture medium:

[0194] (1) in vitro culture fertilized oocytes (obtained from a volunteer couple, wherein oocytes are obtained from female volunteer, and sperms are from male volunteer, and IVF or ICSI is used for in vitro fertilization to obtain fertilized oocytes) to the third day (D3), and further cultur, with or without medium exchange, to the 5th day (D5)/the 6th day (D6);

[0195] (2) When the embryos develop to blastocysts above stage 4, transfer the embryos to at least three droplets of 30 ul blastocyst culture medium (ensuring that there was only one embryo in one droplet), and with gently blowing each droplet, rinse the droplets 2-3 times, then transfer embryos into 10-15 ul microdroplets that have been equilibrated overnight at 37° C., and 5% CO2 and 5% O2, and use a laser drilling instrument to perforate the zona pellucida under a high-power microscope to make the embryos shrink; continue culturing for 3-5 hours or 3-6 hours.

[0196] (3) transfer the culture medium of the short-term cultured blastocyst (about 10-15 microliters) obtained in step (2) to 5 microliters of lysis solution (30 mM Tris-C1, pH7.8, 2 mM EDTA, 20 mM KCl, 0.2% Triton X-100), mark the sample name on the collection tube, and centrifuge for 30 seconds in a microcentrifuge. The sample can immediately be used for the whole genome amplification or stored at −20° C. or −80° C.

[0197] The detection method of the present invention is carried out with reference to the instructions of the Universal Library Preparation Kit (trade name NICS-Inst™, Xukang Medical Technology (Suzhou) Co., Ltd.)

Example 1

[0198] This embodiment relates to a comparison between on-the-day D5 sampling method of the present invention and the conventional D3-D5 sampling method.

[0199] In brief, fertilized oocytes were cultured in vitro to D3, and then the resulted embryos were transferred to a blastocyst culture medium, and were continually cultured to D5. According to the conventional D3-D5 sampling method, the blastocyst culture mediums on D5 were taken as the samples of the “D3-D5 sampling” method. After that, according to the method of the present invention, the D5 blastocysts were transferred to a fresh blastocyst culture medium, and “on-the-day sampling” was performed on the day of D5 according to the general method described above. The CV test results of ‘on-the-day D5 sampling’ were compared with the test results of “D3-D5 sampling” from the same embryos, and compared with the test results of the whole embryos. The results from whole embryos were used as the gold standard. When a test result from a whole embryo presents abnormal chromosome copy number and the test result from culture medium presents normal chromosome copy number, the culture medium test result is judged as false negative. The false-negative rate is the proportion of false negative test results in all test results.

[0200] As shown by a large amount of experimental data, the accuracy of the detection result of the present invention is significantly improved, and the false negative rate is reduced to less than 5%. The false negative rate of the conventional D3-D5 sampling (130 sets of samples) and the false negative rate of the “on-the-day D5 sampling” of the present invention (62 sets of samples) are shown below:

TABLE-US-00001 D3-D5 sampling On-the-Day D5 sampling False negative rate 12.3% 1.3% Accuracy rate 73.1%  91%

[0201] As shown in the above table, by blastocyst culture medium exchange on D5 with a short-term culture before sampling, the false negative rate can be controlled at 1.6%. In contrast, by the D3-D5 sampling method, the maternal-derived interference cannot be effectively controlled, and the false negative rate is 12.3%, prone to cause clinical misdiagnosis.

[0202] Some of the false negative cases caused by D3-D5 are as follows: (the table shows the results of CNV chromosome karyotype analysis using different culture media)

TABLE-US-00002 Test results of D3-D5 sampling Test results of on-the-day D5 sampling 46, XX 46, XN, +9(×3, mos, 70%) 46, XX 45, XN,, −22(×1) 46, XX 46, XN, −3(×1), +13(×3) 46, XX 45, XN, −7(×1) 46, XX 46, XN,, +8(×3, mos*), +9(×3), −13(×1, mos*) 46, XX 45, XN, −6(×1, mos*), −21(×1) 46, XX 47, XN, +22(×3)

[0203] In the present invention, by blastocyst culture medium exchange on D5 with a short-term culture before sampling, the maternal-derived interference can reduced to the greatest extent, as shown in FIG. 2 and FIG. 3. FIGS. 2 and 3 show the chromosome copy number (CN) profiles of a male embryo, respectively obtained by the method of the present invention and by the conventional D3-D5 sampling method. The horizontal axis on the CN profiles is the chromosomes, which are arranged in the order of autosomes 1-22 and sex chromosomes X and Y; the ordinate axis is the copy number. For normal human embryos (diploid), the autosomal copy number should be 2. Sex chromosomes are two copies of X (female), or one copy each of X and Y (male).

[0204] In the D3-D5 sampling method, it is difficult to avoid the phenomenon of maternal-derived interference when collecting culture medium, which may cause inaccurate determination of CNV (Copy number variants). For example, the result of 46XY (male) is interpreted as 46XY/XX (male and female Mosaic), as shown in FIG. 2. In the D5 on-the-day sampling method of the present invention, the culture medium is collected on the day of D5, which can minimize maternal interference and restore the true CNV results, as shown in FIG. 3, where the CNV is interpreted as 46XY (male).

[0205] By comparative experiments, it was also found that when the blastocyst culture medium collected by the present invention is used for gender detection, whether it is an ICSI or IVF embryo has no effect on the accuracy of gender determination compared with the detection by using whole embryos, and there is no paternal interference or maternal interference to cause false negatives. Some cases are as follows:

TABLE-US-00003 Test results of on-the-day D5 Test results of corresponding Fertilization sampling whole-embryo process 46, XX 46, XX ICSI 47, XX, +8(×3) 47, XX, +8(×3) ICSI 46, XY 46, XY ICSI 47, XY, +15(×3) 46, XY ICSI 46, XY 46, XY ICSI 46, XX, +20(×3), −22(×1) 45, XX, −22(×1) ICSI 47, XY, +7(×3) 46, XY ICSI 46, XX 46, XX ICSI 48, XY, +5(×3), +14(×3) 47, XY, +14(×3) IVF 47, XX, +13(×3) 47, XX, +13(×3) IVF 46, XX 46, XX IVF 46, XY 46, XY IVF 47, XY, +22(×3) 47, XY, +22(×3) IVF 45, XY, −7(×1) 46, XY IVF 46, XX 46, XX IVF 46, XY 46, XY IVF

[0206] Therefore, the method of the present invention has very good accuracy and a very low false negative rate.

Comparative Example 1

[0207] The method is the same as in Example 1, the differences are that culture is continued to day 3 (D3) to day4 (D4), and the culture medium is changed on D4, and after 24 or 48 hours of continuous culture, the spent blastocyst culture medium is taken for detection. The test results conducted on 19 sets of samples showed that the accuracy of the test results of embryo health (such as embryo chromosomal abnormality) was 84.2% (16/19), and the false negative rate was 10.5% (2/19). After increasing the sample size, using the method of Comparative Example 1, the results show that the accuracy of this method became smaller with higher false-negative rate.

DISCUSSION

[0208] D4 embryo usually develops into embryo of morula stage, which stage is the critical period for embryo to develop into blastocyst. The stage next to morula-stage is blastocyst stage. The blastocyst forming rate is a key indicator of embryo development. After a blastocyst forms, In vitro operations such as freezing and transplantation can be considered. Thus, clinically embryos at the morula-stage of D4 are usually not subjected to in vitro operations such as medium exchange, so as not to affect the blastocyst formation rate.

[0209] Embryos are observed, graded, and frozen in the blastocyst stage. The operation at the blastocyst stage will not cause damage to the embryos. In the present invention, the blastocyst stage of D5 is selected for laser drilling and in vitro incubation, and therefore, those operations are carried out under the premise of not affecting embryo development.

[0210] All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content 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 the present application.