WO2020135713A1 - 一种利用囊胚培养液检测胚胎健康状况的方法和产品 - Google Patents

一种利用囊胚培养液检测胚胎健康状况的方法和产品 Download PDF

Info

Publication number
WO2020135713A1
WO2020135713A1 PCT/CN2019/129196 CN2019129196W WO2020135713A1 WO 2020135713 A1 WO2020135713 A1 WO 2020135713A1 CN 2019129196 W CN2019129196 W CN 2019129196W WO 2020135713 A1 WO2020135713 A1 WO 2020135713A1
Authority
WO
WIPO (PCT)
Prior art keywords
blastocyst
culture
cultured
module
embryo
Prior art date
Application number
PCT/CN2019/129196
Other languages
English (en)
French (fr)
Inventor
姚雅馨
李文璐
马杰良
陆思嘉
Original Assignee
序康医疗科技（苏州）有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 序康医疗科技（苏州）有限公司 filed Critical 序康医疗科技（苏州）有限公司
Priority to CN201980068400.7A priority Critical patent/CN113286892B/zh
Priority to EP19903766.4A priority patent/EP3904529A1/en
Priority to US17/418,934 priority patent/US20220112549A1/en
Priority to JP2021538406A priority patent/JP2022516543A/ja
Publication of WO2020135713A1 publication Critical patent/WO2020135713A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M43/00—Combinations of bioreactors or fermenters with other apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0604—Whole embryos; Culture medium therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material

Definitions

  • the present invention relates to the fields of biomedicine and molecular cell biology, and in particular, to a method and product for detecting embryo health status using blastocyst culture fluid.
  • PES Preimplantation Genetic Screen detects the chromosome status of embryos cultured in vitro to screen for embryos with normal chromosomes to be placed in the mother's womb, thereby increasing the success rate of conception to about 60%.
  • the biological samples required for various tests are one to several cells collected from embryos cultured in vitro, commonly known as biopsies. The detection of these few cells reflects whether the chromosomes of the entire embryo are normal. Theoretically, the chromosomal state of several cells taken out is the same as that of other cells in the embryo. Examining these cells can know whether the chromosomal state of the embryo is normal. It is generally believed that the absorption of several trophoblast cells at this time will not adversely affect embryonic development.
  • the specific conventional treatment method is: fresh in vitro fertilized eggs are cultured in the culture medium to D3, and the embryos are transferred to the blastocyst culture medium at D3 to continue to culture to D5 or D6, and the blastocysts are removed from D5 or D6 to observe the embryo rating. After the freezing level is reached, the freezing operation is carried out immediately, and the remaining blastocyst culture fluid is the sample to be collected during the test.
  • In vitro fertilization methods are usually selected in IVF and ICSI. IVF is accomplished by co-cultivation of multiple sperm and oocytes, and ICSI is completed by single sperm injection. IVF embryos usually have extra sperm hanging around the zona pellucida. In order to avoid parental DNA interference of sperm, ICSI fertilization is generally adopted in the above conventional processing methods.
  • this method uses D3 to replace the culture medium, and the embryos in the blastocyst culture medium Culture from D3 to D5/D6, and then collect blastocyst culture fluid as the test sample.
  • Vera-Rodriguez M., etc. Origin and composition of cell-free DNA in spent medium from human culture culture during training plant development, Hum Reprod.
  • this document also uses D3 to replace culture
  • this blastocyst culture solution embryos are cultured from D3 to D5/D6, and then blastocyst culture solution is collected as a test sample, and only for ICSI fertilization.
  • CN201510746098.X is also directly cultured to D5/D6 after changing culture medium from D3, and is only for ICSI fertilization.
  • the accuracy of embryo results is greatly improved, but there is still a false negative rate of about 15%, mainly because the test sample is inevitably subject to external interference, especially maternal interference.
  • in vitro operations such as transplantation or freezing can be considered after the blastocysts are encapsulated, so clinically, in vitro operations such as changing the culture medium of embryos during the mulberry stage of D4 are not usually performed, so as not to affect the cyst formation rate.
  • the current detection method is inevitably subject to external interference, especially maternal interference, resulting in a false negative rate that is too high to target both ICSI and IVF samples.
  • the purpose of the present invention is to provide a method and a product suitable for ICSI and IVF that can improve the accuracy and reduce the false negative rate by using the blastocyst culture fluid as the detection sample, thereby detecting the health status of the embryo.
  • the first aspect of the present invention provides a method of using blastocyst culture fluid to detect the blastocyst health status in vitro, which includes the steps of:
  • the first blastocyst culture system includes a de novo cultured blastocyst culture system (that is, culture is started from D1 without changing the medium).
  • the first blastocyst culture system includes the last blastocyst culture system after one or more fluid exchanges (for example, the fluid exchange culture is performed on D2-D3 or D3-D4, for example At D3).
  • "cultivation days are D5-D6" includes D5 ⁇ 12 hours or D6 ⁇ 12 hours, preferably D5 ⁇ 6 hours, more preferably D5 to D5+6 hours.
  • the number of culture days is D5, that is, the 5th day after the start of the culture, and the start of the culture is set to the first day (D1).
  • step (a) the blastocyst culture in vitro is de novo culture (that is, culture is started from D1, and the medium is not changed).
  • step (a) the blastocyst culture in vitro is staged culture.
  • the blastocyst in vitro culture includes first-stage blastocyst culture and second-stage blastocyst culture.
  • the first stage blastocyst culture includes D1 to D3, preferably, D1 to D3 ⁇ 12h, more preferably, D1 to D3 ⁇ 8h, more preferably, D1 to D3 ⁇ 6h , Cultivate with the culture medium of the cleavage stage.
  • the second-stage blastocyst culture includes D3 to D5, and is cultured with blastocyst culture fluid.
  • the T1 time is 2-8h, such as 3-6h, preferably 3-5h.
  • the zona pellucida is perforated in the second blastocyst culture system.
  • the hole diameter of the transparent tape is 10-40 ⁇ m, preferably 10-30 ⁇ m, more preferably 10-20 ⁇ m.
  • the method has one or more of the following characteristics:
  • the signal-to-noise ratio is high, the signal-to-noise ratio S 1 /S 0 >2, preferably, >5, more preferably, >10, where S 1 is the signal from the embryo and S 0 is the signal from the background;
  • the first blastocyst culture system is a single-sperm fertilization culture system (or ICSI culture system).
  • the first blastocyst culture system is a multi-sperm fertilization culture system (or IVF culture system).
  • the blastocyst cultured in vitro is a single-sperm fertilized blastocyst (or ICSI blastocyst).
  • the blastocyst cultured in vitro is a polysperm fertilized blastocyst (or IVF blastocyst).
  • the second blastocyst culture system contains only one blastocyst (embryo).
  • the second blastocyst culture system is a single embryo culture system, and the single embryo culture system contains 10-60 microliters, preferably, 10-50 microliters, or 10- 30 microliters, more preferably, 10-15 microliters of culture medium.
  • the volume of the culture medium taken out is 50-100% of the volume of the culture medium in the single embryo culture system, preferably, 70-100%, more preferably , 80-100%, optimally, 90-100%.
  • step (d) further includes step (e):
  • the health status of the embryo includes: chromosome aneuploidy, mitochondrial copy number, whether the DNA content is normal, and detection of pathogenic genes.
  • the genome analysis method includes NICS-INST amplification method and analysis method.
  • NICS-INST amplification method of NICS-INST
  • amplification method of NICS-INST please refer to the Gene Sequencing Generic Library Kit (trade name NICS-Inst TM , Generic Name: Gene Sequencing Generic Library Kit; English name: Universal Library Preparation Kit) ).
  • Other genome analysis methods known in the art, including amplification methods and analysis methods, are also applicable to the present invention.
  • CN103890191B discloses using MALBAC genome amplification method and the like.
  • the method of genomic analysis is selected from the group consisting of second generation sequencing, nucleic acid chip, immunofluorescence detection, fluorescent PCR detection, first generation sequencing, third generation sequencing, mass spectrometry detection, or a combination thereof.
  • the lysate contains an ingredient selected from the group consisting of Tris buffer, chelating agent, hydrochloride, nonionic surfactant, or a combination thereof.
  • the Tris buffer includes Tris-Cl.
  • the concentration of the Tris buffer is 10-60 mM, preferably 15-50 mM, more preferably 20-45 mM, such as 30 mM.
  • the pH of the Tris buffer is 5-10, preferably, 6-9, and more preferably, 7-8.
  • the chelating agent includes EDTA.
  • the concentration of the chelating agent is 0.2-8 mM, preferably, 0.3-6 mM, more preferably, 0.5-4 mM, such as 2 mM.
  • the hydrochloride salt is selected from the group consisting of KCl, NaCl, or a combination thereof.
  • the concentration of the hydrochloride salt is 5-60 mM, preferably, 8-40 nM, more preferably, 10-40 mM, such as 20 mM.
  • the nonionic surfactant is selected from the group consisting of Triton X-100, Triton X-114, Tween 20, NP40, SDS, or a combination thereof.
  • the concentration of the nonionic surfactant is 0.02-10%, preferably, 0.05-5%, more preferably, 0.1-3%, for example, 0.2%, with the cleavage The total weight of the liquid.
  • step (e) (i) the volume ratio of the culture solution to the lysate is 1:10-10:1, preferably, 1:5-5:1, more preferably Ground, 1:2-2:1.
  • the lyase is selected from the group consisting of proteinase K, Qiagen Protease, pepsin, papain, trypsin, lysozyme, or a combination thereof.
  • the concentration of the lyase is 1-25 ⁇ g/ml, preferably, 5-20 ⁇ g/ml, and more preferably, 10-15 ⁇ g/ml.
  • the added amount of the lyase is 0.1-10 ⁇ l, preferably, 0.5-6 ⁇ l, and more preferably, 0.8-3 ⁇ l.
  • step (e)(ii) includes one or more features selected from the group consisting of:
  • the incubation temperature is 20-70°C, preferably, 30-60°C;
  • the incubation time is 1min-12h, preferably, 10min-6h, more preferably, 30min-2h;
  • the deactivation temperature is 60-100°C, preferably 75-95°C;
  • the inactivation time is 0.5-20 min, preferably 0.8-15 min.
  • genomic analysis is performed by PCR, preferably the PCR reaction tube contains an amplification mixture, 0.5%-20% PCR inhibitor antagonist, 5-20mM dNTP, 5-100 ⁇ M NG and NT primers, 50-200 ⁇ M amplification primers, 0.5-10 unit nucleic acid polymerase, preferably, the PCR inhibitor antagonist is selected from DMSO, betaine, formamide, glycerol and albumin
  • the nucleic acid polymerases are selected from Phi29 DNA polymerase, Bst DNA polymerase, Vent polymerase, Deep Vent polymerase, Klenow Fragment DNA polymerase I, MMLV reverse transcriptase, AMV reverse transcriptase , HIV reverse transcriptase, ultra-fidelity DNA polymerase, Taq polymerase, E. coli DNA polymerase, LongAmp Taq DNA polymerase and OneTaq DNA polymerase one or more.
  • the components of the amplification mixture are 10-25mM Tris-HCl, 5-25mM (NH4) 2SO4, 5-30mM KCl, 0.5-5mM MgSO4, 0.1%-20% DMSO and 0.05- 5% Triton X-100.
  • the components of the amplification mixture are preferably 15 mM Tris-HCl, 15 mM (NH4) 2SO4, 20 mM KCl, 1 mM MgSO4, 5% DMSO, and 2% Triton X-100.
  • the NG and NT primers contain a universal sequence and a variable sequence from the 5'end to the 3'end, wherein the universal sequence is composed of three of the four bases of G, A, C and T Or two components, provided that the universal sequence does not include G and C at the same time; the amplification primer contains the universal sequence and does not contain the variable sequence.
  • variable sequence is selected from the group consisting of: (N)nGGG, (N)nTTT, (N)mTNTNG, (N)xGTGG(N)y, where N is any natural nucleic acid
  • N is any natural nucleic acid
  • n is a positive integer selected from 3-17
  • m is a positive integer selected from 3-15
  • x and y are positive integers selected from 3-13, respectively.
  • step (e)(iii) the thermal cycling procedure of whole genome amplification when performing genome analysis by PCR is as follows:
  • step (e)(iii) the thermal cycling procedure of whole genome amplification when performing genome analysis by PCR is as follows:
  • the annealing temperature is 50 °C for 45s;
  • the method is non-therapeutic and non-diagnostic.
  • the second aspect of the present invention provides a method for preparing a genetic test sample or a chromosome test sample, including the steps of:
  • the cultured blastocyst culture solution is taken out to obtain a cell-free cultured blastocyst culture solution, which is a test sample.
  • the second blastocyst culture system contains only one blastocyst (embryo).
  • the second blastocyst culture system is a single embryo culture system, containing 10-60 microliters, preferably, 10-50 microliters, more preferably, 10-15 microliters of culture medium .
  • the volume of the detection sample is 50-100% of the volume of the culture solution in the second blastocyst culture system, preferably, 70-100%, more preferably , 80-100%, optimally, 90-100%.
  • a third aspect of the present invention provides a kit for detecting blastocyst health using blastocyst culture fluid, including:
  • the first container, the second container, and the third container are different.
  • the cleavage stage culture fluid in the first container is used for D1 to D3, preferably, D3 ⁇ 12h, more preferably, D3 ⁇ 8h, more preferably, D3 ⁇ 6h, Cleavage embryo culture.
  • the first blastocyst culture solution in the second container is used for blastocyst culture of D3-D5.
  • the second blastocyst culture solution in the third container is used for blastocyst culture after D5-D6.
  • the components of the first blastocyst culture fluid and the second blastocyst culture fluid are the same, both of which are G-2 PLUS medium (Vitrolife) or Quinn's Advantage Protein Plus blastocyst medium (SAGE) blastocyst culture fluid .
  • the cleavage stage culture fluid is G-1 PLUS medium (Vitrolife) or Quinn’s Embryo maintenance medium (SAGE) cleavage stage culture fluid.
  • a fourth aspect of the present invention provides a device for assisting in diagnosing the health status of blastocysts, including:
  • a culture module the culture module includes a blastocyst late culture module, the blastocyst late culture module is used to change the medium of D5-D6 cultured blastocysts, and to perform blastocyst late culture for a period of time (T1 );
  • sampling module is used to take out the culture fluid of the blastocyst cultured in the later stage of the blastocyst culture module, and used as a test sample;
  • the detection module is used for genetic testing of the detection sample from the sampling module to obtain the detection result
  • the judgment processing module of the health status of the blastocyst evaluates the health status of the embryo to obtain an evaluation result of the health status of the blastocyst
  • the T1 time is 2-8h, such as 3-6h, preferably 3-5h.
  • FIG. 1 shows a schematic diagram of a conventional sampling method different from the sampling method of the present invention in detecting the exposure time of a sample.
  • Figure 2 shows the results of CNV analysis of the culture fluid collected using the conventional D3-D5 sampling method.
  • Fig. 3 shows the results of CNV analysis of the culture fluid collected on the same day using the D5 sampling method of the present invention.
  • the inventors unexpectedly discovered for the first time that in the 10-20 microliter blastocyst culture system of the present invention, the embryo (blastocyst) is placed in the blastocyst culture solution After culturing to D5-D6 days, the embryos were transferred to fresh blastocyst culture medium for cultivation, and a small amount of culture medium was removed from the fresh blastocyst culture medium for testing.
  • the health status of the obtained embryos (such as (Chromosome aneuploidy, mitochondrial copy number, whether the DNA content is normal, pathogenic gene detection)
  • the detection result has extremely high accuracy, and the false negative rate is extremely low ( ⁇ 8%, preferably, ⁇ 5%) , Can greatly eliminate the risk of contamination and interference of excess sperm and maternal granulosa cells.
  • the inventor has completed the present invention.
  • blastocyst and “embryo” are used interchangeably and refer to the final stage of embryo culture in vitro, which usually forms on the 5th-7th day after egg fertilization.
  • depleted medium refers to a culture medium that has been used, for example, a culture medium separated from a culture system in which embryos have been cultured.
  • de novo culture refers to an embryo culture process in which the culture medium is not replaced from the beginning of the culture process to the end of the culture process.
  • the de novo culture of D1-D5 means that the embryo culture process starts from D1 and continues to D5, and the culture medium is not replaced during this period.
  • staged culture refers to an embryo culture process in which embryos undergo one or more fluid exchanges.
  • D1-D5 culture in stages means that the embryo culture process starts from D1 and continues to D5, but during the embryo undergoes one or more fluid changes.
  • D1-D3 embryos are cultured in the culture medium of the cleavage stage; while in D3-D5, embryos are cultured in blastocyst culture medium.
  • D3-D5 sampling means that one culture broth is used to culture embryos on days D3 to D5, and a sample is collected from the used culture broth (ie, spent culture broth) at D5. Therefore, the culture fluid sample obtained in this sampling manner will have a contact time with the embryo culture (ie, the exposure time of the test sample) of at least about 48 hours or more.
  • “same day” sampling means that, on the day when the culture medium is changed, after a short period of embryo culture, a sample is collected from the used culture medium (ie, the spent culture medium). Therefore, the culture fluid sample obtained in this sampling manner will have a short contact time with the embryo culture (ie, the exposure time of the test sample). In the present invention, it is preferred that the exposure time does not exceed 8 hours, such as 3-6 hours, and more preferably 3-5 hours.
  • lysate refers to a lysis buffer used to decompose proteins and cells in the culture fluid sample.
  • the lysate may or may not contain lyase.
  • the lysate and lyase may be added to the culture fluid sample at the same time. In other embodiments, the lyase is added after the lysate is mixed with the culture fluid sample.
  • test sample in vitro incubation time means that the culture medium taken from the sample has already contacted the embryo in vitro (ie, incubated with the embryo ) Length of time.
  • CNV refers to chromosome copy number variation (Copy number variants). CNV naming adheres to the International Human Cell Genome Naming System (ISCN). See, Shaffer LG, Slovak ML, Campbell LJ (2009): ISCN 2009 international system for human cytogenetic nomenclature, Human Genetics volume 126, Article number: 603 (2009).
  • IVF in vitro fertilization
  • in vitro fertilization in vitro fertilization, in vitro fertilization
  • IVF embryo transfer technology
  • IVF embryo transfer technology
  • Multi-sperm fertilized blastocyst or “IVF blastocyst” refers to a blastocyst obtained by using IVF technology to complete fertilization by co-cultivating multiple sperm and oocytes in vitro.
  • ICSI Intracytoplasmic Sperm Injection
  • Single sperm fertilized blastocyst or “ICSI blastocyst” refers to a blastocyst obtained by using ICSI in the cytoplasm of sperm in the egg.
  • the present invention provides a detection method for genetic testing of depleted medium (that is, culture fluid separated from a culture system where blastocysts have been cultured) after blastocyst culture, thereby identifying The health of the embryo.
  • depleted medium that is, culture fluid separated from a culture system where blastocysts have been cultured
  • the method of performing genetic detection on the "deficient" culture medium after 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, One-generation sequencing, three-generation sequencing, mass spectrometry, or a combination thereof.
  • the detection method includes the following steps:
  • step (d) further includes step (e):
  • the main steps of the method for using the blastocyst culture medium as a test sample to reflect the health status of the embryo include:
  • Oocytes are fertilized by IVF or ICSI. After fertilization, the fertilized eggs are transferred to the culture medium to the D3 cleavage stage, and the embryos are transferred to the blastocyst culture medium at D3 and cultured to D5-D6;
  • step (c) Transfer the original blastocyst culture solution obtained in step (b) to the lysate. After centrifugation, the sample enters the next step of the whole genome amplification step, including but not limited to the NICS-INST amplification method and analysis method To get the test result reflecting the health of the embryo.
  • the invention replaces the culture medium on D5 ⁇ 0.5 days, and after a short period of 3-5 hours of culture, the blastocyst culture medium is obtained as the test sample, the accuracy of the test result is significantly improved, the false negative rate is reduced to less than 5%, and the external source is avoided to the greatest extent. Interference is especially from maternal interference, and both ICSI and IVF fertilization methods are applicable.
  • the principle that the accuracy of the test result is significantly improved is that: first, the exposure time of the test sample is extremely short, and the exposure time of 2-3 days is greatly shortened compared to the conventional operation mode (as shown in FIG. 1), and the maximum avoidance External interference, especially maternal interference, is not restricted by the method of fertilization, which greatly improves the accuracy of the test results.
  • the abnormal fragments will be released into the medium during the cultivation in the conventional manner, which may have an impact on the accuracy; and the sampling of the D5 of the present invention after 3-5 hours of culture replacement makes the embryo in the replacement
  • the culture time in the culture medium is short, and the embryo has completed self-repair in the D3-D5 culture stage before D5 is changed to the culture medium.
  • the abnormal fragments are mainly released in the D3-D5 stage, so the abnormal fragments are released in the detection samples collected by the present invention. The probability is low, so the accuracy of the detection results is improved.
  • no one tried to collect culture fluid of IVF embryos but generally collected culture fluid of embryos produced by ICSI fertilization.
  • the most important factor for exogenous interference is that the detection sample is incubated for too long in vitro.
  • the present invention adopts a short-term incubation medium collection method to avoid external interference such as sperm and granule cells due to too long incubation time. These interferences will cause embryonic The decrease in the specific gravity of DNA affects the accuracy of the results.
  • the amount of the test sample in step (c) is 8-15ul, preferably 10ul.
  • the amount of the new blastocyst culture fluid ie, the second blastocyst culture fluid
  • the blastocyst rating includes selecting embryos that have developed to stages 4 or above as well-developed embryos.
  • the punching operation in step (b) increases the release of blastocyst cavity fluid, that is, the release of free nucleic acids, thereby satisfying the requirement that the initial amount of nucleic acid substance in the test sample is increased under the premise of 3-5 hours of short-term incubation, which is sufficient Subsequent amplification and detection ensure that the success rate of the detection can reach more than 97%.
  • embryo observation, grading and cryopreservation are carried out in the blastocyst stage. It is believed that the operation in the blastocyst stage will not damage the embryo.
  • the present invention also chooses to perform laser drilling and in vitro cultivation in the D5 blastocyst stage, so it does not affect Under the premise of embryonic development.
  • step (b) may further include a step of washing embryos before D5 is changed to a culture medium. This step is helpful to improve the accuracy of the detection result.
  • step (a) after the blastocyst culture solution is replaced by D3, multiple embryos can be mixed and cultured, or single embryo culture can be performed.
  • the invention also provides a method for preparing a gene detection sample or a chromosome detection sample, which includes the steps of:
  • the cultured blastocyst culture solution is taken out to obtain a cell-free cultured blastocyst culture solution, which is a test sample.
  • test sample obtained by the method can be used for genetic testing to identify the health status of the embryo.
  • a preferred aperture diameter of the transparent belt is 10-40 ⁇ m, preferably, 10-30 ⁇ m, and more preferably, 10-20 ⁇ m.
  • the punching operation of the transparent zone increases the release of blastocyst cavity fluid, that is, the release of free nucleic acid, which satisfies the premise that the initial amount of nucleic acid substance in the test sample increases under the premise of 3-5 hours of short-term incubation , Enough for subsequent amplification and detection, the success rate of detection can reach more than 97%.
  • the success rate of detection is also very high, about 70%.
  • kits for detecting embryo health status using blastocyst culture fluid including:
  • a device for assisting diagnosis of the health status of an embryo including:
  • a culture module includes a blastocyst late culture module, and the blastocyst late culture module is used to change the medium of D5-D6 cultured blastocysts and perform blastocyst late culture for T1 time;
  • sampling module is used to take out the culture solution of the blastocyst after culturing T1 time in the blastocyst late culture module, and use it as a test sample;
  • the detection module is used for genetic testing of the detection sample from the sampling module to obtain the detection result
  • a discrimination processing module for the health status of the embryo evaluates the health status of the embryo based on the detection result, thereby obtaining an evaluation result of the health status of the embryo;
  • the invention also relates to the use of the module (a), optionally in combination with modules (b)-(e), in the preparation of equipment for the method for evaluating the blastocyst health of the invention.
  • the present invention can adopt a single embryo culture system, that is, only one embryo is cultured in one culture solution, and the detection result of the health status of the embryos obtained by the system is more accurate.
  • the method of the present invention is a general method, which can be used for single-sperm fertilized embryos, and can also be used for multi-sperm fertilized embryos, preferably multi-sperm fertilized embryos.
  • the transparent tape can be perforated, and the results obtained will be better.
  • the method of the present invention has a very high signal-to-noise ratio.
  • the method of the present invention collects the embryo culture fluid after the embryo is cultured to D5, the operation time is extremely short, the test sample culture time is controllable, and the possibility of external interference is the lowest, in addition, the sample collection is not limited to fertilization
  • the method is a medium collection method that is suitable for IVF and ICSI embryos.
  • the D5 medium replacement short-term culture program of the present invention has the advantage of controlling the incubation time of the test sample in vitro, thereby maximizing the control of exogenous interference such as maternal interference and sperm interference, making the application of the present invention not Limited to ICSI embryos, it can also be applied to IVF embryos.
  • Fertilized eggs in volunteer couples, eggs are obtained from female volunteers, sperm are obtained from male volunteers, and IVF or ICSI is used for in vitro fertilization, that is, fertilized eggs) are cultured in vitro until the third day, Cultivate to 5th/6th day with or without fluid exchange;
  • step (3) Transfer the culture medium of short-term cultured blastocysts (volume is about 10-15 ⁇ l) obtained in step (2) to 5 ⁇ l of lysate (pH 7.8 30 mM Tris-Cl, 2 mM EDTA, 20 mM KCl , 0.2% Triton X-100), mark the sample name on the collection tube with a marker. Centrifuge for 30 seconds in a microcentrifuge. Samples can be immediately entered into the next step of whole genome amplification or stored frozen at -20°C or -80°C.
  • the detection method of the present invention refers to the instruction manual of Gene Sequencing Generic Library Kit (trade name NICS-Inst TM , Generic Name: Gene Sequencing Generic Library Kit; English name: Universal Library Preparation Kit) of Sukang Medical Technology (Suzhou) Co., Ltd. get on.
  • This embodiment relates to a comparison between the D5 sampling method of the present invention and the conventional D3-D5 sampling method.
  • the fertilized egg is cultured to D3 in vitro, and the resulting embryo is transferred to the blastocyst culture medium, and cultured to D5.
  • the blastocyst culture fluid of D5 is taken as the "D3-D5 sampling” sample.
  • D5 blastocysts are transferred to a new blastocyst culture medium, and on the day of D5, according to the description of the previous general method, the day of D5 is sampled. Compare the CV test result of "D3-D5 Sampling" from the same embryo with the test result of "D5 Sampling on the same day” and the test result of the whole embryo.
  • the test result of the whole embryo is abnormal chromosome copy number, and the test result of the culture medium is normal, the test result of the culture medium is judged as a false negative, and the probability of false negatives as a whole is the false negative rate.
  • the false negative rate of D5 replacement blastocyst culture medium after short-term culture sampling can be controlled at 1.6%; while in the D3-D5 sampling method, the maternal interference cannot be effectively controlled, and the false negative rate is 12.3%, easy to cause clinical misdiagnosis.
  • FIG. Figures 2 and 3 show the chromosome copy number (CN) map of a male embryo using the method of the present invention and the conventional D3-D5 sampling method.
  • the horizontal axis of the CN diagram is the chromosome, which is arranged in the order of autosomes 1-22, sex chromosomes X, Y; the ordinate axis is the copy number.
  • the autosomal copy number should be 2
  • the sex chromosomes are two copies of X (female), or one copy of each of X and Y (male).
  • the culture fluid collected on the day of D5 can minimize the interference of maternal sources and restore the true CNV results, as shown in FIG. 3, where the interpretation of CNV is 46XY (male).
  • the method of the present invention has very good accuracy and a very low false negative rate.
  • the method is the same as that in Example 1.
  • the difference is that the culture is changed to D3 to D4, the medium is changed at D4, and after 24 or 48 hours of continuous culture, the blastocyst culture medium is taken for detection.
  • the test results on 19 groups of samples show that the accuracy of the test results of embryo health (such as embryo chromosome abnormality) is 84.2 (16/19)%, and the false negative rate is 10.5 (2/19)%.
  • the method of Comparative Example 1 was used, and the results showed that the accuracy of the method was smaller and the false negative rate was higher.
  • D4 embryos usually develop into mulberry stage embryos, which is the key period for embryos to develop into blastocysts. The next stage of morula embryos is the blastocyst stage.
  • the rate of cyst formation is a key indicator of embryo development.
  • In vitro operations such as freezing are performed, so clinically, in vitro operations such as fluid exchange of embryos during the mulberry stage of D4 are generally not performed, so as not to affect the cyst formation rate.
  • the present invention chooses to perform laser drilling and in vitro culture at the D5 blastocyst stage, so it is carried out on the premise of not affecting embryo development.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Gynecology & Obstetrics (AREA)
  • Reproductive Health (AREA)
  • Developmental Biology & Embryology (AREA)
  • Cell Biology (AREA)
  • Sustainable Development (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种利用囊胚培养液检测胚胎健康状况的方法和产品。具体地，提供了一种体外利用囊胚培养液检测胚胎健康状况的方法，其特征在于，包括步骤：(a)提供第一囊胚培养体系，所述第一囊胚培养体系含有体外培养天数为D5-D6的囊胚；(b)将所述囊胚转移到含新鲜囊胚培养液的第二囊胚培养体系中进行换液培养，所述换液培养的时间为T1，从而获得经培养的囊胚培养液；(c)将所述经培养的囊胚培养液取出，从而获得无细胞的经培养的囊胚培养液，即为检测样本；和(d)对所述检测样本进行基因检测，从而鉴定所述囊胚的健康状况。该方法可去除IVF和ICSI技术过程中多余精子和母源颗粒细胞污染干扰的风险，并可精确的鉴定胚胎的健康状况。

Description

一种利用囊胚培养液检测胚胎健康状况的方法和产品 技术领域
本发明涉及生物医学和分子细胞生物学领域，具体地，涉及一种利用囊胚培养液检测胚胎健康状况的方法和产品。
背景技术
胚胎植入前遗传学筛查(Preimplantation Genetic Screen，PGS)对体外培养胚胎的染色体状态进行检测，以筛选染色体正常的胚胎置入母亲子宫,从而将受孕成功率提高到60％左右。各种检测所需的生物样本都是从体外培养的胚胎中采集的一个至数个细胞、俗称活检，通过对这少量细胞的检测反映整个胚胎的染色体是否正常。理论上讲，吸取出来的几个细胞中的染色体状态与胚胎中其它细胞一致，检测这几个细胞即可了解该胚胎的染色体状态是否正常。一般认为，此时吸取几个滋养层细胞不会对胚胎发育造成不良影响，目前从已出生婴儿的健康状况来看，这一操作也确实没有健康影响。但是，细胞取样时对胚胎操作技术要求较高，如果稍操作不当会导致胚胎严重损伤，损伤过于严重可造成胚胎发育终止；即使良好操作，细胞取样时不可避免地对胚胎造成细胞损失和轻微损伤，尽管现在尚无证据表明细胞损失和轻微损伤会对胚胎发育和出生后健康发生不良影响，但该技术出现的时间尚短(仅数年)，其对人的终生健康是否存在远期影响仍然有待观察；在少数情况下，存在取样获得的几个细胞的染色体状态与胚胎中其它细胞的染色体状态不同的情况，导致检测结果失误。
近期，一种非活检的、不取胚胎细胞，仅利用废弃的囊胚培养液来反映胚胎健康状况的方法得到研究，将原本废弃的囊胚培养液处理后进行检测分析，该检测分析结果可以间接的、且高精确性的反映出囊胚胚胎的健康状态，而且不取任何胚胎本身组分、不存在伦理问题，不会对胚胎带来任何损失、消除健康影响隐患，且操作简单，安全性高，反映胚胎结果的准确性更高。
具体的常规的处理方式为：新鲜的体外受精卵在培养基中培养至D3，在D3将胚胎转移至囊胚培养液中继续培养至D5或D6，于D5或D6取走囊胚观察胚胎评级达到可冷冻级别后立刻实施冷冻操作，剩余的囊胚培养液即检测时所需要收集的样本。临床上选择体外受精的方式通常为IVF和ICSI两种，IVF通过多个精子和卵母细胞共培养完成受精，ICSI为单精子注射完成受精。IVF胚胎的透明带周围通常挂有多余精子，为避免精子的父源性DNA干扰，在上述的 常规处理方式中，一般采用ICSI受精方式。
如Galluzzi L,等(2015),Extracellular embryo genomic DNA and its potential for genotyping applications,Future Science OA 1(4):FSO62描述的方法，该方法利用D3更换培养液，在该囊胚培养液中将胚胎从D3培养至D5/D6，然后采集囊胚培养液作为检测样本。如Vera-Rodriguez M.等，Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development,Hum Reprod.2018 Apr 1；33(4):745-756，该文献也是利用D3更换培养液，在该囊胚培养液中将胚胎从D3培养至D5/D6，然后采集囊胚培养液作为检测样本，并且只针对ICSI受精方式。CN201510746098.X也是D3换培养液后直接培养至D5/D6，并且只针对ICSI受精方式。该常规处理方式与滋养外胚层活检检测方式相比，胚胎结果的准确性大大提高，但是依然有约15％的假阴性率，主要是因为检测样本不可避免的受外源干扰，特别是母源干扰。在一些文献中可以看到稍区别于常规处理方式的方法，如Medium-Based Noninvasive Preimplantation Genetic Diagnosis for Human-Thalassemias-SEA(Medicine(Baltimore).2015 Mar；94(12):e669.)，在D3更换培养基后，在D4再次更换培养基培养至D5/D6、采集该囊胚培养液作为检测样本。如Kuznyetsov V.,Evaluation of a novel non-invasive preimplantation genetic screening approach，PLoS One.2018 May 10；13(5):e0197262，其中一种方法利用新鲜胚胎，也在D4更换培养基培养至D5，收集激光皱缩后的囊胚腔液，该方法只能针对ICSI样本，无法针对IVF样本。这些文献中将体外孵育时间提前到D4，于D5/D6采集，一方面检测样本体外孵育时间为12-36小时，孵育时间过长，依然不可避免的受外源干扰，使得胚胎来源的DNA比重降低，胚胎结果的准确性没有明显提高，依然约15％的假阴性率，且无法同时针对ICSI样本和IVF样本；另一方面在D4对胚胎进行观察或者操作，增加了额外的操作步骤，对胚胎发育会造成不可预知的影响，D4胚胎通常发育为桑葚期胚胎，是胚胎发育成囊胚的关键时期，桑椹胚的下一个时期即囊胚期，而成囊率是胚胎发育的关键指标，囊胚成囊后方可考虑移植或者冷冻等体外操作，因此临床上通常不会在D4的桑葚期对胚胎进行换培养液等体外操作，以免影响成囊率。
然而，目前的检测方法，由于检测样本不可避免的受外源干扰，特别是母源干扰，从而导致假阴性率太高，且无法同时针对ICSI样本和IVF样本。
因此，本领域迫切需要开发一种能够提高准确性、降低假阴性率的利用囊胚培养液作为检测样本，从而检测胚胎健康状况的适用于ICSI和IVF的方法和产品。
发明内容
本发明的目的是提供一种能够提高准确性、降低假阴性率的利用囊胚培养液作为检测样本，从而检测胚胎健康状况的适用于ICSI和IVF的方法和产品。
本发明的第一方面提供了一种体外利用囊胚培养液检测囊胚健康状况的方法，包括步骤：
(a)提供第一囊胚培养体系，所述第一囊胚培养体系含有体外培养天数为D5-D6的囊胚；
(b)将所述囊胚转移到含新鲜囊胚培养液的第二囊胚培养体系中进行换液培养，所述换液培养的时间为T1，从而获得经培养的囊胚培养液；
(c)将所述经培养的囊胚培养液取出，从而获得无细胞的经培养的囊胚培养液，即为检测样本；和
(d)对所述检测样本进行基因检测，从而鉴定所述囊胚的健康状况。
在另一优选例中，所述第一囊胚培养体系包括从头培养的囊胚培养体系(即从D1开始培养，并且未经换液)。
在另一优选例中，所述第一囊胚培养体系包括经历一次或多次换液后的末次囊胚培养体系(例如，所述的换液培养在D2－D3或D3-D4进行，例如在D3进行)。
在另一优选例中，“培养天数为D5-D6”包括D5±12小时或D6±12小时，较佳地D5±6小时，更佳地D5至D5+6小时。
在另一优选例中，所述的培养天数为D5，即从培养开始后的第5天，其中，培养开始时设为第1天(D1)。
在另一优选例中，在步骤(a)中，所述囊胚体外培养为从头培养(即从D1开始培养，并且未经换液)。
在另一优选例中，在步骤(a)中，所述囊胚体外培养为分阶段培养。
在另一优选例中，在步骤(a)中，所述囊胚体外培养包括第一期囊胚培养和第二期囊胚培养。
在另一优选例中，所述第一期囊胚培养包括在D1到D3，较佳地，D1到D3±12h，更佳地，D1到D3±8h，更佳地，D1到D3±6h，用卵裂期培养液进行培养。
在另一优选例中，所述第二期囊胚培养包括在D3到D5，用囊胚培养液进行培养。
在另一优选例中，所述T1时间为2-8h，例如3-6h，较佳地，3-5h。
在另一优选例中，在所述第二囊胚培养体系中对囊胚的透明带进行打孔。
在另一优选例中，所述透明带打孔的孔径为10－40μm，较佳地，10－30μm，更佳地，10－20μm。
在另一优选例中，所述方法具有以下一种或多种特征：
(i)信噪比高，信噪比S 1/S 0＞2，较佳地，＞5，更佳地，＞10，其中S 1为来自胚胎的信号，S 0为来自背景的信号；
(ii)低假阴性率，假阴性率＜8％，较佳地，＜5％，更佳地，＜3％；
(iii)高准确性，准确率≥80％，较佳地，85％≥，更佳地，≥90％。
在另一优选例中，所述第一囊胚培养体系为单精受精的培养体系(或ICSI培养体系)。
在另一优选例中，所述第一囊胚培养体系为多精受精的培养体系(或IVF培养体系)。
在另一优选例中，步骤(a)中，所述体外培养的囊胚为单精受精的囊胚(或ICSI囊胚)。
在另一优选例中，步骤(a)中，所述体外培养的囊胚为多精受精的囊胚(或IVF囊胚)。
在另一优选例中，所述第二囊胚培养体系中仅含有一个囊胚(胚胎)。
在另一优选例中，所述第二囊胚培养体系为单胚胎培养体系，并且在所述单胚胎培养体系中含有10-60微升，较佳地，10-50微升，或10-30微升，更佳地，10－15微升的培养液。
在另一优选例中，所述步骤(c)中，取出的培养液的体积为所述单胚胎培养体系中培养液体积的50-100％，较佳地，70-100％，更佳地，80-100％，最佳地，90-100％。
在另一优选例中，所述步骤(d)中还包括步骤(e)：
(i)将所述培养液与裂解液混合，从而获得含有所述培养液与裂解液的第一混合物；
(ii)将所述第一混合物与裂解酶混合，孵育，之后失活裂解酶，从而获得裂解产物；和
(iii)对所述裂解产物进行基因组分析，从而鉴定所述囊胚(胚胎)的健康状况。
在另一优选例中，所述胚胎的健康状况包括：染色体非整倍体、线粒体拷贝数、DNA含量是否正常、致病基因检测。
在另一优选例中，所述基因组分析方法包括NICS-INST的扩增方法以及分析方法。NICS-INST的扩增方法可以参照序康医疗科技(苏州)有限公司的基因测序通用文库试剂盒(商品名NICS-Inst TM，通用名：基因测序通用文库试剂盒；英文名：Universal Library Preparation Kit)的说明书进行。本领域已知的其它基因组分析方法，包括扩增方法和分析方法，也均适用于本发明中。例如，CN103890191B公开了利用MALBAC基因组扩增方法等。
在另一优选例中，所述基因组分析的方法选自下组：二代测序、核酸芯片、免疫荧光检测、荧光PCR检测、一代测序、三代测序、质谱检测、或其组合。
在另一优选例中，所述裂解液包含选自下组的成分：Tris缓冲液、螯合剂、盐酸盐、非离子型表面活性剂、或其组合。
在另一优选例中，所述Tris缓冲液包括Tris-Cl。
在另一优选例中，所述Tris缓冲液的浓度为10-60mM，较佳地，15-50mM，更佳地，20-45mM，例如30mM。
在另一优选例中，所述Tris缓冲液的pH为5-10，较佳地，6-9，更佳地，7-8。
在另一优选例中，所述螯合剂包括EDTA。
在另一优选例中，所述螯合剂的浓度为0.2-8mM，较佳地，0.3-6mM，更佳地，0.5-4mM,例如2mM。
在另一优选例中，所述盐酸盐选自下组：KCl、NaCl、或其组合。
在另一优选例中，所述盐酸盐的浓度为5-60mM，较佳地，8-40nM，更佳地，10-40mM,例如20mM。
在另一优选例中，所述非离子型表面活性剂选自下组：Triton X-100、Triton X-114、吐温20、NP40、SDS、或其组合。
在另一优选例中，所述非离子型表面活性剂的浓度为0.02-10％，较佳地，0.05-5％，更佳地，0.1-3％，例如，0.2％，以所述裂解液的总重计。
在另一优选例中，所述步骤(e)的(i)中，培养液与裂解液的体积比为1：10－10：1，较佳地，1：5－5：1，更佳地，1：2—2：1。
在另一优选例中，所述裂解酶选自下组：蛋白酶K、Qiagen Protease、胃 蛋白酶、木瓜蛋白酶、胰蛋白酶、溶菌酶、或其组合。
在另一优选例中，所述裂解酶的浓度为1-25μg/ml，较佳地，5-20μg/ml，更佳地，10-15μg/ml。
在另一优选例中，所述裂解酶的添加量为0.1-10μl，较佳地，0.5-6μl，更佳地，0.8-3μl。
在另一优选例中，所述步骤(e)(ii)包括选自下组的一个或多个特征：
(i)孵育温度为20-70℃，较佳地，30-60℃；
(ii)孵育时间为1min－12h，较佳地，10min－6h，更佳地，30min－2h；
(iii)失活温度为60-100℃，较佳地，75-95℃；
(iv)失活时间为0.5-20min，较佳地，0.8-15min。
在另一优选例中，在步骤(e)(iii)中，用PCR进行基因组分析，优选地PCR反应管中含有扩增混合液、0.5％-20％的PCR抑制物对抗剂、5-20mM dNTP、5-100μM NG和NT引物、50-200μM扩增引物、0.5-10单位核酸聚合酶，优选地，所述PCR抑制物对抗剂选自DMSO、甜菜碱、甲酰胺、甘油和白蛋白中的一种或多种，所述核酸聚合酶选自Phi29DNA聚合酶、Bst DNA聚合酶、Vent聚合酶、Deep Vent聚合酶、Klenow Fragment DNA聚合酶I、MMLV反转录酶、AMV反转录酶、HIV反转录酶、超保真DNA聚合酶、Taq聚合酶、E.coli DNA聚合酶、LongAmp Taq DNA聚合酶和OneTaq DNA聚合酶中的一种或多种。
在另一优选例中，所述扩增混合液的成分为10-25mM Tris-HCl，5-25mM(NH4)2SO4，5-30mM KCl，0.5-5mM MgSO4，0.1％-20％DMSO和0.05-5％Triton X-100。
在另一优选例中，所述扩增混合液的成分优选为15mM Tris-HCl，15mM(NH4)2SO4，20mM KCl，1mM MgSO4，5％DMSO和2％Triton X-100。
在另一优选例中，所述NG和NT引物从5’端到3’端包含通用序列和可变序列，其中所述通用序列由G、A、C和T四种碱基中的三种或者两种组成，条件是所述通用序列不同时包括G和C；所述扩增引物包含所述通用序列且不包含所述可变序列。
在另一优选例中，所述可变序列选自下组：(N)nGGG、(N)nTTT，(N)mTNTNG，(N)xGTGG(N)y，其中N为任意的可与天然核酸进行碱基配对的核苷酸，n是选自3-17的正整数，m是选自3-15的正整数，x和y分别是选自3-13的正整数。
在另一优选例中，在步骤(e)(iii)中，用PCR进行基因组分析时的全基因组 扩增的热循环程序如下所述：
(1)在介于90-98℃之间的第一变性温度反应5-20s；
(2)在介于5-15℃之间的第一退火温度反应5-60s，在介于15-25℃之间的第二退火温度反应5-60s，在介于25-35℃之间的第三退火温度反应30-80s，在介于35-45℃之间的第四退火温度反应5-60s，在介于45-55℃之间的第五退火温度反应5-60s；
(3)在介于55-80℃之间的第一延伸温度反应10-150min；
(4)在介于90-98℃之间的第二变性温度反应5-30s；
(5)在介于45-70℃之间的第六退火温度反应10-30s；
(6)在介于60-80℃之间的第二延伸温度反应1-10min；
(7)重复步骤(4)到(6)5至50个循环；
(8)在介于60-80℃之间的温度下继续延伸反应1-10min；
(9)将扩增后的产物在0-5℃下冷藏保存。
在另一优选例中，在步骤(e)(iii)中，用PCR进行基因组分析时的全基因组扩增的热循环程序如下所述：
(1)在第一变性温度95℃下反应10s；
(2)在第一退火温度10℃下反应45s，在第二退火温度20℃下反应45s，在第三退火温度30℃下反应60s，在第四退火温度40℃下反应45s，在第五退火温度50℃下反应45s；
(3)在第一延伸温度62℃下反应90min；
(4)在第二变性温度95℃下反应20s；
(5)在第六退火温度59℃下反应20s；
(6)在第二延伸温度72℃下反应3min；
(7)重复步骤(4)到(6)10至30个循环；
(8)在72℃下继续延伸反应5min；
(9)将扩增后的产物在4℃下冷藏保存。
在另一优选例中，所述方法为非治疗和非诊断性的。
本发明第二方面提供了一种制备基因检测样本或染色体检测样本的方法，包括步骤：
(a)提供第一囊胚培养体系，所述第一囊胚培养体系含有体外培养天数为D5-D6的囊胚；
(b)将所述囊胚转移到含新鲜囊胚培养液的第二囊胚培养体系中进行换液培养，所述换液培养的时间为T1，从而获得经培养的囊胚培养液；
(c)将所述经培养的囊胚培养液取出，从而获得无细胞的经培养的囊胚培养液，即为检测样本。
在另一优选例中，所述第二囊胚培养体系中仅含有一个囊胚(胚胎)。
在另一优选例中，所述第二囊胚培养体系为单胚胎培养体系，含有10-60微升，较佳地，10-50微升，更佳地，10－15微升的培养液。
在另一优选例中，步骤(c)中，所述检测样本的体积为所述第二囊胚培养体系中培养液体积的50-100％，较佳地，70-100％，更佳地，80-100％，最佳地，90-100％。
本发明第三方面提供了一种利用囊胚培养液检测囊胚健康状况的试剂盒，包括：
(i)第一容器，以及位于第一容器的卵裂期培养液；
(ii)第二容器，以及位于第二容器的第一囊胚培养液；
(iii)第三容器，以及位于第三容器的第二囊胚培养液；和
(iv)标签或说明书。
在另一优选例中，所述第一容器、第二容器、第三容器不同。
在另一优选例中，所述位于第一容器的卵裂期培养液用于D1到D3，较佳地，D3±12h，更佳地，D3±8h，更佳地，D3±6h，的卵裂胚培养。
在另一优选例中，所述位于第二容器的第一囊胚培养液用于D3-D5的囊胚培养。
在另一优选例中，所述位于第三容器的第二囊胚培养液用于D5-D6后的囊胚培养。
在另一优选例中，所述第一囊胚培养液和第二囊胚培养液的成分相同，均为G-2 PLUS medium(Vitrolife)或Quinn’s Advantage Protein Plus blastocyst medium(SAGE)囊胚培养液。
在另一优选例中，所述卵裂期培养液为G-1 PLUS medium(Vitrolife)或Quinn’s Embryo maintenance medium(SAGE)卵裂期培养液。
本发明第四方面提供了一种辅助诊断囊胚健康状况的设备，包括：
(a)培养模块，所述培养模块包括囊胚后期培养模块，所述囊胚后期培养模块用于对培养天数为D5-D6的囊胚进行换液，并进行囊胚后期培养一段时间(T1)；
(b)取样模块，所述取样模块用于将囊胚后期培养模块中经囊胚后期培养的 囊胚的培养液取出，并作为检测样本；
(c)检测模块，所述检测模块用于将来自所述取样模块中的检测样本进行基因检测，从而获得检测结果；
(d)囊胚的健康状况的判别处理模块，基于所述检测结果，对胚胎的健康状况进行评价，从而获得囊胚的健康状况的评价结果，
(e)输出模块，所述输出模块用于输出所述的囊胚的健康状况的评价结果。
在另一优选例中，所述T1时间为2-8h，例如3-6h，较佳地，3-5h。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了常规采样方式与本发明采样方法在检测样本暴露时间上不同的示意图。
图2显示了使用常规D3-D5采样法采集的培养液的CNV分析结果。
图3显示了使用本发明的D5当天采样法采集的培养液的CNV分析结果。
具体实施方式
本发明人经过长期广泛而深入的研究，通过大量筛选和测试，首次意外地发现，在本发明的10-20微升的囊胚培养体系中，将胚胎(囊胚)在囊胚培养液中培养至D5-D6天后，将所述胚胎转移到新鲜的囊胚培养液中进行培养，从所述新鲜的囊胚培养液中取出少量培养液进行检测后发现，所得到的胚胎健康状况(如染色体非整倍体、线粒体拷贝数，DNA含量是否正常，致病基因检测)的检测结果具有极高的精确性，并且，假阴性率极低(＜8％，较佳地，＜5％)，可极大程度的排除多余精子和母源颗粒细胞污染干扰的风险。在此基础上，本发明人完成了本发明。
如本文所用，术语“囊胚”、“胚胎”可互换使用，指胚胎体外培养的终末阶段，它通常形成于卵子受精后的第5-7天。
如本文所用，乏培养液(depleted medium)是指已经使用过的培养液，例如，从已经培养过胚胎的培养体系中分离出的培养液。
如本文所用，从头培养是指这样的胚胎培养过程，在该培养过程中从培养过程开始到该培养过程结束不进行培养液的更换。例如，D1-D5的从头培养是指，该胚胎培养过程从D1开始并持续到D5，且期间不进行培养液的更换。
如本文所用，分阶段培养是指这样的胚胎培养过程，在该培养过程中胚胎经历一次或多次的换液。例如，D1-D5分阶段培养是指，该胚胎培养过程从D1开始并持续到D5，但期间胚胎经历一次或多次的换液。例如，在D1-D3，胚胎在卵裂期培养液中培养；而在D3-D5，胚胎更换到囊胚培养液中培养。
如本文所用，“Dx”(其中x＝1,2,3….的正整数)表示，将受精卵在体外培养开始时设为第1天(D1)，Dx是体外培养的第X天。
如本文所用，“D3-D5”采样是指，一个培养液在D3到D5天用于培养胚胎，并在D5从该用过的培养液(即，乏培养液)采集样本。因此，以该采样方式获得的培养液样本，接触胚胎培养物的时间(即，检测样本的暴露时间)将至少为大约48小时或以上。
如本文所用，“当天”采样是指，在更换培养液的当天，经过短时胚胎培养后，从该用过的培养液(即，乏培养液)采集样本。因此，以该采样方式获得的培养液样本，接触胚胎培养物的时间(即，检测样本的暴露时间)将是短时的。在本发明中，优选该暴露时间不超过8小时，例如3-6小时，更优选3-5小时。
在本文中，“裂解液”是指，用于分解培养液样品中蛋白和细胞的裂解缓冲液。在一些实施方案中，裂解液可以包含或不包含裂解酶。在一些实施方案中，可以将裂解液和裂解酶同时加入培养液样品。在另一些实施方案中，在裂解液与培养液样品混合后补充加入裂解酶。
在本文中，“检测样本体外孵育时间”、“检测样本培养时间”和“检测样本暴露时间”可互换使用，是指样本所取自的培养液已经在体外接触胚胎(即，与胚胎孵育)的时间长度。
在本文中，“CNV”是指染色体拷贝数变异(Copy number variants)。CNV命名遵守国际人类细胞基因组命名系统(ISCN)。参见，Shaffer LG,Slovak ML,Campbell LJ(2009):ISCN 2009 an international system for human cytogenetic nomenclature，Human Genetics volume 126,Article number:603(2009)。
IVF和IVF胚胎
IVF指in vitro fertilization(试管婴儿、体外受精),具体指体外受精联合胚胎移植技术(IVF)，又称试管婴儿，是指分别将卵子与精子取出后，置于试管内使其受精，再将胚胎前体—受精卵移植回母体子宫内发育成胎儿的技术。
“多精受精的囊胚”或“IVF囊胚”是指，利用IVF技术，通过多个精子与卵母细胞在体外共培养来完成受精，而得到的囊胚。
ICSI和ICSI胚胎
ICSI(Intracytoplasmic sperm injection)即卵胞浆内单精子显微注射技术，也就是第二代“试管婴儿”，该技术是借助显微操作系统将单一精子注射入卵子内使其受精。“单精受精的囊胚”或“ICSI囊胚”是指，利用卵胞浆内单精子显微注射技术ICSI而获得的囊胚。
检测方法
本发明提供了一种检测方法，所述方法对囊胚进行培养后的乏培养液(depleted medium)(即从已经培养过囊胚的培养体系中分离出的培养液)进行基因检测，从而鉴定胚胎的健康状况。
在本发明中，对囊胚进行培养后的“乏”培养液进行基因检测的方法不受特别的限制，可用常规方法进行检测，如二代测序、核酸芯片、免疫荧光检测、荧光PCR检测、一代测序、三代测序、质谱检测、或其组合。
在一种实施方式中，所述检测方法包括如下步骤：
(a)提供第一囊胚培养体系，所述第一囊胚培养体系含有体外培养天数为D5-D6的囊胚；
(b)将所述囊胚转移到含新鲜囊胚培养液的第二囊胚培养体系中进行换液培养，所述换液培养的时间为T1，从而获得经培养的囊胚培养液；
(c)将所述经培养的囊胚培养液取出，从而获得无细胞的经培养的囊胚培养液，即为检测样本；和
(d)对所述检测样本进行基因检测，从而鉴定所述囊胚的健康状况。
在一优选实施方式中，所述步骤(d)中还包括步骤(e)：
(i)将所述培养液与裂解液混合，从而获得含有所述囊胚培养液与裂解液的 第一混合物；
(ii)将所述第一混合物与裂解酶混合，孵育，失活裂解酶，从而获得裂解产物；和
(iii)对所述裂解产物进行基因组分析，从而鉴定所述胚胎的健康状况。
在一具体的实施方式中，本发明的利用囊胚培养液作为检测样本来反映胚胎健康状况的方法的主要步骤包括：
(a)卵母细胞采用IVF或ICSI方式进行受精，受精后将受精卵转移至培养液中培养至D3卵裂期，在D3将胚胎转移至囊胚培养液，培养至D5-D6；
(b)进行囊胚评级，然后将囊胚发育良好的胚胎在D5±0.5天转移至另一新囊胚培养液中，于高倍镜下利用激光破膜仪在透明带上打孔使得胚胎发生皱缩，于3-5小时后取走囊胚，囊胚进行玻璃化冷冻操作而剩余的原囊胚培养液即为检测时所需要收集的样本；
(c)将步骤(b)中获取的原囊胚培养液转移至裂解液中，离心后，样本进入下一步的全基因组扩增步骤，包含但不限于NICS-INST的扩增方法以及分析方法，得到反映胚胎健康状况的检测结果。
本发明在D5±0.5天更换培养液，经过3-5小时短时培养获取囊胚培养液作为检测样本，检测结果准确性明显提高、假阴性率降低至5％以下，最大程度的避免外源干扰特别是母源干扰，且ICSI和IVF受精方式均可适用。
在本发明中，检测结果准确性明显提高的原理是：首先检测样本的暴露时间极其短，相比常规操作方式2-3天的暴露时间大大缩短(如图1所示)，最大程度的避免外源干扰特别是母源干扰，因此不受受精方式限制，极大提高检测结果准确性。其次，由于胚胎有自我修复的能力，常规方式培养期间会把异常片段释放到培养基中，可能对准确性有影响；而本发明D5换液培养3-5小时后采样使得胚胎在该更换的培养液中培养时间短，且胚胎已在D5换培养液前的D3-D5培养阶段完成自我修复，异常片段主要在该D3-D5阶段释放，所以在本发明采集的检测样本中异常片段释放的概率较低，因此提高检测结果准确性。在本发明之前，未见有人尝试对IVF胚胎进行培养液的收集，而是一般对ICSI受精方式产生的胚胎进行培养液的收集。外源干扰最主要的因素是检测样本体外孵育过长，本发明采用短时孵育的培养液采集方法，避免因孵育时间过长受到精子和颗粒细胞等外源干扰，这些干扰将使得胚胎来源的DNA比重降低从而影响结果的准确性。
所述步骤(c)的检测样本的量为8-15ul，优选10ul。新囊胚培养液(即第二囊胚培养液)的量优选10-15ul。
优选地，囊胚评级包括选择胚胎发育到4期以上为发育良好的胚胎。
步骤(b)的打孔操作，增加了囊胚腔液的释放，也就是游离核酸的释放，从而满足了在3-5小时短时孵育的前提下检测样本的核酸物质起始量增加，足够后续扩增和检测，保证检测的成功率可达97％以上。通常在囊胚期进行胚胎观察、评级以及冻存，认为囊胚期的操作不会对胚胎造成损伤，本发明也同样选择在D5囊胚期进行激光打孔及体外培养，因此是在不影响胚胎发育的前提下进行的。
在本发明中，步骤(b)还可以包括，在D5换培养液前可以增加清洗胚胎的步骤，该步骤对提高检测结果准确性有一定的帮助。
在本发明中，所述步骤(a)在D3更换囊胚培养液后，可多胚胎混合培养,也可以进行单胚胎培养。
样本制备及其检测
本发明还提供了一种制备基因检测样本或染色体检测样本的方法，包括步骤：
(a)提供第一囊胚培养体系，所述第一囊胚培养体系含有体外培养天数为D5-D6的囊胚；
(b)将所述囊胚转移到含新鲜囊胚培养液的第二囊胚培养体系中进行换液培养，所述换液培养的时间为T1，从而获得经培养的囊胚培养液；
(c)将所述经培养的囊胚培养液取出，从而获得无细胞的经培养的囊胚培养液，即为检测样本。
在另一优选例中，通过所述方法获得的检测样本，可以用于基因检测，从而鉴定所述胚胎的健康状况。
透明带打孔
当胚胎扩增至囊胚期，将囊胚置于操作台上，选择远离内细胞团且滋养外胚层稀疏的位置，使用激光束破囊腔，在透明带产生小孔后，让囊胚腔液释放到囊胚培养液中。对透明带的孔径没有特别限制，一种优选的透明带孔径为10－40μm，较佳地，10－30μm，更佳地，10－20μm。
在本发明中，透明带的打孔操作增加了囊胚腔液的释放，也就是游离核酸的释放，满足了在3-5小时短时孵育的前提下，检测样本的核酸物质起始量增加，足够后续扩增和检测，检测的成功率可达97％以上。
如果不进行激光打孔操作，检测的成功率也很高，有70％左右。
试剂盒
在本发明中，提供了一种利用囊胚培养液检测胚胎健康状况的试剂盒，包括：
(i)第一容器，以及位于第一容器的卵裂期培养液；
(ii)第二容器，以及位于第二容器的第一囊胚培养液；
(iii)第三容器，以及位于第三容器的第二囊胚培养液；和
(iv)标签或说明书。
辅助诊断胚胎健康状况的设备
在本发明中，提供了辅助诊断胚胎健康状况的设备，包括：
(a)培养模块，所述培养模块包括囊胚后期培养模块，所述囊胚后期培养模块用于对培养天数为D5-D6的囊胚进行换液并进行囊胚后期培养T1时间；
(b)取样模块，所述取样模块用于将囊胚后期培养模块中培养T1时间后的囊胚的培养液取出，并作为检测样本；
(c)检测模块，所述检测模块用于将来自所述取样模块中的检测样本进行基因检测，从而获得检测结果；
(d)胚胎的健康状况的判别处理模块，所述模块基于所述检测结果，对胚胎的健康状况进行评价，从而获得胚胎的健康状况的评价结果；
(e)输出模块，所述输出模块用于输出所述的胚胎的健康状况的评价结果。
本发明也涉及所述模块(a),任选地结合模块(b)-(e)，在制备用于本发明囊胚健康状况评价方法的设备中的用途。
本发明的主要优点包括：
(1)在本发明中，首次发现，将体外培养天数为D5-D6的囊胚转移到新囊胚培养液中进行短时培养，从经培养的囊胚培养液中分离出所述培养液，并取少量的培养液进行基因检测，所得到的胚胎的健康状况(如染色体是否异常、染色体非整倍体、线粒体拷贝数，DNA含量是否正常、致病基因检测等)的检测结果居然 具有极高的精确性和极低的假阴性率(低于8％，较佳地，低于5％)，可极大程度的排除多余精子和母源颗粒细胞污染干扰的风险。
(2)本发明可以采用单胚胎培养体系，即一个培养液中只培养一个胚胎，该体系所得到的胚胎的健康状况的检测结果更准确。
(3)本发明的方法为通用型方法，可用于单精受精胚胎，也可以用于多精受精胚胎，优选多精受精胚胎。
(4)本发明的方法中可对透明带进行打孔，所得到的结果将更好。
(5)本发明的方法具有很高的信噪比。
(6)本发明的方法在胚胎培养到D5后再进行胚胎培养液的收集操作，操作时间极短，检测样本培养时间可控，受外界干扰的可能性最低，此外样本采集不受限于受精方式，是一种IVF和ICSI胚胎同样适用的培养液采集方法。
(7)本发明的D5换液短时培养方案，其优势在于控制了检测样本体外孵育的时间，从而最大限度的控制了母源干扰和精子干扰等外源性干扰，使得本发明的应用不受限于ICSI胚胎，还可应用于IVF胚胎。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
本发明所用的材料如无特别说明，均为市售产品。
通用方法
囊胚培养液的获得：
(1)受精卵(在志愿者夫妇中，从女性志愿者中获取卵子，从男性志愿者中获取精子，采用IVF或ICSI方式进行体外受精，即得到受精卵)在体外培养至第3天，换液或不换液培养到第5天/第6天；
(2)当胚胎发育到4期以上的囊胚时，将胚胎分别依次转移到至少3个30微升囊胚培养液微滴(保证一个微滴中只有一个胚胎)，每个微滴轻柔吹吸漂洗2-3次，之后再将胚胎转移至37℃，5％CO 2，5％O 2条件下经过夜平衡的微滴10-15ul中，于高倍镜下，利用激光破膜仪在透明带上打孔，使得胚胎发生皱缩；继续培养3-5小时或3-6小时。
(3)将步骤(2)中获取的短时培养囊胚的培养液(体积约为10-15微升)转移至5微升裂解液(pH 7.8的30mM Tris-Cl,2mM EDTA,20mM KCl,0.2％Triton X-100)中，用记号笔在采集管上标记样本名称。微型离心机离心30秒。样本可立即进入下一步全基因组扩增步骤或放入-20℃或-80℃冷冻保存。
本发明的检测方法参照序康医疗科技(苏州)有限公司的基因测序通用文库试剂盒(商品名NICS-Inst TM，通用名：基因测序通用文库试剂盒；英文名：Universal Library Preparation Kit)的说明书进行。
实施例1
本实施例涉及对本发明D5当天采样方法与常规D3-D5采样方法的比较。
简言之，将受精卵在体外培养至D3，将得到的胚胎转移到囊胚培养液中，继续培养到D5。根据常规D3-D5采样法，取D5的囊胚培养液作为“D3-D5采样”样本。之后，根据本发明方法，将D5囊胚转移到新囊胚培养液中，在D5当日，按照前面的通用方法描述，进行D5当天采样。将来自同一胚胎的“D3-D5采样”的CV检测结果与“D5当天采样”检测结果进行比较，并与整胚的检测结果进行比较。以整胚作为金标准，当整胚检测结果为染色体拷贝数异常，而培养液检测结果为正常时，将培养液检测结果判定为假阴性，假阴性占总体的概率为假阴性率。
经大量实验数据证明，本发明检测结果准确性明显提高、假阴性率降低至5％以下。常规D3-D5采样(130组样本)，与本发明D5当天采样(62组样本)的假阴性率如下：
  D3-D5采样 D5当天采样
假阴性率 12.3％ 1.3％
准确率 73.1％ 91％
如上表所示，D5更换新囊胚培养液经短时培养采样，其假阴性率可以控制在1.6％；而在D3-D5采样方法中，对于母源干扰无法有效控制，其假阴性率为12.3％，易造成临床的误诊。
其中，D3-D5造成的假阴性案例，如下所示：(显示了使用不同培养液进行CNV染色体核型分析的结果)
Figure PCTCN2019129196-appb-000001
Figure PCTCN2019129196-appb-000002
本发明D5更换新囊胚培养液经短时培养采样，可最大程度的降低母源干扰，如图2对比图3所示。图2和图3显示了一个男性胚胎应用本发明方法和常规D3-D5采样方法获得的染色体拷贝数(CN)图谱。CN图上横轴为染色体，按照常染色体1-22，性染色体X、Y的顺序依次排列；纵坐标轴为拷贝数，对于正常人胚胎(二倍体)，常染色体拷贝数应为2，性染色体为X两个拷贝(女性)，或X、Y各一个拷贝(男性)。
在D3-D5采样方法中，采集培养液，难以避免母源干扰的现象，可能造成CNV(染色体拷贝数变异，Copy number variants)判读结果的不准确，比如，把46XY(男性)的结果判读成46XY/XX(男女嵌和)，如图2所示。而在本发明D5当天采样方法中，D5当天采集的培养液，可最大程度的降低母源干扰，还原真实CNV结果，如图3，其中CNV的判读为46XY(男性)。
经过对比试验还发现，应用本发明采集的囊胚培养液进行性别检测，无论是ICSI还是IVF胚胎，与应用整胚进行的检测相比，性别判定准确性无影响，不会因为母源干扰或父源干扰，造成假阴性。部分案例，如下所示：
Figure PCTCN2019129196-appb-000003
Figure PCTCN2019129196-appb-000004
因此，本发明的方法具有非常好的准确性和非常低的假阴性率。
对比例1
方法同实施例1，区别在于，培养至D3－D4天，在D4进行换液，继续培养24或48小时后，取囊胚培养液进行检测。在19组样本上进行的检测结果显示，胚胎健康状况(如胚胎染色体异常)的检测结果的准确率为84.2(16/19)％，假阴性率为10.5(2/19)％。增加样本量后，使用该对比例1的方法，结果显示：该方法的准确率更小，假阴性率更高。
讨论
D4胚胎通常发育为桑葚期胚胎，是胚胎发育成囊胚的关键时期，桑椹胚的下一个时期即囊胚期，而成囊率是胚胎发育的关键指标，囊胚成囊后方可考虑移植或者冷冻等体外操作，因此临床上通常不会在D4的桑葚期对胚胎进行换液等体外操作，以免影响成囊率。
在囊胚期进行胚胎观察，评级，以及冻存，对囊胚期的操作不会对胚胎造成损伤。本发明选择在D5囊胚期进行激光打孔及体外培养，因此是在不影响胚胎发育的前提下进行的。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (12)

  1. 一种体外利用囊胚培养液检测囊胚健康状况的方法，其特征在于，包括步骤：
    (a)提供第一囊胚培养体系，所述第一囊胚培养体系含有体外培养天数为D5-D6的囊胚；
    (b)将所述囊胚转移到含新鲜囊胚培养液的第二囊胚培养体系中进行换液培养，所述换液培养的时间为T1，从而获得经培养的囊胚培养液；
    (c)将所述经培养的囊胚培养液取出，从而获得无细胞的经培养的囊胚培养液，即为检测样本；和
    (d)对所述检测样本进行基因检测，从而鉴定所述囊胚的健康状况。
  2. 如权利要求1所述的方法，其特征在于，在步骤(a)中，所述囊胚体外培养为从头培养或分阶段培养。
  3. 如权利要求1所述的方法，其特征在于，在步骤(a)中，所述囊胚体外培养包括第一期囊胚培养和第二期囊胚培养。
  4. 如权利要求3所述的方法，其特征在于，所述第一期囊胚培养包括在D1-D3，用卵裂期培养液进行培养。
  5. 如权利要求3所述的方法，其特征在于，所述第二期囊胚培养包括在D3－D5，用囊胚培养液进行培养。
  6. 如权利要求1所述的方法，其特征在于，所述T1时间为2-8h，例如,3-6h,较佳地，3-5h。
  7. 如权利要求1所述的方法，其特征在于，所述方法具有以下一种或多种特征：
    (i)信噪比高，信噪比S 1/S 0＞2，较佳地，＞5，更佳地，＞10，其中S 1为来自胚胎的信号，S 0为来自背景的信号；
    (ii)低假阴性率，假阴性率＜8％，较佳地，＜5％，更佳地，＜3％；
    (iii)高准确性，准确率≥80％，较佳地，85％≥，更佳地，≥90％。
  8. 一种制备基因检测样本或染色体检测样本的方法，其特征在于，包括步骤：
    (a)提供第一囊胚培养体系，所述第一囊胚培养体系含有体外培养天数为D5-D6的囊胚；
    (b)将所述囊胚转移到含新鲜囊胚培养液的第二囊胚培养体系中进行换液培 养，所述换液培养的时间为T1，从而获得经培养的囊胚培养液；和
    (c)将所述经培养的囊胚培养液取出，从而获得无细胞的经培养的囊胚培养液，即为检测样本。
  9. 一种培养模块在制备用于权利要求1的方法的产品中的用途，其中，所述培养模块包括囊胚后期培养模块，所述囊胚后期培养模块用于对培养天数为D5-D6的囊胚进行换液，并进行囊胚后期培养一段时间(T1)。
  10. 一种辅助诊断囊胚健康状况的设备，其特征在于，包括：
    (a)培养模块，所述培养模块包括囊胚后期培养模块，所述囊胚后期培养模块用于对培养天数为D5-D6的囊胚进行换液，并进行囊胚后期培养一段时间(T1)；
    (b)取样模块，所述取样模块用于将囊胚后期培养模块中经囊胚后期培养的囊胚的培养液取出，并作为检测样本；
    (c)检测模块，所述检测模块用于将来自所述取样模块中的检测样本进行基因检测，从而获得检测结果；
    (d)囊胚的健康状况的判别处理模块，基于所述检测结果，对胚胎的健康状况进行评价，从而获得囊胚的健康状况的评价结果
    (e)输出模块，所述输出模块用于输出所述的囊胚的健康状况的评价结果。
  11. 权利要求1-8的方法，其特征在于，所述方法还包括，在所述换液后，在获取检测样本前，对囊胚的透明带进行打孔。
  12. 权利要求9的用途和权利要求10的设备，其中所述培养模块还包括用于在所述换液后对囊胚的透明带进行打孔的装置。
PCT/CN2019/129196 2018-12-29 2019-12-27 一种利用囊胚培养液检测胚胎健康状况的方法和产品 WO2020135713A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CN201980068400.7A CN113286892B (zh) 2018-12-29 2019-12-27 一种利用囊胚培养液检测胚胎健康状况的方法和产品
EP19903766.4A EP3904529A1 (en) 2018-12-29 2019-12-27 Method and product for detecting embryo health condition by using blastocyst culture solution
US17/418,934 US20220112549A1 (en) 2018-12-29 2019-12-27 Method and product for detecting embryo health condition by using blastocyst culture solution
JP2021538406A JP2022516543A (ja) 2018-12-29 2019-12-27 胚盤胞培養液を使用して胚の健康状態を検出するための方法および製品

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811647225.0 2018-12-29
CN201811647225.0A CN109536581B (zh) 2018-12-29 2018-12-29 一种利用囊胚培养液检测胚胎健康状况的方法和产品

Publications (1)

Publication Number Publication Date
WO2020135713A1 true WO2020135713A1 (zh) 2020-07-02

Family

ID=65831526

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/129196 WO2020135713A1 (zh) 2018-12-29 2019-12-27 一种利用囊胚培养液检测胚胎健康状况的方法和产品

Country Status (5)

Country Link
US (1) US20220112549A1 (zh)
EP (1) EP3904529A1 (zh)
JP (1) JP2022516543A (zh)
CN (2) CN109536581B (zh)
WO (1) WO2020135713A1 (zh)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109536581B (zh) * 2018-12-29 2022-02-01 序康医疗科技（苏州）有限公司 一种利用囊胚培养液检测胚胎健康状况的方法和产品
CN111440857B (zh) * 2020-03-11 2023-03-21 阿吉安(福州)基因医学检验实验室有限公司 用于无创胚胎植入前遗传性检测的方法
CN112582022B (zh) * 2020-07-21 2021-11-23 序康医疗科技（苏州）有限公司 用于无创胚胎移植优先级评级的系统和方法
CN113862303A (zh) * 2020-08-24 2021-12-31 北京希诺谷生物科技有限公司 利用体细胞克隆制备克隆马胚胎的方法
CN114107182A (zh) * 2022-01-07 2022-03-01 徐维海 一种提高囊胚形成率的方法
CN114752552A (zh) * 2022-05-07 2022-07-15 序康医疗科技（苏州）有限公司 冷冻胚胎的复苏、培养、优选和单囊胚移植方法以及用于复苏冷冻胚胎的无创检测方法
CN115678964B (zh) * 2022-11-08 2023-07-14 广州女娲生命科技有限公司 基于胚胎培养液的植入前胚胎的无创筛选方法

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009059139A1 (en) * 2007-10-31 2009-05-07 Christopher William Lipari Assay of nitric oxide metabolites in the culture media of human preimplantation embryos
CN103890191A (zh) 2011-05-27 2014-06-25 哈佛大学校长及研究员协会 单细胞全基因组扩增方法
CN104711362A (zh) * 2015-03-27 2015-06-17 苏州贝康医疗器械有限公司 一种利用囊胚期胚胎细胞进行胚胎染色体异常检测的方法
CN105368936A (zh) * 2015-11-05 2016-03-02 上海序康医疗科技有限公司 一种利用囊胚培养液检测胚胎染色体异常的方法
CN105722995A (zh) * 2013-06-18 2016-06-29 国家医疗保健研究所 用于测定胚胎质量的方法
CN106086199A (zh) * 2016-07-05 2016-11-09 上海序康医疗科技有限公司 一种利用不含透明带的囊胚培养液检测胚胎染色体异常的方法
CN107760773A (zh) * 2017-10-26 2018-03-06 北京中仪康卫医疗器械有限公司 一种对胚胎培养液进行scRRBS分析的方法
CN109536581A (zh) * 2018-12-29 2019-03-29 序康医疗科技（苏州）有限公司 一种利用囊胚培养液检测胚胎健康状况的方法和产品

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080085836A1 (en) * 2006-09-22 2008-04-10 Kearns William G Method for genetic testing of human embryos for chromosome abnormalities, segregating genetic disorders with or without a known mutation and mitochondrial disorders following in vitro fertilization (IVF), embryo culture and embryo biopsy
WO2015114574A1 (en) * 2014-01-30 2015-08-06 Pécsi Tudományegyetem Preimplantation assessment of embryos through detection of free embryonic dna
CN108641929A (zh) * 2018-02-23 2018-10-12 深圳市瀚海基因生物科技有限公司 确定胚胎是否存在异常的方法和系统

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009059139A1 (en) * 2007-10-31 2009-05-07 Christopher William Lipari Assay of nitric oxide metabolites in the culture media of human preimplantation embryos
CN103890191A (zh) 2011-05-27 2014-06-25 哈佛大学校长及研究员协会 单细胞全基因组扩增方法
CN105722995A (zh) * 2013-06-18 2016-06-29 国家医疗保健研究所 用于测定胚胎质量的方法
CN104711362A (zh) * 2015-03-27 2015-06-17 苏州贝康医疗器械有限公司 一种利用囊胚期胚胎细胞进行胚胎染色体异常检测的方法
CN105368936A (zh) * 2015-11-05 2016-03-02 上海序康医疗科技有限公司 一种利用囊胚培养液检测胚胎染色体异常的方法
CN106086199A (zh) * 2016-07-05 2016-11-09 上海序康医疗科技有限公司 一种利用不含透明带的囊胚培养液检测胚胎染色体异常的方法
CN107760773A (zh) * 2017-10-26 2018-03-06 北京中仪康卫医疗器械有限公司 一种对胚胎培养液进行scRRBS分析的方法
CN109536581A (zh) * 2018-12-29 2019-03-29 序康医疗科技（苏州）有限公司 一种利用囊胚培养液检测胚胎健康状况的方法和产品

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
HAITAO WU ET AL: "Medium-Based Noninvasive Preimplantation Genetic Diagnosis for Human a-Thalassemias-SEA", MEDICINE, vol. 94, no. 12, 31 March 2015 (2015-03-31), pages 1 - 7, XP055564337, ISSN: 0025-7974, DOI: 10.1097/MD.0000000000000669 *
JUANJUAN XU ET AL: "Noninvasive Chromosome Screening of Human Embryos by Genome Sequencing of Embryo Culture Medium for in Vitro Fertilization", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 113, no. 42, 18 October 2016 (2016-10-18), pages 11907 - 11912, XP055380709, ISSN: 0027-8424, DOI: 10.1073/pnas.1613294113 *
KUZNYETSOV V: "Evaluation of a novel non-invasive preimplantation genetic screening approach", PLOS ONE, vol. 13, no. 5, 10 May 2018 (2018-05-10), pages e0197262
LIU, DONGYUN ET AL: "Preclinical Trial Study of Non-invasive Embryo Chromosome Screening (NICS)", THE 10TH NATIONAL ACADEMIC CONFERENCE ON GENETIC DISEASE DIAGNOSIS AND PRENATAL DIAGNOSIS; 2016-10-13, 30 November 2016 (2016-11-30), pages 32, XP009521742 *
MEDIUM-BASED NONINVASIVE PREIMPLANTATION GENETIC DIAGNOSIS FOR HUMAN -THALASSEMIAS-SEA, MEDICINE (BALTIMORE, vol. 94, no. 12, March 2015 (2015-03-01), pages e669
VERA-RODRIGUEZ M ET AL.: "Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development", HUM REPROD, vol. 33, no. 4, 1 April 2018 (2018-04-01), pages 745 - 756, XP055525366, DOI: 10.1093/humrep/dey028

Also Published As

Publication number Publication date
US20220112549A1 (en) 2022-04-14
CN113286892B (zh) 2024-02-23
CN113286892A (zh) 2021-08-20
CN109536581A (zh) 2019-03-29
EP3904529A1 (en) 2021-11-03
JP2022516543A (ja) 2022-02-28
CN109536581B (zh) 2022-02-01

Similar Documents

Publication Publication Date Title
WO2020135713A1 (zh) 一种利用囊胚培养液检测胚胎健康状况的方法和产品
AU2016351034B2 (en) Method of detecting chromosome abnormality in embryo by using blastocyst culture
Leaver et al. Non-invasive preimplantation genetic testing (niPGT): the next revolution in reproductive genetics?
Braude et al. Preimplantation genetic diagnosis
US20170044610A1 (en) Compositions and methods for genetic analysis of embryos
McArthur et al. Pregnancies and live births after trophectoderm biopsy and preimplantation genetic testing of human blastocysts
Brouillet et al. Is cell-free DNA in spent embryo culture medium an alternative to embryo biopsy for preimplantation genetic testing? A systematic review
US20080085836A1 (en) Method for genetic testing of human embryos for chromosome abnormalities, segregating genetic disorders with or without a known mutation and mitochondrial disorders following in vitro fertilization (IVF), embryo culture and embryo biopsy
Takeuchi Pre‐implantation genetic testing: past, present, future
WO2018006706A1 (zh) 一种利用不含透明带的囊胚培养液检测胚胎染色体异常的方法
Vagnini et al. Relationship between age and blastocyst chromosomal ploidy analyzed by noninvasive preimplantation genetic testing for aneuploidies (niPGT-A)
De Coster et al. Parental genomes segregate into distinct blastomeres during multipolar zygotic divisions leading to mixoploid and chimeric blastocysts
Shamash et al. Preimplantation genetic haplotyping a new application for diagnosis of translocation carrier’s embryos-preliminary observations of two robertsonian translocation carrier families
Pickering et al. Reliability of detection by polymerase chain reaction of the sickle cell-containing region of the β-globin gene in single human blastomeres
Gavrilov et al. Non-viable human embryos as a source of viable cells for embryonic stem cell derivation
Nagy et al. Scientific and ethical issues of preimplantation diagnosis
Vaccari Non-Invasive Preimplantation Genetic Testing: a Laboratory Review: NIPGTA-a laboratory review
Simpson et al. Overview and Historical Perspective of Preimplantation Genetic Testing
Gosden et al. Micromanipulation in assisted reproductive technology: intracytoplasmic sperm injection, assisted hatching, and preimplantation genetic diagnosis
Imthurn et al. Polar body diagnosis (PBD): an alternative and supplement to preimplantation diagnosis for single embryo transfer
Goldberg et al. Preimplantation diagnosis.
Kuliev et al. Major Components of Preimplantation Genetic Testing
Athalye et al. Preimplantation genetic diagnosis for single gene disorders
Parikh et al. IMPACT OF BIOPSY ON HUMAN EMBRYO CONCERN WITH PREIMPLANTATION GENETIC DIAGNOSIS (PGD): A REVIEW
Ashutosh Placental chimerism in early human pregnancy

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19903766

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021538406

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2019903766

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2019903766

Country of ref document: EP

Effective date: 20210729