WO2018006706A1

WO2018006706A1 - Method for detecting abnormality of embryo chromosomes by using blastocyst culture solution without zona pellucida

Info

Publication number: WO2018006706A1
Application number: PCT/CN2017/089201
Authority: WO
Inventors: 陆思嘉; 蔡立义; 任军; 马婷; 方锐
Original assignee: 序康医疗科技（苏州）有限公司
Priority date: 2016-07-05
Filing date: 2017-06-20
Publication date: 2018-01-11
Also published as: TW201803995A; CN106086199A

Abstract

Provided is a method for detecting the abnormality of embryos chromosomes by using a blastocyst culture solution without zona pellucida. Specifically, provided is an in-vitro non-therapeutic method for detecting the chromosome abnormality of the embryos by using a blastocyst culture solution, comprising the steps of: (a), providing a culture solution from a blastocyst culture system, the zona pellucida of the embryos in the blastocyst culture system being removed before the embryos are cultivated, the embryos being cultivated in the blastocyst system for 3 to 6 days, and preferably, the culture solution being separated from the culture system after 4 days; and (b), carrying out gene detection on the culture solution so as to authenticate whether chromosomes of the embryos are abnormal. By means of the method, contamination and interference risks due to redundant sperms and parent-source granular cells in IVF and ICSI technical procedures can be eliminated, and whether the chromosomes of the embryos are abnormal can be accurately authenticated.

Description

Method for detecting embryo chromosomal abnormality by using blastocyst culture liquid without zona pellucida

Technical field

The present invention relates to the fields of biomedical and molecular cell biology, and in particular to a method for detecting chromosomal abnormalities of an embryo using a blastocyst culture solution containing no zona pellucida.

Background technique

Assisted reproductive technology has become an important means of treating infertility. With the continuous improvement of the technical level, the clinical pregnancy rate of assisted reproduction has also increased, but the embryo implantation rate has not increased significantly. The quality of embryos is the main factor affecting embryo implantation. At present, the selection of high-quality embryos is mainly based on the morphology of embryonic development, and the subjectivity is not enough to fundamentally distinguish the quality of embryos. Studies have shown that even some well-formed high-quality blastocysts are aneuploid, or morphological scores of chromosomal euploid embryos are not high. Therefore, relying solely on embryonic morphology evaluation does not guarantee that the transplanted embryonic chromosome is normal. Numerous studies have shown that a considerable part of embryos derived from in vitro fertilization are chromosomal aneuploidy, especially from older infertile women. Transplantation of aneuploid embryos leads to implantation failure or miscarriage after transplantation, which is also the main reason for the success of IVF.

The use of Pre-implementation Genetic Screen (PGS) to select normal-stained embryos can effectively increase the success rate of IVF embryos and reduce pregnancy abortion. However, the PGS test requires biopsy of the embryo, and 1 to several cells are taken from the embryo for testing, and there is a risk of embryo damage. The current method of examining embryonic chromosomes using blastocyst culture fluids also carries the risk of interference between excess sperm and maternal granule cells in the IVF and ICSI techniques.

In the course of IVF technology, hundreds of millions of sperm are mixed with an egg in an in vitro culture environment. The result is that there are multiple sperm and egg combinations at the same time. Although only one sperm and egg complete normal fertilization, other excess sperm that cannot be fertilized remain in the zona pellucida around the egg cell. In addition, at this time, a large amount of granule cells of maternal origin are adhered to the zona pellucida on the periphery of the egg. The excess sperm remaining in the zona pellucida and the granulosa cells adhering to the surface of the zona pellucida are cultured in vitro in the fertilized egg, and the DNA may be released into the culture medium during the development of the blastocyst, so the DNA in the blastocyst culture solution is easy. Contaminated by DNA from sperm and granulosa cells.

In the ICSI technology process, the technical method of injecting a single sperm into an egg avoids contamination of the blastocyst culture fluid by excess sperm. However, maternal-derived granulosa cells adhering to the zona pellucida will still contaminate the blastocyst culture and interfere with subsequent detection.

Therefore, there is an urgent need in the art to develop a method for removing the risk of contamination of excess sperm and maternal granule cells in the IVF and ICSI techniques, and accurately identifying whether the embryonic chromosome is abnormal.

Summary of the invention

It is an object of the present invention to provide a method for accurately detecting the risk of contamination of excess sperm and maternal granule cells during IVF and ICSI techniques, and for accurately identifying embryonic chromosome abnormalities.

A first aspect of the invention provides a method for detecting embryonic chromosomal abnormalities using blastocyst culture fluid in vitro, comprising the steps of:

(a) providing a culture solution from a blastocyst culture system, wherein the embryo in the blastocyst culture system has removed the zona pellucida before the culture, and the embryo is cultured in the blastocyst culture system for 3-6 days, Preferably, after 4 days, the culture solution is separated from the culture system;

(b) performing genetic testing on the culture solution to identify whether the embryonic chromosome is abnormal.

In another preferred embodiment, the blastocyst culture system contains only one embryo.

In another preferred embodiment, the blastocyst culture system is a single embryo culture system, and there is no transparent band in the single embryo culture system, and contains 10-100 microliters, preferably 15-50 microliters, more Good, 20-35 microliters of culture.

In another preferred embodiment, the volume of the culture solution separated in the step (a) is 30-100%, preferably 40-100%, more preferably 40-100%, more preferably the volume of the culture solution in the single embryo culture system. , 50-100%, optimally, 80-100%.

In another preferred embodiment, the step (b) further comprises the step (c):

(i) mixing the culture solution with the lysate to obtain a first mixture containing the culture solution and the lysate;

(ii) mixing the first mixture with a lyase, incubating, inactivating the lyase, thereby obtaining a cleavage product;

(iii) performing genomic analysis on the lysate to identify whether the embryonic chromosome is abnormal.

In another preferred embodiment, the method of genomic analysis is selected from the group consisting of second generation sequencing, nucleic acid chips, immunofluorescence detection, fluorescent PCR detection, first generation sequencing, third generation sequencing, mass spectrometry detection, or a combination thereof.

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

In another preferred embodiment, the Tris buffer comprises Tris-Cl.

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

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

In another preferred embodiment, the chelating agent comprises EDTA.

In another preferred embodiment, the concentration of the chelating agent is from 0.2 to 8 mM, preferably from 0.5 to 6 mM, more preferably from 1 to 4 mM.

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

In another preferred embodiment, the hydrochloride salt has a concentration of 5 to 60 mM, preferably 8 to 40 nM, more preferably 10 to 40 mM.

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

In another preferred embodiment, the concentration of the nonionic surfactant is from 0.02% to 10%, preferably from 0.05% to 5%, more preferably from 0.1% to 3%, based on the total weight of the lysate .

In another preferred embodiment, in the step (i), the volume ratio of the culture solution to the lysate is from 1:10 to 10:1, preferably from 1:5 to 5:1, and more preferably, 1: 2-2:1.

In another preferred embodiment, the lytic enzyme is selected from the group consisting of proteinase K, Qiagen Protease, pepsin, papain, trypsin, lysozyme, or a combination thereof.

In another preferred embodiment, the lyase is at a concentration of 1 to 25 μg/ml, preferably 5 to 20 μg/ml, more preferably 10 to 15 μg/ml.

In another preferred embodiment, the lyase is added in an amount of 0.1 to 10 μl, preferably 0.5 to 6 μl, more preferably 0.8 to 3 μl.

In another preferred embodiment, the step (c) comprises one or more features selected from the group consisting of:

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

(ii) incubation time is from 1 min to 12 h, preferably from 10 min to 6 h, more preferably from 30 min to 2 h;

(iii) the deactivation temperature is 60-100 ° C, preferably 75-95 ° C;

(iv) the inactivation time is 0.5-20 min, preferably 10-15 min;

In another preferred embodiment, the culture solution is obtained by the following method:

(i) cultivating the fertilized egg embryo for 3-6 days (preferably, 4 days), removing the zona pellucida of the embryo, thereby obtaining an embryo without the zona pellucida;

(ii) transferring the zona pellucida-free embryo of step (i) to a blastocyst culture droplet for 0.5-3 days (preferably, 1-2 days) to obtain a culture medium containing blastocysts;

(iii) The culture solution obtained in the separation step (ii) is a culture solution for identifying whether or not the embryonic chromosome is abnormal.

In another preferred embodiment, the blastocyst culture droplets have a volume of 10 to 100 microliters, preferably 15 to 50 microliters, more preferably 20 to 35 microliters.

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

A second aspect of the present invention provides a method of preparing a genetic test sample or a chromosome test sample, comprising the steps of:

(i) Providing a culture system containing the zona pellucida-removing embryo, which is cultured for 3-6 days, preferably, after 4 days, the liquid is separated from the culture system, that is, the test sample.

In another preferred embodiment, the method further comprises the step (ii): performing genetic testing on the obtained test sample to identify whether the embryo chromosome is abnormal.

In another preferred embodiment, the culture system contains only one embryo.

In another preferred embodiment, the culture system is a single embryo culture system containing 10 to 100 microliters, preferably 15 to 50 microliters, more preferably 20 to 35 microliters of the culture solution.

In another preferred embodiment, in the step (ii), the volume of the test sample is 30-100%, preferably 40-100%, more preferably 50-100, of the volume of the culture solution in the culture system. %, optimally, 80-100%.

In another preferred embodiment, in step (ii), the test sample is centrifuged, and the supernatant is taken for genetic testing to identify whether the embryonic chromosome is abnormal.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

Figure 1 shows that when the conventional ICSI embryos are detected, the NICS test results (1a) are deviated due to interference with granulosa cell contamination, which is inconsistent with the conventional PGS (embryonic lesion biopsy) test result (1b).

Figure 2 shows that the NICS test (2a) results in deviations from the conventional PGS (embryonic lesion biopsy) test results (2b) when the conventional IVF embryos are tested for interference with excess sperm contamination.

Fig. 3 shows that when a normal IVF embryo is detected using the technical scheme of the present invention, the result of the NICS test (3a) is accurate due to the exclusion of interference, which is consistent with the conventional PGS (embryonic lesion biopsy) test result (3b).

Figure 4 shows that when an abnormal IVF embryo is detected using the technical scheme of the present invention, the result of the NICS test (4a) is accurate due to the exclusion of interference, and is consistent with the conventional PGS (embryonic lesion biopsy) test result (4b).

detailed description

After long-term extensive and in-depth research, the inventors discovered through accidental screening and testing for the first time that after removing the zona pellucida, the embryo was cultured in a culture medium of 20-30 microliters using a single embryo culture system. After taking out a small amount of culture solution for detection, it was found that the obtained chromosomal abnormality detection result has extremely high precision, and the risk of interference of excess sperm and maternal granule cell contamination can be largely eliminated. On the basis of this, the inventors completed the present invention.

IVF embryo

IVF refers to in vitro fertilization (IVF), specifically in vitro fertilization combined with embryo transfer technology (IVF), also known as IVF, which refers to the removal of eggs and sperm, placed in a test tube to fertilize, and then Embryonic Precursor - A technique in which a fertilized egg is transplanted back into the mother's uterus to develop into a fetus.

ICSI

ICSI (Intracytoplasmic sperm injection) is the intracytoplasmic single sperm microinjection technique, which is the second generation of "test tube baby". This technique uses a microscopic operating system to inject a single sperm into an egg for fertilization.

Cellophane tape

The outer part of the egg has a coat, the main component of which is glycoprotein, which is secreted by egg cells or other cells. In mammals, this genus is called zona pellucida, which protects the egg and prevents the entry of heterologous sperm.

Detection method

The present invention provides a depleted medium after culturing a blastocyst (ie, The culture solution separated from the culture system is subjected to genetic detection to identify whether the embryonic chromosome is abnormal.

In the present invention, the method of performing genetic detection on the "lean" culture solution (that is, the culture solution separated from the culture system) after culturing the blastocyst is not particularly limited, and can be detected by a conventional method, such as two. Generation sequencing, nucleic acid chips, immunofluorescence detection, fluorescent PCR detection, first generation sequencing, third generation sequencing, mass spectrometry detection, or a combination thereof.

In an embodiment, the detecting method comprises the following steps:

In a preferred embodiment, the step (b) further comprises the step (c):

(i) mixing the culture solution with the lysate to obtain a first mixture containing the blastocyst culture solution and the lysate;

Sample preparation and detection

The invention also provides a method for preparing a genetic test sample or a chromosome test sample, comprising the steps of:

In a preferred embodiment, the method further comprises the step (ii): performing genetic testing on the test sample to identify whether the embryonic chromosome is abnormal.

Transparent belt removal

Methods of removing the zona pellucida include, but are not limited to, mechanical stripping, laser removal, Tyrode liquid digestion, and the like. For a specific method of removing the zona pellucida, see Reference 1 (A comparison of four different techniques of assisted hatching, 2002).

In the present invention, the effect of removing the zona pellucida by the mechanical stripping method is better, and the resulting embryo The tire is more complete and can eliminate the interference of various substances. Therefore, the present invention selects a mechanical stripping method to remove the transparent belt. Specifically, in the present invention, the transparent belt is removed by a conventional mechanical stripping method and a commercially available apparatus.

The main advantages of the invention include:

(1) In the present invention, after completely removing the zona pellucida, the embryo is cultured in 20-30 μl of the culture solution, and a small amount of the culture solution is detected, and the obtained chromosomal abnormality detection result is extremely high. Accuracy can greatly eliminate the risk of interference from excess sperm and maternal granule cell contamination.

(2) The present invention adopts a single embryo culture system, that is, only one embryo is cultured in one culture liquid droplet, and the detection result of the chromosomal abnormality obtained by the system is more accurate.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually in accordance with conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight and parts by weight.

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

Qiagen Protease: crude protease, purchased from Kaijie.

General method

Obtainment of culture fluid

(1) fertilized eggs (in the volunteer couple, the eggs are obtained from the female volunteers, the sperm are obtained from the male volunteers, and the in vitro fertilization, that is, the fertilized eggs are obtained) are cultured in vitro until the third day to the sixth day, preferably On the 4th day, when the embryo develops to the morula, the blastomere is completely fused, and a large gap is created between the embryo and the zona pellucida.

(2) Using a mechanical stripping method (such as PIZO and other mechanical methods) to remove the transparent band around the embryo, and gently suck the embryo with a 135 micron stripping needle to completely separate the zona pellucida from the embryo.

(3) Transfer at least 3 embryos with clear zona pellucida to at least 3 30 microliter blastocyst culture droplets (guaranteed only one embryo in one droplet), each droplet gently rinsing and rinsing 2-3 After that, the embryos were transferred to 37 ° C, 5% CO ₂ , 5% O ₂ , and the culture was continued to the blastocysts in overnight equilibrated droplets (30 μl).

(4) Transfer the blastocyst culture medium (ie, the spent culture solution) in step (3) (about 24-30 μl in volume) to 30 μl of lysate (30 mM Tris-Cl, pH 7.8, 2 mM EDTA, 20mM KCl, 0.2% Triton In X-100), mark the sample name on the collection tube with a marker. Centrifuge in a microcentrifuge for 30 seconds. Samples can be immediately taken to the next whole genome amplification step or cryopreserved at -20 °C or -80 °C.

Whole genome amplification of trace DNA in blastocyst culture

(1) The mixture of the blastocyst culture solution and the lysate was melted at room temperature.

(2) Add 1 μl of lyase (12.5 μg/ml Qiagen Protease) to the tube and mix by pipetting up and down.

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

(4) The tube was placed at 80 ° C for 10 minutes to inactivate the lyase.

(5) Remove 10 μl of the lysate from the tube and add it to a PCR reaction tube.

(6) 60 μl of the preamplification mixture was added to the PCR reaction tube.

(7) The PCR reaction tube is placed in a PCR instrument for pre-amplification, and the thermal cycle program is:

(8) Add 60 μl of the amplification mixture to the PCR reaction tube (the composition of the amplification mixture is 10-25 mM Tris-HCl, 5-25 mM (NH ₄ ) ₂ SO ₄ , 5-30 mM KCl, 0.5 -5 mM MgSO ₄ , 0.1%-20% DMSO and 0.05-5% Triton X-100. Preferably, the composition of the amplification mixture is 15 mM Tris-HCl, 15 mM (NH ₄ ) ₂ SO ₄ , 20 mM KCl, 1 mM MgSO ₄ , 5% DMSO and 2% Triton X-100).

(9) The PCR reaction tube is placed in a PCR machine for exponential amplification, and the thermal cycle program is:

NICS method

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

(4) The tube was placed at 80 ° C for 10 minutes to inactivate the lyase.

(6) 60 μl of the preamplification mixture was added to the PCR reaction tube.

(10) The amplified DNA product is subjected to second generation sequencing by a conventional method to identify whether the chromosomal state of the embryo is normal.

Sample processing and sample acquisition

Sample: Five fertilized eggs were taken from five pairs of volunteer couples (including eggs from female volunteers, sperm from male volunteers, and in vitro fertilization to obtain fertilized eggs).

Sample processing and sample acquisition:

(1) The fertilized egg is cultured in vitro from day 3 to day 6, preferably, on day 4, that is, the embryo develops to the morula, and when the blastomere is completely fused, the embryo and the zona pellucida are larger at this time. Void.

(4) Take 30 μl (volume) (replenished) culture solution (ie, spent culture) (volume about 24-30 μl) and transfer it to 30 μl of lysate (30 mM pH 7.8) Tris-Cl, 2mM EDTA, 20mM In KCl, 0.2% Triton X-100), a sample of the culture medium from which the zona pellucida was removed was obtained, centrifuged in a microcentrifuge for 30 seconds, and subjected to the next whole genome amplification step or frozen at -20 ° C or -80 ° C.

Example 1

Using the detection method of the present invention (ie, exfoliating the embryonic zona pellucida at the morula stage, and then changing the liquid culture), the blastocyst biopsy method and the blastocyst culture solution are respectively performed on the two IVF embryos (samples C and D) The method of detection assesses its chromosomal status. The blastocyst culture solution detection method obtained the same results as the blastocyst cell detection method.

The results of the second-generation sequencing data showed that in the C sample, the blastocyst culture solution detection method (Fig. 3a) and the blastocyst cell detection method (Fig. 3b) detected normal chromosomes. In the D sample, both the blastocyst culture solution detection method (Fig. 4a) and the blastocyst cell detection method (Fig. 4b) detected the deletion of the chromosome 2 partial segment and the amplification of the chromosome 4 partial segment.

The fertilized egg of the C sample is implanted into the uterus of the husband and the mother, and the embryo with normal chromosomal state and normal development can be obtained.

Comparative example 1

The blastocyst culture and blastocyst biopsies of an ICSI embryo were separately examined using the conventional NICS method (i.e., only liquid exchange, without zona pellucida culture) to assess their chromosomal status. The results of blastocyst culture fluid assays were evaluated using blastocyst biopsy cell assay results as a standard.

The results of the second-generation sequencing showed that compared with the blastocyst biopsy cell detection method (Fig. 1b), the blastocyst culture solution detection method (Fig. 1a) showed granulocyte contamination, resulting in false positive amplification of the X chromosome, and No. 6 Amplification variants of chromosomes and chromosome 16 cannot be detected.

Comparative example 2

The blastocyst culture and blastocyst biopsies of an IVF embryo were separately examined using the conventional NICS method (i.e., only for liquid exchange, without zona pellucida culture) to assess their chromosomal status. The results of blastocyst culture fluid assays were evaluated using blastocyst biopsy cell assay results as a standard.

The results of the second-generation sequencing showed that compared with the blastocyst biopsy cell detection method (Fig. 2b), the blastocyst culture solution detection method (Fig. 2a) showed sperm contamination, resulting in false positive X chromosome copy number deletion, chromosome 1 deletion. Failure to check out, the results of the two methods are inconsistent.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

An in vitro non-therapeutic method for detecting chromosomal abnormalities of an embryo using a blastocyst culture solution, comprising the steps of:

(a) providing a culture solution from a blastocyst culture system, wherein the embryo in the blastocyst culture system has removed the zona pellucida before the culture, and the embryo is cultured in the blastocyst culture system for 3-6 days, Preferably, after 4 days, the culture solution is separated from the culture system;

(b) performing genetic testing on the culture solution to identify whether the embryonic chromosome is abnormal.
The method according to claim 1, wherein said blastocyst culture system is a single embryo culture system, and said single embryo culture system has no zona pellucida, and contains 10-100 microliters, preferably, 15 - 50 microliters, more preferably, 20-35 microliters of culture medium.
The method according to claim 1, wherein the volume of the culture solution separated in the step (a) is 30-100%, preferably 40-, of the volume of the culture solution in the single embryo culture system. 100%, more preferably, 50-100%, optimally, 80-100%.
The method of claim 1 wherein said step (b) further comprises the step (c):

(i) mixing the culture solution with the lysate to obtain a first mixture containing the culture solution and the lysate;

(ii) mixing the first mixture with a lyase, incubating, inactivating the lyase, thereby obtaining a cleavage product;

(iii) performing genomic analysis on the lysate to identify whether the embryonic chromosome is abnormal.
The method according to claim 4, wherein 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 combination.
The method of claim 4 wherein said lysate is selected from the group consisting of a Tris buffer, a chelating agent, a hydrochloride, a nonionic surfactant, or a combination thereof.
The method according to claim 4, wherein in the step (i), the volume ratio of the culture solution to the lysate is from 1:10 to 10:1, preferably from 1:5 to 5:1. More preferably, 1:2-2:1.
The method according to claim 4, wherein the lyase is at a concentration of 1 to 25 μg/ml, preferably 5 to 20 μg/ml, more preferably 10 to 15 μg/ml.
A method for preparing a genetic test sample or a chromosome test sample, comprising the steps of:

(i) Providing a culture system containing the zona pellucida-removing embryo, which is cultured for 3-6 days, preferably, after 4 days, the liquid is separated from the culture system, that is, the test sample.
The method of claim 9, wherein the method further comprises the step of (ii): performing genetic testing on the obtained test sample to identify whether the embryonic chromosome is abnormal.