Method for performing DedscRRBS-PGS analysis on embryo culture solution

A culture solution and pre-embryo implantation technology, applied in the fields of biomedicine and molecular cell biology, can solve the problems of inability to provide more reference for embryo development potential, embryo damage, and injury to embryos, etc.

Inactive Publication Date: 2019-04-23
北京中科遗传与生殖医学研究院有限责任公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] (1) Biopsy sampling has high requirements on embryo operation technology. Even if the operation is good, it will cause some damage to the embryo. If the operation is wrong, it is likely to damage the embryo and cause serious consequences; In some cases, a small number of 3-5 cells may not fully represent the embryo, resulting in misjudgment;
[0009] (2) The traditional PGS only evaluates from the chromosome state, ignoring the very important epigenetic changes in the early embryo, and cannot provide more reference for a more comprehensive evaluation of the embryonic developmental potential; CN105368936A uses the waste liquid after embryo culture as th

Method used

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  • Method for performing DedscRRBS-PGS analysis on embryo culture solution
  • Method for performing DedscRRBS-PGS analysis on embryo culture solution
  • Method for performing DedscRRBS-PGS analysis on embryo culture solution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1, DedscRRBS-PGS sequencing analysis of chromosome status and DNA methylation status

[0083] The single sperm injection embryo samples were taken, and the corresponding blastocyst trophoblast biopsy cells and blastocyst culture medium were subjected to PGS and DedscRRBS-PGS sequencing to analyze the chromosome status and DNA methylation status. The specific operations were as follows:

[0084] 1. Acquisition of blastocyst culture medium:

[0085] The specific operation is carried out by the embryo laboratory of the assisted reproduction center of the hospital according to the standard process:

[0086] Qualified MⅡ stage human mature egg cells were selected (obtained by the CITIC Xiangya Reproductive and Genetic Specialist Hospital with the knowledge of the provider), and the granulosa cells attached to the egg cells were completely removed as much as possible, and then healthy human sperm were selected (with the knowledge of the provider, obtained by (obtaine...

Embodiment 2

[0122] Embodiment 2, the groping of different enzyme cutting selections

[0123] The method is basically the same as in Example 1, except that the restriction sites used for library construction are MspI, XmaI (NEB, R0180L), TaqI (Thermo Scientific TM , ER0671) for various combinations:

[0124] The enzyme digestion combination includes MspI / XmaI / TaqI, MspI / TaqI and MspI single enzyme digestion.

[0125] The results of the combined sequencing data of various restriction enzymes are shown in Table 1. image 3 In order to statistically analyze the sequencing results of multiple enzyme digestion libraries in Table 1, the genome comparison rate, redundancy ratio, and genome coverage ratio per M (million) data volume all reflect that under the same sequencing throughput, effective The ability of the data; the SD value represents the standard deviation obtained by establishing a baseline based on normal cells. The smaller the SD value, the closer to the standard baseline, and the mo...

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Abstract

The invention discloses a method for performing DedscRRBS-PGS analysis on an embryo culture solution. The invention provides a method for performing simplifying representative bisulfite sequencing andchromosomal aneuploidy status analysis on the embryo culture solution, and the method comprises the steps of 1) constructing a DNA library by using a blastocyst culture solution as a sample, and in the process of constructing the DNA library, using two restriction enzymes to enrich CpG sites by digesting genomic DNA of the blastocyst culture solution; 2) conducting CT conversion on the DNA library with bisulfite to obtain a conversion product; 3) using the conversion product for preparing a methylation sequencing library, following by high-throughput sequencing, and analyzing DNA methylationand a chromosomal aneuploidy status based on a sequencing result. The blastocyst culture solution is used as a raw material, a double enzyme digestion simplifying representative bisulfite sequencing technology is adopted, and the development potential of an embryo is comprehensively evaluated from multiple angles.

Description

technical field [0001] The invention relates to the fields of biomedicine and molecular cell biology, in particular to a method for DedscRRBS-PGS analysis of embryo culture fluid. Background technique [0002] Test-tube baby technology was born in 1978. It is a medical invention with cross-age significance to the history of human reproduction. It is considered as a "milestone in the development of modern medicine". In 2010, its founder Robert G. Edwards won the Nobel Prize in Medicine and Physiology . Over the past 40 years, millions of test-tube babies have been born around the world, bringing good news to countless families with difficult fertility. [0003] Test-tube baby technology involves multiple disciplines such as obstetrics and gynecology, andrology, reproductive physiology, genetics, embryology and developmental biology, and must also rely on laboratory technology and clinical disciplines. At present, the main basis for screening embryos is morphological criteri...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/6869C12Q1/6888C40B50/06
CPCC12Q1/6806C12Q1/6869C12Q1/6888C40B50/06C12Q2525/191C12Q2535/122
Inventor 费嘉张癸荣金治平蔡旺庭王云云刘威达
Owner 北京中科遗传与生殖医学研究院有限责任公司
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