Method for performing DedscRRBS-PGS analysis on embryo culture solution

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...

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 the material, Trying to achieve non-invasive testing, but still only analyzing the chromosome status;

[0010] (3) Whether it is single-cell whole-genome bisulfite sequencing (scWGBS) or single-enzyme digestion (Msp I)-based reduced expression bisulfite sequencing (scRRBS), the genome coverage rate is relatively low, and the effective data obtained The amount is relatively small; and when the PGS analysis is performed, the SD value is large, and a clear result of chromosome aneuploidy cannot be obtained;

[0011] (4) There are many studies on methylation in embryos and TE cells, but there is no formal report on the methylation of DNA from embryos in culture medium

Images