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miRNA molecular markers for predicting the quality of early embryos in human assisted reproductive technology and their applications

A molecular marker, embryo technology, applied in the field of molecular biology, can solve the problem of no prediction, and achieve the effect of great social and economic benefits

Active Publication Date: 2022-03-15
华中科技大学同济医学院生殖医学中心
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on miRNA molecular markers in the culture medium of in vitro cultured embryos that predict the quality of early embryos in human assisted reproductive technology

Method used

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  • miRNA molecular markers for predicting the quality of early embryos in human assisted reproductive technology and their applications
  • miRNA molecular markers for predicting the quality of early embryos in human assisted reproductive technology and their applications
  • miRNA molecular markers for predicting the quality of early embryos in human assisted reproductive technology and their applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] [Example 1] Sample collection and grouping

[0025] Embryo culture fluid samples were obtained from the Reproductive Medicine Center of Tongji Medical College of Huazhong University of Science and Technology, the Reproductive Center of Wuhan Union Medical College Hospital, and the Reproductive Center of Wuhan Tongji Hospital. The research was reviewed and approved by the Ethics Committee and informed consent was obtained from the patients. In the process of in vitro fertilization, oocytes are usually placed in 30-50ul droplets containing 5,000-10,000 sperm, and the sperm and eggs are co-cultured for 18-20 hours to allow in vitro fertilization to occur, observe the shape and number of pronuclei, and double-pronucleus The normal embryos were placed in the commercial embryo culture medium G1-plus (Vitrolife, Sweden), which is specially used for the growth and development of cleavage stage embryos. After continuous culture for 3 days, the culture medium used for IVF-ET embry...

Embodiment 2

[0026] [Example 2] Extraction of total RNA and miRNA library construction

[0027]Total RNA was extracted from the embryo culture fluid specimens of group A and group B respectively, and the total RNA extracted was quantified and inspected by Nanodrop instrument (Thermo Company, USA). Add 3' and 5' adapters to the RNA respectively, amplify by RT-PCR, and recover the target band of 145-160 bp by electrophoresis to obtain the miRNA library.

Embodiment 3

[0028] [Example 3] RNA-Seq high-throughput sequencing to screen differential miRNAs

[0029] Qubit (Invitrogen, USA) quantified the miRNA library, used the Start cBot instrument for cluster generation, and then sequenced with Hiseq2500 (Illumnia, USA) to obtain raw data, and calculated miRNA expression. Use DESeq software to analyze the differential expression of the two groups of samples, calculate the expression level, the mean value within the group and the differential expression multiple between the groups, and further calculate the log2 (difference multiple) value, when the P value between the two groups is ≤0.05 and the log2 (difference multiple) )≥1, it is considered that the expression of the miRNA is different between the two groups. Through the above differential expression analysis of the sequencing data, combined with the GO clustering analysis of the specific biological functions of miRNA target genes, 18 differentially expressed miRNAs related to biological func...

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Abstract

The invention discloses a miRNA molecular marker for predicting early embryo quality in human assisted reproductive technology and its application. It belongs to the technical field of molecular biology. The inventors found that after IVF / ICSI-ET assisted reproductive therapy, the expression levels of hsa-miR-26b-5p and hsa-miR-21-5p in the in vitro embryo culture fluid of patients with successful pregnancy were significantly lower than those of those with failed pregnancy. patient. Evaluate the quality of embryos cultured in vitro by detecting its expression level in embryo culture medium, so as to pre-select high-quality embryos to reduce the number of embryos transferred during assisted reproductive therapy and improve the implantation rate of embryos, so as to reduce the probability of multiple pregnancy in patients and avoid The goal of patients undergoing assisted reproductive cycle treatment again not only has the advantages of non-invasive diagnosis, but also has greater social and economic benefits.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to specific miRNA molecular markers in embryo culture fluid for predicting the quality of early embryos in human assisted reproductive technology and applications thereof. Background technique [0002] Infertility is one of the most concerned reproductive health problems worldwide, affecting approximately 15% of couples of reproductive age. In recent years, the rapid development of assisted reproductive technology has brought many conveniences to the treatment of infertility. Assisted reproductive techniques (assisted reproductive techniques, ARTs) mainly include a series of in vitro operations on human germ cells (sperm and eggs) and early embryos, such as in vitro fertilization (in vitro fertilization, IVF), intracytoplasmic sperm injection , ICSI) and embryo transfer (embryo transfer, ET). The main goal of ARTs is to increase the pregnancy rate of single transfer and re...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888
CPCC12Q1/6888C12Q2600/178C12Q2600/158
Inventor 熊承良方芳李自立
Owner 华中科技大学同济医学院生殖医学中心
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