CN-119464472-B - Marker for predicting copy number variation of egg cells and application thereof

Abstract

The invention discloses a marker for predicting copy number variation of an egg cell and application thereof, wherein the marker comprises a combination of CLEC11A, P HB, EFEMP2, IL32, FTL, FLNA, COL A3, ACPP and APOO proteins. By adopting the marker, the expression level of the secretory protein of the granulosa cells can be determined through the transcriptome sequencing of the granulosa cells, and the genome CNV condition of the corresponding ovum cells can be deduced. The detection object of the technical scheme is waste granulosa cells in the assisted reproduction technology, so that the method does not have any influence on oocytes, is noninvasive, is used for detecting transcriptome and methylation groups from single cell level, has the characteristic of high flux, and can reflect the difference between COC at single cell level due to the fact that all granulosa cells and oocytes are paired one by one.

Inventors

• SONG SHI
• ZHANG CONG
• YANG MING
• YAN LIYING
• QIAO JIE

Assignees

• 北京大学第三医院（北京大学第三临床医学院）

Dates

Publication Date

20260508

Application Date

20241016

Claims (4)

1. 1. Use of a combination of protein markers in granulosa cells, CLEC11A, P HB, EFEMP2, IL32, FTL, FLNA, COL A3, ACPP and APOO protein, wherein CLEC11A, P HB, EFEMP2, IL32, FTL and FLNA are low-expressed and COL6A3, ACPP and APOO are high-expressed, to predict that a copy number abnormality exists in an egg cell corresponding to the granulosa cell, for the preparation of a diagnostic reagent for predicting an egg cell copy number abnormality.
2. 2. The use according to claim 1, the diagnostic reagent being for assessing the expression level of the protein marker combination CLEC11A, P HB, EFEMP2, IL32, FTL, FLNA, COL6A3, ACPP and APOO in granulosa cells.
3. 3. The use according to claim 1, wherein the diagnostic reagent is for assessing female fertility.
4. 4. The use according to claim 3, wherein the female is a female over 35 years old.

Description

Marker for predicting copy number variation of egg cells and application thereof Technical Field The invention belongs to the technical field of reproduction, and particularly relates to a marker for predicting copy number variation of egg cells and application thereof. Background Egg cell quality is a fundamental indicator for the assessment of fertility in women. Unlike other mammals, the proportion of abnormal human egg cell copy numbers is high, which is particularly evident in women of high childbearing age (generally women older than 35 years). An abnormal chromosomal copy number of an egg will directly lead to abnormal embryo development after fertilization, leading to pregnancy failure. The clinical auxiliary reproduction technology firstly obtains the fertilized eggs which can be transplanted by selecting healthy egg cells through in vitro fertilization, so that the chromosome copy number of the egg cells is effectively predicted, and the elimination of abnormal egg cells is critical to the success or failure of the auxiliary reproduction technology. There are studies currently on the availability of chromosomal abnormality information for egg cells or early embryo by high throughput sequencing of polar bodies and PGTs, but the procedures are complex and expensive. In addition, research reports that the follicular microenvironment during the development process of the egg cells has an important influence on the development of the egg cells, wherein the granular cells are in direct contact with the egg cells in a space structure, and signal factors and nutrient substances secreted by the granular cells directly act on the egg cells, so that the method has a key effect on the normal development and maturation of the egg cells. Abnormal granulosa cells will cause imbalance in their secreted signal factors, which in turn affect the quality of the egg cells. In Anderson,R.A.,et al."Cumulus gene expression as a predictor of human oocyte fertilisation,embryo development and competence to establish apregnancy."Reproduction 138.4(2009):629-637, researchers first clarified the feasibility of using granulosa cells to assess the developmental potential of an egg. Experimental study 674 COC samples were obtained from 75 women involved in ICSI, and granulosa cells were further isolated for analysis of gene expression by quantitative RT-PCR. The study shows that the egg cells corresponding to the GREM1 are more suitable for the subsequent ICSI and transplantation (49% vs 33%, P < 0.02), and the egg cells corresponding to the BDNF with lower expression have higher normal fertilization rate. Due to the classical qPCR assay adopted by the authors, only six genes (HAS 2, BDNF, GREM1, PTGS2, TNFAIP6 and PTX 3) were supported, with low throughput and few potential targets that could be screened. In addition, the authors themselves indicated that GREM1 expression in granulosa cells was not significantly related to pregnancy rate, except that GREM1 expression was clearly responsible for embryo transfer rate and cryopreservation options, and the method in this article was not related to predicting egg Copy Number Variation (CNV). In Assidi,M.,et al."Biomarkers of human oocyte developmental competence expressed in cumulus cells before ICSI:a preliminary study."Journal of Assisted Reproduction&Genetics 28.2(2011):173-188, the authors first established that the morphological properties of granulosa cells can be used to reflect the mass of egg cells. These morphologically good COCs are associated with their pregnancy outcome. The differential expression genes in the granulosa cells corresponding to the successfully pregnant and the pregnant failure egg cells are further determined by a gene expression chip method, potential granulosa cell markers capable of predicting the quality and the pregnancy ending of the egg cells are screened, and the expression levels of DPP8, HIST1H4C, UBQLN1, CALM1, NRP1 and PSMD6 in the granulosa cells are further verified by qPCR and can be used as markers for evaluating the development potential of the egg cells. The paper also predicts the development potential of the egg cells through the granular cells, adopts a detection method of a high-flux gene expression chip, solves the problem of small number of genes which can be potentially screened, but the detection index of the research on the development potential of the egg cells is still small, the quality of the egg cells is evaluated on one side only from pregnancy or not, the physiological state of the egg cells is also ambiguous, the paper does not propose a method for detecting the CNV of the egg cells through the granular cells, and the molecular mechanism of the influence of specific granular cells on the function of the egg cells is not clarified. Therefore, how to obtain a marker for predicting the abnormal egg cell copy number as an evaluation index of the fertility of clinical females is still a technical problem to be solved by the