CN111778326A - Gene marker combination for endometrial receptivity assessment and application thereof - Google Patents
Gene marker combination for endometrial receptivity assessment and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of biological medicines, and discloses a gene marker combination for evaluating endometrial receptivity. The invention provides a nucleic acid composition, which comprises DNA and RNA corresponding to 515 endometrium receptivity related characteristic genes. The invention also discloses application of the gene marker combination in preparation of endometrial receptivity detection products. The invention comprises a molecular biology method for evaluating the endometrial receptivity based on the gene expression condition of uterine biopsy sample cells, the method can accurately and effectively judge the endometrial receptivity period, and the success rate of in-vitro fertilization or embryo implantation can be obviously achieved through accurate guidance of infertility patients.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a gene marker combination for endometrial receptivity assessment and application thereof.
Background
According to the data issued by the China Association of population and the State family planning Commission, the infertility rate of couples of childbearing age in China is increased from 2.5-3% before 20 years to about 12-15% in recent years, and the number of patients exceeds 5000 ten thousand. 5000 ten thousand patients have 50% of women, 40% of men and 10% of couples with both common reasons. And the number of infertile couples is increasing with the reasons of environmental pollution, late childbearing age, life pressure, etc.
Studies have shown that changes in endometrial receptivity may be one of the major factors leading to reproductive disorders. Endometrial receptivity refers to the ability of the endometrium to accept an embryo and refers to the endometrium being in a state that allows for embryo localization, adhesion, and invasion. This period is also known as the window of implantation (WOI) of the endometrium, and corresponds to 20 to 24 days of the menstrual cycle or 6 to 8 days after ovulation in normal adult women. However, in the field of in vitro fertilization or embryo transfer, women who have repeated transfer failures or suffer from secondary infertility often have time nodes of WOI that shift forward or backward, and if judged by normal calculation methods, risk of transfer failure. Therefore, the accurate and effective evaluation method of the endometrial receptivity has important significance for diagnosis and treatment of patients with reproductive disorders.
Currently, the evaluation means of endometrial receptivity in clinical practice is mainly morphological evaluation. The evaluation indexes include three main categories: a. ultrasound modalities such as endometrial thickness, echogenicity, and blood flow; b. tissue biopsy morphology such as gland size, interstitial density and blood vessel abundance; c. endometrial pinocytosis. However, morphological evaluation cannot make an accurate judgment of WOI, and the technology is theoretically controversial.
Therefore, in the face of the increasingly severe infertility onset situation caused by the deterioration of social environment, an accurate and effective endometrial receptivity assessment method is urgently needed in the field, can help patients find the embryo planting window period, and has great significance for improving the success rate of in vitro fertilization/embryo transplantation and improving the social population structure.
Disclosure of Invention
The invention aims to solve the technical problems that the prior art can not accurately and effectively evaluate the endometrial receptivity and is difficult to help the infertility patients to find the optimal WOI.
In order to solve the above problems, in one aspect of the present invention, there is provided a nucleic acid composition comprising DNA, RNA corresponding to an endometrial receptivity-related characteristic gene; the specific characteristic genes related to the evaluation of endometrial receptivity are shown in Table 1.
TABLE 1515 genes related to endometrial receptivity assessment
The invention provides a combination product comprising a plurality of polynucleotides or fragments thereof, wherein the polynucleotides can be differentially expressed to different degrees in different periods of endometrial receptivity (such as early stage, middle stage and later stage of receptivity), and the polynucleotides comprise characteristic genes related to endometrial receptivity evaluation with serial numbers 1-515.
Preferably, the nucleic acid composition further comprises a hybridization probe of the characteristic gene related to endometrial receptivity.
Preferably, the nucleic acid composition further comprises an amplification primer or a detection probe of the characteristic gene related to endometrial receptivity.
The nucleic acid composition of the present invention can be prepared by a method of artificially synthesizing according to the sequence.
The endometrial receptivity related characteristic gene can be obtained by a method of artificial sequence synthesis, and can also be obtained by screening according to the following steps: obtaining endometrium or uterine cavity fluid as a sample, and sequencing after constructing a gene library; obtaining high-quality gene sequencing data through quality control analysis; calculating the TPM value of each gene and transcription of each sample; carrying out SVM prediction; screening to obtain the characteristic gene related to endometrial receptivity.
In another aspect of the present invention, there is also provided a use of the above-mentioned combination product comprising a plurality of polynucleotides or fragments thereof for preparing a product for detecting endometrial receptivity, i.e., a use of the nucleic acid composition for preparing a medicament for detecting endometrial receptivity.
Preferably, the medicament comprises a product for detecting endometrial receptivity by RNA sequencing, gene chip or real-time quantitative PCR.
Preferably, said application comprises the following steps:
extracting nucleic acid in the sample;
aligning nucleic acids in a sample with the nucleic acid composition; and/or
Hybridizing the nucleic acid in the sample with the detection probe of the nucleic acid composition.
Preferably, the use further comprises the step of amplifying the nucleic acid in the sample.
The product for detecting the endometrial receptivity comprises: the product for detecting and evaluating the endometrial receptivity by RNA sequencing, gene chip and real-time quantitative PCR.
The product for detecting the endometrial receptivity by RNA sequencing comprises a messenger RNA sequence of a characteristic gene sequence related to the endometrial receptivity and a corresponding detection primer and/or probe.
The product for detecting the endometrial receptivity by using the gene chip comprises a hybridization probe of a characteristic gene sequence related to the endometrial receptivity.
The product for detecting the endometrial receptivity by using the real-time quantitative PCR comprises a messenger RNA sequence for specifically amplifying a characteristic gene sequence related to the endometrial receptivity and a corresponding detection primer and/or probe.
In another aspect of the invention, a kit for endometrial receptivity detection is also provided, the kit comprising a messenger RNA sequence specific for a characteristic gene sequence associated with endometrial receptivity and corresponding detection primers and/or probes.
Preferably, the kit comprises an amplification primer or a detection probe of the characteristic gene related to endometrial receptivity.
Preferably, the kit further comprises a positive control of the characteristic gene related to endometrial receptivity, or a negative control, an amplification reagent, a detection reagent, instructions and the like.
Preferably, the kit of the invention can be used for detecting the expression condition of the characteristic gene sequence related to the endometrial receptivity in the biopsy sample cells of the detected object, then a model is constructed by combining the information of the up-regulation or the down-regulation of the gene expression with algorithms such as SVM (support vector machine), Random Forest, KNN (K nearest neighbor) and the like, the endometrial receptivity of the detected object is evaluated, and the actual WOI time node of the patient is accurately judged, so that the success rate of in vitro fertilization/embryo planting is improved.
In the present invention, the term "characteristic gene marker" means a gene useful for the assessment of endometrial receptivity, and the change in the expression level of this group of genes is closely linked to the endometrial receptivity period.
In the present invention, the term "polynucleotide" refers to a gene and a genomic fragment obtained by artificial synthesis and processing, and is a single-stranded or double-stranded nucleotide compound.
In the present invention, the term "biopsy sample cells" refers in particular to endometrial biopsy tissue, uterine lavage fluid and uterine fluid cells and exfoliated cell samples thereof.
In the present invention, the term "primer" means an oligonucleotide capable of initiating primer extension synthesis product in the presence of an appropriate reaction when paired with one strand of DNA. To maximize amplification efficiency, the primer is preferably single-stranded. The length of the primer depends on many factors, including: application area, temperature used, template reaction conditions, other reagents and primer sources. The skilled person can design the primers autonomously according to the breast cancer signature genes and specific requirements of the present invention.
The term "probe" refers to an oligonucleotide molecule capable of binding to all or part of a particular nucleotide sequence. The probe may be directly or indirectly labeled.
The invention is based on the quantitative analysis of the whole gene expression profile of somatic cells, compares the gene expression difference of the early stage of endometrial receptivity, the receptivity period and the late stage of receptivity by the quantitative analysis of the gene expression status, screens out 515 characteristic gene combinations related to the receptivity of endometrium from the whole gene expression profile by a machine learning method, quantitatively detects the relative expression quantity data of characteristic genes in the biopsy sample cells of a detected person by technologies such as RNA sequencing, gene chips, fluorescence quantitative PCR and the like, constructs an endometrial receptivity evaluation model by adopting SVM, obtains a decision score (decision score) of the sample, and determines the receptivity period according to the decision score. The invention is mainly used for evaluating the endometrial receptivity based on the molecular biology technology, and the characteristic gene marker has high sensitivity and strong specificity, and the accuracy is over 80 percent. Moreover, the invention can draw materials for multiple times and detect multiple endometrial receptions, greatly improves the accuracy and has wide application prospect.
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In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a flow chart of sample testing and data analysis in the present invention.
FIG. 2 is the screening process of the gene markers of the endometrial receptivity related characteristics in the invention.
FIG. 3 is a cluster map (a) and a sample correlation map (b) of the gene markers of the endometrial receptivity-related characteristics in the present invention.
FIG. 4 is a GO enrichment map (a) and a KEGG enrichment map (b) of the related characteristic gene markers of endometrial receptivity in the invention.
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Example 1 sample Collection, transport, quality control
1. The subject or volunteer is sampled by a gynecologist or professional qualified technician, the sample comprising not less than 1mg of endometrial tissue and not less than 4uL of uterine fluid. Immediately after the biopsy is completed, the sample is added to a storage tube containing RNA storage solution (Qiagen RNA Later) and stored at-20 ℃ or-80 ℃.
2. Sample transportation: the samples were transported to the designated laboratory using ice bags or dry ice.
3. Sample quality control: the quality control content comprises sample information, sample integrity, measured temperature when the sample is received and the like.
4. Specific sample collection and grouping are shown in table 2.
TABLE 2 number of samples of endometrial receptions at different periods
Example 2RNA sequencing
1. Total RNA extraction was performed on endometrial biopsy samples.
2. RNA integrity was analyzed using Agilent2100, and RIN >7.0, 28S/18S >1.2 were passed. The concentration and purity of RNA are accurately and quantitatively detected by using the Qubit, the OD260/OD280 is between 1.8 and 2.2, and the RNA is qualified when the extraction amount is more than 2 ug.
3. The mRNA is enriched and purified by poly (A) purification or rRNA removal purification.
4. RNA Library construction was performed using the KAPA Stranded RNA-Seq Library Preparation Kit.
5. Library fragment distribution was analyzed using Agilent2100 and library concentration was accurately quantified using Qubit.
6. Sequencing on an illumina Hiseq2000 machine.
Example 3 endometrial receptivity assessment-related characteristic Gene marker screening
1. And (3) data quality control: and (3) after the original data are downloaded, performing quality control on the sequencing result through fastp software, wherein the quality control standard is that the read length is not less than 75, and the rest parameters are default (the original data are called RawData, and the data after quality control are called CleanData).
2. Ribosome data deletion: the rRNA content can reflect the quality of the database establishment to a certain extent, so before comparing the reference genome, the sorterna is used for comparing the CleanData to the ribosome database, the read on the comparison is removed, and the default screening parameter of the de-rRNA CleanData is obtained and is 1 e-5.
3. Reference genome alignment: comparing de-rRNA CleanData to a reference genome or transcriptome, counting the comparison efficiency, and performing randomness analysis and insert analysis according to the comparison result, wherein the comparison software is STAR, and default parameters are used for comparison.
4. Quantification of gene expression: based on the comparison result, the gene or transcriptome level expression quantification is carried out by using featurepopulations software, and a gene quantitative expression matrix is obtained after the quantification value is standardized by adopting a TPM (trans Per Million) algorithm.
5. Differential expression analysis: based on the quantitative results, differential expression analysis was performed using edgeR to screen for differentially expressed genes with a screening threshold of FDR < ═ 0.05 and | log2FC | >1.
6. And (3) feature screening: and (3) performing feature screening on the gene expression matrix based on a machine learning method to obtain candidate endometrial receptivity related gene markers.
7. Constructing a model: model training is carried out on the candidate markers by respectively using SVM, Randomforest and KNN models based on 10 x 10-fold cross validation, an optimal prediction model is constructed based on parameter tuning, and test results are shown in Table 3.
From the test results, it can be judged that the SVM model constructed based on 166 gene sets has very good performance, the test accuracy reaches 85.1%, and the 166 gene sets are shown in Table 4.
TABLE 3 model training of different machine learning algorithms for candidate markers
Note:CI:cumulative importance,0.5 means CI not less than 0.5；T:modelprediction accuracy for training set；V:model prediction accuracy forvalidation sets；RF:Random Forest；SVM:support vector method；KNN:k-NearestNeighbor。
TABLE 4 166 Gene sets for SVM model construction
Example 4 endometrial receptivity assessment and receptivity prediction
1. Grouping samples: 60 endometrium biopsy samples of 20 female patients with infertility between 23 and 39 years old or multiple embryo transfer failures are selected as study objects. All study individuals underwent sample collection after intervention in the hospital's natural or artificial period. All clinical study procedures were ethical reviewed by the hospital and informed consent was signed with the volunteer informed of the true condition.
2. Samples were collected, transported, and quality controlled according to the procedure of example 1.
3. The samples were RNA sequenced according to the procedure of example 2.
4. Original off-machine data of 60 clinical samples are obtained, gene expression conditions of biopsy sample cells are analyzed, and then an expression quantity matrix of endometrial receptivity related characteristic gene markers is output. The expression matrix is substituted into the prediction model of the invention, so that the endometrial receptivity period of the individual to which the sample belongs can be accurately judged, the embryo implantation is guided, the pregnancy outcome is recorded, and the detailed results are shown in table 3. According to clinical follow-up data, the embryo implantation clinical cases guided by the technology of the invention find out proper embryo planting periods and succeed in pregnancy.
Table 560 clinical specimens endometrial receptivity prediction results and pregnancy outcome
Therefore, the prediction of the method is consistent with the pregnancy result, and the accuracy rate reaches more than 80%.
The above description is only for the specific embodiments of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present disclosure should be covered within the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.
Claims (10)
1. A nucleic acid composition comprising DNA or RNA corresponding to a gene characteristic of endometrial receptivity;
the related characteristic gene of endometrial receptivity comprises one or more of the following genes:
2. the nucleic acid composition of claim 1, wherein said endometrial receptivity associated signature genes comprise at least the following genes:
3. the nucleic acid composition of claim 1, further comprising a hybridization probe for a gene characteristic of endometrial receptivity.
4. Use of a nucleic acid composition according to claim 1, for the preparation of a medicament for the detection of endometrial receptivity.
5. The use of claim 4, wherein the medicament comprises a product for detecting endometrial receptivity using RNA sequencing, gene chip or real-time quantitative PCR.
6. The use according to claim 4, characterized in that said use comprises the following steps:
extracting nucleic acid in the sample;
aligning nucleic acids in a sample with a nucleic acid composition of claim 1; and/or
Hybridizing nucleic acids in the sample to detection probes of the nucleic acid composition of claim 1.
7. The use of claim 6, further comprising the step of amplifying nucleic acids in the sample.
8. The use of claim 6 or 7, wherein said use further comprises: the nucleic acid composition of claim 1 expressing up-or down-regulated information and modeling in conjunction with support vector machine, random forest, or K-nearest neighbor algorithms to assess endometrial receptivity of a subject.
9. A kit for detecting endometrial receptivity, which is characterized in that the kit comprises the amplification primer or the detection probe of the endometrial receptivity-related characteristic gene as claimed in claim 1.
10. The kit for detecting endometrial receptivity as claimed in claim 9, wherein said kit further comprises a positive control for the gene for a characteristic related to endometrial receptivity.
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| CN112662758A (en) * | 2021-02-07 | 2021-04-16 | 成都西囡妇科医院有限公司 | miRNA marker related to auxiliary diagnosis of endometrial receptivity and application thereof |
| CN113621695A (en) * | 2021-04-13 | 2021-11-09 | 深圳市锦欣医疗科技创新中心有限公司 | Marker for endometrial receptivity of RIF patient, application thereof and detection kit |
| CN113621695B (en) * | 2021-04-13 | 2024-04-09 | 深圳市锦欣医疗科技创新中心有限公司 | Marker of endometrial receptivity of RIF patient, application of marker and detection kit |
| CN113416774A (en) * | 2021-06-08 | 2021-09-21 | 上海交通大学医学院附属仁济医院 | Use of long non-coding RNA HOXA11-AS AS biomarker |
| CN114517232A (en) * | 2022-03-15 | 2022-05-20 | 苏州亿康医学检验有限公司 | Method, model and marker for judging endometrial receptivity in noninvasive mode |
| CN115873937A (en) * | 2022-08-15 | 2023-03-31 | 苏州市立医院 | Biomarker for predicting occurrence of repeated planting failure and application thereof |
| CN115873937B (en) * | 2022-08-15 | 2023-07-14 | 苏州市立医院 | Biomarker for predicting occurrence of repeated planting failure and application thereof |
| CN115976200A (en) * | 2023-03-21 | 2023-04-18 | 北京大学第三医院（北京大学第三临床医学院） | Kit for evaluating endometrial receptivity-related recurrent abortion risk and application thereof |
| CN115976200B (en) * | 2023-03-21 | 2023-06-30 | 北京大学第三医院（北京大学第三临床医学院） | Kit for evaluating recurrent abortion risk related to endometrial receptivity and application of kit |
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Denomination of invention: Gene marker combinations and their applications for evaluating endometrial receptivity Effective date of registration: 20231201 Granted publication date: 20211022 Pledgee: Industrial Bank Co.,Ltd. Shanghai Changning sub branch Pledgor: BASETRA MEDICAL TECHNOLOGY CO.,LTD. Registration number: Y2023310000796 |