阅读说明：本技术 凋亡相关基因在反复种植失败中的应用 (Application of apoptosis-related gene in repeated planting failure ) 是由 曲迅 徐小飞 董召刚 南兆棣 于 2021-07-28 设计创作，主要内容包括：本发明提供凋亡相关基因在反复种植失败中的应用,属于生物医药和分子生物学技术领域。本发明通过从GEO数据库中调取子宫内膜RNA-seq相关数据,在GSE92324队列筛选出一系列差异基因,最终确定三个凋亡相关基因即CSRNP1、BIRC8和ELAPOR1的异常表达与反复种植失败紧密相关,且经过验证队列(GSE71835)进行验证,证明上述凋亡相关基因对反复种植失败具有良好的诊断价值,因此可作为反复种植失败的预测诊断标志物和潜在治疗靶点,从而在反复种植失败的诊断、治疗中发挥重要作用,具有良好的实际推广应用之价值。(The invention provides application of apoptosis-related genes in repeated planting failure, belonging to the technical field of biomedicine and molecular biology. According to the invention, a series of differential genes are screened in a GSE92324 queue by calling endometrial RNA-seq related data from a GEO database, and finally, the abnormal expressions of three apoptosis related genes, namely CSRNP1, BIRC8 and ELAPOR1 are determined to be closely related to the repeated planting failure, and the apoptosis related genes are proved to have good diagnostic value on the repeated planting failure through verification of a verification queue (GSE71835), so that the apoptosis related genes can be used as a prediction diagnostic marker and a potential treatment target point of the repeated planting failure, thereby playing an important role in diagnosis and treatment of the repeated planting failure and having good value of practical popularization and application.)

1. Application of apoptosis-related genes as markers in preparation of products for diagnosing, monitoring and/or predicting repeated planting failure;

wherein the apoptosis-related genes at least comprise any one or more of CSRNP1, BIRC8 and ELAPOR 1.

2. A product comprising a substance for detecting expression of apoptosis-related genes, said product being for use in diagnosing, monitoring and/or predicting recurrent plant failure;

wherein the apoptosis-related genes at least comprise any one or more of CSRNP1, BIRC8 and ELAPOR 1.

3. The product of claim 2, wherein the substance for detecting the expression of the apoptosis-related gene comprises a substance for detecting the expression level of the apoptosis-related gene by RT-PCR, real-time quantitative PCR, in situ hybridization, gene chip, and gene sequencing;

the product comprises a primer, a probe, a chip, a nucleic acid membrane strip, a preparation or a kit for detecting the expression level of the apoptosis-related gene in a sample to be detected.

4. The product of claim 2, wherein the samples to be tested are human samples and non-human samples;

preferably, the test sample comprises endometrial tissue and/or cells of the subject.

5. A system for diagnosing, monitoring and/or predicting recurrent planting failure, the system comprising:

i) an analysis module, the analysis module comprising: a detection substance for determining the expression level of a gene selected from the group consisting of apoptosis-related genes in a test sample of a subject, and;

ii) an evaluation module comprising: determining whether the subject has recurrent planting failure based on the apoptosis-related gene expression level determined in i);

wherein the apoptosis-related gene comprises any one or more of CSRNP1, BIRC8 and ELAPOR 1.

6. The system of claim 5, wherein the evaluation module of step ii) specifies an evaluation process comprising:

(ii) the expression level of the apoptosis-related gene in the test sample of the subject is up-regulated and/or down-regulated as compared to a reference, then the subject is or is candidate for a repeat planting failure patient; otherwise, the subject is not or is not candidate for a repeat planting failure patient.

7. The system of claim 5, wherein the test sample comprises endometrial tissue and/or cells of the subject.

8. A method for diagnosing, monitoring and/or predicting recurrent planting failure, the method comprising:

A) isolating a test sample from a subject;

B) detecting the expression level of the apoptosis-related gene in a sample to be tested of the subject, and;

C) comparing the expression level of the apoptosis-related gene in the sample with the expression level of the apoptosis-related gene in the reference;

wherein the apoptosis-related gene comprises any one or more of CSRNP1, BIRC8 and ELAPOR 1;

preferably, the expression level in the sample is up-regulated and/or down-regulated, then the subject is or is candidate for a repeat planting failure patient; otherwise, the subject is not or is not candidate for a repeat planting failure patient.

9. The application of the apoptosis-related gene as a target point in the treatment of the repeated planting failure and/or the screening of the medicines for the repeated planting failure;

the apoptosis-related gene comprises any one or more of CSRNP1, BIRC8 and ELAPOR 1.

10. A method of screening for a drug that fails to repeatedly plant, the method comprising:

a) treating a system expressing and/or containing an apoptosis-related gene with a candidate substance; setting a parallel control without candidate substance treatment;

b) detecting the expression level of said apoptosis-related gene in the system after step a) is completed; if the expression level of the apoptosis-related gene in the system treated with the candidate substance is significantly decreased and/or increased as compared to a parallel control, the candidate substance may serve as a candidate recurrent planting failure drug;

the apoptosis-related gene comprises any one or more of CSRNP1, BIRC8 and ELAPOR 1.

Technical Field

The invention belongs to the technical field of biomedicine and molecular biology, and particularly relates to application of apoptosis-related genes in repeated planting failure.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Recurrent Implantation Failure (RIF) means that an infertility patient undergoes multiple In Vitro Fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles and transplants multiple high quality embryos without embryo implantation or clinical pregnancy. The molecular mechanisms of embryo planting have not been elucidated so far, and the reasons are complex and involve several aspects, including in particular the following aspects: maternal factors include psychological factors, abnormalities of the anatomical structure of the reproductive system, abnormal development and function of endometrium, frequent uterine contraction, thrombophilia, abnormal immune function of maternal-fetal interface, etc.; factors in the embryo include defects in the genetic material of the embryo, hatching of the embryo and poor developmental potential; and other relevant factors (patient management, clinical management and quality control, laboratory management and quality control), etc. The occurrence of RIF may be due to a single factor or due to a combination of multiple factors, and there are some unknown reasons. Therefore, the exploration of the etiology and pathogenesis of RIF is the key to the prevention and treatment of RIF.

Apoptosis (apoptosis) is an important life process widely existing in the biological world, and is an active cell death process of multicellular animals for regulating the development of organisms, maintaining homeostasis and carrying out gene regulation. The apoptosis phenomenon is widely existed in the individual generation process and immune mechanism, and is closely related to anticancer, anti-aging, AIDS prevention and treatment, etc., which arouses more and more research interests of scientists. During molecular biological studies of apoptosis, a variety of genes have been found to be involved in the regulation of apoptosis, including the Bcl-2 family and Bax, p53, Fas and FasL, among others. However, at present, the research on the role of apoptosis-related genes in the repeated planting failure is still less, so that more effective markers for diagnosing and monitoring the repeated planting failure are needed to be searched, and a foundation is laid for the research and development of diagnosis and treatment of RIF and related medicines.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the application of the apoptosis-related gene in the repeated planting failure. The invention discovers for the first time that the expression levels of apoptosis-related genes such as CSRNP1, BIRC8 and ELAPOR1 are closely related to repeated planting failure, and the sensitivity and the specificity of the diagnosis of the apoptosis-related genes in RIF are higher, so that the apoptosis-related genes such as CSRNP1 can be used as a prediction diagnosis marker of the repeated planting failure, thereby completing the invention.

Specifically, the invention relates to the following technical scheme:

in a first aspect of the invention, there is provided the use of an apoptosis-related gene as a marker in the manufacture of a product for repeated plant failure diagnosis (aided diagnosis), monitoring and/or prognosis.

Wherein the apoptosis-related genes at least comprise any one or more of CSRNP1, BIRC8 and ELAPOR 1.

According to the invention, the research shows that apoptosis-related genes such as CSRNP1 and the like are highly expressed in endometrial cells of patients who fail to plant repeatedly. And further drawing a characteristic curve (ROC curve) of the operation of the testee and calculating the area under the curve (AUC), which indicates that the apoptosis-related genes such as CSRNP1 and the like have a diagnostic effect, and the diagnostic sensitivity and specificity of the apoptosis-related genes such as CSRNP1 and the like in repeated planting failure are high. Particularly, the combination of the three components has the area under the curve AUC of 0.917(0.615to 0.986), the sensitivity and the specificity of 100.0 (95% CI: 54.1-100.0) and 83.3 (95% CI: 36.1-97.2), and has extremely high diagnostic value.

In a second aspect of the invention, there is provided a product comprising a substance as described above for detecting expression of apoptosis-related genes for use in diagnosing (aiding diagnosis), monitoring and/or predicting recurrent plant failure.

The substance for detecting the expression of the apoptosis-related gene includes, but is not limited to, substances for detecting the expression level of the apoptosis-related gene by RT-PCR, real-time quantitative PCR, in situ hybridization, gene chip and gene sequencing.

The product comprises but is not limited to a primer, a probe, a chip, a nucleic acid membrane strip, a preparation or a kit for detecting the expression level of the apoptosis-related gene in a sample to be detected.

The test sample may be a human sample, and more specifically, the test sample includes endometrial tissue and/or cells of the subject.

In a third aspect of the present invention, there is provided a system for diagnosing (aiding diagnosis), monitoring and/or predicting recurrent plant failure, the system comprising:

i) an analysis module, the analysis module comprising: a detection substance for determining the expression level of the above apoptosis-related gene in a test sample of a subject, and;

ii) an evaluation module comprising: determining whether the subject has recurrent planting failure based on the apoptosis-related gene expression level determined in i);

the specific evaluation process of the evaluation module in the step ii) comprises the following steps:

(ii) the expression level of the apoptosis-related gene in the test sample of the subject is up-regulated and/or down-regulated as compared to a reference, then the subject is or is candidate for a repeat planting failure patient; otherwise, the subject is not or is not candidate for a repeat planting failure patient.

In a fourth aspect of the invention, there is provided a method for diagnosing (aiding diagnosis), monitoring and/or predicting recurrent plant failure, the method comprising:

A) isolating a test sample from a subject;

B) detecting the expression level of the apoptosis-related gene in a sample to be tested of the subject, and;

C) comparing the expression level of the apoptosis-related gene in the sample with the expression level of the apoptosis-related gene in the reference;

wherein, an up-regulation and/or down-regulation of the expression level in the sample compared to the level in the reference is indicative of or is a candidate for a repeat planting failure patient; otherwise, the subject is not or is not candidate for a repeat planting failure patient.

In a fifth aspect of the present invention, the apoptosis-related gene is provided as a target for use in treatment of recurrent implantation failure and/or screening of drugs for recurrent implantation failure.

In a sixth aspect of the present invention, there is provided a method for screening for a drug with recurrent planting failure, the method comprising:

a) treating a system expressing and/or containing said apoptosis-related gene with a candidate substance; setting a parallel control without candidate substance treatment;

b) detecting the expression level of said apoptosis-related gene in the system after step a) is completed; if the expression level of the apoptosis-related gene in the system treated with the candidate substance is significantly decreased and/or increased as compared to the parallel control, the candidate substance can be used as a candidate recurrent planting failure drug.

The beneficial technical effects of one or more technical schemes are as follows:

according to the technical scheme, a series of differential genes are screened in a GSE92324 queue by calling endometrial RNA-seq related data from a GEO database, abnormal expressions of three apoptosis-related genes, namely CSRNP1, BIRC8 and ELAPOR1 are finally determined to be closely related to repeated planting failure, and the apoptosis-related genes are proved to have good diagnostic value on the repeated planting failure through verification of a verification queue (GSE71835), so that the apoptosis-related genes can be used as a prediction diagnostic marker and a potential treatment target point of the repeated planting failure, play an important role in diagnosis and treatment of the repeated planting failure and have good value of practical popularization and application.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a graph of cluster analysis of differential apoptosis-related genes in GSE92324 cohort according to an embodiment of the present invention.

FIG. 2 is a graph of cluster analysis of differentially apoptotic-related genes in GSE71835 cohort according to an embodiment of the present invention.

FIG. 3 is a graph of differential apoptosis-related gene expression in GSE92324 cohorts according to the present invention.

FIG. 4 is a graph of differential apoptosis-related gene expression in GSE71835 cohort in accordance with an embodiment of the present invention.

FIG. 5 is a graph showing the diagnostic effect of the combination of CSRNP1, BIRC8 and ELAPOR1 on RIF in the examples of the present invention.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. The experimental procedures, if specific conditions are not indicated in the following detailed description, are generally in accordance with conventional procedures and conditions of molecular biology within the skill of the art, which are fully explained in the literature. See, e.g., Sambrook et al, "molecular cloning: the techniques and conditions described in the laboratory Manual, or according to the manufacturer's recommendations.

The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions not specified in the examples are specified, they are generally according to the conventional conditions, or according to the conditions recommended by the sales companies; materials, reagents and the like used in examples were commercially available unless otherwise specified.

The term "expression level" refers to the amount of a gene product present in vivo or in a sample at a particular time point. The expression level can be measured/quantified/detected, for example, by protein or mRNA expressed by the gene. The expression level can be quantified, for example, as follows: normalizing the amount of the gene product of interest present in the sample with the total amount (total protein or mRNA) of the same type of gene product in the same sample or reference sample (e.g., a sample obtained from the same individual at the same time or a fraction of the same size (weight, volume) of the same sample), or determining the amount of the gene product of interest/defined sample size (weight, volume, etc.). The expression level can be measured or detected by any method known in the art, such as a method for direct detection and quantification of a gene product of interest (e.g., mass spectrometry), or a method for indirect detection and measurement of a gene product of interest that generally works by binding the gene product of interest to one or more different molecules or detection devices (e.g., primers, probes, antibodies, protein scaffolds) specific for the gene product of interest. Also known to the skilled person is the determination of the level of gene copy, which also includes the determination of the absence or presence of one or more fragments (e.g. by nucleic acid probes or primers, such as quantitative PCR, Multiplex ligation-dependent probe amplification (MLPA) PCR).

The terms "indicator" and "marker" are used interchangeably herein and refer to a sign or signal of a condition or to monitor a condition. Such "disorder" refers to a biological state of a cell, tissue or organ, or to a health and/or disease state of an individual. The indicator may be the presence or absence of molecules including, but not limited to, peptides, proteins, and nucleic acids, or may be a change in the level or pattern of expression of such molecules in a cell, or tissue, organ, or individual. The indicator can be a sign of the occurrence, development or presence of a disease in an individual or of further progression of such a disease. The indicator may also be a sign of the risk of developing a disease in the individual.

The term "up-regulation", "increase" or "increase" of the level of an indicator means that the level of such indicator is reduced in a sample compared to a reference.

The term "down-regulation", "reduction" or "decrease" of the level of an indicator refers to a decrease in the level of such indicator in a sample compared to a reference.

The term "kit" as used herein refers to a collection of the above-mentioned components, preferably provided separately or in a single container. The container also preferably contains instructions for carrying out the method of the invention. Examples of these components of the kit and methods of use thereof have been given in the present specification. Preferably, the kit comprises the above components in a ready-to-use formulation. Preferably, the kit may additionally comprise instructions, such as a user's manual for adjusting the components (e.g., the concentration of the detection agent) and for interpreting the results of any assay with respect to the diagnosis provided by the methods of the invention. In particular, such a manual may comprise information for assigning the amount of a determined gene product to a diagnostic type. Details are found elsewhere in this specification. Furthermore, such user manual may provide instructions on the correct use of the kit components for determining the amount of the respective biomarker. The user manual may be provided in paper or electronic form (e.g., stored on a CD or CD ROM). The invention also relates to the use of said kit in any method according to the invention.

The term "system" as used herein relates to a system of devices comprising at least the above-mentioned devices operatively interconnected to allow a diagnosis to be made. Preferred means for determining the methylation state or amount of a gene product and means for making a comparison are disclosed above in connection with the methods of the invention. How the devices are operatively contacted will depend on the type of device included in the apparatus. For example, in the case of the application of a device for the automated determination of the methylation state or amount of a gene product, the data obtained by the automated operating device can be processed by, for example, a computer program to establish a diagnosis. Preferably, in this case, the apparatus is comprised in a single device. Thus, the device may comprise an analysis module for determining the methylation status or amount of a gene product in a sample and an evaluation module for processing the resulting data for diagnosis. Preferred detection devices are disclosed above in connection with embodiments relating to the methods of the present invention. In this case, the devices are effectively connected so that the user of the system combines the results of the determination of the quantities and their diagnostic values together owing to the instructions and explanations given in the manual. In such embodiments the device may be presented as a separate apparatus and preferably packaged together as a kit. Those skilled in the art will know how to contact the device without further inventive skill. Preferred devices are those that can be applied without the specific knowledge of a skilled clinician, such as test strips or electronic devices that only require loading of a sample. The results can be output as parametric diagnostic raw data, preferably given as absolute or relative quantities. It will be appreciated that these data will need to be interpreted by a clinician. However, expert system devices are also contemplated where the output contains processed diagnostic raw data, the interpretation of which does not require a specialized clinician. Further preferred devices comprise an analysis module/device (e.g. biosensor, array, solid support coupled to a ligand specifically recognizing a polypeptide, plasmon surface resonance device, NMR spectrometer, mass spectrometer, etc.) or an evaluation module/device as mentioned above according to the method of the invention.

In a specific embodiment of the invention, the apoptosis-related gene is used as a marker in the preparation of a product for repeated plant failure diagnosis (auxiliary diagnosis), monitoring and/or prediction.

Wherein the apoptosis-related genes at least comprise any one or more of CSRNP1, BIRC8 and ELAPOR 1; the kit can also comprise other related genes and/or proteins which can be used for diagnosis (auxiliary diagnosis), monitoring and/or prediction of repeated planting failure, and is beneficial to further improving the specificity and sensitivity of detection through the optimized combination of different markers, thereby providing support for a clinician to accurately diagnose the repeated planting failure and timely adopt a prevention and treatment scheme.

According to the invention, the research shows that apoptosis-related genes such as CSRNP1 and the like are highly expressed in endometrial cells of patients who fail to plant repeatedly. And further drawing a characteristic curve (ROC curve) of the operation of the testee and calculating the area under the curve (AUC), which indicates that the apoptosis-related genes such as CSRNP1 and the like have a diagnostic effect, and the diagnostic sensitivity and specificity of the apoptosis-related genes such as CSRNP1 and the like in repeated planting failure are high. Particularly, the combination of the three components has the area under the curve AUC of 0.917(0.615to 0.986), the sensitivity and the specificity of 100.0 (95% CI: 54.1-100.0) and 83.3 (95% CI: 36.1-97.2), and has extremely high diagnostic value.

In yet another embodiment of the present invention, there is provided a product comprising the above-mentioned substance for detecting apoptosis-related gene expression for use in diagnosis (diagnosis assistance), monitoring and/or predicting recurrent plant failure.

The substance for detecting the expression of the apoptosis-related gene includes, but is not limited to, substances for detecting the expression level of the apoptosis-related gene by RT-PCR, real-time quantitative PCR, in situ hybridization, gene chip and gene sequencing.

The product comprises but is not limited to a primer, a probe, a chip, a nucleic acid membrane strip, a preparation or a kit for detecting the expression level of the apoptosis-related gene in a sample to be detected.

The test sample can be a human sample and a non-human sample, and more particularly, the test sample comprises endometrial tissues and/or cells of a subject.

In yet another embodiment of the present invention, a system for diagnosing (aiding diagnosis), monitoring and/or predicting recurrent plant failure is provided, the system comprising:

i) an analysis module, the analysis module comprising: a detection substance for determining the expression level of the above apoptosis-related gene in a test sample of a subject, and;

ii) an evaluation module comprising: determining whether the subject has recurrent planting failure based on the expression level of the apoptosis-related gene determined in i).

The apoptosis-related genes comprise any one or more of CSRNP1, BIRC8 and ELAPOR1, and experiments prove that CSRNP1 and ELAPOR1 are abnormally up-regulated in patients with repeated planting failure, and BIRC8 is abnormally down-regulated in patients with repeated planting failure.

In still another embodiment of the present invention, the test sample comprises endometrial tissue and/or cells of the subject.

In still another embodiment of the present invention, the detection material includes, but is not limited to, materials for detecting expression levels of apoptosis-related genes in RT-PCR, real-time quantitative PCR, in situ hybridization, gene chip and gene sequencing.

In yet another embodiment of the present invention, the analysis module further comprises a substance for detecting other biomarkers that are suitable for detecting recurrent planting failure.

In another embodiment of the present invention, the specific evaluation process of the evaluation module includes:

(ii) the expression level of the apoptosis-related gene in the test sample of the subject is up-regulated and/or down-regulated as compared to a reference, then the subject is or is candidate for a repeat planting failure patient; otherwise, the subject is not or is not candidate for a repeat planting failure patient.

Where a "reference" can be a suitable control sample, e.g., a sample from a normal healthy subject that has no symptoms associated with recurrent graft failure and has no abnormal physiological and pathological findings, a reference can also be a sample from the same subject prior to exhibiting symptoms of the disorder or disease or prior to diagnosing recurrent graft failure. The reference can be a labeled sample, e.g., a sample comprising material or data from samples of healthy subjects who have no symptoms of recurrent planting failure, nor associated physiological and pathological findings.

The system for diagnosing (auxiliary diagnosis), monitoring and/or predicting recurrent plant failures of the present invention may be a virtual device as long as the functions of the analysis module and the evaluation module can be realized. The analysis module can comprise various detection reagent materials and/or detection instrument equipment and the like; the evaluation module may be any computing instrument, module or virtual device capable of analyzing and processing the detection result of the analysis module to obtain the evaluation condition of the recurrent planting failure morbidity risk, for example, various possible detection results and the corresponding morbidity risk condition may be formulated into a corresponding data chart in advance, and the detection result of the detection unit is compared with the data chart to obtain the evaluation result of the recurrent planting failure morbidity risk.

In yet another embodiment of the present invention, a method for diagnosing (aiding diagnosis), monitoring and/or predicting recurrent plant failure is provided, the method comprising:

A) isolating a test sample from a subject;

B) detecting the expression level of the apoptosis-related gene in a sample to be tested of the subject, and;

C) comparing the expression level of the apoptosis-related gene in the sample with the expression level of the apoptosis-related gene in the reference;

(ii) up-and/or down-regulation of expression levels in the sample compared to levels in a reference, then the subject is or is candidate for a repeat planting failure patient; otherwise, the subject is not or is not candidate for a repeat planting failure patient.

Where a "reference" can be a suitable control sample, e.g., a sample from a normal healthy subject that has no symptoms associated with recurrent graft failure and has no abnormal physiological and pathological findings, a reference can also be a sample from the same subject prior to exhibiting symptoms of the disorder or disease or prior to diagnosing recurrent graft failure. The reference can be a labeled sample, e.g., a sample comprising material or data from samples of healthy subjects who have no symptoms of recurrent planting failure, nor associated physiological and pathological findings.

In another embodiment of the present invention, the apoptosis-related gene is used as a target for treatment and/or screening of recurrent implantation failure drugs.

In another embodiment of the present invention, the recurrent medical treatment is a drug for preventing and/or treating recurrent medical treatment.

In another embodiment of the present invention, there is provided a method for screening for a drug with recurrent graft failure, the method comprising:

a) treating a system expressing and/or containing said apoptosis-related gene with a candidate substance; setting a parallel control without candidate substance treatment;

b) detecting the expression level of said apoptosis-related gene in the system after step a) is completed; if the expression level of the apoptosis-related gene in the system treated with the candidate substance is significantly decreased and/or increased as compared to the parallel control, the candidate substance can be used as a candidate recurrent planting failure drug.

In yet another embodiment of the present invention, the system may be a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

In yet another embodiment of the present invention, the cells in the cell system may be endometrial cells;

in yet another embodiment of the present invention, the tissue in the tissue system may be endometrial tissue;

in yet another embodiment of the present invention, the organ in the organ system may be placenta, uterus;

in another embodiment of the present invention, the animal in the animal system may be a mammal, such as rat, mouse, guinea pig, rabbit, monkey, human, etc.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Examples

1. Expression and clinical cohort

GEO database (https:// www.ncbi.nlm.nih.gov/GEO /): RIF data set GSE92324, RIF verification set GSE 71835.

Wherein, GSE92324 queue sample information:

1.1 conditions are included:

RIF group: age 20-40 years, past history of pregnancy (primary infertility), unsuccessful for at least 2 cycles of IVF or unsuccessful for at least 2 transplants of suboptimal embryos.

Control group: the egg donor treated with COS has fertility, and is a control group, i.e. female who has at least normal parturition 2 years before the group, and is a healthy egg donor with fertility (age 20-40 years).

1.2 exclusion conditions:

polycystic ovary syndrome, endometriosis, abnormal hormone levels, autoimmune patients, male infertility, while controls exclude women undergoing oral contraceptives or any hormone treatment prior to delivery or oocyte transfer.

1.3 protocol:

(1) all subjects (including RIF and control) had normal hormone levels (follitropin FSH, luteinizing hormone LH, estradiol E2, progesterone P, prolactin PRL, anti-mullerian hormone AMH), ultrasound showed a "triple line" in the intima, a thickness of > 8mm, and normal ovarian response in the previous IVF cycle.

(2) All subjects used a "long regimen" to down-regulate the pituitary gland, i.e. from day 21 of the previous menstrual cycle, the subcutaneous injection of GnRH-a class drugs (0.5mg/0.5ml) was initiated, and endogenous FSH and LH secreted by the pituitary gland were suppressed, thereby suppressing endogenous LH-surges and premature ovulation. On day 3 of the current menstrual cycle, HMG (75 IU each containing FSH and LH) is injected intramuscularly every day to promote follicular growth. Ovarian responsiveness is judged by monitoring the size of the follicles by ultrasound, and the drug dose is adjusted accordingly. When at least 3 follicles reach 18-20mm in diameter, intramuscular injection of HCG (10000IU) is administered to induce ovulation

The maturation of the ova should be comprehensively judged by combining the E2 level of the serum (each dominant follicle corresponds to the estrogen level of 250-300 pg/ml). Follicle puncture and ovum aspiration are carried out 34-36 hours after HCG trigger by ultrasonic monitoring, 4 days of progestogen (50mg/ml) is given to both groups after ovum aspiration to serve as corpus luteum support, and all embryos are frozen. The RIF group underwent embryo transfer in the next menstrual cycle.

(3) At the embryo implantation window, i.e., intramembranous biopsies 6-7 days after HCG intramuscular injection, tissues were taken for RNA-seq.

2. The mean gene expression levels of RIF patients in the GSE92324 cohort and a control group are compared, t test is carried out, and the difference genes are screened by taking the difference multiple more than 1.5 times and the p value less than 0.05 as threshold values to obtain 1497 difference expression genes.

3. Comparing with the apoptosis-related genes obtained from GO database (https:// genentology. org /) to obtain 11 differentially expressed genes (AEN, AIFM1, BIRC3, BIRC8, CSRNP1, CSRNP3, DDIAS, DNASE2B, ELAPOR1, ENDOG and HRK) belonging to the apoptosis-related genes, the results are shown in FIG. 1 and FIG. 3.

4. The endometrial RNA-seq data were continuously downloaded from the GEO database (https:// www.ncbi.nlm.nih.gov/GEO /), 6 cases of Repetitive Implantation Failure (RIF) and control (GSE71835) were used as validation cohorts, the expression of 11 apoptosis-differential genes were analyzed, and further screened to yield 3 differentially expressed genes (BIRC8, CSRNP1, ELAPOR1), see FIG. 2 and FIG. 4.

The diagnostic value of ROC analysis is shown in Table 3, the area under the combined curve of the three is 0.917(0.615to 0.986), the sensitivity and specificity are 100.0(54.1-100.0) and 83.3(36.1-97.2), and the result is shown in FIG. 5, which shows that the kit has good diagnostic effect and high diagnostic sensitivity and specificity.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.