Biomarker for evaluating endometrial aplastic disorder, product and application

Abstract

The invention provides a biomarker for evaluating endometrial aplastic, a product and application, and belongs to the technical field of biology. Wherein the biomarker is microRNA-21-5p (micro Ribonucleic Acid-21-5p). The biomarker for evaluating endometrial aplastic provided by the invention has the advantages that the expression quantity of the biomarker in the endometrium of a normal pregnant woman and the expression quantity of the endometrium of a thin endometrium (TE) patient are obviously different; and an efficient, rapid, high-specificity and high-sensitivity detection and evaluation means can be provided for early discovery and diagnosis of TE patients, targeted intervention and embryo transplantation guidance.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a biomarker, product and application for evaluating endometrial regeneration disorder. Background Art

[0002] In the biomedical field, with the development of science and technology, biological detection technology has become an important tool for disease diagnosis and treatment. Biological detection technology covers a wide range of means and methods, aiming to achieve early diagnosis, progression monitoring and efficacy evaluation of diseases by detecting biomarkers. These technologies include a variety of high-throughput analysis methods such as genomics, transcriptomics, proteomics, metabolomics, and various biosensors and detection platforms developed based on these methods. The development of biological detection technology has not only greatly improved the sensitivity and specificity of disease detection, but also promoted the development of personalized medicine, making it possible to provide precise treatment for specific patients and specific conditions.

[0003] In the field of assisted reproductive technology (ART), although this technology has brought hope to many infertile patients, its success rate is still limited by many factors. Among them, thin endometrium (TE) is one of the key factors affecting the success rate of ART. Existing studies and clinical guidelines show that the incidence of thin endometrium is quite high in infertile women, and the ART failure rate caused by it cannot be ignored. At present, the evaluation of TE mainly relies on morphological indicators, such as ultrasound detection of endometrial thickness and pathological analysis of endometrial biopsy. Although these methods can provide information on the status of the endometrium to a certain extent, they have certain limitations.

[0004] Existing technologies have obvious deficiencies in the detection and treatment of thin endometrium. First, although ultrasound detection is a non-invasive, intuitive and accurate evaluation method, it can only measure the thickness of the endometrium and cannot provide an in-depth understanding of its functional state and pathophysiological mechanisms. Secondly, although endometrial biopsy can provide objective information on tissue cell morphology, it is an invasive operation and difficult to perform frequently, especially for patients with refractory TE. It is even more difficult to obtain endometrial samples. In addition, existing drug treatment regimens are generally not effective for refractory TE and cannot effectively improve the receptivity of the endometrium. Existing evaluation methods and treatment strategies cannot accurately find effective therapeutic targets, resulting in unsatisfactory management and treatment of TE.

[0005] In summary, existing technologies have many deficiencies in the detection and treatment of TE, including: the inability to non-invasively and accurately evaluate the functional status of the endometrium; limited application of invasive detection methods; poor efficacy of existing treatment options for refractory TE; and lack of clear molecular mechanisms and effective therapeutic targets. These problems have seriously restricted the success rate of ART and the treatment effect of patients, and new technical means and strategies are urgently needed to improve the diagnosis and treatment of thin endometrium. Summary of the invention

[0006] To solve the above problems, the present invention provides a biomarker for evaluating endometrial regenerative disorders, wherein the biomarker is: microRNA-21-5p;

[0007] Preferably, the AccID of the biomarker is hsa-miR-21-5p.

[0008] In addition, to solve the above problems, the present invention also provides a product, including the biomarkers for evaluating endometrial regenerative disorders as described above.

[0009] Preferably, the product includes a reagent, a test kit, a test paper, a chip or an instrument.

[0010] In order to solve the above problems, the present invention also provides a use of the above-mentioned biomarker in the preparation of a product for evaluating endometrial regenerative disorders.

[0011] Preferably, the evaluation method comprises:

[0012] Using the product to detect the expression amount of the biomarker in the product in the exosomes derived from intrauterine fluid of normal fertile women as the normal expression amount, and deriving the corresponding evaluation reference threshold value according to the normal expression amount;

[0013] Using the product to detect the expression amount of the biomarker in the exosomes derived from the intrauterine effusion of the TE patient to be tested, as the target expression amount;

[0014] An evaluation result is obtained based on the target expression amount and the evaluation reference threshold.

[0015] Preferably, obtaining the evaluation result according to the target expression amount and the evaluation reference threshold value includes:

[0016] comparing the target expression amount with the evaluation reference threshold;

[0017] If the target expression amount is not less than the evaluation reference threshold, the evaluation result is negative;

[0018] If the target expression amount is less than the evaluation reference threshold, the evaluation result is positive;

[0019] Preferably, the evaluation reference threshold is 1.06.

[0020] Preferably, the biomarker is screened by the following method:

[0021] RNA-seq technology was used to detect the expression levels of all microRNAs in the exosomes of the proliferative uterine effusion in the normal fertility group and the TE patient group, and the results were obtained.

[0022] According to the measurement results, the markers to be screened in the TE patient group are screened based on the absolute value difference of the expression amount in the endometrium, and the biomarker is screened out from the markers to be screened.

[0023] Preferably, the RNA-seq technique is used to detect the expression levels of all microRNAs in the exosomes of the proliferative uterine effusion in the normal fertile group and the TE patient group, and the measurement results obtained include:

[0024] The uterine fluid of the proliferative phase of normal fertile women and TE patients is collected respectively as samples of the normal fertile group and the TE patient group;

[0025] Separating and extracting samples from the normal fertility group and the TE patient group to obtain exosomes;

[0026] Performing morphological identification on the exosomes;

[0027] Extracting the exosomes to obtain a total RNA sample;

[0028] The genes of all markers in the total RNA samples of the normal fertility group and the TE patient group were respectively subjected to microRNA-seq assay to obtain the assay results including the expression level of each marker.

[0029] Preferably, the method of screening the markers to be screened in the TE patient group based on the absolute value difference of the expression amount in the endometrium according to the measurement results, and screening the biomarker from the markers to be screened includes:

[0030] Based on the determination results, screening out markers whose absolute expression difference fold is greater than or equal to 2 times as target markers;

[0031] The target marker is analyzed using a bioinformatics analysis method, and the biomarker is obtained according to the analysis result.

[0032] Preferably, the bioinformatics analysis method comprises:

[0033] At least one of differential gene GO functional analysis, KEGG pathway analysis and target gene prediction analysis.

[0034] The present invention provides a biomarker, product and application for evaluating endometrial aplastic disorder. The biomarker is: microRNA-21-5p. The biomarker for evaluating endometrial aplastic disorder provided by the present invention has a significant difference in the expression level of the endometrium of normal fertile women and TE patients, and can provide an efficient, fast, highly specific and sensitive non-diagnostic detection and evaluation method for early detection and diagnosis of TE patients, targeted intervention and guidance of embryo transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a schematic diagram of a method for evaluating the use of biomarkers in the preparation of products for evaluating endometrial aplastic disorders in the embodiments of the present application;

[0036] Figure 2 This is a schematic diagram of a screening method for the use of biomarkers in the preparation of products for evaluating endometrial aplastic disorders in the embodiments of the present application;

[0037] Figure 3 This is a schematic flow chart of the refinement of step S300 in the screening method for the use of biomarkers in preparing products for evaluating endometrial aplastic disorder in the embodiments of the present application;

[0038] Figure 4 This is a heat map of differentially expressed genes of exosome microRNA derived from uterine effusion of normal fertile women and TE patients in Example 1 of the present application;

[0039] Figure 5 This is a statistical diagram of the differentially expressed genes of the physical markers in Example 1 of the present application.

[0040] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0041] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] In the embodiment of the present application, a biomarker for evaluating endometrial aplastic disorder is provided, wherein the biomarker is: microRNA-21-5p;

[0043] Furthermore, the AccID of the biomarker is hsa-miR-21-5p.

[0044] As mentioned above, microRNA-21-5p (miR-21-5p) is a microRNA molecule that belongs to the non-coding RNA family and is widely present in eukaryotic organisms. It plays an important regulatory role in a variety of biological processes, including cell growth, differentiation, apoptosis, and metabolism. Specifically, miR-21-5p regulates gene expression by binding to the mRNA molecules of target genes, inhibiting their translation or promoting their degradation.

[0045] The AccID of miR-21-5p is hsa-miR-21-5p, where "hsa" stands for Homo sapiens, indicating that this is a miRNA molecule found in humans. Current studies have shown that miR-21-5p is abnormally expressed in a variety of diseases, such as cancer, cardiovascular disease, and inflammation, and is therefore considered an important biomarker.

[0046] In the examples of this application, microRNA-21-5p is used for the first time as a biomarker for evaluating endometrial aplastic disorders because the abnormal expression of miR-21-5p in endometrial cells affects cell proliferation, adhesion, angiogenesis, and fibrosis, which are essential for the health and function of the endometrium. Therefore, miR-21-5p is expected to be used as a diagnostic marker for TE. By detecting its expression level, the state of the endometrium can be non-invasively and accurately evaluated, and a new target for treatment can be provided.

[0047] In summary, the biomarkers for evaluating endometrial aplastic disorders provided in the examples of this application have significant differences in the expression levels of the endometrium of normal fertile women and TE patients, which can provide an efficient, fast, highly specific and sensitive non-diagnostic detection and evaluation method for early detection and diagnosis, targeted intervention and guidance of embryo transplantation of TE patients. As an important biomarker, miR-21-5p will have broad application prospects in the diagnosis, treatment, repair and correlation research of TE, especially in assisted reproductive technology, and its value is even greater in the research and application of TE.

[0048] In addition, a product is provided in an embodiment of the present application, comprising the biomarker for evaluating endometrial regenerative disorder as described above.

[0049] Furthermore, the product includes a reagent, a test kit, a test paper, a chip or an instrument.

[0050] As mentioned above, reagents refer to chemical substances used in biological or chemical experiments. When detecting biomarkers such as miR-21-5p, reagents may include various reagents used to extract, purify and detect RNA. For example, common reagents include lysate, washing solution, reverse transcriptase, nucleic acid amplification reagents (such as PCR reagents), etc.

[0051] As mentioned above, the test kit is a set of products containing multiple reagents and tools designed for specific biological detection tasks. For the evaluation of endometrial aplastic disorders and the detection of miR-21-5p, the kit may include RNA extraction reagents, cDNA synthesis reagents, qPCR reagents, standards, positive and negative controls, etc. The kit provides a systematic and standardized operating process to facilitate users to perform detection.

[0052] As mentioned above, a test strip is a portable detection tool that usually contains a specific reaction area and displays the test results through color changes or other visual signals. Test strips for miR-21-5p may use specific probes or antibodies to capture and detect target miRNAs, providing a quick and easy means of detection.

[0053] As mentioned above, a biochip is a small device that integrates multiple detection units and can detect multiple biomarkers simultaneously. The chip used for miR-21-5p detection may contain a large number of probe arrays to detect the presence and expression level of the target miRNA through hybridization technology. Chip technology has the characteristics of high throughput, high sensitivity and high specificity.

[0054] The above-mentioned instruments refer to the equipment used to perform biological detection experiments. These instruments may include real-time quantitative PCR instruments (qPCR), nucleic acid sequencers, microarray scanners, nano flow cytometers, etc. For the detection of miR-21-5p, qPCR instruments are often used to quantitatively analyze its expression level, sequencers are used for high-throughput analysis, and microarray scanners are used to detect signals on biochips.

[0055] These products play an important role in the field of biological testing. By providing various technical means, they help researchers and clinicians accurately evaluate the condition of endometrial regenerative disorders and improve the accuracy of diagnosis and treatment.

[0056] In addition, the present application also provides an example of the use of the above-mentioned biomarker in the preparation of a product for evaluating endometrial regenerative disorders.

[0057] Furthermore, the evaluation method is used for non-diagnostic purposes. This non-diagnostic evaluation is used to draw probabilistic conclusions about correlation. It is not for the direct purpose of obtaining disease diagnosis results or health status, but is used to further explore and study the correlation between the status of TE patients and the status of normal fertile women. The evaluation results obtained cannot directly reflect the diagnosis results or health status of the subjects, but need to be further comprehensively evaluated in combination with other clinical indicators.

[0058] refer to Figure 1 , the method for evaluating a product for evaluating endometrial regeneration disorder comprises:

[0059] Step S1, using the product to detect the expression amount of the biomarker in the product in the exosomes derived from intrauterine fluid of normal fertile women as the normal expression amount, and deriving the corresponding evaluation reference threshold value according to the normal expression amount;

[0060] As mentioned above, this step is used to determine the normal expression level and the reference threshold for evaluation. By detecting the expression level of miR-21-5p in the uterine fluid of normal fertile women, a normal baseline expression level is established. This baseline level is used for subsequent comparison to determine the condition of TE patients.

[0061] Specifically, a group of uterine effusion samples from normal fertile women can be collected. The expression of miR-21-5p in these samples is detected using the product (such as a reagent, a kit, a test paper, a chip or an instrument), thereby obtaining a group of miR-21-5p expression data of normal fertile women. The mean and standard deviation of these data are determined by statistical analysis, and an evaluation reference threshold (usually the mean ± 1 or 2 standard deviations) is established.

[0062] Among them, statistical analysis can use software tools (such as SPSS, R language) to calculate the mean and standard deviation and determine the evaluation reference threshold.

[0063] Step S2, using the product to detect the expression amount of the biomarker in the exosomes derived from the intrauterine effusion of the TE patient to be tested, as the target expression amount;

[0064] The above steps are used to determine the target expression level, and detect the expression level of miR-21-5p in the intrauterine fluid of TE patients as the target expression level for comparison with the baseline level.

[0065] Specifically, uterine effusion samples of TE patients to be tested are collected, and the product is used to detect the expression level of miR-21-5p in these samples, thereby obtaining the expression data of miR-21-5p in TE patients as the target expression level.

[0066] Among them, ensure the consistency of experimental conditions and methods to ensure the comparability of results.

[0067] Step S3, obtaining an evaluation result according to the target expression level and the evaluation reference threshold.

[0068] By comparing the target expression level and the evaluation reference threshold, the evaluation results of the endometrial regeneration status of TE patients were obtained.

[0069] The target expression levels of TE patients were compared with the normal assessment reference threshold, thereby determining whether TE patients had endometrial regeneration disorder and quantifying the extent of the disorder.

[0070] It should be noted that the expression levels of different biomarkers in the endometrium of TE and normal fertile women are very different, so the expression level cannot be judged by simply detecting the absolute value of the expression level of a certain differential gene through conventional techniques.

[0071] The use of the biomarkers provided in the examples of the present application in the preparation of products for evaluating endometrial aplastic disorders can evaluate the endometrial regeneration status of TE patients through this evaluation method and provide certain correlation information. The advantages of this method are its non-invasiveness, accuracy and repeatability. The expression level of the biomarker miR-21-5p is used to achieve early evaluation, which provides a basis for subsequent comprehensive evaluation based on other clinical indicators and further treatment.

[0072] For further reference, Figure 2 , the step S3, obtaining an evaluation result according to the target expression amount and the evaluation reference threshold, comprises:

[0073] Step S31, comparing the target expression level with the evaluation reference threshold;

[0074] Step S32, if the target expression amount is not less than the evaluation reference threshold, the evaluation result is negative;

[0075] Step S33, if the target expression amount is less than the evaluation reference threshold, the evaluation result is positive;

[0076] Furthermore, the evaluation reference threshold is 1.06.

[0077] In the above, the target expression amount of the subject is compared with the normal evaluation reference threshold. Referring to Table 1, if the target expression amount is greater than or equal to the evaluation reference threshold, the evaluation result is negative, and a negative evaluation result indicates that the endometrial regeneration may be in a normal state, or the subject's endometrial regeneration is in a normal state with a high probability; if the target expression amount is less than the evaluation reference threshold, the evaluation result is positive, which may indicate endometrial regeneration disorder, or the subject has a high probability of endometrial regeneration disorder.

[0078] Table 1. Correspondence between the comparison results of the evaluation reference threshold and the evaluation results

[0079] NO. Is it less than the reference threshold? Evaluation results 1 yes Positive 2 no Negative

[0080] In the embodiments of the present application, the biomarker microRNA-21-5p has a specificity of significant difference in the expression level in the endometrium of normal fertile women and TE patients, and a comprehensive evaluation is performed through statistics. The difference in expression level is used as an evaluation indicator to evaluate the endometrial regeneration and repair status of the subjects. This can provide an efficient and fast evaluation method for TE patients, so that TE patients can further combine other clinical indicator data according to the evaluation results to achieve early detection and diagnosis, targeted intervention and guidance of embryo transplantation. The evaluation method in this application provides an efficient, fast, highly specific and sensitive detection and evaluation method.

[0081] For further reference, Figure 3 , the biomarker is screened by the following method:

[0082] Step S100, using RNA-seq technology to detect the expression levels of all microRNAs in the exosomes of the proliferative uterine effusion in the normal fertility group and the TE patient group, and obtain the measurement results;

[0083] As mentioned above, RNA sequencing technology (RNA-seq) is a high-throughput sequencing technology used to determine the sequence information and expression levels of RNA molecules in a sample. This technology can provide a detailed view of the transcriptome (i.e., the collection of all actively transcribed RNAs in cells under specific conditions), including mRNA, non-coding RNA (such as lncRNA, miRNA, etc.) and other types of RNA molecules.

[0084] As mentioned above, RNA sequencing (RNA-seq) technology was used to comprehensively detect and quantify the expression levels of all miRNAs contained in exosomes of intrauterine fluid in normal fertile women and TE patient groups, providing basic data for subsequent screening work.

[0085] Specifically, we first collect uterine fluid samples from normal fertile women and TE patients, extract exosomes from the samples, extract RNA from the exosomes, use RNA-seq technology to sequence the extracted RNA, and detect the expression levels of all miRNAs in the samples, thereby obtaining the expression spectrum data of all miRNAs in the two groups (normal fertile group and TE patient group), including the expression level of each miRNA.

[0086] RNA-seq technology has high throughput and high sensitivity, and can comprehensively and accurately detect the expression levels of all miRNAs in a sample. In this step, comprehensive miRNA expression data is provided, which is helpful for comprehensive analysis and screening of miRNAs related to TEs.

[0087] Step S200, according to the measurement results, based on the absolute value difference of the expression amount in the endometrium, the markers to be screened in the TE patient group are screened, and the biomarkers are screened from the markers to be screened.

[0088] As mentioned above, by comparing the miRNA expression levels in the normal fertility group and the TE patient group, the miRNAs that were significantly upregulated or downregulated in TE patients were screened out to find potential biomarkers.

[0089] Specifically, differential expression analysis can be performed on RNA-seq data to calculate the difference in expression of each miRNA in the two groups, and miRNAs with significant fold change in expression are selected as markers to be screened, thereby obtaining biomarkers.

[0090] This method of detecting miRNA expression and screening biomarkers by comparing normal fertile women and thin endometrium (TE) patients has high sensitivity and specificity, is systematic and comprehensive, and is objectively data-driven, ensuring the accuracy and reliability of the screening results. RNA-seq technology and differential expression analysis provide a comprehensive miRNA expression profile, and screen out significantly different miRNAs (such as miR-21-5p) through fold change and p-value. These markers have important diagnostic and evaluation value in clinical practice, and can achieve non-invasive, rapid and accurate detection, providing strong support for the diagnosis and treatment of TE.

[0091] The present invention uses RNA-Seq transcriptomics technology for the first time to screen endometrial regeneration and repair genes that are differentially expressed in exosomes derived from intrauterine fluid of normal fertile women and TE patients, opening up a new way for the screening of TE endometrial regeneration and repair markers, and providing an efficient, fast, highly specific and sensitive detection and evaluation method for the early detection and diagnosis of TE patients.

[0092] Furthermore, in step S100, the expression levels of all microRNAs in the exosomes of the proliferative uterine effusion in the normal fertile group and the TE patient group are detected by RNA-seq technology to obtain the measurement results, including:

[0093] Step S110, collecting uterine effusions of normal fertile women and TE patients during the proliferative period as samples of the normal fertile group and the TE patient group;

[0094] As mentioned above, the collection of uterine fluid in the proliferative phase of normal fertile women and TE patients can be carried out through a disposable sterile human catheter. Specifically, the uterine cavity can be flushed with sterile saline and the uterine fluid therein can be collected into a container.

[0095] Step S120, separating and extracting samples from the normal fertility group and the TE patient group respectively to obtain exosomes;

[0096] As mentioned above, separation and extraction can be performed by centrifugation, and purified exosomes can be obtained by gradient centrifugation.

[0097] Step S130, performing morphological identification on the exosomes;

[0098] As mentioned above, morphological identification can be used to identify the particle size, specific surface markers and integrity of the purified exosomes to ensure the quality of the product.

[0099] Among them, the identification methods must include NTA, WB and TEM at the same time.

[0100] As mentioned above, NTA (Nanoparticle Tracking Analysis): Nanoparticle tracking analysis is a technology based on the principles of Brownian motion and dynamic light scattering. NTA measures the size and concentration of nanoparticles by tracking their movement trajectory in liquid.

[0101] As mentioned above, WB (Western Blot): Protein blotting technology is used to detect the presence and expression level of specific proteins in a sample. WB separates proteins by electrophoresis and then transfers them to a membrane to identify the target protein through specific antibodies.

[0102] As mentioned above, TEM (Transmission Electron Microscopy): transmission electron microscopy technology, which uses electron beams to penetrate samples and form images to observe the ultrastructure of samples.

[0103] Step S140, extracting the exosomes to obtain a total RNA sample;

[0104] As described above, exosomes are extracted to obtain a total RNA sample. Specifically, the total RNA in the exosomes can be extracted using a miRNeasy Mini Kit, for example, including:

[0105] (1) Lysis of exosomes: Add lysis buffer and incubate at room temperature;

[0106] (2) Phase separation: Add chloroform, incubate at room temperature, centrifuge and collect the supernatant;

[0107] (3) RNA precipitation: add ethanol, centrifuge, and discard the supernatant;

[0108] (4) Washing RNA: Wash by centrifugation through RNeasy Mini columns;

[0109] (5) RNA elution: Pass the RNA through an RNeasy Mini column, add RNase-free water, centrifuge, and collect the liquid to obtain the total RNA sample.

[0110] Step S150, performing microRNA-seq measurement on the genes of all markers in the total RNA samples of the normal fertility group and the TE patient group, respectively, to obtain the measurement results including the expression level of each marker.

[0111] The genes of all markers in the samples of the normal fertility group and the TE patient group were respectively measured and analyzed by microRNA-seq, the difference multiples of the expression levels of each biomarker in the two groups of samples were compared, and the measurement results were obtained.

[0112] Further, the step S200, according to the measurement results, screens the markers to be screened in the TE patient group based on the absolute value difference of the expression amount in the endometrium, and screens the biomarker from the markers to be screened, including:

[0113] Step S210, based on the measurement results, screening out markers whose absolute expression difference fold is greater than or equal to 2 times as target markers;

[0114] Step S220, analyzing the target marker using a bioinformatics analysis method, and obtaining the biomarker according to the analysis result.

[0115] Furthermore, the bioinformatics analysis method includes:

[0116] At least one of differential gene GO functional analysis, KEGG pathway analysis and target gene prediction analysis.

[0117] As mentioned above, among the markers with an absolute expression difference greater than or equal to 2 times, bioinformatics analysis methods such as differential gene GO function analysis, KEGG pathway analysis and target gene prediction analysis were combined to reveal the characteristics of the biomarker and screen out a biomarker associated with thin endometrial aplasia.

[0118] As mentioned above, GO functional analysis of differentially expressed genes, namely Gene Ontology (GO) functional analysis, is a tool for annotating gene and protein functions. The GO database provides standardized descriptions of genes, which are divided into three main categories: biological process, cellular component, and molecular function.

[0119] The above-mentioned KEGG Pathway analysis, namely Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analysis, is a tool for understanding the role of genes and molecules in metabolic pathways and signal transduction pathways. The KEGG database collects a large number of biological pathway maps that describe cellular processes, metabolic pathways, signal transduction pathways, etc.

[0120] As mentioned above, target gene prediction analysis is to predict the relationship between miRNA and its regulated target gene. miRNA regulates gene expression by binding to target gene mRNA, inhibiting its translation or promoting its degradation.

[0121] The present invention is further described below by means of specific examples, but it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0122] Embodiment 1:

[0123] This example performed biomarker screening.

[0124] 1. Experimental samples:

[0125] According to the inclusion criteria, in this example, 4 normal fertile women and 4 TE patients were recruited, and the basic information of the patients is shown in the following table:

[0126] Table 2. Basic information of patients

[0127] Group Normal fertility group (n=4) TE group (n=4) p Age (years) 31.47±1.51 31.33±1.68 0.817 <![CDATA[BMI(kg / m 2 )]]> 21.16±1.12 21.47±1.73 0.891 FSH (mIu / mL) 5.21±1.95 4.94±2.01 0.477 LH (mIu / mL) 4.75±1.70 5.01±1.85 0.594 <![CDATA[E2(pg / mL)]]> 41.26±14.49 45.19±18.26 0.368 PRL (ng / mL) 12.57±5.60 13.10±6.21 0.721

[0128] 2. Experimental methods:

[0129] (1) Sample collection: The samples in the experiment were uterine effusions in the proliferative phase of 4 TE patients and 4 normal fertile women listed in Table 1. Disposable sterile human catheters were used to flush the uterine cavity with 3 mL of sterile saline, and the uterine fluid was collected in sterile centrifuge tubes as samples of the normal fertile group and TE patient group.

[0130] (2) Exosome separation and extraction: Ultra-high-speed centrifugation was used to obtain purified exosomes by step-by-step centrifugation according to the conditions of gradient centrifugation (300 g, 2000 g, 10000 g, 120000 g, and 120000 g);

[0131] (3) Exosome morphology identification: The exosomes separated and extracted in step (2) are identified by NTA, WB, TEM and other methods to determine the particle size, specific surface markers and integrity of the exosomes;

[0132] (4) Extraction of exosome RNA: The total RNA in exosomes was extracted using miRNeasy Mini Kit. The specific steps are as follows:

[0133] 1) Add 700 μl QIAzol lysis buffer to the exosomes, mix well, and incubate at room temperature (15-25°C) for 5 min;

[0134] 2) Add 140 μl of chloroform, shake vigorously for 15 seconds, and incubate at room temperature for 2 to 3 minutes;

[0135] 3) After centrifugation at 12000g for 15 min at 4°C, collect the supernatant;

[0136] 4) Add 1.5 times the volume of 100% ethanol and mix well;

[0137] 5) Pipette 700 μl of sample into a 2 mL collection tube, centrifuge at room temperature, 8000 g, 15 s, and discard the supernatant;

[0138] 6) Repeat step 5);

[0139] 7) Pipette 500 μl of RPE buffer into the RNeasy Mini column, centrifuge at room temperature, ≥8000 g, 15 s, and discard the supernatant;

[0140] 8) Pipette 500 μl of RPE buffer into the RNeasy Mini column and centrifuge at room temperature, ≥8000 g, for 2 min;

[0141] 9) Transfer the RNeasy Mini column to a new 1.5 mL collection tube, pipette 30-50 μl of RNase-free water onto the membrane of the RNeasy Mini column, centrifuge at room temperature, ≥8000 g, 1 min, and collect the RNA liquid.

[0142] (5) performing microRNA-seq assay on the genes of all markers in the total RNA samples of the normal fertility group and the TE patient group, respectively, to obtain the assay results including the expression level of each marker.

[0143] (6) Based on the measurement results, among the markers whose absolute expression value difference is greater than or equal to 2 times, bioinformatics analysis methods such as differential gene GO function analysis, KEGG pathway analysis and target gene prediction analysis are combined to reveal the characteristics of the biomarkers, and markers related to thin endometrial aplasia are screened as biomarkers.

[0144] 3. Experimental results:

[0145] Compared with the normal fertile group, a total of 306 differentially expressed genes in the uterine effusion-derived exosomes were identified in the TE patient group, with an absolute expression difference of less than 1.0 times, including 142 up-regulated genes and 164 down-regulated genes. Figure 4 This is the heat map of the above differentially expressed genes.

[0146] The differentially expressed genes obtained in the above steps were screened to identify a biomarker that can reflect the differential characteristics of endometrial regeneration and repair between normal fertile women and TE patients. The expression ratio of this biomarker is shown in the following table. Figure 5 Statistical results of differential gene expression of one of the above-mentioned endometrial regeneration and repair biomarkers were analyzed to expand the sample size.

[0147] Table 3. Endometrial regeneration and repair biomarkers and the logarithm of the ratio of their absolute expression levels in normal fertile women and TE group

[0148] serial number Acc_ID (gene number) <![CDATA[log2FC (logarithm of the expression ratio)]]> P-value 1 hsa-miR-21-5p -1.07314915515631 0.012

[0149] Embodiment 2:

[0150] This example evaluates endometrial regenerative disorder based on the biomarkers screened in Example 1.

[0151] 1. Experimental samples:

[0152] According to the inclusion criteria, in this example, one normal fertile woman (sample A) and one TE patient (sample B) were recruited, and disposable sterile human insemination tubes were used. After flushing the uterine cavity with 3 mL of sterile saline, the uterine cavity fluid was collected in a sterile centrifuge tube as sample A and sample B, respectively.

[0153] The above samples are processed as follows:

[0154] (1) Extraction of exosomes: Purified exosomes were obtained by step-by-step centrifugation according to the conditions of gradient centrifugation (300 g, 2000 g, 10000 g, 120000 g and 120000 g) using ultrahigh-speed centrifugation method.

[0155] (2) Exosome morphology identification: The isolated and extracted exosomes were identified by NTA, WB, TEM and other methods to determine the particle size, specific surface markers and integrity of the exosomes;

[0156] (3) Extraction of exosome RNA: The total RNA in exosomes was extracted using miRNeasy Mini Kit. The specific steps are the same as the extraction process in Implementation Case 1;

[0157] 2. Experimental methods:

[0158] (1) The product (biomarker: microRNA-21-5p) is used to detect the expression level of the biomarker in the product of normal fertile female intrauterine fluid, which is used as the normal expression level, and the corresponding evaluation reference threshold is obtained based on the normal expression level; in this embodiment, the evaluation reference threshold is 1.06.

[0159] (2) using the product to detect the expression level of the biomarker in the exosomes derived from the intrauterine effusion of the TE patient to be tested, as the target expression level;

[0160] (3) Obtaining an evaluation result based on the target expression level and the evaluation reference threshold.

[0161] Specifically, the evaluation method is:

[0162] comparing the target expression amount with the evaluation reference threshold;

[0163] If the target expression amount is less than the evaluation reference threshold, the evaluation result is positive;

[0164] If the target expression amount is not less than the evaluation reference threshold, the evaluation result is negative.

[0165] 3. Experimental results and analysis:

[0166] (1) Based on the expression level of miR-21-5p in exosomes derived from intrauterine fluid in normal fertile population, SPSS27.0 statistical software was used to analyze the reference value range, that is, the evaluation reference threshold was 1.06.

[0167] (2) Sample test results:

[0168] Table 4. Test results

[0169] Sample No. Sample A Sample B miR-21-5p expression 1.81 0.23 Comparison with evaluation reference threshold ＞1.06 ＜1.06 result Negative Positive

[0170] According to the measurement results, the expression level of miR-21-5p in the exosomes derived from uterine effusion of a normal fertile woman (sample A) was 1.81, and the expression level of miR-21-5p in the exosomes derived from uterine effusion of a TE patient (sample B) was 0.23.

[0171] (3) Data analysis process:

[0172] Referring to Table 4, the following conclusions are drawn based on the comparative analysis of the reference threshold of 1.06:

[0173] Sample A: The expression level of miR-21-5p in the exosomes derived from uterine effusion in normal fertile women was 1.81>1.06, and the result was negative, suggesting that the patient was not a thin endometrium patient;

[0174] Sample B: The expression level of miR-21-5p in the exosomes derived from intrauterine fluid of TE patients was 0.23<1.06, and the result was positive, suggesting that the patient had thin endometrium.

[0175] (4) Analysis:

[0176] It can be seen from the above results in this embodiment that by detecting the expression level of miR-21-5p in the patient's intrauterine fluid-derived exosomes, the functional state of the endometrium can be accurately evaluated, and non-invasive, rapid and accurate detection can be achieved, providing strong support for the diagnosis and treatment of TE.

[0177] The above are preferred implementation modes and corresponding embodiments of the present invention. It should be pointed out that, for those skilled in the art, without departing from the creative concept of the present invention, several modifications and improvements may be made, including but not limited to the adjustment of proportions, processes, dosages and reaction vessels, which all fall within the scope of protection of the present invention.

Claims

1. A biomarker for evaluating endometrial regenerative disorders, characterized in that: The biomarkers are: microRNA-21-5p; Preferably, the AccID of the biomarker is hsa-miR-21-5p.

2. A product, characterized in that: Comprising the biomarker for assessing endometrial regenerative disorder as described in claim 1.

3. The product according to claim 2, characterized in that: The products include reagents, test kits, test strips, chips or instruments.

4. Use of the biomarker as claimed in claim 1 in the preparation of a product for evaluating endometrial regenerative disorders.

5. Use of the biomarker according to claim 4 in the preparation of a product for evaluating endometrial aplastic disorder, characterized in that: The evaluation methods include: Using the product to detect the expression amount of the biomarker in the product in the exosomes derived from intrauterine fluid of normal fertile women as the normal expression amount, and deriving the corresponding evaluation reference threshold value according to the normal expression amount; Using the product to detect the expression amount of the biomarker in the exosomes derived from the intrauterine effusion of the TE patient to be tested, as the target expression amount; An evaluation result is obtained based on the target expression amount and the evaluation reference threshold.

6. Use of the biomarker according to claim 5 in the preparation of a product for evaluating endometrial aplastic disorder, characterized in that: The obtaining of the evaluation result according to the target expression amount and the evaluation reference threshold value includes: comparing the target expression amount with the evaluation reference threshold; If the target expression amount is not less than the evaluation reference threshold, the evaluation result is negative; If the target expression amount is less than the evaluation reference threshold, the evaluation result is positive; Preferably, the evaluation reference threshold is 1.

06.

7. Use of the biomarker according to claim 4 in the preparation of a product for evaluating endometrial aplastic disorder, characterized in that: The biomarkers were screened by the following method: RNA-seq technology was used to detect the expression levels of all microRNAs in the exosomes of the proliferative uterine effusion in the normal fertility group and the TE patient group, and the results were obtained. According to the measurement results, the markers to be screened in the TE patient group are screened based on the absolute value difference of the expression amount in the endometrium, and the biomarker is screened out from the markers to be screened.

8. Use of the biomarker according to claim 7 in the preparation of a product for evaluating endometrial aplastic disorder, characterized in that: The RNA-seq technology is used to detect the expression levels of all microRNAs in the exosomes of the proliferative uterine effusion in the normal fertility group and the TE patient group, and the measurement results are obtained, including: The uterine fluid of the proliferative phase of normal fertile women and TE patients is collected respectively as samples of the normal fertile group and the TE patient group; Separating and extracting samples from the normal fertility group and the TE patient group to obtain exosomes; Performing morphological identification on the exosomes; Extracting the exosomes to obtain a total RNA sample; The genes of all markers in the total RNA samples of the normal fertility group and the TE patient group were respectively subjected to microRNA-seq assay to obtain the assay results including the expression level of each marker.

9. Use of the biomarker according to claim 7 in the preparation of a product for evaluating endometrial aplastic disorder, characterized in that: The method of screening the markers to be screened in the TE patient group based on the absolute value difference of the expression amount in the endometrium according to the measurement results, and screening the biomarker from the markers to be screened includes: Based on the determination results, screening out markers whose absolute expression difference fold is greater than or equal to 2 times as target markers; The target marker is analyzed using a bioinformatics analysis method, and the biomarker is obtained according to the analysis result.

10. Use of the biomarker according to claim 9 in the preparation of a product for evaluating endometrial aplastic disorder, characterized in that: The bioinformatics analysis method comprises: At least one of differential gene GO functional analysis, KEGG pathway analysis and target gene prediction analysis.

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