Marker related to different time points in embryo implantation process as well as screening method and application thereof

Abstract

The invention discloses markers related to different time points in an embryo implantation process as well as a screening method and application of the markers. The molecular marker comprises the following components: hsa-miR-1249 to 3p, hsa-miR-1260a, hsa-miR-1332 to 5p, hsa-miR-154 to 3p, hsa-miR-154 to 5p, hsa-miR-202 to 3p, hsa-miR-202 to 5p, hsa-miR-299 to 3p, hsa-miR-328 to 3p, hsa-miR-335 to 3p, and the like. Based on the expression of miRNA in a maternal plasma sample, the invention develops a method and a molecular marker for identifying the dynamic change of maternal plasma miRNA in an embryo implantation process, and provides a basis for further knowing the dynamic change of human endometrial tissue WOI before and after.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology and relates to a marker related to different time points in the embryo implantation process and a screening method and application thereof. Background Art

[0002] In Vitro Fertilization-Embryo Transfer (IVF-ET) is an effective form of assisted reproductive technology, which allows eggs to be fertilized in vitro and developed into early embryos before being implanted into the woman's uterus. Implantation failure is the most common cause of IVF-ET failure, which is mainly affected by two aspects: embryo quality and endometrial implantation window. Preimplantation genetic testing (PGT) technology can help transplant euploid embryos with a higher chance of live birth, while those with repeated implantation failure (RIF) need to further consider endometrial factors.

[0003] The window of implantation (WOI) is a short and specific time window in the endometrial cycle, during which the endometrium is receptive to the attachment, growth and invasion of the blastocyst. It usually occurs between the 19th and 24th days of the menstrual cycle, which is the 7th day after the peak of luteinizing hormone (LH) in the natural cycle (LH+7) or the 5th day after the administration of progesterone in the artificial cycle (P+5). The early delay of WOI in some women usually leads to implantation failure and pregnancy loss. Since Noyes first established the classic histological criteria in 1975, the evaluation of the endometrial implantation window has gradually transitioned to omics research with the development of high-throughput biotechnology. In 2009, researchers identified five genes upregulated in WOI as new biomarkers of the endometrial implantation window based on endometrial biopsy samples. In 2017, researchers used the expression of 184 genes related to endometrial proliferation and maternal immune response to accurately classify endometrial samples from fertile and infertile groups. However, the clinical translation of the above endometrial biopsy studies is limited by the complexity and invasiveness of sample collection.

[0004] As a transcriptional regulator of gene expression, miRNA is highly stable in plasma. Endometrial cells secrete and transport a large amount of miRNA to tissue sites through the blood to exert their effects. Many studies have shown that miRNA expression in the blood is related to embryo implantation outcomes, and there are specific miRNAs expressed in people with abnormal WOI, such as CN112469836A discloses a method for determining endometrial status using a sample from a woman, such as an endometrial specimen, the method comprising: (a) performing analysis on an endometrial sample from a woman to determine the microRNA (miRNA) expression profile of the endometrial sample, wherein the miRNA expression profile includes multiple miRNAs, such as the expression levels of 167 miRNAs having sequences of SEQ ID NO: 1 to SEQ ID NO: 167, respectively; and (b) analyzing the miRNA expression profile using, for example, a computer algorithm to obtain a receptivity prediction score. The above method uses endometrial curettage to evaluate the endometrial receptivity, but this method is invasive and will also cause changes in the endometrial environment during the biopsy of endometrial tissue, which has limitations in clinical use.

[0005] In summary, the development of markers related to the endometrial implantation window period can non-invasively and accurately identify the endometrial implantation window period, which is of great significance in the field of embryo transplantation. Summary of the invention

[0006] In view of the deficiencies in the prior art and actual needs, the present invention provides a marker associated with different time points in the embryo implantation process, a screening method and application thereof, identifies miRNAs that change at different time points during the embryo transfer cycle based on maternal plasma samples, describes the change model of these miRNAs during the embryo implantation process, and identifies the dynamic changes of the endometrial receptive phase at the plasma miRNA level, which is stable and non-invasive.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a marker associated with different time points during embryo implantation, the marker comprising: hsa-miR-1249-3p, hsa-miR-1260a, hsa-miR-132-5p, hsa-miR-154-3p, hsa-miR-154-5p, hsa-miR-202-3p, hsa-miR-202-5p, hsa-miR-299-3p, hsa-miR-328-3p, hsa-miR-335-3p, hsa-miR-33a-5p, hsa-miR-340-3p, hsa-miR-369-3p, hsa-miR-369-5p. p, hsa-miR-376c-3p, hsa-miR-379-3p, hsa-miR-381-3p, hsa-miR-382-3p, hsa-miR-4286, hsa-miR-4433b-5p, hsa-miR-485-3p, hsa-miR-493-5p, h sa-miR-495-3p, hsa-miR-513a-5p, hsa-miR-513c-5p, hsa-miR-539-3p, hsa-miR-654-3p, hsa-miR-6747-3p, hsa-miR-744-3p, hsa-miR-7977, hsa- miR-10527-5p, hsa-miR-107, hsa-miR-25-3p, hsa-miR-331-5p, hsa-miR-3684, hsa-miR-3691-5p, hsa-miR-3939, hsa-miR-423-5p, hsa-miR-4732- 5p, hsa-miR-484, hsa-miR-5003-3p, hsa-miR-5010-5p, hsa-miR-532-5p, hsa-miR-636, hsa-miR-6715a-3p, hsa-miR-6767-5p, hsa-miR-6781-5p, h sa-miR-6804-5p, hsa-miR-6805-5p, hsa-miR-6850-5p, hsa-miR-6894-5p, hsa-miR-125b-5p, hsa-miR-143-3p, hsa-miR-143-5p, hsa-miR-145-3p, hsa-miR-145-5p, hsa-miR-181c-5p, hsa-miR-190a-5p, hsa-miR-199b-5p, hsa-miR-218-5p, hsa-miR-338-3p, hsa-miR-450a-5p, hsa-miR-504-5p,hsa-miR-551a, hsa-miR-582-3p, hsa-miR-615-3p, hsa-miR-1-3p, hsa-miR-11400, hsa-mi R-1185-1-3p, hsa-miR-12136, hsa-miR-124-3p, hsa-miR-127-5p, hsa-miR-133a-3p, hsa-m iR-136-3p, hsa-miR-199a-5p, hsa-miR-205-5p, hsa-miR-206, hsa-miR-208b-3p, hsa-miR- 216a-5p, hsa-miR-299-5p, hsa-miR-323a-3p, hsa-miR-329-3p, hsa-miR-370-3p, hsa-miR- 382-5p, hsa-miR-409-3p, hsa-miR-410-3p, hsa-miR-411-3p, hsa-miR-411-5p, hsa-miR-4 33-3p, hsa-miR-487a-3p, hsa-miR-494-3p, hsa-miR-499a-5p, hsa-miR-5100, hsa-miR-518 The nucleic acid sequences are SEQ ID NO.1 to SEQ ID NO.104.

[0009] In the present invention, marker molecules at different time points during embryo implantation were developed based on maternal plasma miRNA, 104 miRNAs that changed at 5 time points during the embryo transfer cycle were identified, and the dynamic changes of the endometrial receptive period were identified at the plasma miRNA level. This method is stable and non-invasive, and provides a basis for further understanding the dynamic changes of human endometrial tissue before and after WOI, which is beneficial for judging the endometrial implantation window period and guiding the timing of embryo transfer in assisted reproductive technology.

[0010] In a second aspect, the present invention provides the use of the markers related to different time points in the embryo implantation process and the detection reagents thereof as described in the first aspect in the preparation and analysis of endometrial implantation window period products.

[0011] In a third aspect, the present invention provides a kit for analyzing the endometrial implantation window period, the kit comprising reagents for detecting the expression levels of the markers associated with different time points during embryo implantation as described in the first aspect.

[0012] In a fourth aspect, the present invention provides the use of the markers associated with different time points in the embryo implantation process and the detection reagents thereof as described in the first aspect in analyzing the endometrial implantation window period for non-disease diagnosis purposes.

[0013] In a fifth aspect, the present invention provides a method for screening markers associated with different time points in the embryo implantation process according to the first aspect, the method comprising the following steps:

[0014] (1) Quantitative analysis of miRNA in samples from women undergoing embryo transfer;

[0015] (2) Analyze the dynamic changes of miRNA during embryo implantation.

[0016] The miRNA includes the markers associated with different time points during embryo implantation as described in claim 1.

[0017] Preferably, the sample comprises any one of serum, plasma, uterine fluid, uterine lavage fluid, vaginal exfoliated cells, vaginal secretions or endometrial biopsy products, or a combination of at least two thereof.

[0018] Preferably, step (1) comprises the following steps:

[0019] (1-1) Sequence the miRNA in the sample, remove the 3' end adapter, sequences without adapter insertion, sequences containing polyA, polyT, sequences with only AT or CG, sequences with a single base accounting for more than 80% of a sequence, and sequences with a length of less than 18 nt after the adapter, and remove duplicates;

[0020] (1-2) Align the deduplicated sequences to the Rfam database, obtain annotations for the aligned sequences, and extract the sequences aligned to miRNA and those not aligned as valid data;

[0021] (1-3) Quantify valid data and identify known miRNAs;

[0022] (1-4) The expression level of known miRNA is standardized according to formula (1);

[0023] miRNA i RPM = miRNA i count / total miRNAcount ×10 6 Formula (1);

[0024] Among them, miRNA i count For each known miRNA expression level, miRNA i RPM The expression level of each known miRNA is standardized, total miRNA count It is the sum of the expression levels of all known miRNAs.

[0025] It is understood that the commonly used miRNA sequencing methods and corresponding expression analysis methods and software in the art are applicable to the present invention.

[0026] Preferably, step (2) comprises the following steps:

[0027] (2-1) using differential analysis software to calculate the differential levels of miRNA expression between different time points, and obtain the differentially expressed miRNAs between samples at any two time points;

[0028] (2-2) calculating the correlation between the expression level of the differentially expressed miRNA identified in step (2-1) and different implantation periods, and expressing it as miRNA significance;

[0029] (2-3) Clustering and time series analysis are performed on the miRNA significance obtained in step (2-2) to obtain miRNA expression profiles with different changes during embryo implantation.

[0030] Preferably, the time point includes: day 1-9 after the peak of luteinizing hormone in women undergoing embryo transfer.

[0031] In the present invention, the 1st to 2nd day after the peak of luteinizing hormone (LH) appears is marked as D0, the 3rd day is marked as D3, the 5th day is marked as D5, the 7th day is marked as D7, and the 9th day is marked as D9.

[0032] Preferably, the time points include: day 1, day 3, day 5, day 7 and day 9 after the peak of luteinizing hormone in women undergoing embryo transfer.

[0033] In a sixth aspect, the present invention provides a method for analyzing the endometrial implantation window period for the purpose of non-disease diagnosis, the method comprising:

[0034] The expression levels of the markers related to different time points in the embryo implantation process described in the first aspect in the female sample to be tested are detected, and the changing trends of the expression levels are analyzed to determine the dynamic changes of the endometrium before and after WOI.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention identified the miRNA changes at five time points during the embryo transfer cycle based on maternal plasma samples, and recognized the dynamic changes of the endometrial receptive period at the plasma miRNA level. It is stable and non-invasive, and provides a basis for further understanding the dynamic changes of human endometrial tissue before and after WOI, which is beneficial for judging the endometrial implantation window period and guiding the embryo transfer time in assisted reproductive technology, as well as being applied to basic research related to the endometrium for purposes other than disease diagnosis and / or treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a graph of the number of differentially expressed miRNAs at any two time points;

[0038] Figure 2 This is the MFUZZ clustering result diagram. DETAILED DESCRIPTION

[0039] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0040] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0041] Example 1

[0042] In this example, miRNA in plasma samples was quantitatively analyzed.

[0043] 1. Preparation and loading of plasma samples

[0044] (1) Peripheral blood samples were collected from 63 infertile women who had only fallopian tube factors and underwent natural cycle frozen-thaw blastocyst transfer. Peripheral blood was collected from each patient at five time points (D0, D3, D, D7, and D9, with D0 being one day after the LH peak, recorded as the ovulation day). 5 mL of peripheral blood samples were centrifuged at 4°C for 10 min with a centrifugal force of 2000×g. The plasma was then aliquoted and stored at -80°C for subsequent experimental use.

[0045] (2) The absorbance was measured at 414 nm using a Thermo Scientific Nanodrop One spectrophotometer, and plasma samples with a ΔCq (quantification cycle) of ≤0.2 were selected as non-hemolyzed samples. For samples with a ΔCq (quantification cycle) of miR-23a and miR-451 of <7, samples with a ΔCq (quantification cycle) of >0.2 were selected as non-hemolyzed samples;

[0046] 2. Extraction and library construction of miRNA

[0047] (1) Plasma miRNA was extracted using the miRNeasy Serum / Plasma Advanced Kit (Qiagen);

[0048] (2) Use the TruSeq small RNA library preparation kit and HiSeq2500 (Illumina, San Diego, USA) system to construct and sequence miRNA libraries.

[0049] 3. Analysis of miRNA offline data

[0050] (1) The raw data of plasma miRNA sequencing were filtered using cutadapt software (v3.4): 3' end adapters, sequences without adapter insertions, sequences containing polyA or polyT, sequences with only AT or CG, sequences with a single base (A / T / C / G) accounting for more than 80% of a sequence, and sequences with a length of less than 18 nt after adapter removal; and the filtered sequences were deduplicated;

[0051] (2) Use bowtie software (v1.34.0) to align the quality-controlled sequences to the Rfam database, obtain annotations for the aligned sequences, and select the sequences aligned to miRNA and those not aligned as the subsequent valid data;

[0052] (3) Use miRDeep2 (v0.1.3) to quantify valid data and identify known miRNAs;

[0053] (4) The known miRNA quantification results were standardized according to the following formula (1);

[0054] miRNA i RPM =miRNA i count / total miRNA count ×10 6 Formula (1).

[0055] Example 2

[0056] This example analyzes the dynamic changes of miRNA in plasma samples.

[0057] 1. Differential miRNA Analysis

[0058] The DESeq2 software (v1.34.0) was used to calculate the difference in miRNA expression between samples at different time points. The miRNAs with CPM (Counts per million) values ​​≤ 0.5 in more than 20% of the samples were deleted. The Wald test was used to perform hypothesis testing to obtain the significant p value. The Benjamini-Hochberg method was used to perform multiple hypothesis testing to obtain the Q value. The miRNAs with Q ≤ 0.02 were extracted to obtain the differentially expressed miRNAs between samples at different sampling time points. Figure 1 As shown, the number of differentially expressed miRNAs between any two time points is displayed.

[0059] 2. Identification of the dynamic expression profile of miRNA during implantation

[0060] (1) Calculate the correlation between the expression level of the differentially identified miRNAs and different implantation periods (D0, D3, D5, D7, and D9), and record it as the significance of miRNAi;

[0061] (2) Cluster the significance of miRNAs using MFUZZ (v2.32.0) to obtain four clustering results (Cluster1-4). Among them, Cluster 1 includes hsa-miR-1249-3p, hsa-miR-1260a, hsa-miR-132-5p, hsa-miR-154-3p, hsa-miR-154-5p, hsa-miR-202-3p, hsa-miR-202-5p, hsa-miR-299-3p, hsa-miR-328-3p, hsa-miR-335-3p, hsa-miR-33a-5p, hsa-miR-340-3p, hsa-miR-369-3p, hsa-miR-369-5p, hsa-miR-376c-3p, hsa-miR-379-3p, hsa-miR-381-3p, hsa-miR-382-3p, hsa-miR-4286, hsa-miR-4433b-5p, hsa-miR-485-3p, hsa-miR-493-5p, hsa-miR-495-3p, hsa-miR-513a-5p, hsa-miR-513c-5p, hsa-miR-539-3p, hsa-miR-654-3p, hsa-miR-6747-3p, hsa-miR-744-3p, hsa-miR-7977; Cluster 2 includes hsa-miR-10527-5p, hsa-miR-107, hsa-miR-25-3p, hsa-miR-331-5p, hsa-miR-3684, hsa-miR-3691-5p, hsa-miR-3939, hsa-miR-423-5p, hsa-miR-4732-5p, hsa-miR-484, hsa-miR-5003-3p, hsa-miR-5010-5p, hsa-miR-532-5p, hsa-miR-636, hsa-miR-6715a-3p, hsa-miR-6767-5p, hsa-miR-6781-5p, hsa-miR-6804-5p, hsa-miR-6805-5p, hsa-miR-6850-5p, hsa-miR-6894-5p;Cluster 3 includes hsa-miR-125b-5p, hsa-miR-143-3p, hsa-miR-143-5p, hsa-miR-145-3p, hsa-miR-145-5p, hsa-miR-181c-5p, hsa-miR-190a-5p, hsa-miR-199b-5p, hsa-miR-218-5p, hsa-miR-338-3p, hsa-miR-450a-5p, hsa-miR-504-5p, hsa-miR-551a, hsa-miR-582-3p, hsa-miR-615-3p; Cluster 4 includes hsa-miR-1-3p, hsa-miR-11400, hsa-miR-1185-1-3p, hsa-miR-12136, hsa-miR-124-3p, hsa-miR-127-5p, hsa-miR-133a-3p, hsa-miR-136-3p, hsa-miR-199a-5p, hsa-miR-205-5p, hsa-miR-206, hsa-miR-208b-3p, hsa-miR-216a-5p, hsa-miR-299-5p, hsa-miR-323a-3p, hsa-miR-329-3p, hsa-miR-370-3p, hsa-miR-382-5p, hsa-miR-409-3p, hsa-miR-410-3p, hsa-miR-411-3p, hsa-miR-411-5p, hsa-miR-433-3p, hsa-miR-487a-3p, hsa-miR-494-3p, hsa-miR-499a-5p, hsa-miR-5100, hsa-miR-518e-5p, hsa-miR-519a-5p, hsa-miR-519b-5p, hsa-miR-519c-5p, hsa-miR-522-5p, hsa-miR-523-5p, hsa-miR-524-5p, hsa-miR-543, hsa-miR-548ag, hsa-miR-654-5p, hsa-miR-758-3p. Such as; Figure 2As shown in the figure, the cluster center is the weighted sum of all cluster members, indicating the overall change trend of this type of miRNAs. The ordinate represents the significance of miRNA. The significance value of miRNA represents the correlation between the miRNA and the time point, indirectly reflecting the expression level of miRNA at the time point. The higher the value, the higher the expression level. The expression of miRNAs in Cluster 1 decreased on D3 and gradually increased in the following time; the expression of miRNAs in Cluster 2 increased on D3 and gradually decreased in the following time; the expression of miRNAs in Cluster 3 decreased on D3 and D5, and maintained a high expression level on D7 and D9; the expression of miRNAs in Cluster 4 decreased on D3 and increased on D5, and its expression remained at a relatively consistent level from D5 to D9.

[0062] In summary, this application identified 104 miRNAs that changed at five time points during the embryo transfer cycle based on maternal plasma samples, and described the change model of these miRNAs during embryo implantation. The dynamic changes of the endometrial receptive period were identified at the plasma miRNA level, which is stable and non-invasive, and provides a basis for further understanding the dynamic changes of human endometrial tissue before and after WOI, which is beneficial for determining the endometrial implantation window period and guiding the timing of embryo transfer in assisted reproductive technology.

[0063] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A marker associated with different time points during embryo implantation, characterized in that: The markers include: hsa-miR-1249-3p, hsa-miR-1260a, hsa-miR-132-5p, hsa-miR-154-3p, hsa-miR-154-5p, hsa-miR-202-3p, hsa-miR-202-5p, hsa-miR-299-3p, hsa-miR-328-3p, hsa-miR-335-3p, hsa-miR-33a-5p, hsa-miR-340-3p, hsa-miR-369-3p, hsa-miR-369-5p, hsa-miR-376c-3p, hsa-miR-379-3p, hsa-miR-381-3p, hsa-miR-382-3p, hsa-miR-4286, hsa-miR-4433b-5p, hsa-miR-485-3p, hsa-miR-493-5p, hsa-miR-495-3p, hsa-miR-513a-5p, hsa-miR-513c-5p, hsa-miR-539-3p, hsa-miR-654-3p, hsa-miR-6747-3p, hsa-miR-744-3p, hsa-miR-7977, hsa-miR-10527-5p, hsa-miR-107, hsa-miR-25-3p, hsa-miR-331-5p, hsa-miR-3684, hsa-miR-3691-5p, hsa-miR-3939, hsa-miR-423-5p, hsa-miR-4732-5p, hsa-miR-484, hsa-miR-5003-3p, hsa-miR-5010-5p, hsa-miR-532-5p, hsa-miR-636, hsa-miR-6715a-3p, hsa-miR-6767-5p, hsa-miR-6781-5p, hsa-miR-6804-5p, hsa-miR-6805-5p, hsa-miR-6850-5p, hsa-miR-6894-5p, hsa-miR-125b-5p, hsa-miR-143-3p, hsa-miR-143-5p, hsa-miR-145-3p, hsa-miR-145-5p, hsa-miR-181c-5p, hsa-miR-190a-5p, hsa-miR-199b-5p, hsa-miR-218-5p, hsa-miR-338-3p, hsa-miR-450a-5p, hsa-miR-504-5p, hsa-miR-551a, hsa-miR-582-3phsa-miR-615-3p, hsa-miR-1-3p, hsa-miR-11400, hsa-miR-1185-1-3p, hsa-miR-12136, hsa-miR-124-3p, hsa-miR-127-5p, hsa-miR-133a-3p, hsa-miR-136-3p, hsa-miR-199a-5p, hsa-miR-205-5p, hsa-miR-206, hsa-miR-208b-3p, hsa-miR-216a-5p, hsa-miR-299-5p, hsa-miR-323a-3p, hsa-miR-329-3p, hsa-miR-370-3p, hsa-miR-382-5p, hsa-miR-409-3p, hsa-miR-410-3p, hsa-miR-411-3p, hsa-miR-411-5p, hsa-miR-433-3p, hsa-miR-487a-3p, hsa-miR-494-3p, hsa-miR-499a-5p, hsa-miR-5100, hsa-miR-518e-5p, hsa-miR-519a-5p, hsa-miR-519b-5p, hsa-miR-519c-5p, hsa-miR-522-5p, hsa-miR-523-5p, hsa-miR-524-5p, hsa-miR-543, hsa-miR-548ag, hsa-miR-654-5p and hsa-miR-758-3p., 2. Use of the markers associated with different time points in the embryo implantation process and their detection reagents as described in claim 1 in the preparation and analysis of endometrial implantation window period products.

3. A kit for analyzing the endometrial implantation window period, characterized in that: The kit comprises reagents for detecting the expression levels of the markers associated with different time points during embryo implantation as claimed in claim 1.

4. Use of the markers associated with different time points in the embryo implantation process and the detection reagents thereof as claimed in claim 1 in analyzing the endometrial implantation window period for non-disease diagnosis purposes.

5. A method for screening markers associated with different time points during embryo implantation according to claim 1, characterized in that: The method comprises the following steps: (1) Quantitative analysis of miRNA in samples from women undergoing embryo transfer; (2) Analyze the dynamic changes of miRNA during embryo implantation; The miRNA includes the markers associated with different time points during embryo implantation as described in claim 1.

6. The method for screening markers associated with different time points during embryo implantation according to claim 5, characterized in that: The sample includes any one of serum, plasma, uterine fluid, uterine lavage fluid, vaginal exfoliated cells, vaginal secretions or endometrial biopsy products, or a combination of at least two of them.

7. The method for screening markers associated with different time points during embryo implantation according to claim 5 or 6, characterized in that: Step (1) comprises the following steps: (1-1) Sequence the miRNA in the sample, remove the 3' end adapter, sequences without adapter insertion, sequences containing polyA, polyT, sequences with only AT or CG, sequences with a single base accounting for more than 80% of a sequence, and sequences with a length of less than 18 nt after the adapter, and remove duplicates; (1-2) Align the deduplicated sequences to the Rfam database, obtain annotations for the aligned sequences, and extract the sequences aligned to miRNA and those not aligned as valid data; (1-3) Quantify valid data and identify known miRNAs; (1-4) The expression level of known miRNA is standardized according to formula (1); miRNA iRPM = miRNA icount / total miRNA count ×10 6 Formula (1): Among them, miRNA icount For each known miRNA expression level, miRNA iRPM The expression level of each known miRNA is standardized, total miRNA count It is the sum of the expression levels of all known miRNAs.

8. The method for screening markers associated with different time points during embryo implantation according to claim 5, characterized in that: Step (2) comprises the following steps: (2-1) using differential analysis software to calculate the differential levels of miRNA expression between different time points, and obtain the differentially expressed miRNAs between samples at any two time points; (2-2) calculating the correlation between the expression level of the differentially expressed miRNA identified in step (2-1) and different implantation periods, and expressing it as miRNA significance; (2-3) Clustering and time series analysis are performed on the miRNA significance obtained in step (2-2) to obtain miRNA expression profiles with different changes during embryo implantation.

9. The method for screening markers associated with different time points during embryo implantation according to claim 8, characterized in that: The time points include: days 1-9 after the peak of luteinizing hormone in women undergoing embryo transfer.

10. The method for screening markers associated with different time points during embryo implantation according to claim 9, characterized in that: The time points include: day 1, day 3, day 5, day 7 and day 9 after the peak of luteinizing hormone in women undergoing embryo transfer.

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