A combination of genetic markers for detecting implantation window shift in the endometrium and its application.

Abstract

This invention discloses a combination of gene markers for detecting the embryo implantation window (WOI) shift in the endometrium. The gene markers comprise characteristic gene sequences related to WOI shift, as shown in SEQ ID NO.1 to SEQ ID NO.10. This invention can accurately and effectively determine whether there is a time shift in the WOI of the endometrium, and provide guidance on whether the subject's WOI should be adjusted accordingly, which can significantly improve the success rate of in vitro fertilization-embryo implantation (IVF-ET).

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and more specifically, to a combination of gene markers for detecting the offset state of the endometrial embryo implantation window (WOI) and their applications. Background Technology

[0002] Assisted reproductive technology (ART), especially in vitro fertilization-embryo transfer (IVF-ET), is currently recognized as an effective means of treating infertility. However, data shows that the success rate of embryo transfer using the traditional IVF-ET protocol is only about 40%, and a considerable number of patients experience recurrent implantation failure (RIF), that is, more than three embryo implantations, without achieving clinical pregnancy.

[0003] Studies have found that approximately two-thirds of embryo implantation failures are caused by misjudging the patient's WOI (Whole Window of Implantation). Traditionally, the WOI in the uterine lining is believed to open between days 19 and 21 of the menstrual cycle. However, in women with recurrent implantation failures or secondary infertility, the WOI often shifts earlier or later. If IVF-ET is performed according to traditional protocols, i.e., on day 7 of a natural cycle (LH+7) or day 5 of an artificial cycle (P+5), there is a risk of embryo transfer failure. Therefore, developing a method to accurately determine whether the WOI has shifted is of great significance for the diagnosis and treatment of patients with reproductive disorders.

[0004] Currently, clinical methods such as histological examination, ultrasound examination, and molecular marker detection can roughly locate a patient's WOI (Wood Occlusion Injection). However, these techniques cannot accurately determine the WOI deviation status, and there are still some controversies regarding their theoretical basis. Histological examination, using pinocytosis as a marker, has disadvantages including: ① it is an invasive procedure that can cause damage to the endometrium; ② there is still controversy in the industry regarding whether pinocytosis can be used as an implantation marker; ③ it relies on the experience of pathologists, leading to subjective interpretation and a lack of objectivity; ④ the results have poor reproducibility, and this test cannot be performed during the same cycle, limiting its prognostic value. Ultrasound imaging, with markers including endometrial thickness, echo type, and submucosal blood flow, suffers from a lack of unified interpretation criteria. The significance of a single parameter in ER assessment is not significant and remains controversial, requiring a comprehensive evaluation of multiple parameters, resulting in poor reliability and stability. Molecular marker detection, including cytokines and homeobox genes, has limitations such as poor reproducibility and high variability when using a single molecular marker as an assessment standard.

[0005] Therefore, faced with the reproductive medicine challenge of recurrent implantation failure and the increasingly severe situation of infertility, researchers urgently need to find a technical method to accurately determine the state of WOI (Woman Endometrial Instantaneous ... Summary of the Invention

[0006] The purpose of this invention is to address the technical problem that existing technologies cannot accurately determine whether the endometrial embryo implantation window (WOI) of women intending to undergo IVF-ET has shifted over time, making it difficult for clinicians to determine whether the traditional embryo implantation window (natural cycle LH+7 days or artificial cycle P+5 days) is suitable for embryo transfer. This invention provides a combination of gene markers or nucleic acid compositions for detecting the shift status of the endometrial embryo implantation window (WOI), thereby improving the success rate of IVF-ET and improving pregnancy outcomes for infertile patients (mainly RIF patients).

[0007] Another object of the present invention is to provide the application of the aforementioned combination of gene markers or nucleic acid composition in the preparation of a detection system product for detecting the WOI shift status of the endometrium.

[0008] To address the above problems, the present invention provides a combination of gene markers for detecting the embryo implantation window offset status of the endometrium, wherein the combination of gene markers consists of endometrial WOI offset-related characteristic gene sequences shown in SEQ ID NO.1 to SEQ ID NO.10.

[0009] The specific combinations of gene markers are shown in Table 1.

[0010] Table 1. Combinations of 10 characteristic genes associated with endometrial embryo implantation window shift.

[0011]

[0012] The present invention provides a nucleic acid composition comprising a combination of multiple polynucleotides or fragments thereof, wherein the multiple polynucleotides exhibit differential expression to varying degrees in the endometrium during the luteal phase, and the sequences of the multiple polynucleotides are endometrial WOI offset-related characteristic gene sequences shown in SEQ ID NO.1 to SEQ ID NO.10.

[0013] The six characteristic gene sequences (DPP4, CXCR1, CXCR2, OSM, LCN2, TNFRSF10C) shown in SEQ ID NO.1 to SEQ ID NO.6 were relatively upregulated in the WOI forward shift group and relatively downregulated in the WOI backward shift group.

[0014] The four characteristic gene sequences (TM4SF4, CES4A, LRRC1, and SLC25A48) shown in SEQ ID NO.7 to SEQ ID NO.10 were relatively downregulated in the WOI forward shift group and relatively upregulated in the WOI backward shift group.

[0015] The present invention also provides the use of the combination of said gene markers or the said nucleic acid composition in the preparation of products for detecting the WOI shift status of the endometrium.

[0016] Preferably, the product for detecting the WIO shift status of the endometrium includes products that use RNA sequencing (RNA-seq), microarray, or quantitative real-time PCR (qRT-PCR) to detect and evaluate the WIO shift status of the endometrium.

[0017] The product for detecting endometrial WII shift state using RNA sequencing contains messenger RNA sequences of characteristic gene sequences related to endometrial WII shift state as shown in SEQ ID NO.1 to SEQ ID NO.10, as well as corresponding detection primers and / or probes.

[0018] The product for detecting endometrial WIO shift status using gene chips contains hybridization probes with characteristic gene sequences related to endometrial WIO shift status as shown in SEQ ID NO.1 to SEQ ID NO.10.

[0019] The product for detecting endometrial WII shift status using real-time quantitative PCR contains messenger RNA sequences that specifically amplify the characteristic gene sequences related to endometrial WII shift status shown in SEQ ID NO.1 to SEQ ID NO.10, as well as corresponding detection primers and / or probes.

[0020] The present invention also provides a kit for detecting endometrial WOI shift status, the kit comprising messenger RNA sequences specifically targeting the endometrial WOI shift-related characteristic gene sequences shown in SEQ ID NO.1 to SEQ ID NO.10, as well as corresponding detection primers and / or probes.

[0021] When using the kit, firstly, based on the expression level data of the endometrial WOI shift-related characteristic gene sequences shown in SEQ ID NO.1 to SEQ ID NO.10 in endometrial cells, a prediction model is constructed by combining SVM, Random Forest, and KNN algorithms. Then, the kit is used to detect the expression level data of the characteristic gene sequences in the biopsy sample cells of the subject, and the prediction model is used for analysis to determine whether there is a time shift in the subject's endometrial WOI, which is classified as normal WOI, forward WOI, and backward WOI.

[0022] The combination of gene markers for detecting the offset state of the endometrial embryo implantation window (WOI) described in this invention has the following advantages:

[0023] 1) The gene biomarker combination for detecting the offset status of the endometrial embryo implantation window (WOI) described in this invention consists of 10 characteristic gene sequences shown in SEQ ID NO.1 to SEQ ID NO.10. It can be used as a molecular method to accurately determine the offset status of the endometrial embryo implantation window, thereby improving the success rate of IVF-ET and improving pregnancy outcomes for infertile patients (mainly RIF patients).

[0024] 2) This invention is mainly based on molecular biology techniques and transcriptome analysis methods to accurately determine the WOI shift status of the endometrium. Its characteristic gene markers have high sensitivity and specificity, and the prediction accuracy is 87.5%.

[0025] 3) Using the kit of the present invention, the expression of endometrial WOI shift-related characteristic gene sequences shown in SEQ ID NO.1 to SEQ ID NO.10 in the biopsy sample cells of the subject can be detected. The characteristic gene expression data of the sample can be analyzed by using a prediction model to accurately and objectively determine the endometrial WOI shift status of the subject, and provide the best embryo implantation advice to clinicians in order to improve the success rate of IVF-ET.

[0026] 4) This invention can perform characteristic gene expression data analysis on endometrial samples collected from the same patient at multiple time points within the same menstrual cycle, greatly improving accuracy and having broad application prospects. Attached Figure Description

[0027] Figure 1 This is a Venn diagram showing the differentially expressed genes among samples with different WOI offset states in this invention.

[0028] Figure 2 This is a flowchart of the sample detection and data analysis process of the present invention. Detailed Implementation

[0029] Embodiments of the present invention will now be described with reference to the accompanying drawings. Elements and features described in one drawing or embodiment of the invention may be combined with elements and features shown in one or more other drawings or embodiments. It should be noted that, for clarity, representations and descriptions of components or processes unrelated to the invention and known to those skilled in the art have been omitted from the drawings and description.

[0030] In this invention, the term "characteristic gene markers" refers to genes that can be used to assess the WOI shift status of the endometrium, and changes in the expression levels of these genes are closely related to the endometrial receptivity period.

[0031] In this invention, the term "polynucleotide" specifically refers to genes and genomic fragments obtained through artificial synthesis and processing, and is a single-stranded or double-stranded nucleotide compound.

[0032] In this invention, the term "biopsy sample cells" specifically refers to endometrial biopsy tissue, uterine irrigation fluid, and uterine fluid cells and their exfoliated cell samples.

[0033] In this invention, the term "primer" refers to an oligonucleotide that, when paired with one strand of DNA, can initiate primer extension to synthesize a product in the presence of a suitable reaction. To maximize amplification efficiency, primers are preferably single-stranded. The length of the primer depends on many factors, including: the application field, the temperature used, the template reaction conditions, other reagents, and the source of the primer. Those skilled in the art can design primers independently according to the breast cancer characteristic genes and specific requirements of this invention.

[0034] In this invention, the term "probe" refers to an oligonucleotide molecule that can bind to all or part of a specific nucleotide sequence. Probes can be labeled directly or indirectly.

[0035] This invention, based on quantitative analysis of somatic cell whole-genome expression profiles, quantitatively analyzes the gene expression status of endometrial samples, comparing gene expression differences in endometrial samples with forward-shifted WOI, normal WOI, and backward-shifted WOI. Using machine learning, it screens 10 characteristic gene combinations related to endometrial WOI shift from the whole-genome expression profile. Based on the relative expression levels of these characteristic genes, it constructs a high-precision prediction model using SVM, Random Forest, and KNN algorithms. RNA sequencing technology is used to detect the relative expression levels of these characteristic genes in the patient's endometrial samples. The model predicts the decision score of the sample, and the decision score is used to determine whether the patient's WOI has a time shift, providing a rational implantation recommendation based on the patient's specific situation.

[0036] Example 1

[0037] A group of infertile patients who were planning to undergo in vitro fertilization-embryo transfer (IVF-ET) due to fallopian tube or male factors underwent hormone replacement therapy (HRT). On day P+5 (the day of the first dose of progesterone was recorded as day P+0), tissue biopsies were performed, endometrial tissue samples were collected on day P+5, RNA was extracted and RNA sequencing was performed.

[0038] Infertile patients who successfully underwent personalized embryo transfer (pET) and achieved clinical pregnancy were selected and divided into three groups according to the implantation timing: ① WOI pre-implantation group, IVF-ET performed on day P+3 or P+4 with successful implantation; ② normal group, IVF-ET performed on day P+5 with successful implantation; ③ post-implantation group, IVF-ET performed on day P+6 or P+7 with successful implantation. Subsequently, differential gene expression analysis was performed pairwise between samples from each group to obtain a list of differentially expressed genes (DEGs).

[0039] like Figure 1 As shown, the differential expression analysis of "forward shift group vs. normal group", "backward shift group vs. normal group" and "forward shift group vs. backward shift group" yielded the intersection of DEG, which represents the 10 characteristic genes related to WOI shift in this invention. Compared to their expression in the endometrium with normal WOI, SEQ ID NO.7–SEQ ID NO.10 showed differential expression in WOI-forward and WOI-backward shift samples. Compared to the normal WOI group samples, the six characteristic gene sequences shown in SEQ ID NO.1–SEQ ID NO.6 were relatively upregulated in the WOI-forward shift group samples and relatively downregulated in the WOI-backward shift group samples; while the four characteristic gene sequences shown in SEQ ID NO.7–SEQ ID NO.10 were relatively downregulated in the WOI-forward shift group samples and relatively upregulated in the WOI-backward shift group samples. The differential expression of characteristic genes in different group samples is shown in Table 2.

[0040] Table 2. Differential expression of 10 characteristic genes in WOI shifted forward and backward samples (relative to normal WOI samples).

[0041]

[0042] * A gene differential expression fold increase (FC) > 0 indicates that the gene expression level in this sample is relatively upregulated compared to its expression level in endometrium with normal WOI; a gene differential expression fold increase (FC) < 0 indicates that the gene expression level in this sample is relatively downregulated compared to its expression level in endometrium with normal WOI.

[0043] Example 2: Sample Collection, Transportation, and Quality Control

[0044] Samples are taken from the subject or volunteer by a gynecologist or qualified technician. The samples include more than 5 mg of endometrial tissue and more than 10 μL of uterine fluid. After the biopsy, the samples are immediately added to a storage tube containing RNA preservation solution (QiagenRNA Later) and stored at -20°C or -80°C.

[0045] Sample transportation: Samples are transported to the designated laboratory using ice packs or dry ice.

[0046] Sample quality control: Quality control includes sample information, sample integrity, and measured temperature upon receipt of the sample.

[0047] The specific sample collection details and grouping are shown in Table 3.

[0048] Table 3. Statistics on the number of endometrial samples collected in different WOI offset groups.

[0049]

[0050] Example 3: RNA Sequencing

[0051] 1. RNA was extracted using the Tiangen RNAprep Pure Animal Tissue Total RNA Extraction Kit (centrifuge column type).

[0052] 2. RNA integrity was analyzed using an Agilent 2100 analyzer. A RIN > 7.0 and a 28S / 18S ratio > 1.2 were considered acceptable. RNA concentration and purity were accurately quantified using Qubit. An OD260 / OD280 ratio between 1.8 and 2.2 and an RNA extraction yield greater than 2 μg were considered acceptable.

[0053] 3. Use Novizan Library Preparation The mRNA Capture Beads kit is used to enrich and purify mRNA.

[0054] 4. RNA library preparation was performed using the KAPA Stranded RNA-Seq Library Preparation Kit.

[0055] 5. Use Agilent 2100 to analyze the distribution of library fragments and use Qubit to accurately quantify the library concentration.

[0056] 6. Use Illumina Hiseq2000 for sequencing.

[0057] Example 4: Determination of expression levels of WOI-shift-related characteristic genes in endometrial samples

[0058] 1. Data quality control: After the raw data is processed, the sequencing results are quality controlled using the FASTP software. The quality control standard is that the read length is not less than 75, and the other parameters are left as default (the raw data is called RawData, and the data after quality control is called CleanData).

[0059] 2. Ribosome Data Deletion: The rRNA content can reflect the quality of library construction to a certain extent. Therefore, before aligning with the reference genome, sortmerna is used to align CleanData to the ribosome library, removing the aligned reads to obtain de-rRNA. The default filter parameter for CleanData is 1e-5.

[0060] 3. Reference genome alignment: Align de-rRNA CleanData to the reference genome or transcriptome, calculate alignment efficiency, and perform randomness and insertion analysis based on the alignment results. The alignment software is STAR, and the alignment uses default parameters.

[0061] 4. Gene expression quantification: Based on the alignment results, featureCounts software was used to quantify gene or transcriptome-level expression, and the TPM (Trans Per Million) algorithm was used to standardize the quantified values ​​to obtain the gene expression matrix.

[0062] 5. Differential expression analysis: Based on the quantitative results, edgeR was used to perform differential expression analysis and screen differentially expressed genes. The screening threshold was FDR <= 0.05 and |log2FC| >= 1.

[0063] 6. Feature screening: Based on machine learning methods, feature screening is performed on the gene expression matrix to obtain candidate endometrial WOI offset state related gene markers.

[0064] 7. Model Construction: Based on 10×10 fold cross-validation, SVM, Random Forest, and KNN were used to train the model on the candidate markers. The best prediction model was constructed based on parameter tuning. The test results are shown in Table 4.

[0065] Table 4. Model training results for candidate markers using different machine learning algorithms.

[0066] Model Training set prediction accuracy Test set prediction accuracy SVM 93.62％ 87.50％ RF 100％ 93.75％ kNN 100％ 93.75％

[0067] Example 5: Determining the WOI offset status of the endometrium

[0068] Figure 2 This is a flowchart illustrating the sample detection and data analysis process of this invention. (According to...) Figure 2 The flowchart shown is analyzed as follows:

[0069] 1. Sample Enrollment: Eighty biopsy samples of endometrial tissue were collected from 80 patients aged 25–39 years with recurrent implantation failure, 5 days after P (120 hours after progesterone administration). All participants underwent artificial cycle intervention at a specialized hospital. All clinical research procedures were subject to ethical review by the hospital, and informed consent forms were obtained from the volunteers after they were fully informed of the full details.

[0070] 2. Samples were collected, transported, and quality controlled according to the procedure in Example 2.

[0071] 3. Perform RNA sequencing on the sample according to the procedure in Example 3.

[0072] 4. Raw data from 80 P+5 endometrial tissue samples were obtained. After analyzing the gene expression status of the biopsy sample cells, an expression matrix of characteristic gene markers related to endometrial WOI shift was output. Substituting this expression matrix into the prediction model of this invention can accurately determine whether the WOI of the individual to which the sample belongs has experienced a time shift. Based on the WOI shift status, the embryo implantation plan can be adjusted in a timely manner, and pregnancy outcomes can be recorded. Detailed results are shown in Table 5.

[0073] According to follow-up results, 87.5% (70 / 80) of the embryo implantation clinical cases guided by the technology described in this invention achieved successful pregnancies.

[0074] Table 5. Predicted WOI offset status and pregnancy outcomes of 80 P+5 endometrial tissue samples.

[0075]

[0076]

[0077]

[0078]

[0079] The present invention uses a constructed model to interpret endometrial gene expression profile data, making the detection biomarker more objective and reliable. Existing studies have shown that the expression of endometrial genes in each menstrual cycle follows a specific pattern, and the detection results remain consistent within 29-40 months, ensuring the reproducibility of the detection results.

[0080] While the invention and its advantages have been described in detail, it should be understood that various changes, substitutions, and modifications can be made without departing from the spirit and scope of the invention as defined by the appended claims. Furthermore, the scope of this application is not limited to the specific embodiments of the processes, apparatus, means, methods, and steps described in the specification. Those skilled in the art will readily understand from the disclosure of this invention that existing and future processes, apparatus, means, methods, or steps that perform substantially the same function or obtain substantially the same results as the corresponding embodiments described herein can be used according to the invention. Therefore, the appended claims are intended to include such processes, apparatus, means, methods, or steps within their scope.

Claims

1. A combination of gene markers for detecting endometrial embryo implantation window shift, characterized in that, The genetic marker combination consists of the endometrial WOI offset-related characteristic gene sequences shown in SEQ ID NO.1 to SEQ ID NO.10, wherein the sequences shown in SEQ ID NO.1 to SEQ ID NO.10 are as follows: SEQ ID NO.1ENSG00000197635 SEQ ID NO.2ENSG00000163464 SEQ ID NO.3ENSG00000180871 SEQ ID NO.4ENSG00000099985 SEQ ID NO.5ENSG00000148346 SEQ ID NO.6ENSG00000173535 SEQ ID NO.7ENSG00000169903 SEQ ID NO.8ENSG00000172824 SEQ ID NO.9ENSG00000137269 SEQ ID NO.10ENSG00000145832.

2. A nucleic acid composition, characterized in that, A combination product comprising multiple polynucleotides, wherein the sequences of the multiple polynucleotides are the endometrial WOI offset-related characteristic gene sequences shown in SEQ ID NO.1 to SEQ ID NO.10; The sequences represented by SEQ ID NO.1 to SEQ ID NO.10 are shown below: SEQ ID NO.1ENSG00000197635 SEQ ID NO.2ENSG00000163464 SEQ ID NO.3ENSG00000180871 SEQ ID NO.4ENSG00000099985 SEQ ID NO.5ENSG00000148346 SEQ ID NO.6ENSG00000173535 SEQ ID NO.7ENSG00000169903 SEQ ID NO.8ENSG00000172824 SEQ ID NO.9ENSG00000137269 SEQ ID NO.10ENSG00000145832.

3. The use of the gene marker combination of claim 1 or the nucleic acid composition of claim 2 in the preparation of a product for detecting the WOI shift status of the endometrium.

4. The application according to claim 3, characterized in that, The products for detecting the WIO shift status of the endometrium include those that use RNA sequencing, gene chips, and real-time quantitative PCR to detect and evaluate the WIO shift status of the endometrium.

5. The application according to claim 4, characterized in that, The product for detecting endometrial WII shift state using RNA sequencing contains messenger RNA sequences of characteristic gene sequences related to endometrial WII shift state as shown in SEQ ID NO.1 to SEQ ID NO.10, as well as corresponding detection primers and / or probes.

6. The application according to claim 4, characterized in that, The product for detecting endometrial WIO shift status contains hybridization probes with characteristic gene sequences related to endometrial WIO shift status as shown in SEQ ID NO.1 to SEQ ID NO.

10.

7. The application according to claim 4, characterized in that, The product for detecting endometrial WII shift status contains messenger RNA sequences that specifically amplify the characteristic gene sequences related to endometrial WII shift status shown in SEQ ID NO.1 to SEQ ID NO.10, as well as corresponding detection primers and / or probes.

8. A kit for detecting the WOI offset state of the endometrium, characterized in that, The kit contains messenger RNA sequences specifically targeting the combination of gene markers of claim 1, as well as corresponding detection primers and / or probes.

9. The reagent kit according to claim 8, characterized in that, When using the kit, firstly, based on the expression level data of the endometrial WOI shift-related characteristic gene sequences shown in SEQ ID NO.1 to SEQ ID NO.10 in endometrial cells, a prediction model is constructed by combining SVM, Random Forest, and KNN algorithms. Then, the kit is used to detect the expression level data of the characteristic gene sequences in the biopsy sample cells of the subject, and the prediction model is used for analysis to determine whether there is a time shift in the subject's endometrial WOI, which is classified as normal WOI, forward WOI, and backward WOI.

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