CN116287219A - Gene marker combination for detecting endometrial embryo implantation window deviation state and application thereof - Google Patents

Abstract

The invention discloses a gene marker combination for detecting the deviation state of an endometrial embryo implantation window, which comprises embryo implantation window deviation related characteristic gene sequences shown in SEQ ID NO. 1-SEQ ID NO. 10. The invention can accurately and effectively judge whether the time shift occurs in the embryo implantation Window (WOI) of the endometrium, and accordingly gives instruction advice on whether the WOI of the testee is correspondingly regulated, thereby remarkably improving the success rate of in vitro fertilization-embryo implantation (IVF-ET).

Description

Gene marker combination for detecting endometrial embryo implantation window deviation state and application thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to a gene marker combination for detecting a WOI (WOI) offset state of an endometrial embryo implantation window and application thereof.

Background

Assisted Reproductive Therapy (ART), particularly in vitro fertilization-embryo transfer (IVF-ET) techniques, are currently accepted as an effective means of treating infertility. However, the data show that the success rate of embryo transfer using the traditional IVF-ET protocol is only about 40%, and that a significant portion of patients experience repeated planting failures (recurrent implantation failure, RIF), i.e., experience more than 3 embryo planting, and that clinical pregnancy has not yet been achieved.

It was found that about 2/3 of the failure cases resulted from misjudged patients' embryo implantation Windows (WOIs). In traditional cognition, WOIs of female endometrium are open at 19-21 days of menstrual cycle, however, repeated transplantation failure or female suffering secondary infertility often occurs with forward or backward movement of time node of WOIs, for example, IVF-ET is implemented according to traditional embryo implantation scheme, i.e. on natural cycle lh+7 days or artificial cycle p+5 days, and there is a risk of embryo transplantation failure. Therefore, the detection method capable of accurately judging whether the WOI of the endometrium deviates or not has important significance for diagnosis and treatment of patients with reproductive disorder.

At present, the method of tissue morphology detection, ultrasonic detection, molecular marker detection and the like can be used for roughly positioning the WOI of a patient in clinic, but the technical methods cannot accurately judge the WOI deviation state, and a certain dispute exists in technical theory. Histomorphometric assays with pinocytosis as a marker, disadvantages include: (1) invasive detection can cause certain damage to the endometrium; (2) the industry is controversial as to whether pinocytosis can be used as an implant marker; (3) subjective interpretation is dependent on experience judgment of pathology specialists, and objectivity is lacking; (4) the reproducibility of the results is poor and the assay cannot be transplanted in the current cycle and has limited prognostic value. The ultrasonic imaging detection has the defects that the marker comprises endometrium thickness, echo type, subintimal blood flow and the like, the unified discrimination standard is lacking, the significance of single parameters on ER evaluation is not obvious and is controversial, the comprehensive evaluation of a plurality of parameters is required to be combined, and the reliability and the stability are poor. Molecular marker detection, wherein the markers comprise cytokines, homologous frame genes and the like, and have the defects of poor repeatability, large variability and the like when a single molecular marker is used as an evaluation standard.

Therefore, in the face of the reproductive medicine problem of repeated planting failure and the increasingly severe infertility disease situation, researchers are urgently required to find a technical method for accurately judging the WOI shift state of the endometrium, so that the WOI can be timely regulated by patients, and the method has great significance for improving the success rate of IVF-ET and improving the pregnancy outcome of infertility patients.

Disclosure of Invention

The invention aims to solve the technical problems that whether an endometrial embryo implantation Window (WOI) of a female who is in a proposed IVF-ET is time-shifted cannot be accurately judged in the prior art, and a clinician is difficult to be helped to determine whether a traditional embryo implantation window (natural period LH+7 days or artificial period P+5 days) is suitable for embryo transplantation, and provides a gene marker combination or a nucleic acid composition for detecting the shift state of the endometrial embryo implantation window (window of implantation, WOI), so that the success rate of IVF-ET is improved for an infertility patient (mainly RIF patient) and the pregnancy outcome of the infertility patient is improved.

It is another object of the present invention to provide the use of said gene marker combination or nucleic acid composition for the preparation of a detection system product for detecting the WOI shift status of endometrium.

In order to solve the problems, the invention provides a gene marker combination for detecting the endometrial embryo implantation window deviation state, wherein the gene marker combination is an endometrial WOI deviation related characteristic gene sequence shown in SEQ ID NO. 1-SEQ ID NO. 10.

Wherein specific gene marker combinations are shown in table 1.

TABLE 1 10 combinations of characteristic genes related to endometrial embryo implantation window shift

The invention provides a nucleic acid composition, which comprises a combination product of a plurality of polynucleotides or fragments thereof, wherein the polynucleotides can be differentially expressed in different degrees in the endometrium of luteal phase, and the sequences of the polynucleotides are the endometrium WOI offset related characteristic gene sequences shown in SEQ ID NO. 1-SEQ ID NO. 10.

The 6 characteristic gene sequences (DPP 4, CXCR1, CXCR2, OSM, LCN2 and TNFRSF 10C) shown in SEQ ID No. 1-SEQ ID No.6 are relatively up-regulated in the WOI forward group sample and relatively down-regulated in the WOI backward group sample.

The 4 characteristic gene sequences (TM 4SF4, CES4A, LRRC1 and SLC25A 48) shown in SEQ ID No. 7-SEQ ID No.10 are relatively down-regulated in the WOI forward group sample and relatively up-regulated in the WOI backward group sample.

The invention also provides the application of the gene marker combination or the nucleic acid composition in preparing a product for detecting the WOI shift state of endometrium.

Preferably, the product for detecting the WOI shift status of endometrium comprises: products for detection and evaluation of endometrial WOI shift status by RNA sequencing (RNA-seq), gene chip (microarray), and real-time quantitative PCR (qRT-PCR).

The product for detecting the endometrial WOI deviation state by using RNA sequencing comprises messenger RNA sequences of the endometrial WOI deviation state related characteristic gene sequences shown in SEQ ID NO. 1-SEQ ID NO.10 and corresponding detection primers and/or probes.

The product for detecting the endometrial WOI deviation state by using the gene chip comprises hybridization probes of the endometrial WOI deviation state related characteristic gene sequences shown in SEQ ID NO. 1-SEQ ID NO. 10.

The product for detecting the endometrial WOI deviation state by using the real-time quantitative PCR comprises a messenger RNA sequence for specifically amplifying the endometrial WOI deviation state related characteristic gene sequences shown in SEQ ID NO. 1-SEQ ID NO.10 and corresponding detection primers and/or probes.

The invention also provides a kit for detecting the endometrial WOI shift state, which comprises a messenger RNA sequence specific to the endometrial WOI shift related characteristic gene sequences shown in SEQ ID NO. 1-SEQ ID NO.10 and corresponding detection primers and/or probes.

When the kit is used, firstly, a prediction model is constructed based on the expression quantity data of the characteristic gene sequences related to the endometrial WOI offset shown in SEQ ID NO. 1-SEQ ID NO.10 in endometrial cells in combination with SVM, random Forest and KNN algorithms, then the expression quantity data of the characteristic gene sequences in the biopsy sample cells of a tested person are detected by the kit, and the prediction model is utilized to analyze, so that whether the endometrial WOI of the tested person has time offset or not is determined, and the time offset is classified into normal WOI, forward WOI and backward WOI.

The gene marker combination for detecting the deviation state of the endometrial embryo implantation window (window of implantation, WOI) has the following advantages:

1) The gene marker combination for detecting the endometrial embryo implantation window (window of implantation, WOI) deviation state is 10 characteristic gene sequences shown in SEQ ID NO. 1-SEQ ID NO.10, can be used as a molecular method for accurately judging the endometrial embryo implantation window deviation state, and can improve the success rate of IVF-ET for sterile patients (mainly RIF patients) and the pregnancy ending of the sterile patients.

2) The invention realizes accurate judgment of the WOI shift state of endometrium based on molecular biology technology and transcriptome analysis method, and has the characteristics of high sensitivity, strong specificity and 87.5% prediction accuracy of the marker of the characteristic gene.

3) The kit can detect the expression condition of the endometrial WOI offset related characteristic gene sequences shown in SEQ ID NO. 1-SEQ ID NO.10 in the biopsy sample cells of the testee, analyze the characteristic gene expression data of the sample by using a prediction model, accurately and objectively judge the endometrial WOI offset state of the testee, and provide the optimal embryo implantation suggestion for clinicians so as to improve the success rate of IVF-ET.

4) The invention can analyze the characteristic gene expression data of the endometrium samples collected at a plurality of time points in the same menstrual cycle of the same patient, greatly improves the accuracy and has wide application prospect.

Drawings

FIG. 1 is a diagram of a gene Venn differentially expressed between samples of different WOI shift states according to the present invention;

FIG. 2 is a flow chart of sample detection and data analysis according to the present invention.

Detailed Description

Embodiments of the present invention are described below 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 the illustration and description of components or processes known to those skilled in the art, which are not related to the present invention, have been omitted in the drawings and description for the sake of clarity.

In the present invention, the term "signature gene marker" means a gene which can be used in the evaluation of the WOI shift status of the endometrium, and the change in the expression level of the gene is closely related to the endometrial tolerance period.

In the present invention, the term "polynucleotide" refers specifically to a single-stranded or double-stranded nucleotide compound, which is obtained by artificial synthesis and processing of genes and genome fragments.

In the present invention, the term "biopsy sample cells" refers in particular to endometrial biopsy tissue, uterine cavity lavage fluid and uterine cavity fluid cells and exfoliated cell samples thereof.

In the present invention, the term "primer" means an oligonucleotide capable of initiating primer extension in the presence of a suitable reaction to synthesize a product when paired with one strand of DNA. To maximize amplification efficiency, the primer is preferably single stranded. The length of the primer depends on a number of factors, including: application fields, temperatures employed, template reaction conditions, other reagents and primer sources. The skilled artisan can autonomously design primers according to the breast cancer trait genes and specific requirements of the present invention.

In the present invention, the term "probe" refers to an oligonucleotide molecule capable of binding to all or part of a specific nucleotide sequence. The probes may be labeled directly or indirectly.

On the basis of quantitative analysis of whole body cell gene expression profiles, the embodiment of the invention compares the gene expression differences of the WOI forward movement, the WOI normal and the WOI backward movement type endometrium by quantitatively analyzing the gene expression condition of an endometrium sample, screens out 10 characteristic gene combinations related to the WOI deviation of the endometrium from the whole gene expression profiles by a machine learning method, and constructs a high-precision prediction model based on the relative expression quantity data of the characteristic genes and combined with SVM, random Forest and KNN algorithms. The relative expression quantity data of the characteristic genes in the endometrial sample of the subject is detected through an RNA sequencing technology, a decision score (decision score) of the sample is obtained through model prediction, finally whether the WOI of the patient has time offset or not is determined according to the decision score, and reasonable implantation suggestions are given for the actual situation of the subject.

Example 1

A batch of infertility patients undergoing in vitro fertilization-embryo transfer (IVF-ET) due to oviduct or male factor were subjected to Hormone Replacement Therapy (HRT), tissue biopsies were taken on day P+5 (the first administration of progesterone was taken when the diary was P+0), P+5 endometrial tissue samples were collected, RNA was extracted and RNA sequencing was performed.

Sterile patients from which personalized embryo transfer (pET) was successfully performed and clinical pregnancy was achieved were selected and divided into 3 groups according to implantation timing, including: (1) WOI forward group, develop IVF-ET and implant successfully on P+3 or P+4 days; (2) normal group, IVF-ET was developed and implanted successfully on day P+5; (3) the group was moved backward, IVF-ET was developed and implanted successfully on either P+6 or P+7 days. Then, gene differential expression analysis is carried out between every two groups of samples, and a Differential Expression Gene (DEG) list is obtained.

As shown in fig. 1, the DEG intersection is obtained by the difference analysis of the "forward group vs normal group", "backward group vs normal group" and "forward group vs backward group", which is the 10 characteristic genes related to WOI shift in the present invention. SEQ ID No.7 to SEQ ID No.10 are differentially expressed in WOI advanced and WOI advanced samples relative to the expression in woI normal endometrium. Relative to the WOI normal group sample, 6 characteristic gene sequences shown in SEQ ID NO. 1-SEQ ID NO.6 are relatively up-regulated in the WOI forward group sample and relatively down-regulated in the WOI backward group sample; the 4 characteristic gene sequences shown in SEQ ID No. 7-SEQ ID No.10 are relatively down-regulated in the WOI forward group sample and relatively up-regulated in the WOI backward group sample. The differential expression of the signature genes in the different panel samples is shown in Table 2.

TABLE 2 statistics of differential expression of 10 signature genes in WOI Forward and backward samples (relative to WOI Normal samples)

* Fold differential expression (FC) >0 of the gene, i.e., demonstrates that the amount of expression of the gene in the sample is relatively up-regulated relative to that in WOI-normal endometrium; fold differential expression (FC) <0 of the gene, i.e., indicating that the amount of expression of the gene in this sample is relatively down-regulated relative to that in WOI-normal endometrium.

Example 2 sample collection, transport, quality control

The subject or volunteer is sampled by a gynecologist or professional qualification technician, the sample comprising greater than 5mg of endometrial tissue, greater than 10uL of uterine fluid. Immediately after the biopsy was completed, the sample was added to a storage tube containing RNA storage solution (Qiagen RNA Later) and stored at-20℃or-80 ℃.

Sample transportation: the samples were transported to the designated laboratory using ice bags or dry ice.

Sample quality control: the quality control content comprises sample information, sample integrity, measured temperature when the sample is received, and the like.

The specific sample collection and grouping are shown in table 3.

Table 3 statistics of the number of endometrial samples collected in groups of different WOI shift states

EXAMPLE 3RNA sequencing

1. RNA was extracted using the tenna prep Pure animal tissue total RNA extraction kit (centrifugation column).

2. RNA integrity was analyzed using Agilent2100, and RIN >7.0, 28S/18S >1.2 was acceptable. The concentration and purity of RNA are accurately and quantitatively detected by using Qubit, the OD260/OD280 is between 1.8 and 2.2, and the extraction quantity of RNA is more than 2 ug.

3. Using Northene Library Preparation

mRNA Capture Beads the mRNA is enriched and purified by the kit.

4. RNA pooling was performed using KAPA Stranded RNA-Seq Library Preparation Kit.

5. Library fragment distribution was analyzed using Agilent2100 and library concentrations were accurately quantified using Qubit.

6. Sequencing was performed using an illuminea Hiseq2000 on-machine.

Example 4 measurement of WOI offset-related characteristic Gene expression in endometrial samples

1. And (3) data quality control: after the original data is taken off, the quality control is carried out on the sequencing result through fastp software, the quality control standard is that the read length is not less than 75, and the rest parameters are default (the original data is called RawData, and the data after quality control is called CleanData).

2. Ribosome data deletion: rRNA content can reflect the quality of library construction to a certain extent, so that clearData is compared to a ribosome library by using sortmer before comparison of reference genome, and read on the comparison is removed, so that the default screening parameter of de-rRNA clearData is 1e-5.

3. Reference genome alignment: the de-rRNA CleanData is aligned to a reference genome or transcriptome, the alignment efficiency is counted, and the alignment software is STAR according to the randomness analysis and the insert analysis of the alignment result, and the default parameters are used for alignment.

4. Quantification of gene expression: based on the comparison result, the featurescents software is used for carrying out gene or transcriptome level expression quantification, and a TPM (Trans Per Million) algorithm is adopted for normalizing the quantitative value to obtain a gene quantitative expression matrix.

5. Differential expression analysis: based on the quantitative results, differential expression analysis was performed using edge, screening for differentially expressed genes, with a screening threshold of FDR < = 0.05 and |log2fc| > =1.

6. Feature screening: and (3) based on a machine learning method, screening the characteristics of the gene expression matrix to obtain candidate endometrial WOI offset state related gene markers.

7. Model construction: model training is carried out on candidate markers based on 10 multiplied by 10 fold cross validation by using SVM, random Forest and KNN respectively, an optimal prediction model is built based on parameter tuning, and test results are shown in table 4.

Table 4 model training conditions of different machine learning algorithms for candidate markers

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

Example 5 determination of endometrial WOI offset status

FIG. 2 is a flow chart of sample detection and data analysis according to the present invention. The detection analysis according to the flow chart shown in fig. 2 is as follows:

1. sample entry: 80 biopsy samples taken from 80 patients with repeated failure of planting between 25 and 39 years old, P+5 days (120 hours after progesterone administration), of endometrial tissue, were selected as subjects. All study individuals performed sample collection at a professional hospital manual cycle dry prognosis. All clinical study procedures passed the ethical review of the hospital and informed consent was signed with the volunteer informed of the true condition.

2. Samples were collected, transported and quality controlled according to the procedure of example 2.

3. Samples were RNA sequenced according to the procedure of example 3.

4. And obtaining original off-machine data of 80 P+5 endometrial tissue samples, analyzing the gene expression condition of the biopsy sample cells, and outputting an expression quantity matrix of the endometrial WOI offset related characteristic gene markers. By substituting the expression quantity matrix into the prediction model, whether the WOI of the individual to which the sample belongs is subjected to time migration or not can be accurately judged, the embryo implantation scheme is timely adjusted according to the WOI migration state, the pregnancy ending is recorded, and the detailed result is shown in the table 5.

According to the follow-up result, 87.5% (70/80) of successful pregnancy is realized through the embryo implantation clinical case guided by the technology.

TABLE 5 WOI shift status prediction results and pregnancy outcome for 80 P+5 endometrial tissue samples

The detection marker uses the endometrium gene expression profile data as the interpretation basis by using the construction model, so that the detection marker is more objective and reliable; the existing research shows that the expression of the endometrial genes in each menstrual cycle follows a specific rule, the detection result is consistent within 29-40 months, and the repeatability of the detection result is ensured.

Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, means, method and steps described in the specification. Those of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, the appended claims are intended to include within their scope such processes, apparatuses, means, methods, or steps.

Claims (9)

1. The gene marker combination for detecting the endometrial embryo implantation window deviation state is characterized by being an endometrial WOI deviation related characteristic gene sequence shown in SEQ ID NO. 1-SEQ ID NO. 10.

2. A nucleic acid composition comprising a combination of a plurality of polynucleotides having the sequences set forth in SEQ ID No.1 to SEQ ID No.10 and having a WOI shift-related characteristic gene sequence of endometrium, or a fragment thereof.

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

4. The use according to claim 3, wherein the product for detecting the WOI shift status of the endometrium comprises: and (3) detecting and evaluating the WOI shift state of the endometrium by using RNA sequencing, a gene chip and real-time quantitative PCR.

5. The use according to claim 4, wherein the product for detecting the deviation status of endometrial WOI by RNA sequencing comprises the messenger RNA sequences of the characteristic gene sequences related to the deviation status of endometrial WOI as shown in SEQ ID No.1 to SEQ ID No.10 and corresponding detection primers and/or probes.

6. The use according to claim 4, wherein the product for detecting the deviation state of endometrial WOI by using the gene chip comprises hybridization probes of the characteristic gene sequences related to the deviation state of endometrial WOI shown in SEQ ID No.1 to SEQ ID No. 10.

7. The use according to claim 4, wherein the product for detecting the deviation status of endometrial WOI by real-time quantitative PCR comprises a messenger RNA sequence specifically amplifying the gene sequences related to the deviation status of endometrial WOI as shown in SEQ ID No.1 to SEQ ID No.10, and corresponding detection primers and/or probes.

8. A kit for detection of the WOI shift status of endometrium, characterized in that the kit comprises a messenger RNA sequence specific for a combination of gene markers according to claim 1 and corresponding detection primers and/or probes.

9. The kit according to claim 8, wherein in use, the kit is firstly based on the expression level data of the characteristic gene sequences related to the endometrial WOI shift shown in SEQ ID No.1 to SEQ ID No.10 in endometrial cells, a prediction model is constructed by combining with SVM, random Forest and KNN algorithms, then the expression level data of the characteristic gene sequences in the biopsy sample cells of the subject is detected by using the kit, and analysis is performed by using the prediction model to determine whether the endometrial WOI of the subject has time shift, and is classified into WOI normal, WOI forward shift and WOI backward shift.

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