CN115873937A

CN115873937A - Biomarker for predicting occurrence of repeated planting failure and application thereof

Info

Publication number: CN115873937A
Application number: CN202210973641.XA
Authority: CN
Inventors: 黄伯贤; 陆嘉逢; 郭灵; 李红; 刘振兴; 钱春凤
Original assignee: Suzhou Municipal Hospital
Current assignee: Suzhou Municipal Hospital
Priority date: 2022-08-15
Filing date: 2022-08-15
Publication date: 2023-03-31
Anticipated expiration: 2042-08-15
Also published as: CN115873937B

Abstract

The invention discloses a biomarker NSUN2 for predicting repeated planting failure occurrence in an assisted reproduction technology. The effect of the expression level or methylation level of NSUN2 on the occurrence of recurrent planting failures was disclosed, and it was found that the adhesion efficiency of embryonal balls significantly decreased after NSUN2 overexpression or hypermethylation. The discovery of the invention provides a new thought and theoretical basis for the prediction, diagnosis and prognosis of diseases related to the adhesion, planting and implantation of embryos.

Description

Biomarker for predicting occurrence of repeated planting failure and application thereof

Technical Field

The invention belongs to the technical field of endometrial function detection, and particularly relates to a biomarker for predicting repeated planting failure and application of the biomarker in endometrial receptivity detection.

Background

Failure in repeated planting (recovery Implantation failure) refers to failure of embryo transplantation of <40 years old and more than or equal to 3 times in the past; or more than or equal to 2 embryo transplantation failures, and the number of the transplanted embryos is more than or equal to 4 high-scoring embryo in the cleavage stage (I or II grade D3 embryo), or more than or equal to 2 high-scoring blastocysts (Gardner blastocyst score AA/AB/BA/BB), or 2 high-scoring embryo in the cleavage stage and 1 high-scoring blastocyst.

Pregnancy is the result of successful implantation of the embryo into the endometrium, followed by sufficient decidualization of the endometrium, and placental formation. Each step requires the involvement of numerous cytokines and physiological processes to achieve the balance necessary for pregnancy development. Implantation is the combination of dynamic interactions between the normally functioning endometrium and blastocyst. Implantation requires the involvement of a variety of physiological processes, requiring adhesion of the embryo, invasion of trophoblasts and homeostasis of immune regulation. Successful implantation depends to a large extent on the proper structure and adequate tolerability of the endometrium. The period of high embryo implantation rate is called implantation window (the window of implantation WOI), which depends on the high tolerance of endometrium. Endometrial receptivity is the result of many physiological and molecular mechanisms, and during embryo transfer it is very important to recognize WOI and maintain endometrial receptivity.

After about half a month after embryo transplantation, negative detection of HCG in blood indicates implantation failure. Due to the complexity of the embryo implantation process, RIF needs to be evaluated from multiple layers. Although the exact mechanism of occurrence of RIF has not been fully elucidated, numerous studies have shown that imbalances in hormone levels, angiogenesis and immunoregulatory disorders, and certain genetic polymorphisms are associated with the occurrence of RIF. RiF risk factors mainly include female age, BMI, stress, male and female smoking, and the occurrence causes mainly include embryonic factors, endometrial receptivity, diseases and other comprehensive factors. Embryo factors include chromosomal or genetic abnormalities, embryo culture conditions, zona pellucida sclerosis, type and technique of embryo transfer. The factors influencing the receptivity of endometrium mainly comprise immune factors, infection factors, expression of related cell molecules, uterine cavity abnormality and the like. Existing studies find NK cells and T cells in cellular immunity; IL1a, IL1b, IL6, IL15 of the interleukin family; leukemia Inhibitory Factor (Leukemia Inhibitory Factor, LIF) vascular endothelial growth Factor VEGF; homeobox gene a10 (HOXA 10); forkhead transcription factors FOXO1 and KLF12; the claudin family protein 4 (CLDN 4) and the like are involved in the regulation of the RIF generation mechanism.

RNA methylation is one of common chemical modification modes of RNA, belongs to the category of epigenetics, and researches in recent years find that RNA methylation is involved in regulation and control of various biological functions and is related to pathogenesis of various diseases. Currently, m6A (N6-methylaldenosine, 6-methyladenine) and m5C (5-methylcytosine ) are being studied more. m5C is widely found in a variety of RNAs, including cytoplasmic and mitochondrial ribosomal RNAs (cytoplasmic and mitogenic ribosomal RNAs) and transfer RNAs (trnas), as well as messenger RNAs (mrnas), enhancer RNAs (enrhancer RNAs), and many non-coding RNAs (ncrnas). In eukaryotes, m5C methylation is catalyzed by an enzyme of the NOL1/NOP2/SUN domain (NSUN) family (NSUN 1-7) and the DNA methyltransferase homolog DNMT 2. In recent years, numerous studies have revealed a variety of molecular functions of m5C in RNA processing, such as mRNA export, RNA stability, translation, and long-distance transport of plant RNA. In addition, m5C modification of RNA plays an important regulatory role in mitochondrial activity, stress response, gametogenesis and embryogenesis, neural and brain development, and the development and migration of various tumors.

In conclusion, at present, no exact index can predict the occurrence of repeated planting failure. Therefore, a marker capable of accurately predicting the occurrence of the repeated planting failure is urgently needed, and a theoretical basis is provided for diagnosis and treatment of the occurrence of the repeated planting failure.

Disclosure of Invention

In order to solve the problems, the inventor finds that NSUN2 can be used as a biomarker for predicting the occurrence of repeated planting failure by finding that the increase of NSUN2 expression level causes the decrease of embryo adhesion level through multi-angle research.

In particular, the invention provides the use of the NSUN2 gene or protein as a biomarker for the preparation of a reagent for Recurrent Implantation Failure (RIF), embryo implantation failure risk prediction and/or endometrial receptivity assessment.

In a second aspect, the invention provides the use of a reagent for detecting the NSUN2 gene or protein in the preparation of a kit for Recurrent Implantation Failure (RIF), prediction of risk of embryo implantation failure and/or assessment of endometrial receptivity.

In certain embodiments, the reagents detect the level of transcription of the gene by one or a combination of real-time fluorescent quantitative PCR and high-throughput sequencing.

Preferably, the detection reagent of the NSUN2 gene comprises a primer and/or a probe for NSUN2 real-time fluorescent quantitative PCR detection; more preferably, the forward primer and the reverse primer of the real-time fluorescent quantitative PCR detection of NSUN2 are respectively shown in SEQ ID NO:1 (acctgctcaagaccacacag) and SEQ ID NO:2 (TGGCTTGATGGACGAGCAGGTA).

In certain embodiments, the reagent detects the level of the protein by one or a combination of BCA protein quantification, immunohistochemistry, western blotting, and ELISA.

Preferably, the reagent for detecting the NSUN2 protein comprises an NSUN 2-specific antibody.

In certain embodiments, the reagent detects the level of methylation of the gene by high throughput sequencing.

In certain embodiments, the test sample of the kit is endometrial tissue from a RIF patient; preferably endometrial tissue at the window of embryo implantation.

In a third aspect, the present invention provides a Recurrent Implantation Failure (RIF), embryo implantation failure risk prediction and/or endometrial receptivity assessment kit, comprising: a reagent for detecting the transcriptional level of the NSUN2 gene, a reagent for detecting the methylation level of the NSUN2 gene and/or a reagent for detecting the protein level of the NSUN2 gene.

In a fourth aspect, the invention provides a method of assessing endometrial receptivity in a endometrial tissue sample for non-disease diagnosis and treatment purposes, said method comprising detecting the NSUN2 gene transcript level, NSUN2 gene methylation level and/or NSUN2 protein level in the tissue; using the NSUN2 transcription level, NSUN2 gene methylation level or NSUN2 protein level value of the patient with repeated planting failure or embryo implantation failure as a control; higher NSUN2 gene transcript levels, NSUN2 gene methylation levels, and/or NSUN2 protein than controls indicate poor endometrial tissue intima compatibility.

A fifth aspect of the invention provides a system for Recurrent Implantation Failure (RIF), prediction of risk of embryo implantation failure, and/or assessment of endometrial receptivity, comprising: 1) Reagents for detecting the transcriptional level of NSUN2, the methylation level of NSUN2, and/or the protein level of NSUN 2; 2) The device comprises a data input module, a data comparison module and a conclusion output module.

In some embodiments, the data input module is configured to input a transcriptional level value of NSUN2 in the test sample.

In some embodiments, the data comparison module is configured to compare the transcription level value of the test sample NSUN2 with a control value; the control values are the transcriptional level of NSUN2, the methylation level of the NSUN2 gene or the NSUN2 protein level in patients with recurrent planting failures or embryonic bed failures.

In some embodiments, the conclusion output module is configured to output the conclusion according to the following criteria: if the transcriptional level of NSUN2, the methylation level of the NSUN2 gene, or the NSUN2 protein level in the sample of the subject is higher than or close to the control value, the subject is a recurrent implantation failure or is candidate for embryo implantation failure or poor endometrial receptivity.

Compared with the prior art, the invention has the beneficial effects that:

the invention discovers the biomarker NSUN2 of repeated planting failure (RIF) for the first time. The expression level or methylation level of NSUN2 affects the risk of occurrence of repeated planting failure, and the adhesion efficiency of embryoid balls is significantly reduced after NSUN2 is overexpressed or hypermethylated. The invention provides a new thought and theoretical basis for the prediction, diagnosis and prognosis of diseases related to the adhesion, planting and implantation of embryos.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

figure 1BCA assay of NSUN2 protein levels in RIF patients.

FIG. 2 real-time quantitative PCR detection of mRNA levels in RIF patients.

FIG. 3 high throughput sequencing measures mRNA levels and methylation levels in RIF patients. FIG. 3A shows the difference in expression of m 5C-related writers in the RIF group compared to the CTRL group; figure 3B shows a heatmap analysis of the global methylation levels of genes between the RIF and CTRL groups. Figure 3C shows the number of sites modified by m5C in the RIF and CTRL groups compared. Figure 3D shows a comparison of the base factors of the m5C modifications in the RIF and CTRL groups.

FIG. 4 Effect of overexpression of NSUN2 on embryo adhesion efficiency. FIG. 4A shows comparison of the adhesion of the embryonic balls in the NSUN2 overexpression group to the control group. Fig. 4B shows statistics of the efficiency of embryosphere adhesion for the NSUN2 over-expression group versus the control group. Figure 4C shows the comparison of the efficiency of adhesion of embryonic spheres to NSUN2 over-expressed versus control.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings of the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without any inventive step, are within the scope of protection of the invention.

Unless defined otherwise, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.

The invention is illustrated below with reference to specific examples.

Example 1BCA assay for NSUN2 protein levels in RIF patients

3 patients with Recurrent Implantation Failure (RIF) (designated RIF1, RIF2, RIF 3) and 3 cases of control (designated Ctrl1, ctrl2, ctrl 3) secreted mid-term endometrium were collected from the cases of clinically assisted reproductive treatment. After grinding the tissue, the tissue is lysed by using a lysis solution, and then protein in the inner membrane tissue is extracted, and the protein concentration is determined by BCA. The protein concentration was diluted to 2. Mu.g/. Mu.L, and an equal amount of the protein was subjected to protein electrophoresis. After membrane transfer, blocking was performed with 5% skim milk, followed by overnight incubation at 4 ℃ in a shaker using primary antibody to NSUN2 and GAPDH.

And incubating with the second antibody of the same species on the next day, and preparing a developing solution for developing and photographing. As a result, as shown in fig. 1, it was found that the protein expression level of NSUN2 was significantly higher in endometrial tissue of patients who failed in repeated planting than in the control group.

Example 2 real-time quantitative PCR detection of mRNA levels in RIF patients

3 patients with Recurrent Implantation Failure (RIF) and 3 cases of the control group with middle-stage endometrial secretion were collected from the cases of clinical adjuvant reproduction therapy. After grinding the tissue using a cryomill, cells were lysed by adding Trizol to release RNA from the cells. The RNA precipitate obtained is eluted by a proper amount of enzyme-free water through chloroform extraction, isopropanol precipitation, ethanol washing and air drying, and the concentration is measured. The obtained RNA was subjected to reverse transcription to obtain a single-stranded DNA template, which was then subjected to Q-PCR to measure the mRNA expression levels of NSUN2 and GAPDH. The results are shown in fig. 2, where the mRNA expression level of NSUN2 in the secretory endometrial tissue of patients with recurrent implantation failures was significantly higher than that of the control group.

The primer probe sequence of Q-PCR is:

NSUN2(human)F：ACCTGGCTCAAAGACCACACAG(SEQ ID NO：1)；

NSUN2(human)R：TGGCTTGATGGACGAGCAGGTA(SEQ ID NO：2)；

GAPDH(human)F:GTCTCCTCTGACTTCAACAGCG(SEQ ID NO：3)；

GAPDH(human)R：ACCACCCTGTTGCTGTAGCCAA(SEQ ID NO：4)。

example 3 high throughput sequencing for mRNA levels and methylation levels in RIF patients

3 patients with Recurrent Implantation Failure (RIF) and 3 cases in the control group were collected from the cases clinically treated for assisted reproduction and RNA was extracted after secreting endometrium in the middle stage. mRNA was isolated from total RNA and further BSP experiments were performed to convert the C base of mRNA that was m5C modified to U base. And constructing an RNA library, and performing high-throughput methylation sequencing after quality control is qualified. Results as shown in fig. 3, the results of the sequencing by methylation of the intima tissue showed that the expression level of NSUN2 was significantly increased in the patients with failed repeated planting, while the overall methylation level of the intima was significantly increased in the patients with failed repeated planting. Specifically, FIG. 3A shows the difference in expression of m 5C-related writers in the RIF group versus the CTRL group; figure 3B shows a heatmap analysis of the global methylation levels of genes between the RIF and CTRL groups. Figure 3C shows the number of sites modified by m5C in the RIF and CTRL groups compared. Figure 3D shows a comparison of the base factors of the m5C modifications in the RIF and CTRL groups.

Example 4 Effect of overexpression of NSUN2 on embryo adhesion efficiency

NSUN2 was overexpressed on endometrial cancer cell lines (Ishikawa), plated using Ishikawa on day one in 6-well plates with a plating cell count of 3.0X 10^ 5. After 24 hours of plating, plasmid transfection was performed, NSUN2 overexpression plasmid was transfected into 3 wells, and another 3 wells used pcdna3.1 as a control, serum-free DMEM was used for 48 hours, and then serum-containing DMEM was replaced, and simultaneously suspension culture was performed using human chorionic carcinoma cell line (Bewo), and cytosphere mock blastocyst with the same diameter and size were selected for planting.

The same number of mock embryonic balls were inoculated into NSUN2 over-expressed Ishikawa cells and control cells. After 2 hours of co-culture, the dish was gently shaken, and the detached cell pellet was washed away with dPBS. 4% paraformaldehyde was fixed for 30 minutes at room temperature. Residual adherent cell pellet counts were performed under microscope and results are shown in figure 4, where the adhesion efficiency of embryosphere was significantly lower in NSUN2 over-expressed tissues than in control group. Specifically, FIG. 4A shows comparison of the adhesion of the embryonic spheres in the NSUN2 overexpression group compared to the control group. Fig. 4B shows statistics of the efficiency of embryosphere adhesion for the NSUN2 over-expression group versus the control group. Figure 4C shows the comparison of the efficiency of adhesion of embryonic spheres to NSUN2 over-expressed versus control.

While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and the description is given here only for clarity, and those skilled in the art should integrate the description, and the embodiments may be combined appropriately to form other embodiments understood by those skilled in the art.

Claims

Use of the NSUN2 gene or protein as a biomarker for the preparation of an agent for Recurrent Implantation Failure (RIF), risk prediction of embryo implantation failure and/or endometrial receptivity assessment.
Use of a detection reagent for the NSUN2 gene or protein for the preparation of a kit for Recurrent Implantation Failure (RIF), risk prediction of embryo implantation failure and/or assessment of endometrial receptivity.
3. The use according to claim 1 or 2, wherein the reagent detects the transcription level of the gene by one or a combination of real-time fluorescent quantitative PCR and high-throughput sequencing; preferably, the detection reagent of the NSUN2 gene comprises a primer and/or a probe for NSUN2 real-time fluorescent quantitative PCR detection; more preferably, the forward primer and the reverse primer of the real-time fluorescent quantitative PCR detection of NSUN2 are respectively shown in SEQ ID NO:1 (acctgctcaagaccacacag) and SEQ ID NO:2 (TGGCTTGATGGACGAGCAGGTA).
4. The use according to claim 1 or 2, wherein the agent detects the level of said protein by one or a combination of several of BCA protein quantification, immunohistochemistry, western blotting and ELISA; preferably, the reagent for detecting the NSUN2 protein comprises an NSUN 2-specific antibody.
5. Use according to claim 1 or 2, wherein said reagent detects the methylation level of said gene by high throughput sequencing.
6. The use according to any one of claims 1 to 5, wherein the test sample of the kit is endometrial tissue from a RIF patient; preferably endometrial tissue at the window of embryo implantation.
7. A Recurrent Implantation Failure (RIF), embryo implantation failure risk prediction and/or endometrial receptivity assessment kit, comprising: a reagent for detecting the transcriptional level of the NSUN2 gene, a reagent for detecting the methylation level of the NSUN2 gene and/or a reagent for detecting the protein level of the NSUN2 gene.
8. A method for assessing endometrial receptivity in endometrial tissue samples for non-disease diagnostic and therapeutic purposes, said method comprising detecting the level of NSUN2 gene transcription, NSUN2 gene methylation, and/or NSUN2 protein levels in the tissue; using the NSUN2 transcription level, NSUN2 gene methylation level or NSUN2 protein level value of the patient with repeated planting failure or embryo bed failure as a control; higher levels of NSUN2 gene transcription, NSUN2 gene methylation, and/or NSUN2 protein than controls indicate poor endometrial tissue intima tolerance.
9. A system for Recurrent Implantation Failure (RIF), embryo implantation failure risk prediction, and/or endometrial receptivity assessment, comprising: 1) Reagents for detecting the transcriptional level of NSUN2, the methylation level of NSUN2, and/or the protein level of NSUN 2; 2) The device comprises a data input module, a data comparison module and a conclusion output module.
10. The system of claim 9, wherein the data input module is configured to input a transcription level value of NSUN2 in the sample to be tested; the data comparison module is used for comparing the transcription level value of the NSUN2 sample to be detected with a comparison value; the control value is the transcriptional level, the methylation level or the protein level of NSUN2 of a patient with repeated planting failure or embryo bed failure; the conclusion output module is used for outputting the conclusion according to the following standards: if the transcriptional level of NSUN2, the methylation level of the NSUN2 gene, or the NSUN2 protein level in the sample of the subject is higher than or close to the control value, the subject is a recurrent implantation failure or is candidate for embryo implantation failure or endometrial receptions.