WO2023176923A1 - Implantability detection method and implantation failure prediction method - Google Patents

Abstract

The purpose of the present invention is to provide a novel implantability detection method and a novel implantation failure prediction method. The present invention provides an implantability detection method or an implantation failure prediction method including a step for measuring the expression level of a gene in an endometrial tissue sample from a test subject, said gene being a PRC2-associated gene, an EZH2-associated gene, and/or a SUZ12-associated gene. The methods according to the present invention are advantageous in terms of conveniently and accurately implementing detection of implantability or prediction of an implantation failure in the test subject.

Description

Methods for detecting implantation potential and predicting implantation failure References to related applications

This application benefits from the priority of Japanese Patent Application No. 2022-42463 (filing date: March 17, 2022), which is an earlier Japanese application, and the entire disclosure thereof is incorporated herein by reference. considered to be part of

The present invention relates to a method for detecting implantation ability and a method for predicting implantation failure.

Infertility is a worldwide problem, and in Japan, as women continue to advance into society, the number of couples getting married later in life and the age of childbearing is increasing, and the number of couples undergoing infertility treatment is increasing. As a treatment for infertility, if pregnancy does not occur with conventional infertility treatments such as the timing method or artificial insemination, or if it is considered difficult to conceive with general infertility treatments, in vitro fertilization and embryo transfer (assisted reproductive technology (ART)) are recommended. IVF-ET) will be carried out. The number of ART treatment cycles is increasing year by year, reaching 458,000 cycles and 60,000 births in 2019, with approximately 1 in 14 babies conceived and born as a result of ART treatment. As described above, while the importance of ART in infertility treatment is increasing, repeated implantation failure (implantation disorder), in which pregnancy does not result even after repeated embryo transfers, has become a major problem in reproductive medicine (non-patent References 1 and 2).

On the other hand, implantation failure is defined by repeated unsuccessful implantation of a good embryo into the uterus, and cannot be diagnosed unless a fertilized egg is implanted multiple times. However, until now there has been no method for diagnosing the state of the endometrium or predicting implantation failure before embryo transfer. In addition, it is not possible to functionally evaluate implantation ability using endometrial dating using endometrial tissue or measurement of endometrial thickness using ultrasonic tomography, which is performed on infertile patients. There was no index to evaluate implantation ability.

Pirtea P,, et al., Life (Basel). 2021 Dec 27;12(1):39. Franasiak JM,, et al., Fertil Steril. 2021 Dec;116(6):1436-1448.

An object of the present invention is to provide a novel method for detecting implantation ability and a novel method for predicting implantation failure.

The present inventors have recently classified infertility patients into a group of patients who received assisted reproductive technology and became pregnant (control group) and a group who did not become pregnant despite receiving assisted reproductive technology (implantation disorder group). As a result of comparing the gene expression levels in the endometrial tissue specimens during the implantation stage of the groups based on the amount of RNA expression, it was confirmed that there were differences in the gene groups related to PRC2. The present inventors also found that trimethylation of the 27th lysine residue of histone H3 (H3K27me3) by Ezh2 in the uterus is involved in the implantation process, and that deletion of Ezh2 causes implantation failure. I found out what's going on. The present inventors further compared the RNA expression levels of genes at the uterine implantation site (6th day of pregnancy) of Ezh2 knockout mice and control mice, and found that RNA expression increased in Ezh2 knockout mice. We found that H3K27me3 upstream of the 5' side of the gene was significantly decreased, and among these, H3K27me3 was particularly decreased in the region near the transcription start site of Ccnd2, which is related to the cell cycle and whose expression level had increased. Ta. The present invention is based on these findings.

According to the present invention, the following inventions are provided.
[1] A method for detecting or diagnosing implantation ability, or a method for predicting implantation failure, comprising the step of measuring the expression level of a gene in an endometrial tissue sample of a test subject, wherein the gene is PRC2 A detection method, diagnostic method, or prediction method that is a related gene, an EZH2-related gene, and/or a SUZ12 gene.
[2] PRC2-related genes are (1) OSR1 gene, (2) SCUBE1 gene, (3) HOXA13 gene, (4) MEGF10 gene, (5) TRPM6 gene, (6) SPINK2 gene, (7) RSPO3 gene, (8) The detection method, diagnosis method, or prediction method according to [1] above, which is one or more genes selected from the group consisting of the LGI2 gene and (9) the MAL gene.
[3] The detection method, diagnostic method, or prediction method according to [1] or [2] above, wherein the EZH2-related gene is (10) EZH2 gene and/or (11) CCND2 gene.
[4] The detection according to any one of [1] to [3] above, further comprising the step of measuring the expression level of genes other than genes (1) to (11) in the endometrial tissue specimen of the test subject. Method or diagnostic or predictive method.
[5] Genes other than genes (1) to (11) are (12) SLC46A2 gene, (13) AZGP1 gene, (14) BRINP2 gene, (15) RDH12 gene, (16) SST gene, (17) PENK The detection method, diagnostic method, or prediction method according to [4] above, which is one or more genes selected from the group consisting of a gene and (18) CHI3L2 gene.
[6] The method according to any one of [1] to [5] above, further comprising the step of determining the presence or absence of implantation ability using the gene expression level in the endometrial tissue specimen of the test subject as an index. Detection or diagnosis or prediction methods.
[7] The detection method, diagnostic method, or prediction method according to any one of [1] to [6] above, in which the expression level of the gene is quantified by the amount of RNA expression.
[8] The detection method, diagnostic method, or prediction method according to any one of [1] to [7] above, wherein the test subject is an infertile patient.
[9] A method for determining clinical pregnancy potential through infertility treatment, the method comprising the step of measuring the expression level of a gene in an endometrial tissue specimen of a test subject, the method comprising: measuring the expression level of a gene in an endometrial tissue specimen of a test subject, wherein the gene is a PRC2-related gene, an EZH2-related gene gene and/or SUZ12 gene.
[10] A method for evaluating the implantation process for detecting or predicting an abnormality in the implantation process, the method comprising the step of measuring the expression level of a gene in an endometrial tissue specimen of a test subject, the method comprising: is a PRC2-related gene, an EZH2-related gene, and/or a SUZ12 gene.
[11] A diagnostic marker for implantation ability using the expression level of a gene in an endometrial tissue sample of a test subject, a predictive marker for implantation failure, a marker for determining clinical pregnancy potential through infertility treatment, or an implantation process A diagnostic marker, predictive marker, determination marker, or evaluation marker, wherein the gene is a PRC2-related gene, an EZH2-related gene, and/or a SUZ12 gene.
[12] As a marker for diagnosis or detection of implantation ability, as a marker for predicting implantation failure, as a marker for determining clinical pregnancy potential by infertility treatment, or as a marker for implantation process Use of a gene as a marker for the evaluation of a gene, said gene being a PRC2-related gene, an EZH2-related gene and/or a SUZ12 gene.
[13] A diagnostic kit for implantation ability, a kit for predicting implantation failure, a kit for determining clinical pregnancy potential through infertility treatment, or A diagnostic kit, prediction kit, determination kit, or implantation process evaluation kit, wherein the gene is a PRC2-related gene, an EZH2-related gene, and/or a SUZ12 gene.
[14] Measuring the gene expression level in the endometrial tissue sample of the test subject, and determining the presence or absence of implantation ability using the gene expression level in the endometrial tissue sample of the test subject as an index. and a step of administering treatment for implantation disorder to the test subject determined to be incapable of implantation, wherein the gene is a PRC2-related gene, A therapeutic method using EZH2-related genes and/or SUZ12 genes.

The present invention is advantageous in that it is possible to easily and accurately detect the implantation ability of a test subject or predict implantation failure. The present invention is also advantageous in that it can be used to determine whether or not to continue infertility treatment among infertility patients undergoing infertility treatment.

FIG. 1 shows a schematic diagram of trimethylation of the 27th lysine residue of histone H3 (H3K27me3) by PRC2. Figure 2 shows the endometrial tissues of infertility patients in a group of patients who became pregnant through embryo transfer after specimen collection (control group) and a group of patients who did not become pregnant through embryo transfer after specimen collection (implantation disorder group). The results of RNA-Seq of the specimen are shown. RPKM (Reads Per Kilobase of exon per Million mapped reads) on the vertical axis indicates the number of reads for each gene when the total number of reads is 1 million, and pregnancy and non-pregnancy on the horizontal axis indicate the control group and implantation disorder group. Each is shown below. Figure 3 shows the endometrial tissues of infertility patients in a group of patients who became pregnant through embryo transfer after specimen collection (control group) and a group of patients who did not become pregnant through embryo transfer after specimen collection (implantation failure group). The results of RNA-Seq of the specimen are shown. RPKM (Reads Per Kilobase of exon per Million mapped reads) on the vertical axis indicates the number of reads for each gene when the total number of reads is 1 million, and pregnancy and non-pregnancy on the horizontal axis indicate the control group and implantation disorder group. Each is shown below. Figures 4A and B show intrauterine infertility patients in a group of patients who became pregnant by embryo transfer after sample collection (control group) and in a group of patients who did not become pregnant by embryo transfer after sample collection (implantation failure group). The results of RNA-Seq of membrane tissue samples are shown. RPKM (Reads Per Kilobase of exon per Million mapped reads) on the vertical axis indicates the number of reads for each gene when the total number of reads is 1 million, and pregnancy and non-pregnancy on the horizontal axis indicate the control group and implantation disorder group. Each is shown below. Figure 5 shows the endometrial tissues of infertility patients in a group of patients who became pregnant through embryo transfer after specimen collection (control group) and a group of patients who did not become pregnant through embryo transfer after specimen collection (implantation failure group). The results of RNA-Seq of the specimen are shown. RPKM (Reads Per Kilobase of exon per Million mapped reads) on the vertical axis indicates the number of reads for each gene when the total number of reads is 1 million, and pregnancy and non-pregnancy on the horizontal axis indicate the control group and implantation disorder group. Each is shown below. FIG. 6 shows the results of immunostaining of human endometrial tissue during the implantation period using EZH2 antibody. The scale bar is 100 μm, le indicates luminal epithelium, ge indicates glandular epithelium, and s indicates stroma. Figure 7 shows an overview of human and mouse hormonal dynamics during early pregnancy. The vertical axis shows the blood concentrations of estrogen and progesterone, and the horizontal axis shows the number of days from the day of ovulation. "Implantation window" refers to "the period during which the uterus retains the ability to implant." FIG. 8 shows an overview of the mouse implantation process from blastocyst activation to placentation. The horizontal axis shows the number of days since pregnancy. FIG. 9 shows the expression pattern of the Ezh2 gene on day 1 (A), day 4 (B), day 6 (C), and day 8 (D) in the pregnant uterus of WT mice. The scale bar is 200 μm, le is the luminal epithelium, ge is the glandular epithelium, s is the stroma, de is the decidua in which the stroma has differentiated and proliferated, AM is the opposite side of the blood vessel, and M is the side of the blood vessel. FIG. 10A shows the results of Ezh2 immunostaining in the uterus (day 4) of Ezh2 uControl and Ezh2 uKO, respectively. FIG. 10B shows the results of Western blotting of the uterine implantation site (day 5) of Ezh2 uControl and Ezh2 uKO, respectively. FIG. 11 shows the number of fetuses (number of littermates) (A), photographs of cesarean-sectioned embryos (B), and number of litters (C) in Ezh2 uControl and Ezh2 uKO, respectively. Figures 12A and B show photographs of blastocysts (scale bar is 100 μm) and the number of blastocysts in each uterus (day 4) of Ezh2 uControl and Ezh2 uKO, respectively. FIG. 12C shows Ki67 immunostaining of each uterus (day 4) of zh2 uControl and Ezh2 uKO (scale bar is 50 μm, le indicates luminal epithelium, ge indicates glandular epithelium, and s indicates stroma). Figures 13A and B show photographs of implantation sites (arrowhead: implantation site, arrow: ovary) and the number of implantation sites in each uterus (day 5) of Ezh2 uControl and Ezh2 uKO, respectively. Figures 14A and B show photographs of implantation sites (arrowhead: implantation site, arrow: ovary) and the number of implantation sites in each uterus (6th day) of Ezh2 uControl and Ezh2 uKO, respectively. Figure 15 shows HE staining (A), CK8 immunostaining (B), and the state of endometrial luminal epithelium (C) in each uterus (day 6) of Ezh2 uControl and Ezh2 uKO (scale bar is 100 μm, AM indicates the opposite side of the blood vessel, M indicates the blood vessel side, and * indicates the embryo). FIG. 16 shows HE staining (A) and the state of the implantation site (B) in each uterus (day 8) of Ezh2 uControl and Ezh2 uKO (scale bar is 200 μm, arrowheads indicate embryos). FIG. 17 shows three-dimensional images of each uterus (6th day) of Ezh2 uControl and Ezh2 uKO. The scale bar is 200 μm, Lumen indicates the lumen, Gland indicates the gland, and * indicates the embryo. FIG. 18 shows the results of Chip-seq of DNA extracted by chromatin immunoprecipitation (Chip) using H3K27me antibody at the implantation site of each uterus (6th day) of Ezh2 uControl and Ezh2 uKO. FIG. 19 shows the results of RT-qPCR in each uterus (day 6) of Ezh2 uControl and Ezh2 uKO. FIG. 20 shows analysis of bromodeoxyuridine (BrdU) uptake ability (bottom) and immunostaining of phosphorylated histone H3 (pHH3) (top) in each uterus of Ezh2 uControl and Ezh2 uKO (day 6). The region surrounded by a broken line indicates a region where pHH3 is not expressed, the scale bar is 200 μm, AM indicates the opposite side of the blood vessel, M indicates the blood vessel side, * indicates the embryo, and Nu indicates the nucleus. FIG. 21 shows SA-β-gal (senescence-associated beta-galactosidase) staining in each uterus (day 8) of Ezh2 uControl and Ezh2 uKO.

Specific description of the invention

<<Definition>>
"PRC2" means polycomb repressive complex 2, which is one of two classes of polycomb-group proteins (PcGs). As shown in the schematic diagram of Figure 1, PRC2 is composed of multiple proteins, including EZH2 (enhancer of zeste homolog 2), EED (embryonic ectoderm development), SUZ12 (suppressor of zeste homolog 12), and RbAp46/48 (retinoblastoma (Rb)-associated protein 46/48) is considered to be the main component (R. Margueron, et al., Nature 469, 343-349 (2011).), and constitutes nucleosomes, which are the constituent units of chromatin. The 27th lysine residue of histone H3 in the histone octamer is trimethylated to suppress target gene expression. In this specification, the state in which the 27th lysine residue of histone H3 is methylated is sometimes referred to as "H3K27me3."

"EZH2" is a methyltransferase that plays a central role in the methylation activity of PRC2 as one of the proteins that constitute PRC2, and is an enzyme that catalyzes the addition of a methyl group to the 27th lysine residue of histone H3. It is.

"SUZ12" is one of the proteins that constitute PRC2, and together with EZH2, it functions in methylation of the 27th lysine residue of histone H3 (H3K27me), and is involved in transcriptional repression of target genes.

"OSR1" is "oxidative stress-responsive kinase 1" and is related to PRC2 in that the expression of the OSR1 gene is regulated by H3K27me3.

"SCUBE1" is "signal peptide, CUB domain and EGF-like domain containing 1" and is related to PRC2 in that the expression of SCUBE1 gene is regulated by H3K27me3. .

"HOXA13" is "homeobox protein HOX-A13" and is related to PRC2 in that the expression of the HOXA13 gene is regulated by H3K27me3.

"MEGF10" is "Multiple EGF Like Domains 10" and is related to PRC2 in that the expression of MEGF10 gene is regulated by H3K27me3.

"TRPM6" is "Transient Receptor Potential Cation Channel Subfamily M Member 6" and is related to PRC2 in that the expression of the TRPM6 gene is regulated by H3K27me3.

"SPINK2" is "Serine protease inhibitor Kazal-type 2" and is related to PRC2 in that the expression of the SPINK2 gene is regulated by H3K27me3.

"RSPO3" is "R-spondin 3" and is related to PRC2 in that the expression of the RSPO3 gene is regulated by H3K27me3.

"LGI2" is "leucine-rich repeat LGI family member 2" and is related to PRC2 in that the expression of the LGI2 gene is regulated by H3K27me3.

"MAL" is "myelin and lymphocyte protein" and is related to PRC2 in that the expression of the MAL gene is regulated by H3K27me3.

"CCND2" is also referred to as "cyclin D2", belongs to the cyclin family, forms a complex as a regulatory subunit of CDK4 or CDK6, and its activation is required for cell cycle G1/S transition.

"SLC46A2" is "Solute Carrier Family 46 Member 2".

"AZGP1" is "Zinc alpha-2-glycoprotein 1 (Alpha-2-Glycoprotein 1, Zinc protein").

"BRINP2" is "BMP/retinoic acid inducible neural specific protein 2".

"RDH12" is "retinol dehydrogenase 12" and is expressed in the human endometrial stroma during the implantation period.

"SST" is "somatostatin" and is expressed in the human endometrial stroma after pregnancy.

"PENK" is "proenkephalin" and is expressed in the uterus of cows, rhesus monkeys, and mice during the implantation stage.

"CHI3L2" is "chitinase 3 like protein 2".

"Implantation," which is the starting point of pregnancy, refers to the state in which an organic bond is established between the embryo and the endometrium, and the "implantation process" refers to the scutellum in which the fertilized egg repeatedly divides and develops. It refers to a series of events in which a cyst adheres to the luminal epithelium of the mucosal tissue called the endometrium (embryo adhesion), then invades the interstitium of the endometrium (embryo invasion), and forms a villus structure. In the present invention, "having the ability to implant" means having the ability to lead to implantation, and "not having the ability to implant" means not having the ability to lead to implantation.

In the present invention, "implantation failure" refers to a state in which pregnancy does not result even after multiple embryo implantations into the uterus using embryos with good morphology (Repeated Implantation Failure). The causes of implantation failure can be broadly divided into "embryonic factors" and "uterine factors." Embryonic factors are often caused by chromosomal abnormalities (aneuploidy) in fertilized eggs. Preimplantation genetic testing for aneuploidy (PGTA), which is currently undergoing clinical research in Japan with the aim of improving pregnancy outcomes and reducing miscarriage rates, can improve the pregnancy rate per embryo transfer. (T. Sato et al., Hum Reprod 34, 2340-2348 (2019)). On the other hand, various uterine factors have been suggested, including chronic endometritis, abnormal uterine flora, and immunological abnormalities.

"Infertility" refers to a condition in which a man and a woman of reproductive age are unable to become pregnant despite having sexual intercourse without contraception for a certain period of time (approximately one year). In addition, if there is a clear cause of infertility, it may be considered infertility regardless of the period of infertility.

In the present invention, "infertility treatment" is used to include "general infertility treatment" and "assisted reproductive technology." "General infertility treatments" include timing methods and artificial insemination. "Assisted reproductive technology (ART)" refers to all treatments or methods that involve handling human eggs, sperm, or embryos in order to establish a pregnancy, and includes in vitro fertilization and embryo transfer (ART). Infertility treatment methods include IVF-ET (in vitro fertilization and embryo transfer), intracytoplasmic sperm injection and embryo transfer (ICSI-ET), and frozen/thawed embryo transfer.

In the present invention, the "endometrial tissue specimen" may be a specimen collected during the implantation period or a specimen collected at a time other than the implantation period, but is preferably collected during the implantation period. This is a sample that was tested. Here, the implantation period may be based on an ovulation cycle or a hormone replacement cycle. Furthermore, in the present invention, an endometrial tissue sample of a subject (human) undergoing infertility treatment collected 6 to 8 days after the ovulation day (or 6 to 8 days after administration of progestin) is used. be able to.

The "subject" in the present invention includes mammals including humans, and preferably humans. The "subject" in the present invention includes a normal subject (healthy person), but is preferably an infertile patient (including a subject diagnosed with infertility and a subject suspected of being infertile).

<<Marker gene>>
In the present invention, genes (marker genes) that are indicators of implantation ability or implantation failure include at least PRC2-related genes, EZH2-related genes, and/or SUZ12 genes.

The PRC2-related genes used as indicators in the present invention include at least one or more genes selected from the group consisting of the OSR1 gene, the SCUBE1 gene, the HOXA13 gene, the MEGF10 gene, and the TRPM6 gene. In this specification, "one or more genes selected from the group consisting of the OSR1 gene, SCUBE1 gene, HOXA13 gene, MEGF10 gene, and TRPM6 gene" is referred to as "gene a of the present invention" or "gene a." There is. The gene a of the present invention is characterized in that the expression level of gene a in endometrial tissue specimens of subjects with implantation disorders is compared with the expression level of gene a in endometrial tissue specimens of subjects without implantation disorders. It is characterized by low In particular, gene a of the present invention can be such that the gene expression level in endometrial tissue specimens of subjects with implantation disorders is 1/2 or less compared to subjects without implantation disorders. .

In addition to the gene a of the present invention, the PRC2-related genes that serve as indicators in the present invention include at least one or more genes selected from the group consisting of the SPINK2 gene, RSPO3 gene, LGI2 gene, and MAL gene. In this specification, "one or more genes selected from the group consisting of the SPINK2 gene, RSPO3 gene, LGI2 gene, and MAL gene" may be referred to as "gene b of the present invention" or "gene b." The expression level of gene b in endometrial tissue samples of subjects with implantation disorders is compared with the expression level of gene b in endometrial tissue samples of subjects without implantation disorders. It is characterized by high In particular, gene b of the present invention can be such that the gene expression level in endometrial tissue specimens of subjects with implantation disorders is twice or more compared to subjects without implantation disorders.

EZH2-related genes that serve as indicators in the present invention include at least the "EZH2 gene" and/or the "CCND2 gene." In this specification, the "EZH2 gene" is sometimes referred to as "gene c of the present invention" or "gene c," and the "CCND2 gene" is sometimes referred to as "gene d of the present invention" or "gene d." The expression level of gene c in endometrial tissue samples of subjects with implantation disorders is compared with the expression level of gene c in endometrial tissue samples of subjects without implantation disorders. It is characterized by low The expression level of gene d in endometrial tissue samples of subjects with implantation disorders is compared with the expression level of gene d in endometrial tissue samples of subjects without implantation disorders. It is characterized by high

The "SUZ12 gene" that serves as an indicator in the present invention is defined as the expression level of the SUZ12 gene in endometrial tissue specimens from subjects with implantation disorders compared to that in endometrial tissue specimens from subjects without implantation disorders. It is characterized by a low expression level. In this specification, the "SUZ2 gene" may be referred to as the "gene e of the present invention" or "gene e."

Genes that serve as indicators in the present invention include at least the SLC46A2 gene and/or the AZGP1 gene in addition to the genes a, b, c, d, and e of the present invention. In this specification, "SLC46A2 gene and/or AZGP1 gene" may be referred to as "gene f of the present invention" or "gene f." The expression level of gene f in endometrial tissue samples of subjects with implantation disorders is compared with the expression level of gene f in endometrial tissue samples of subjects without implantation disorders. It is characterized by low In particular, the gene f of the present invention can be such that the gene expression level in endometrial tissue specimens of subjects with implantation disorders is 1/2 or less compared to subjects without implantation disorders. .

In addition to the genes a, b, c, d, e, and f of the present invention, the gene serving as an indicator in the present invention is one selected from the group consisting of the BRINP2 gene, RDH12 gene, SST gene, PENK gene, and CHI3L2 gene. It contains at least one species or two or more genes. In this specification, "one or more genes selected from the group consisting of BRINP2 gene, RDH12 gene, SST gene, PENK gene, and CHI3L2 gene" is referred to as "gene g of the present invention" or "gene g". There is. The expression level of gene g in endometrial tissue samples of subjects with implantation disorders is compared with the expression level of gene g in endometrial tissue samples of subjects without implantation disorders. It is characterized by high In particular, the gene g of the present invention can be such that the gene expression level in the endometrial tissue specimen of a subject with an implantation disorder is twice or more compared to that of a subject without an implantation disorder. In this specification, any of the genes a, b, c, d, e, f, and g of the present invention may be referred to as the "gene of the present invention."

<<Method for detecting implantation ability>>
According to a first aspect of the present invention, a method for detecting implantation potential is provided. According to the detection method of the present invention, implantation ability can be detected using the expression level of a gene in an endometrial tissue specimen of a test subject as an index. That is, the detection method of the present invention is characterized in that the expression level of the gene is associated with the presence or absence of implantation ability of the test subject.

In the detection method of the present invention, first, (A) one or more genes of the present invention selected from the group consisting of a, b, c, d, e, f, and g in an endometrial tissue specimen of a test subject; A step of measuring the expression level of two or more genes (genes of the present invention) can be carried out. The gene expression level can be measured by a known method, for example, a method for measuring the amount of RNA expression or protein expression can be used. Furthermore, the detection method of the present invention measures the expression level of the gene of the present invention in an endometrial tissue sample collected from a test subject, so it can be called an in vitro detection method for implantation ability.

Methods for measuring RNA expression include RNA-seq using a next-generation sequencer, quantitative PCR (RT-qPCR), digital PCR, microarray, and fluorescence in situ hybridization (FISH), but these methods are rapid and RT-qPCR and digital PCR are preferred from the viewpoint of easy detection.

Methods for measuring protein expression include enzyme immunoassays (EIA and ELISA), radioimmunoassays (RIA), fluorescence immunoassays (FIA), fluorescence polarization immunoassays (FPIA), chemiluminescence immunoassays, and mass immunoassays using antibodies and aptamers. Examples include analytical methods, but EIA and ELISA are preferred from the viewpoint of rapid and simple detection.

In the detection method of the present invention, (B) the expression level of the gene of the present invention measured in step (A) is used as an index to determine the presence or absence of implantation ability of the test subject from whom the endometrial tissue sample was collected; The method may further include the step of evaluating.

When the genes of the present invention are genes a, c, e, and f, step (B) includes (B-1-1) the expression level of the genes of the present invention in the endometrial tissue specimen of the test subject and the predetermined and (B-1-2) when the expression level of the gene of the present invention in the endometrial tissue specimen of the test subject is below the reference value or lower than the reference value. This can be carried out by a step of determining that the test subject does not have the ability to implant. In the present invention, the term "incapable of implantation" is used to include "there is a possibility that there is no implantation ability, or there is a low possibility that there is no implantation ability."

In step (B-1-2), the implantation ability of the test subject is determined when the expression level of the gene of the present invention in the endometrial tissue sample of the test subject is equal to or higher than the reference value. It can also be determined that there is. In the present invention, the term "having implantation potential" is used to include "possibly having implantation potential, or highly likely to have implantation potential."

When the genes of the present invention are genes b, d, and g, step (B) includes (B-2-1) the expression level of the gene of the present invention in the endometrial tissue specimen of the test subject and a predetermined reference. (B-2-2) When the expression level of the gene of the present invention in the endometrial tissue sample of the test subject is equal to or higher than the reference value, the test subject This can be carried out by a step of determining that the patient is incapable of implantation.

In step (B-2-2), the implantation ability of the test subject is determined when the expression level of the gene of the present invention in the endometrial tissue sample of the test subject is below the reference value or lower than the reference value. It can also be determined that there is.

In the present invention, the "reference value" can be calculated and determined from the measured value of the expression level of the gene of the present invention in an endometrial tissue specimen of a subject with implantation potential (normal subject). Such subjects are preferably infertility patients who have undergone infertility treatment and become pregnant, but they may also be healthy subjects who have the ability to implant. In the present invention, the reference value also refers to the gene of the present invention in an endometrial tissue specimen of a subject who does not have implantation ability, that is, a subject who did not become pregnant even after undergoing infertility treatment among infertile patients (subject with implantation failure). can be calculated and determined from the measured value of the expression level. In the method for determining the reference value described above, the average value, median value, percentile value, maximum value, or minimum value of the measured values of the normal subject group or the implantation disorder subject group can be used. The percentile value can be selected to be any value, for example, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 85, 90 or 95. The number of normal subjects and implantation disorder subjects when calculating the reference value is preferably plural, for example, 2 or more, 5 or more, 10 or more, 20 or more, 50 or more, or 100 or more. be able to. Note that the reference value is a numerical value that distinguishes the presence or absence of implantation ability or the possibility of implantation failure when implementing the present invention, and in this sense, it can be referred to as a cutoff value or a boundary value.

In the present invention, the reference value also includes the measured value of the expression level of the gene of the present invention in an endometrial tissue specimen of a subject with implantation potential (normal subject), and the measured value of the expression level of the gene of the present invention in an endometrial tissue specimen of a subject with implantation potential (implantation disordered subject). It can also be calculated based on the measured value of the expression level of the gene of the present invention in an endometrial tissue sample. For example, the expression level of the gene of the present invention in endometrial tissue specimens is measured for the implantation disorder target group and the normal target group, and the obtained measurement values are used to determine the ROC (Receiver Operating Characteristic Curve). Reference values can be set by performing statistical analysis such as curve)) analysis. Creation of an ROC curve and setting of a reference value based on the ROC curve are well known, and can be set appropriately by those skilled in the art from the viewpoint of sensitivity and specificity.

In the present invention, when using a combination of multiple genes of the present invention as an indicator, or when using a gene of the present invention in combination with other clinical variables as an index, the measured value of the expression level of multiple genes serving as an index It is also possible to set a single reference value for. For example, instead of the measured value of the expression level of one type of gene, the reference value can be calculated using the sum, average value, ratio, etc. of the measured value of the expression level of multiple types of genes, or After weighting the measured values of the expression levels of the genes, the total value, average value, ratio, etc. can be calculated, and the reference value can be calculated using the calculated values. When using the reference value calculated in this way in the present invention, the measured values of the expression levels of multiple genes in the endometrial tissue specimen of the test subject are processed in the same manner as the reference value calculation method. The determination can be made by comparing the obtained numerical value with a predetermined reference value.

In step (B) of the detection method of the present invention, for example, one or two genes selected from the group consisting of gene a, gene c, gene e, and gene f of the present invention in the endometrial tissue specimen of the test subject. The expression level of the gene in the species or more is lower than the average expression level of the gene in the normal subject group, or about 0.9 times or less, about 0.85 times or less, about 0. .8 times or less, about 0.75 times or less, about 0.7 times or less, about 0.65 times or less, about 0.6 times or less, about 0.55 times or less, about 0.5 times or less, about 0. If it is 45 times or less, about 0.4 times or less, or about 0.35 times or less, it can be determined that the test subject does not have implantation ability.

In step (B) of the detection method of the present invention, for example, one or more genes selected from the group consisting of gene b, gene d, and gene g of the present invention in the endometrial tissue specimen of the test subject. The expression level of the gene is higher than the average expression level of the gene in the normal subject group, or about 1.05 times or more, about 1.1 times or more, about 1.2 times as compared to the average value. or more, about 1.3 times or more, about 1.4 times or more, about 1.5 times or more, about 1.6 times or more, about 1.7 times or more, about 1.8 times or more, about 1.9 times or more , about 2.0 times or more, about 2.5 times or more, or about 3 times or more, it can be determined that the test subject does not have implantation ability.

The detection method of the present invention detects 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, and 11 genes of the present invention. The above can be carried out in combination of 12 or more, 13 or more, 14 or more, 15 or more, or 16 or more, and for example, the expression level of a certain number or more of the genes of the present invention is controlled. If it is different (higher or lower) compared to , it can be determined that the test subject does not have implantation ability (or has implantation ability).

In the detection method of the present invention, the presence or absence of implantation ability can be detected by measuring the gene level of the endometrial tissue specimen of the test subject in combination with the measurement of other clinical variables. Clinical variables to be combined include, for example, the number of CD138-positive cells in the endometrial stroma in terms of the inflammatory state of the endometrium. By using the gene of the present invention in combination with such clinical variables, detection accuracy can be further improved. Here, improving detection accuracy means that when ROC analysis is used, the area under the ROC curve (AUC) improves.

According to the detection method of the present invention, the implantation ability of a test subject can be detected or evaluated. Therefore, the detection method of the present invention can be used as an auxiliary for diagnosis of implantation ability, and the determination of whether a test subject has implantation ability can be made by combining other findings as the case may be. Ultimately, it can be done by a doctor. For example, in the present invention, for test subjects who have been determined to not have (or may not have) the ability to implant, a doctor may refer to other findings and determine whether the test subject has the ability to implant. (or may not have implantation ability). That is, the detection method of the present invention can be rephrased as a method for assisting in the diagnosis of implantation potential or a method for assisting in the diagnosis of implantation potential.

According to the detection method of the present invention, implantation potential can be quantitatively detected or evaluated based on an endometrial tissue specimen collected from a test subject. That is, the detection method of the present invention is advantageous in that implantation ability can be detected or evaluated simply and accurately while reducing the burden on the patient. Therefore, the detection method of the present invention can be rephrased as a biological sample analysis method for detecting or evaluating implantation ability. The detection method of the present invention is also advantageous in that it can be used to determine whether or not to continue infertility treatment for infertility patients undergoing infertility treatment.

According to another aspect of the present invention, a method for diagnosing implantation potential is also provided. According to the diagnostic method of the present invention, the presence or absence of implantation ability in a subject can be diagnosed using the expression level of a gene in an endometrial tissue specimen as an index. The diagnostic method of the present invention measures the expression level of the gene of the present invention in an endometrial tissue sample collected from a test subject, so it can be called an in vitro diagnostic method for implantation ability. In the diagnostic method of the present invention, similarly to the detection method of the present invention, the step (A') of measuring the expression level of a gene in an endometrial tissue specimen of a test subject is carried out. In the diagnostic method of the present invention, the expression level of the gene of the present invention measured in step (B') (A') is used as an index to determine the presence or absence of implantation ability in the test subject from whom the endometrial tissue sample was collected. It may further include a step of determining or evaluating. The steps (A') and (B') correspond to the steps (A) and (B), respectively, and can be carried out according to the description of the detection method of the present invention.

<<How to predict implantation failure>>
According to a second aspect of the present invention, a method for predicting implantation failure is provided. According to the prediction method of the present invention, implantation failure can be predicted using the expression level of a gene in an endometrial tissue specimen of a test subject as an index. That is, the prediction method of the present invention is characterized by associating the expression level of a gene with the prediction of implantation failure in a test subject.

In the prediction method of the present invention, first, (C) one or more genes of the present invention selected from the group consisting of a, b, c, d, e, f, and g in an endometrial tissue specimen of a test subject; A step of measuring the expression level of two or more genes (genes of the present invention) can be carried out. Measurement of gene expression level can be carried out according to the description of the detection method of the present invention. Furthermore, the prediction method of the present invention measures the expression level of the gene of the present invention in an endometrial tissue sample collected from a test subject, so it can be called an in vitro prediction method for implantation failure.

In the prediction method of the present invention, the possibility of implantation failure in the test subject from whom the endometrial tissue sample was collected is determined using the expression level of the gene of the present invention measured in step (D) (C) as an index. Alternatively, the method may further include a step of evaluating.

When the genes of the present invention are genes a, c, e, and f, step (D) includes (D-1-1) the expression level of the genes of the present invention in the endometrial tissue specimen of the test subject and the predetermined and (D-1-2) when the expression level of the gene of the present invention in the endometrial tissue specimen of the test subject is below the reference value or lower than the reference value. This can be carried out by determining that there is a possibility that the test subject has an implantation disorder. In the present invention, the expression "there is a possibility of implantation disorder" is used to include "there is a high possibility of implantation disorder."

In step (D-1-2), if the expression level of the gene of the present invention in the endometrial tissue specimen of the test subject is equal to or higher than the reference value, the test subject is diagnosed with implantation failure. It can also be determined that there is no possibility. In the present invention, "there is no possibility of implantation disorder" is used to include "there is a low possibility of implantation disorder."

When the genes of the present invention are genes b, d, and g, step (D) comprises (D-2-1) the expression level of the gene of the present invention in the endometrial tissue specimen of the test subject and a predetermined reference. (D-2-2) If the expression level of the gene of the present invention in the endometrial tissue sample of the test subject is equal to or higher than the reference value, the test subject This can be carried out by a step of determining that there is a possibility of implantation disorder.

In step (D-2-2), if the expression level of the gene of the present invention in the endometrial tissue sample of the test subject is below the reference value or lower than the reference value, the test subject is diagnosed with implantation failure. It can also be determined that there is no possibility.

In step (D) of the prediction method of the present invention, for example, one or two genes selected from the group consisting of gene a, gene c, gene e, and gene f of the present invention in the endometrial tissue specimen of the test subject. The expression level of the gene in the species or more is lower than the average expression level of the gene in the normal subject group, or about 0.9 times or less, about 0.85 times or less, about 0. 8 times or less, about 0.75 times or less, about 0.7 times or less, about 0.65 times or less, about 0.6 times or less, about 0.55 times or less, about 0.5 times or less, about 0.45 If it is less than 2 times, about 0.4 times or less, or about 0.35 times or less, it can be determined that the test subject is likely to have implantation disorder.

In step (D) of the prediction method of the present invention, for example, one or more genes selected from the group consisting of gene b, gene d, and gene g of the present invention in the endometrial tissue specimen of the test subject. The expression level of the gene is higher than the average expression level of the gene in the normal subject group, or about 1.05 times or more, about 1.1 times or more, about 1.2 times as compared to the average value. or more, about 1.3 times or more, about 1.4 times or more, about 1.5 times or more, about 1.6 times or more, about 1.7 times or more, about 1.8 times or more, about 1.9 times or more , about 2.0 times or more, about 2.5 times or more, or about 3 times or more, it can be determined that the test subject is likely to have implantation disorder.

The prediction method of the present invention can detect 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, and 11 genes of the present invention. The above can be carried out in combination of 12 or more, 13 or more, 14 or more, 15 or more, or 16 or more, and for example, the expression level of a certain number or more of the genes of the present invention is controlled. The test subject can be determined to have a possibility of implantation disorder (or not to have a possibility of implantation disorder) if it is different (higher or lower) compared to .

In the prediction method of the present invention, prediction of implantation failure can be performed by combining measurement of gene levels of endometrial tissue specimens of test subjects with measurement of other clinical variables. Clinical variables to be combined include, for example, the number of CD138-positive cells in the endometrial stroma in terms of the inflammatory state of the endometrium. Prediction accuracy can be further improved by using the gene of the present invention in combination with such clinical variables. Here, improving prediction accuracy means that when ROC analysis is used, the area under the ROC curve (AUC) improves.

According to the prediction method of the present invention, it is possible to predict or evaluate implantation disorder in a test subject. Therefore, the prediction method of the present invention can be used as an auxiliary for the diagnosis of implantation failure, and in some cases, the prediction method may be used in combination with other findings to determine whether or not a test subject is likely to have implantation failure. , ultimately can be performed by a doctor. For example, in the present invention, for a test subject determined to have a possibility of implantation disorder, a doctor can refer to other findings and determine that the test subject may have an implantation disorder. . That is, the prediction method of the present invention can be rephrased as a method for assisting in the diagnosis of implantation disorders or a method for assisting in the diagnosis of implantation disorders.

According to the prediction method of the present invention, the possibility of implantation failure can be quantitatively predicted or evaluated based on the endometrial tissue sample collected from the test subject. That is, the prediction method of the present invention is advantageous in that implantation failure can be easily and accurately predicted or evaluated while reducing the burden on the patient. Therefore, the prediction method of the present invention can be rephrased as a biological sample analysis method for detecting or evaluating implantation failure. The prediction method of the present invention is also advantageous in that it can be used to determine whether or not to continue infertility treatment in patients undergoing infertility treatment.

In addition to the above, the prediction method of the present invention can be implemented according to the description of the detection method of the present invention.

<<Method for determining clinical pregnancy potential through infertility treatment>>
According to a third aspect of the present invention, a method for determining clinical pregnancy potential through infertility treatment is provided. According to the determination method of the present invention, the clinical possibility of pregnancy due to infertility treatment can be determined using the expression level of a gene in an endometrial tissue specimen of a test subject as an index. That is, the determination method of the present invention is characterized by associating the gene expression level with the clinical pregnancy potential of the test subject due to infertility treatment.

In the determination method of the present invention, first, (E) one or more genes of the present invention selected from the group consisting of a, b, c, d, e, f, and g in an endometrial tissue specimen of a test subject; A step of measuring the expression level of two or more genes (genes of the present invention) can be carried out. Measurement of gene expression level can be carried out according to the description of the detection method of the present invention. Furthermore, since the determination method of the present invention measures the expression level of the gene of the present invention in endometrial tissue samples collected from test subjects, it can be said to be an in vitro method for determining clinical pregnancy potential through infertility treatment.

In the determination method of the present invention, the expression level of the gene of the present invention measured in step (F) (E) is used as an index to determine the clinical pregnancy potential due to infertility treatment of the test subject from whom the endometrial tissue sample was collected. The method may further include the step of evaluating.

When the genes of the present invention are genes a, c, e, and f, step (F) includes (F-1-1) the expression level of the genes of the present invention in the endometrial tissue specimen of the test subject and the predetermined and (F-1-2) when the expression level of the gene of the present invention in the endometrial tissue specimen of the test subject is below the reference value or lower than the reference value. This can be carried out by evaluating that there is no possibility of clinical pregnancy in the test subject. In the present invention, the term "there is no possibility of clinical pregnancy" is used to include "the possibility of clinical pregnancy is low."

In step (F-1-2), if the expression level of the gene of the present invention in the endometrial tissue specimen of the test subject is equal to or higher than the reference value, clinical pregnancy of the test subject is determined. It can also be evaluated as a possibility. In the present invention, "there is a possibility of clinical pregnancy" is used to include "there is a high possibility of clinical pregnancy."

When the genes of the present invention are genes b, d, and g, step (F) comprises (F-2-1) the expression level of the gene of the present invention in the endometrial tissue specimen of the test subject and a predetermined reference. (F-2-2) If the expression level of the gene of the present invention in the endometrial tissue specimen of the test subject is equal to or higher than the reference value, the test subject This can be carried out by assessing that there is no clinical possibility of pregnancy.

In step (F-2-2), if the expression level of the gene of the present invention in the endometrial tissue sample of the test subject is below the reference value or lower than the reference value, the clinical pregnancy of the test subject is determined. It can also be determined that there is a possibility.

In step (F) of the determination method of the present invention, for example, one or two genes selected from the group consisting of gene a, gene c, gene e, and gene f of the present invention in the endometrial tissue specimen of the test subject. The expression level of the gene in the species or more is lower than the average expression level of the gene in the normal subject group, or about 0.9 times or less, about 0.85 times or less, about 0. 8 times or less, about 0.75 times or less, about 0.7 times or less, about 0.65 times or less, about 0.6 times or less, about 0.55 times or less, about 0.5 times or less, about 0.45 A subject can be assessed as not having clinical pregnancy potential if the amount is less than or equal to about 0.4 times or less than about 0.35 times.

In step (F) of the determination method of the present invention, for example, one or more genes selected from the group consisting of gene b, gene d, and gene g of the present invention in the endometrial tissue specimen of the test subject. The expression level of the gene is higher than the average expression level of the gene in the normal subject group, or about 1.05 times or more, about 1.1 times or more, about 1.2 times as compared to the average value. or more, about 1.3 times or more, about 1.4 times or more, about 1.5 times or more, about 1.6 times or more, about 1.7 times or more, about 1.8 times or more, about 1.9 times or more , about 2.0 times or more, about 2.5 times or more, or about 3 times or more, the subject can be evaluated as not having clinical pregnancy potential.

The determination method of the present invention can detect 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, and 11 genes of the present invention. The above can be carried out in combination of 12 or more, 13 or more, 14 or more, 15 or more, or 16 or more, and for example, the expression level of a certain number or more of the genes of the present invention is controlled. If it is different (higher or lower) compared to , it can be determined that there is no possibility (or possibility) of clinical pregnancy due to the infertility treatment of the subject.

In the determination method of the present invention, determination of clinical pregnancy potential through infertility treatment can be performed by combining measurement of gene levels of endometrial tissue specimens of test subjects with measurement of other clinical variables. Clinical variables to be combined include, for example, the number of CD138-positive cells in the endometrial stroma in terms of the inflammatory state of the endometrium. By using the gene of the present invention in combination with such clinical variables, the determination accuracy can be further improved. Here, improving the determination accuracy means that when ROC analysis is used, the area under the ROC curve (AUC) improves.

According to the determination method of the present invention, it is possible to determine the clinical pregnancy potential of a test subject due to infertility treatment. Therefore, the determination method of the present invention can be used as an auxiliary for diagnosing the clinical possibility of pregnancy due to infertility treatment. In some cases, in combination with other findings, a doctor may ultimately be able to perform this. For example, in the present invention, for a test subject who has been determined to have a possibility of clinical pregnancy due to infertility treatment, a doctor refers to other findings and determines whether the test subject has a possibility of clinical pregnancy due to infertility treatment. It can be determined that That is, the determination method of the present invention can be rephrased as a method for assisting in diagnosing clinical pregnancy potential through infertility treatment or a method for assisting in diagnosing clinical pregnancy potential through infertility treatment.

According to the determination method of the present invention, clinical pregnancy potential due to infertility treatment can be quantitatively determined based on endometrial tissue specimens collected from test subjects. That is, the determination method of the present invention is advantageous in that it can easily and accurately determine the clinical possibility of pregnancy due to infertility treatment while reducing the burden on the patient. Therefore, the determination method of the present invention can be rephrased as a biological sample analysis method for determining the clinical possibility of pregnancy through infertility treatment. The determination method of the present invention is also advantageous in that it can be used to determine whether or not to continue infertility treatment in patients undergoing infertility treatment.

In addition to the above, the determination method of the present invention can be implemented according to the description of the detection method and prediction method of the present invention.

<<Evaluation method of implantation process>>
According to a fourth aspect of the present invention, an implantation process evaluation method for detecting or predicting an abnormality in the implantation process is provided. According to the evaluation method of the present invention, the implantation process can be evaluated in order to detect or predict abnormalities in the implantation process using the gene expression level in the endometrial tissue specimen of the test subject as an index. . That is, the evaluation method of the present invention is characterized by associating the gene expression level with an abnormality in the implantation process of the test subject. According to the evaluation method of the present invention, it can be used to detect or predict an abnormality in the "implantation process," preferably, it can be used to detect or predict an abnormality in the "process from embryo adhesion to embryo invasion." be able to. Here, in the present invention, "abnormality in the implantation process" refers to an adverse effect on pregnancy, typically resulting in abnormal embryo attachment or abnormal embryo infiltration after embryo attachment. Say something.

In the evaluation method of the present invention, first, (G) one or more genes of the present invention selected from the group consisting of a, b, c, d, e, f, and g in an endometrial tissue specimen of a test subject; A step of measuring the expression level of two or more genes (genes of the present invention) can be carried out. Measurement of gene expression level can be carried out according to the description of the detection method of the present invention. Furthermore, the evaluation method of the present invention measures the expression level of the gene of the present invention in endometrial tissue specimens collected from test subjects, so it can be called an in vitro evaluation method for the implantation process.

In the evaluation method of the present invention, the expression level of the gene of the present invention measured in step (H) (G) is used as an index to determine the presence or absence of abnormalities in the implantation process of the test subject from whom the endometrial tissue sample was collected. The method may further include a step of determining.

When the genes of the present invention are genes a, c, e, and f, step (H) includes (H-1-1) a predetermined expression level of the gene of the present invention in the endometrial tissue specimen of the test subject; and (H-1-2) when the expression level of the gene of the present invention in the endometrial tissue specimen of the test subject is below the reference value or lower than the reference value. This can be carried out by determining that there is an abnormality in the implantation process of the test subject. In the present invention, "there is an abnormality in the implantation process" is used to include "there is a possibility that there is an abnormality in the implantation process, or there is a high possibility that there is an abnormality in the implantation process." shall be.

In step (H-1-2), if the expression level of the gene of the present invention in the endometrial tissue specimen of the test subject is equal to or higher than the reference value, the implantation process of the test subject is affected. It can also be determined that there is no abnormality. In addition, in the present invention, "there is no abnormality in the implantation process" is used to include "there is a possibility that there is no abnormality in the implantation process, or there is a high possibility that there is no abnormality in the implantation process." shall be.

When the genes of the present invention are genes b, d, and g, step (H) includes (H-2-1) the expression level of the gene of the present invention in the endometrial tissue specimen of the test subject and a predetermined reference. (H-2-2) When the expression level of the gene of the present invention in the endometrial tissue specimen of the test subject is equal to or higher than the reference value, the test subject This can be carried out by a step of determining that there is an abnormality in the implantation process of the patient.

In step (H-2-2), if the expression level of the gene of the present invention in the endometrial tissue specimen of the test subject is below the reference value or lower than the reference value, the implantation process of the test subject is It can also be determined that there is no abnormality.

In step (H) of the evaluation method of the present invention, for example, one or two genes selected from the group consisting of gene a, gene c, gene e, and gene f of the present invention in the endometrial tissue specimen of the test subject. The expression level of the gene in the species or more is lower than the average expression level of the gene in the normal subject group, or about 0.9 times or less, about 0.85 times or less, about 0. 8 times or less, about 0.75 times or less, about 0.7 times or less, about 0.65 times or less, about 0.6 times or less, about 0.55 times or less, about 0.5 times or less, about 0.45 If it is less than 0.4 times, or less than about 0.35 times, it can be determined that there is an abnormality in the implantation process of the test subject.

In step (H) of the evaluation method of the present invention, for example, one or more genes selected from the group consisting of gene b, gene d, and gene g of the present invention in the endometrial tissue specimen of the test subject. The expression level of the gene is higher than the average expression level of the gene in the normal subject group, or about 1.05 times or more, about 1.1 times or more, about 1.2 times as compared to the average value. or more, about 1.3 times or more, about 1.4 times or more, about 1.5 times or more, about 1.6 times or more, about 1.7 times or more, about 1.8 times or more, about 1.9 times or more , about 2.0 times or more, about 2.5 times or more, or about 3 times or more, it can be determined that there is an abnormality in the implantation process of the test subject.

The evaluation method of the present invention can evaluate the genes of the present invention using 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, and 11 or more genes. The above can be carried out in combination such as 12 or more, 13 or more, 14 or more, 15 or more, or 16 or more, for example, the expression level of a certain number or more of the genes of the present invention is controlled. It can be determined that there is an abnormality (or no abnormality) in the implantation process of the test subject if it is different (higher or lower) compared to .

In the evaluation method of the present invention, the evaluation of the implantation process can be performed by combining the measurement of the gene level of the endometrial tissue specimen of the subject with the measurement of other clinical variables. Clinical variables to be combined include, for example, the number of CD138-positive cells in the endometrial stroma in terms of the inflammatory state of the endometrium. By using the gene of the present invention in combination with such clinical variables, the determination accuracy can be further improved. Here, improving the determination accuracy means that when ROC analysis is used, the area under the ROC curve (AUC) improves.

According to the evaluation method of the present invention, it can be used to detect or predict abnormalities in the implantation process of a test subject. Therefore, the evaluation method of the present invention can be used as an auxiliary for diagnosing abnormalities in the implantation process, and may be combined with other findings in some cases to determine whether a test subject has an abnormality in the implantation process. Ultimately, this can be done by a doctor. For example, in the present invention, for a test subject who has been evaluated as having an abnormality in the implantation process, a doctor can refer to other findings and determine that the test subject has an abnormality in the implantation process. That is, the evaluation method of the present invention can be rephrased as a method for assisting in diagnosing an abnormality in the implantation process or a method for assisting in diagnosing an abnormality in the implantation process.

According to the evaluation method of the present invention, it is possible to quantitatively evaluate the implantation process based on the endometrial tissue specimen collected from the test subject. That is, the evaluation method of the present invention is advantageous in that it can easily and accurately evaluate the implantation process to detect or predict abnormalities in the implantation process while reducing the burden on the patient. Therefore, the evaluation method of the present invention can be rephrased as a biological sample analysis method for detecting or predicting abnormalities in the implantation process. The evaluation method of the present invention is also advantageous in that it can be used to determine whether or not to continue infertility treatment in patients undergoing infertility treatment.

In addition to the above, the evaluation method of the present invention can be implemented according to the description of the detection method, prediction method, and determination method of the present invention.

<<Treatment method for implantation disorder>>
A test subject who is detected as having no implantation ability by the detection method of the present invention or a test subject who is diagnosed as having no implantation ability by the diagnostic method of the present invention may be treated for implantation disorder. can. Accordingly, another aspect of the present invention also includes the steps of: (A) measuring the expression level of a gene in an endometrial tissue sample of a test subject; A step of determining the presence or absence of implantation ability using the gene expression level as an index, and (N) a step of administering treatment for implantation disorder to the test subject determined to have no implantation ability in step (B). Provided is a method for treating implantation disorders, comprising: In the treatment method of the present invention, the step of detecting implantation ability (ie, the steps (A) and (B)) can be carried out according to the description of the detection method of the present invention and the diagnostic method of the present invention. Furthermore, treatment for implantation disorders can be carried out by infertility treatment.

<<Diagnostic markers of implantation ability and predictive markers of implantation failure>>
According to a fifth aspect of the present invention, a marker for use in diagnosing or detecting implantation ability, a marker for use in predicting implantation failure, and a marker for clinical pregnancy resulting from infertility treatment, comprising the gene of the present invention. Markers are provided for use in determining feasibility and in evaluating the implantation process. The present invention also provides a marker for use in diagnosing or detecting implantation potential, a marker for use in predicting implantation failure, a marker for use in determining clinical pregnancy potential through infertility treatment, or an implantation process. Provided is the use of the gene of the present invention as a marker for use in the evaluation of. In the present invention, the presence and amount of diagnostic markers, detection markers, predictive markers, determination markers, and evaluation markers are used to diagnose the presence or absence of implantation ability, predict the possibility of implantation failure, and determine the clinical possibility of pregnancy through infertility treatment. Substances that serve as indicators for the determination of implantation and evaluation of the implantation process, respectively, and are used for diagnosis and detection of implantation ability, prediction of implantation failure, determination of clinical pregnancy possibility through infertility treatment, evaluation of the implantation process, etc. Each can be used as a marker. That is, according to the present invention, the gene of the present invention can be used as a marker for diagnosing or detecting implantation ability, as a marker for predicting implantation failure, or as a marker for determining clinical pregnancy potential through infertility treatment or evaluating the implantation process. It can be used as a marker.

In addition to the above, the marker of the present invention can be carried out according to the description of the detection method, prediction method, determination method, and evaluation method of the present invention.

<<Diagnostic kit for implantation ability and prediction kit for implantation failure>>
According to a sixth aspect of the present invention, there is provided a kit for use in diagnosing or detecting implantation potential, comprising a means for quantifying the expression level of the gene of the present invention in endometrial tissue, and a kit for use in predicting implantation failure. Kits are provided for use in determining clinical fertility through infertility treatment, and kits for use in evaluating the implantation process. The kit of the present invention typically includes a kit for diagnosing or detecting implantation ability, a kit for predicting implantation failure, and a kit for predicting infertility, which is performed according to the detection method, prediction method, determination method, or evaluation method of the present invention. This kit is for determining clinical pregnancy potential due to treatment or for evaluating the implantation process. Quantification of the expression level of the gene of the present invention can be carried out according to the method for measuring the gene expression level described in the detection method of the present invention.

In addition to the above, the kit of the present invention can be implemented according to the descriptions of the detection method, prediction method, determination method, and evaluation method of the present invention.

The present invention will be explained more specifically based on the following examples, but the present invention is not limited to these examples.

Example 1: Study using human endometrial tissue (1)
In Example 1, endometrial tissue from infertile patients was collected, and a group of infertile patients (control group) who received assisted reproductive technology (embryo transfer) and became pregnant after collection, and a group of infertile patients who received assisted reproductive technology (embryo transfer) after collection. We compared the RNA expression levels in endometrial tissue samples from a group of infertile patients who did not become pregnant (implantation disorder group).

(1) Method A: Endometrial tissue samples Implantation-stage uterine samples were collected from 80 infertile patients attending the University of Tokyo Hospital, with informed consent, for investigation of the causes of infertility. A portion of the membrane specimen was used as an endometrial tissue specimen. When collecting endometrial tissue, hormones were administered in the same manner as during embryo transfer as assisted reproductive therapy, and endometrium was collected during the implantation period (7th day of progesterone administration) due to the hormone replacement cycle. In addition, a follow-up investigation was conducted to determine whether or not a clinical pregnancy occurred due to frozen-thawed embryo transfer performed during the infertility treatment cycle as an assisted reproductive technology after sample collection. In addition, patients who receive early embryo transfer treatment using in vitro fertilization as assisted reproductive technology should have the embryo transferred 4 days before the estimated implantation date, and patients who receive blastocyst transfer treatment as assisted reproductive technology should have the embryo transferred four days before the estimated implantation date. Embryo transfer was performed two days before the estimated bed stage.

B RNA extraction and RNA-Seq
RNA extraction from endometrial tissue specimens was performed using RNeasy Plus Mini Kit (Qiagen). A total of 80 specimens were collected, including a group of infertile patients (control group) (43 patients) who became pregnant through embryo transfer after specimen collection, and a group of infertile patients (implantation disorder group) (37 patients) who did not become pregnant through embryo transfer after specimen collection. RNA-seq was performed at Macrogen Japan.

C. Comparative analysis of RNA expression levels Raw read files obtained by RNA-Seq were aligned on the human genome sequence (GRCh38/hg38) using Hisat2 version 2.1.0, and each feature count function in the Subread tool was used to The number of reads on the gene locus was counted. In order to compare the expression level between the control group and the implantation disorder group, the read count file was submitted to DESeq2 version 1.16.1, and genes whose expression level changed by more than 2 times were classified as Differential expressed genes (DEGs). It was evaluated as Note that EZH2 and CCND2, which are EZH2-related genes, were included in the analysis although no change in their expression levels was observed to be more than twofold. These genes were then compared with two types of databases (Epigenetic Roadmap project (National Institutes of Health), ChEA 2016) using Enrichr.

(2) Results The results were as shown in Figures 2 to 5. In the implantation disorder group, compared to the control group, regarding the expression of PRC2-related genes, it was confirmed that the expression levels of OSR1, SCUBE1, HOXA13, MEGF10, and TRPM6 were decreased, and the expression of genes other than PRC2-related genes was confirmed. A decrease in the expression levels of SLC46A2 and AZGP1 was confirmed (Figure 2). On the other hand, in the implantation disorder group, compared to the control group, an increase in the expression levels of SPINK2, RSPO3, LGI2, and MAL was confirmed, as well as an increase in the expression of genes other than PRC2-related genes. An increase in the expression levels of BRINP2, RDH12, SST, PENK, and CHI3L2 was confirmed (Figure 3). Furthermore, in the implantation disorder group, compared to the control group, regarding the expression of EZH2-related genes, a decrease in the expression level of EZH2 and an increase in the expression level of CCND2 were confirmed (FIG. 4A and FIG. 5). Furthermore, regarding the expression of the SUZ2 gene, a decrease in the expression level of SUZ12 was confirmed in the implantation disorder group compared to the control group (FIG. 4B). These results showed that the expression levels of all genes differed between the control group and the implantation disorder group (p<0.5), indicating that these genes serve as indicators of implantation disorder.

Example 2: Study using human endometrial tissue (2)
In Example 2, the expression level of EZH2, one of the constituent molecules of PRC2, was investigated in human endometrial tissue during the implantation stage.

As a result of immunostaining the human endometrium during the implantation stage using an EZH2 antibody, expression of EZH2 was confirmed mainly in the endometrial glandular epithelium, and weak expression was also observed in the endometrial luminal epithelium and stroma. (Figure 6).

Example 3: Study using genetically modified mice (1)
In Example 3, the expression pattern of Ezh2 in the uterus during the implantation stage was examined using mice whose female hormone dynamics during the implantation stage are similar to those of humans.

Although there is a structural difference between the human uterus, which is monocornuate, and the mouse uterus, which is bicornuate, it still contains the sex steroid hormones estradiol-17β (E ₂ ) and progesterone (P ₄ ), which are important during the implantation period. ) are similar in humans and mice (Figure 7). Additionally, humans and mice have similar characteristics in that the period from ovulation to implantation is close to each other, and that trophoblast cells (trophoblasts), the main cells that make up the placenta, violently infiltrate the endometrium. .

Figure 8 shows an overview of the mouse implantation process. Activation of the blastocyst occurs on the fourth day of pregnancy, and at midnight the embryo adhesion reaction to the uterus begins, and the stimulation is transmitted from the endometrial luminal epithelium to the stroma, and in the endometrial stroma, decidua transformation begins. At the same time, a pit-like structure (crypt) in the endometrium begins to form, near the bottom of which the embryo can be seen. After the fifth day of pregnancy, the luminal epithelium disappears and trophoblast cells (trophoblasts) infiltrate into the interstitium. Thereafter, at the stage where placentation by trophoblasts progresses, endometrial stromal cells become decidualized. On the eighth day of pregnancy, decidualization is complete and leads to placenta formation. In the Examples in this specification using mice, the study focused mainly on the implantation period (6th day of pregnancy).

As wild type mice (WT), female C57BL/6 mice (7 to 12 weeks old) were mated with fertile males (WT) to become pregnant. Here, the day when the vaginal plug was recognized (the day of pregnancy) was defined as "day 1" (in the examples of this specification, when "X day" is stated, it is the X day after the day when the vaginal plug was recognized). ``Day X'' is synonymous with ``Day X of pregnancy.'') Then, expression analysis of Ezh2 in the uterus at the implantation site on "day 1," "day 4," "day 6," and "day 8" of WT was performed by immunostaining.

It is expressed in the endometrial epithelium (Figure 9A) on the "1st day", in both the endometrial epithelium and stroma on the "4th day" (Figure 9B), and on the "6th day" it is expressed in the endometrial stroma (sloughed off). It was confirmed that it was expressed in the endometrial stroma (decidua) on the blood vessel side on "8th day" (Fig. 9D). These results showed that Ezh2 is expressed in the mouse uterus during the implantation stage and that the expression site changes depending on the pregnancy stage.

Example 4: Study using genetically modified mice (2)
In Example 4 and later, the effect of Ezh2 expressed in the uterus on pregnancy was examined using mice in which Ezh2 was selectively knocked out in the uterus.

Mice in which Ezh2 in the uterus was selectively knocked out were created using the Cre-loxP system using the following procedure. Ezh2-loxP mice (C57BL/6) and Pgr-Cre mice (C57BL/6/129SV) were crossed to generate mice in which uterine Ezh2 was selectively knocked out (Ezh2 uKO). Cre-negative littermates for Ezh2 uKO were used as control mice (Ezh2 uControl). In addition, C57BL/6 wild type mice were used as wild type mice (WT) (male mice for mating and female mice for Ezh2 expression analysis).

In Ezh2 uKO, compared to Ezh2 uControl, Ezh2 expression was clearly decreased in both the endometrial epithelium and stroma in the uterus on day 4 (Fig. 10A), and the expression of Ezh2 was clearly decreased in both the endometrial epithelium and stroma on day 4 of the uterus (Figure 10A), Western blotting of the site also confirmed that the expression of Ezh2 was significantly reduced (FIG. 10B). Furthermore, although Ezh2 uKO did not result in complete infertility, it was confirmed that the number of offspring produced was reduced compared to Ezh2 uControl (Figure 11A). Furthermore, when a caesarean section was performed on the "19th day", no abnormality was observed in the embryonic growth of Ezh2 uKO in the late pregnancy, but the number of fetuses on the "19th day" decreased as well as the number of offspring ( Figure 11B), no increase in intrauterine fetal death was observed in Ezh2 uKO (Figure 11C). Furthermore, no abnormality was observed in the morphology or number of blastocysts of Ezh2 uKO (FIGS. 12A and B). These results showed that ovulation and preimplantation embryonic development were normal in Ezh2 uKO.

On the other hand, in the "fourth day" endometrium of WT mice, proliferation-differentiation switching (PDS) occurs, in which luminal epithelial cell proliferation stops and interstitial cell proliferation increases. However, as a result of immunostaining for Ki67, a cell proliferation marker, it was confirmed that cell proliferation in the endometrial luminal epithelium was not suppressed and continued in the Ezh2 uKO uterus (FIG. 12C).

Example 5: Study using genetically modified mice (3)
On the ``fifth day'' when the adhesion reaction between the uterus and the embryo occurs, the number of embryo attachment sites was investigated by taking advantage of the fact that the implantation site is stained blue when Chicago blue dye is injected into the tail vein. No difference in the number of blue-stained areas was confirmed between Ezh2 uControl and Ezh2 uKO (FIG. 13).

Next, on the 6th day, when embryo infiltration into the endometrial stroma and decidualization of the endometrial stroma was observed, Chicago blue dye was administered in the same manner as on the 5th day, but Ezh2 No difference in the number of implantation sites was confirmed between uControl and Ezh2 uKO (Figure 14).

On the other hand, normally on the 6th day, the endometrial luminal epithelium around the embryo disappears and trophoblast infiltration is observed, but based on the results of HE staining and CK8 immunostaining, in Ezh2 uKO, the uterus around the embryo It was confirmed that the intimal luminal epithelium remained, the morphology of the epithelium was serrated, and no (or only minimal) invasion of trophoblasts was observed (FIGS. 15A and B). Furthermore, when examining the percentage of endometrial luminal epithelium surrounding the embryo disappearing at the implantation site of Ezh2 uControl and Ezh2 uKO, it was found that endometrial luminal epithelium remained in approximately 80% of cases of Ezh2 uKO ( Figure 15C). In Ezh2 uKO, the percentage of endometrial luminal epithelium remaining on "6th day" and the percentage of decrease in the number of offspring born at delivery are almost the same, and endometrial luminal epithelium remains on "6th day". It was suggested that the occurrence of impaired embryonic invasion is the main cause of decreased fertility.

In addition, when frozen sections of the implantation site were stained with HE and the development of the embryo after implantation was evaluated, embryonic development was impaired and implantation failure occurred in approximately 80% of the implantation sites in Ezh2 uKO (Fig. 16A and B), the frequency of which was considered to be equivalent to the phenotype of impaired embryo invasion and reduced number of offspring born on "6th day".

Example 6: Study using genetically modified mice (4)
The implantation site on the "6th day" was visualized using tissue transparency and three-dimensional image construction technology. Typically, at the implantation site on day 6, an implantation chamber is formed consisting of the embryo, the crypt of the endometrial lumen, and the endometrial glands leading to the crypt. . In Ezh2 uControl, the crypt was deeply formed, and the glandular structure extended toward the region near the embryo of the crypt, which was a normal finding. On the other hand, in Ezh2 uKO, the formation of the crypt, the decidualized endometrial stroma surrounding it, and the glandular structure toward the crypt were poorly formed, and it was confirmed that the formation of the implantation chamber was insufficient (Fig. 17).

Example 7: Study using genetically modified mice (5)
On "day 6", the implantation sites of Ezh2 uControl and Ezh2 uKO uteruses were used for chromatin immunoprecipitation (ChIP) using anti-H3K27me3 antibody, and ChIP-seq was performed on the extracted DNA. Then, the state of H3K27me3 in the gene whose expression level was increased in Ezh2 uKO in RNA-seq of the uterine implantation site on "6th day" was compared between Ezh2 uControl and Ezh2 uKO.

Comparing the status of H3K27me3 for gene groups whose expression levels increased in RNA-seq, it was confirmed that H3K27me3 modification at the 5' upstream of each gene was significantly reduced in Ezh2 uKO compared to Ezh2 uControl. (Figure 18A: Tag density plot, Figure 18B: Heatmap). In addition, since RNA-seq showed that the expression of Ccnd2 and Cdkn2b, which are involved in the cell cycle, was increased in Ezh2 uKO, analysis of the state of H3K27me3 in the Ccnd2 and Cdkn2b gene regions revealed that the expression of H3K27me3 in the vicinity of the transcription start point of each gene was increased. A decrease in H3K27me3 was confirmed in the region (FIG. 18C). These results showed that Ezh2 controls H3K27me3 and regulates gene expression related to the cell cycle.

Furthermore, when we compared the expression levels of Ccnd2 and Cdkn2b mRNA in the uterus on "6th day" by RT-qPCR, it was consistent with the RNA-Seq results, and Ezh2 uKO showed that the cell cycle was more affected than Ezh2 uControl. It was confirmed that the related mRNA expression levels of Ccnd2 and Cdkn2b were increased (FIG. 19A). On the other hand, the mRNA expression levels of Cdk4, Cdk6, and Cdkn1a, which are strongly related to these genes, were only slightly increased for Cdk4 (FIG. 19B).

These results and the results of Example 1 (FIG. 5) showed that an increase in the expression level of Ccnd2 in endometrial tissue can be used as a predictive indicator for detection of implantation ability and implantation failure.

Example 8: Study using genetically modified mice (6)
Regarding the state of the cell cycle in the Ezh2 uKO uterus, we analyzed the ability to incorporate bromodeoxyuridine (BrdU) to detect the S phase, and immunostained for phosphorylated histone H3 (pHH3), a marker for the G2 to M phase. went.

In Ezh2 uControl, pHH3 expression was not observed in the area corresponding to the decidualized endometrial stroma, and in Ezh2 uKO, the area where pHH3 was not expressed was clearly small (Fig. 20, top). ), no difference was observed in the expression region of BrdU between the two (Figure 20, bottom). These results indicate that in the decidualized endometrial stroma, the transition from G2 to M phase is normally stopped, leading to differentiation without cell division, but these results indicate that cell division does not occur in Ezh2 uKO. It was inferred that the cells did not stop, making it difficult for cell differentiation to occur.

The decidual cells, which are produced by the differentiation of endometrial stromal cells during pregnancy, are often large and polyploid cells, and cell proliferation is stopped and physiological functions are maintained even though they decline. It is known that cells enter a state of differentiation (a state called cellular senescence). Therefore, when we performed SA-β-gal (senescence-associated beta-galactosidase) staining, which is a marker of cellular senescence, on the 8th day, we found that in Ezh2 uKO, cellular senescence in the decidualized endometrial stroma was attenuated. It was confirmed that this was the case (Figure 21). From these results, it was inferred that in Ezh2 uKO, terminal differentiation did not proceed due to an abnormality in the cell cycle of the endometrial stroma, and eventually functional failure occurred due to insufficient decidualization.

Claims (14)

A method for detecting implantation ability or predicting implantation failure, the method comprising the step of measuring the expression level of a gene in an endometrial tissue sample of a test subject, wherein the gene is a PRC2-related gene or an EZH2-related gene. and/or SUZ12 gene, a detection method or a prediction method. PRC2-related genes are (1) OSR1 gene, (2) SCUBE1 gene, (3) HOXA13 gene, (4) MEGF10 gene, (5) TRPM6 gene, (6) SPINK2 gene, (7) RSPO3 gene, (8) The detection method or prediction method according to claim 1, which is one or more genes selected from the group consisting of LGI2 gene and (9) MAL gene. The detection method or prediction method according to claim 1 or 2, wherein the EZH2-related gene is (10) EZH2 gene and/or (11) CCND2 gene. The detection method or prediction method according to claim 1 or 2, further comprising the step of measuring the expression level of genes other than genes (1) to (11) in the endometrial tissue specimen of the test subject. Genes other than genes (1) to (11) include (12) SLC46A2 gene, (13) AZGP1 gene, (14) BRINP2 gene, (15) RDH12 gene, (16) SST gene, (17) PENK gene, and ( 18) The detection method or prediction method according to claim 4, which is one or more genes selected from the group consisting of CHI3L2 genes. The detection method or prediction method according to claim 1 or 2, further comprising the step of determining the presence or absence of implantation ability using the expression level of a gene in the endometrial tissue specimen of the test subject as an index. The detection method or prediction method according to claim 1 or 2, wherein the expression level of the gene is quantified by the amount of RNA expression. The detection method or prediction method according to claim 1 or 2, wherein the test subject is an infertility patient. A method for determining clinical pregnancy potential through infertility treatment, the method comprising the step of measuring the expression level of a gene in an endometrial tissue specimen of a test subject, the method comprising: determining whether the gene is a PRC2-related gene, an EZH2-related gene and/or Or, the determination method is SUZ12 gene. A method for evaluating the implantation process for detecting or predicting an abnormality in the implantation process, the method comprising the step of measuring the expression level of a gene in an endometrial tissue specimen of a test subject, the method comprising: measuring the expression level of a gene in an endometrial tissue specimen of a test subject, the method comprising: measuring the expression level of a gene in an endometrial tissue specimen of a test subject, the method comprising: gene, EZH2-related gene and/or SUZ12 gene. A diagnostic marker for implantation ability based on the expression level of a gene in an endometrial tissue sample of a test subject, a predictive marker for implantation failure, a marker for determining clinical pregnancy potential through infertility treatment, or a marker for evaluating the implantation process. A diagnostic marker, predictive marker, determination marker, or evaluation marker, wherein the gene is a PRC2-related gene, an EZH2-related gene, and/or a SUZ12 gene. As a marker for the diagnosis or detection of implantation potential, as a marker for predicting implantation failure, as a marker for determining clinical fertility through infertility treatment, or for evaluating the implantation process. Use of a gene as a marker for, said gene being a PRC2-related gene, an EZH2-related gene and/or a SUZ12 gene. A diagnostic kit for implantation ability, a kit for predicting implantation failure, a kit for determining clinical pregnancy potential through infertility treatment, or implantation process, comprising a means for quantifying the expression level of a gene in an endometrial tissue sample of a test subject. A diagnostic kit, a prediction kit, a determination kit, or an evaluation kit for the implantation process, wherein the gene is a PRC2-related gene, an EZH2-related gene, and/or a SUZ12 gene. a step of measuring the expression level of a gene in an endometrial tissue sample of the test subject; and a step of determining the presence or absence of implantation ability using the gene expression level in the endometrial tissue sample of the test subject as an index. , a method for treating implantation disorders, the method comprising the step of administering treatment for implantation disorders to the test subject who has been determined to have no implantation capacity, wherein the gene is a PRC2-related gene or an EZH2-related gene. and/or the SUZ12 gene.

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