JP7475626B2 - Method for selecting mammalian embryos using biomarkers - Google Patents

Description

(1) Published in a poster presentation at the "Bioresource Genome Analysis Center Research Report Session" on February 15, 2019. (2) Published on page 6 of the "15th Tokai ART Conference Program Abstracts" distributed by the "Tokai ART Conference" on February 24, 2019. (3) Published in an oral presentation at the "15th Tokai ART Conference Program" on February 24, 2019. (4) Published on page 63 of the "Bioresource Genome Analysis Center Newsletter No. 6" mailed by the "Tokyo University of Agriculture Bioresource Genome Analysis Center" on April 9, 2019. (5) Published on the website of the "Tokyo University of Agriculture Bioresource Genome Analysis Center" on May 10, 2019. (6) Published in the "The Journal of Reproduction and (7) Published on page 27 of the "Abstracts of the 12th Japanese ET Practitioners Network Training Workshop at the 3rd Japanese Embryo Transfer Technology Research Society Conference" distributed by the "Japan Embryo Transfer Technology Research Society Secretariat" on August 28, 2019. (8) Published in an oral presentation at the "3rd Japanese Embryo Transfer Technology Research Society Conference (Wakayama Conference)" on August 30, 2019. (9) Published in a poster presentation at the "112th Japanese Society of Reproductive Biology Conference" on September 5, 2019.

The present invention relates to a method for evaluating mammalian embryos; a method for selecting mammalian embryos; a kit for evaluating and/or selecting mammalian embryos; a biomarker for evaluating and/or selecting mammalian embryos; a method for identifying genes for evaluating and/or selecting mammalian embryos; and the like.

Currently, one in six couples in developed countries suffer from infertility. For this reason, in addition to general infertility treatments such as artificial insemination (AI), there is a growing trend for advanced infertility treatments (ART) such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), but the success rate in Japan remains low at less than 10% (Non-Patent Document 1). Recently, the decline in conception rates has also become a major problem in the livestock industry. In particular, cows have a long gestation period, so a single miscarriage results in a large economic loss.

It is believed that many embryos that do not progress to maintaining pregnancy suffer from developmental insufficiency during cleavage or early miscarriage after implantation. One of the main causes is thought to be a decline in the quality of gametes and embryos due to late marriage and external environmental stress. Embryo quality has traditionally been assessed based on differences in embryo morphology and division rate, but this is often based on personal perception, is not very accurate, and lacks scientific evidence.

In recent years, in search of more objective indicators, methods have been developed that involve detailed imaging of embryos to detect chromosomal abnormalities (Non-Patent Document 2). However, such methods require a process in which mRNA encoding a fluorescent protein is synthesized in a test tube and then injected into a fertilized egg, making it difficult to apply to human embryos, for which injection of nucleic acids is not permitted. There has been a demand for a method to assess the quality of embryos (fertilized eggs) at an early stage that is less invasive and based on scientific evidence.

Meanwhile, a method has been reported for detecting the presence or absence of a maternal pathogenic gene in a fertilized egg by performing genetic testing on the polar body that is generated during the meiosis of the egg (Patent Document 1).

Japanese Patent Application Laid-Open No. 2-86800

HumReprod. 2016 Jul;31(7):1588-609. Hum Reprod. 2009Oct;24(10):2490-9.

The objective of the present invention is to provide a method for evaluating and selecting high-quality embryos while keeping damage to the embryos relatively low, and a method for identifying and using biomarkers for evaluating and selecting high-quality embryos.

In order to solve the above problems, the present inventors analyzed gene transcription products in a fertilized egg and gene transcription products in the second polar body released from the fertilized egg, and found that the majority of gene transcription products in the second polar body were maternal transcription products of the fertilized egg. In addition, in order to identify maternal transcription products that can evaluate the quality of an embryo, they attempted to identify them from gene transcription products in the second polar body, and 113 types of genes (i.e., normal development embryo marker genes) were identified as transcription products that were significantly increased in the second polar body derived from a normally developed embryo compared to the second polar body derived from an abnormally developed embryo. In addition, 80 types of genes (i.e., abnormal development embryo marker genes) were identified as transcription products that were significantly increased in the second polar body derived from an abnormally developed embryo compared to the second polar body derived from a normally developed embryo. In addition, it was confirmed that normal development embryos and abnormal development embryos can be distinguished from each other using the levels of the transcription products of these normal development embryo marker genes and abnormal development embryo marker genes as indicators. The present invention was completed based on these findings.

That is, the present invention is as follows.
[1] A method for evaluating a mammalian embryo, comprising the following steps (a) and (b-1):
(a) detecting a transcription product of one or more genes selected from the following [group of marker genes for normal development embryos] and [group of marker genes for abnormal development embryos] in a polar body derived from an in vitro fertilized mammalian fertilized egg;
[Normal embryo development marker genes]
Cdk2ap1, Trappc12, Kif13a, Redrum, Enpep, Gart, Rogdi, 2310003L06Rik, Rabl3, F730016J06Rik, Prdx3, Xpnpep1, Abcc5, 4933431E20Rik, Spire2, Mgme1, Ak9, Sipa1, Narf, Tmem147, Rimbp2, Dlgap1, Cd209f, Itpr3, Atxn7l1, T hap3, Dr1, Casq2, Dnah11, Nsun5, Tek, Sec1, Zc3h7a, Vgll1, Nfkbid, Rin2, Ube2m, Zfp954, Fkbp9, Rrp1, Otx1, Ap2a2, Dpep2, Ces1d, Upp1, Zg16, Adam12, Phf2, Phrf1, Slc43a2, Gm13977, Mfsd8, Cotl1, Efcab3, Commd4, Kantr, Rnf2 12, Ankmy2, Popdc3, Arpc1a, Npdc1, Sema6a, Etfb, D030040B21Rik, Ttc21b, Plekhg5, Nat8f1, Zscan18, 5330439K02Rik, Rbfa, Zfp408, Aadacl3, Oit1, Tmem120a, Traf5, Krt8, Cd200, Ostm1, Zc3h12d, Ammecr1, AI413582, Commd5, Prkdc, Tmprss2, Prkcsh, Wdr74, Nme1, Tex15, Zfp563, Gpn3, F5, Mib2, Gm4926, Sbspon, Nup155, Pbx4, Defb48, Ddx20, Itga4, Pcsk5, Dtna, Epcam, Gm28940, Gm44196, Eif3g, Polg2, Gm364, Mtch2, Htt, Ing5, Gm42945, Aknad1, Ehbp1
[Embryonic abnormality marker genes]
Dmd, Tyk2, Nyap2, Mettl22, Manea, Nudt18, Lair1, Slamf1, March3, Ulk2, AI429214, Zfp938, Wdr59, Spryd7, Grin2b, Fsd2, Ciao3, Zhx1, Grm1, Adm, Ugt2b38, T bc1d25, Lrrc61, Fam132b, Exoc6b, Reps2, Cdh24, Fech, Agpat1, Plcb3, A230056P14Rik, Spsb4, Dmrtc1b, Raver2, 4921531C22Rik, Tbc1d31, Nutm1, Nek11, Morc 2a, Trhr2, Aacs, Atp5o, Sirt5, Id3, Zfp507, AW551984, Lias, Usp20, Wfdc1, Tubd1, Zmym6, Klf10, Mfsd11, Nudt15, Nisch, Hipk3, Tatdn3, Tyw3, Ubl4a, Atp6ap1 l, Snx8, Gm12364, Ndufa7, Zfp341, Col4a1, Zfp595, G2e3, Slc15a2, Sgsm1, Parp4, Ciao1, Fam20b, Acvr2b, Ttc25, Mau2, Sybu, Kdm3b, AY512915, Gm13136, Trmt6
(b-1) when the transcription product of the gene detected in step (a) is a transcription product of the [group of normal development embryo marker genes], if the level of the transcription product of the gene detected in step (a) is higher than the level in a polar body derived from an embryo whose development does not progress to the blastocyst stage, then the fertilized egg is evaluated as an embryo that will develop normally or an embryo that is likely to develop normally;
a step of evaluating, when the transcription product of the gene detected in step (a) is a transcription product of the [group of abnormal development embryo marker genes], the fertilized egg as an embryo that will develop normally or is likely to develop normally, when the level of the transcription product of the gene detected in step (a) is lower than the level in a polar body derived from an embryo whose development does not progress to the blastocyst stage;
[2] A method for selecting a mammalian embryo, comprising the following steps (a) and (b-2):
(a) detecting a transcription product of one or more genes selected from the following [group of marker genes for normal development embryos] and [group of marker genes for abnormal development embryos] in a polar body derived from an in vitro fertilized mammalian fertilized egg;
[Normal embryo development marker genes]
Cdk2ap1, Trappc12, Kif13a, Redrum, Enpep, Gart, Rogdi, 2310003L06Rik, Rabl3, F730016J06Rik, Prdx3, Xpnpep1, Abcc5, 4933431E20Rik, Spire2, Mgme1, Ak9, Sipa1, Narf, Tmem147, Rimbp2, Dlgap1, Cd209f, Itpr3, Atxn7l1, T hap3, Dr1, Casq2, Dnah11, Nsun5, Tek, Sec1, Zc3h7a, Vgll1, Nfkbid, Rin2, Ube2m, Zfp954, Fkbp9, Rrp1, Otx1, Ap2a2, Dpep2, Ces1d, Upp1, Zg16, Adam12, Phf2, Phrf1, Slc43a2, Gm13977, Mfsd8, Cotl1, Efcab3, Commd4, Kantr, Rnf2 12, Ankmy2, Popdc3, Arpc1a, Npdc1, Sema6a, Etfb, D030040B21Rik, Ttc21b, Plekhg5, Nat8f1, Zscan18, 5330439K02Rik, Rbfa, Zfp408, Aadacl3, Oit1, Tmem120a, Traf5, Krt8, Cd200, Ostm1, Zc3h12d, Ammecr1, AI413582, Commd5, Prkdc, Tmprss2, Prkcsh, Wdr74, Nme1, Tex15, Zfp563, Gpn3, F5, Mib2, Gm4926, Sbspon, Nup155, Pbx4, Defb48, Ddx20, Itga4, Pcsk5, Dtna, Epcam, Gm28940, Gm44196, Eif3g, Polg2, Gm364, Mtch2, Htt, Ing5, Gm42945, Aknad1, Ehbp1
[Embryonic abnormality marker genes]
Dmd, Tyk2, Nyap2, Mettl22, Manea, Nudt18, Lair1, Slamf1, March3, Ulk2, AI429214, Zfp938, Wdr59, Spryd7, Grin2b, Fsd2, Ciao3, Zhx1, Grm1, Adm, Ugt2b38, T bc1d25, Lrrc61, Fam132b, Exoc6b, Reps2, Cdh24, Fech, Agpat1, Plcb3, A230056P14Rik, Spsb4, Dmrtc1b, Raver2, 4921531C22Rik, Tbc1d31, Nutm1, Nek11, Morc 2a, Trhr2, Aacs, Atp5o, Sirt5, Id3, Zfp507, AW551984, Lias, Usp20, Wfdc1, Tubd1, Zmym6, Klf10, Mfsd11, Nudt15, Nisch, Hipk3, Tatdn3, Tyw3, Ubl4a, Atp6ap1 l, Snx8, Gm12364, Ndufa7, Zfp341, Col4a1, Zfp595, G2e3, Slc15a2, Sgsm1, Parp4, Ciao1, Fam20b, Acvr2b, Ttc25, Mau2, Sybu, Kdm3b, AY512915, Gm13136, Trmt6
(b-2) when the transcription product of the gene detected in step (a) is a transcription product of the [group of normally developing embryo marker genes], selecting the fertilized egg when the level of the transcription product of the gene detected in step (a) is higher than the level in a polar body derived from an embryo whose development does not progress to the blastocyst stage;
selecting the fertilized egg when the level of the transcription product of the gene detected in step (a) is the amount of transcription product of the [group of abnormal development embryo marker genes], is lower than the level in a polar body derived from an embryo whose development does not progress to the blastocyst stage;
[3] The method according to [1] or [2] above, wherein the gene is Cdk2ap1 or Dmd.
[4] The method according to any one of [1] to [3] above, wherein the polar body is a second polar body.
[5] The method according to any one of [1] to [4] above, wherein in the step (a), the transcription product of the gene is detected by a sequencing method using a next-generation sequencer.
[6] A kit for evaluating and/or selecting a mammalian embryo, comprising a reverse primer and/or a probe that hybridizes to the transcription product of one or more genes selected from the following [group of marker genes for normal development embryos] and [group of marker genes for abnormal development embryos] in a polar body derived from an in vitro fertilized mammalian fertilized egg, and/or a primer set and/or a probe consisting of a forward primer and a reverse primer that hybridize to the cDNA of the gene.
[Normal embryo development marker genes]
Cdk2ap1, Trappc12, Kif13a, Redrum, Enpep, Gart, Rogdi, 2310003L06Rik, Rabl3, F730016J06Rik, Prdx3, Xpnpep1, Abcc5, 4933431E20Rik, Spire2, Mgme1, Ak9, Sipa1, Narf, Tmem147, Rimbp2, Dlgap1, Cd209f, Itpr3, Atxn7l1, T hap3, Dr1, Casq2, Dnah11, Nsun5, Tek, Sec1, Zc3h7a, Vgll1, Nfkbid, Rin2, Ube2m, Zfp954, Fkbp9, Rrp1, Otx1, Ap2a2, Dpep2, Ces1d, Upp1, Zg16, Adam12, Phf2, Phrf1, Slc43a2, Gm13977, Mfsd8, Cotl1, Efcab3, Commd4, Kantr, Rnf2 12, Ankmy2, Popdc3, Arpc1a, Npdc1, Sema6a, Etfb, D030040B21Rik, Ttc21b, Plekhg5, Nat8f1, Zscan18, 5330439K02Rik, Rbfa, Zfp408, Aadacl3, Oit1, Tmem120a, Traf5, Krt8, Cd200, Ostm1, Zc3h12d, Ammecr1, AI413582, Commd5, Prkdc, Tmprss2, Prkcsh, Wdr74, Nme1, Tex15, Zfp563, Gpn3, F5, Mib2, Gm4926, Sbspon, Nup155, Pbx4, Defb48, Ddx20, Itga4, Pcsk5, Dtna, Epcam, Gm28940, Gm44196, Eif3g, Polg2, Gm364, Mtch2, Htt, Ing5, Gm42945, Aknad1, Ehbp1
[Embryonic abnormality marker genes]
Dmd, Tyk2, Nyap2, Mettl22, Manea, Nudt18, Lair1, Slamf1, March3, Ulk2, AI429214, Zfp938, Wdr59, Spryd7, Grin2b, Fsd2, Ciao3, Zhx1, Grm1, Adm, Ugt2b38, T bc1d25, Lrrc61, Fam132b, Exoc6b, Reps2, Cdh24, Fech, Agpat1, Plcb3, A230056P14Rik, Spsb4, Dmrtc1b, Raver2, 4921531C22Rik, Tbc1d31, Nutm1, Nek11, Morc 2a, Trhr2, Aacs, Atp5o, Sirt5, Id3, Zfp507, AW551984, Lias, Usp20, Wfdc1, Tubd1, Zmym6, Klf10, Mfsd11, Nudt15, Nisch, Hipk3, Tatdn3, Tyw3, Ubl4a, Atp6ap1 l, Snx8, Gm12364, Ndufa7, Zfp341, Col4a1, Zfp595, G2e3, Slc15a2, Sgsm1, Parp4, Ciao1, Fam20b, Acvr2b, Ttc25, Mau2, Sybu, Kdm3b, AY512915, Gm13136, Trmt6
[7] The kit according to [6] above, wherein the gene is Cdk2ap1 or Dmd.
[8] The kit according to [6] or [7] above, characterized in that the polar body is a second polar body.
[9] A biomarker for evaluating and/or selecting a mammalian embryo, comprising one or more genes selected from the following [group of marker genes for normal development embryos] and [group of marker genes for abnormal development embryos]:
When the gene is a gene of the "group of normal development embryo marker genes," the level of the transcription product of the gene in a polar body derived from an in vitro fertilized mammalian zygote is higher than the level in a polar body derived from an embryo that does not progress to the blastocyst stage;
When the gene is a gene of the "abnormal development embryo marker gene group," the level of the transcription product of the gene in a polar body derived from an in vitro fertilized mammalian fertilized egg is lower than the level in a polar body derived from an embryo that does not progress to the blastocyst stage.
[Normal embryo development marker genes]
Cdk2ap1, Trappc12, Kif13a, Redrum, Enpep, Gart, Rogdi, 2310003L06Rik, Rabl3, F730016J06Rik, Prdx3, Xpnpep1, Abcc5, 4933431E20Rik, Spire2, Mgme1, Ak9, Sipa1, Narf, Tmem147, Rimbp2, Dlgap1, Cd209f, Itpr3, Atxn7l1, T hap3, Dr1, Casq2, Dnah11, Nsun5, Tek, Sec1, Zc3h7a, Vgll1, Nfkbid, Rin2, Ube2m, Zfp954, Fkbp9, Rrp1, Otx1, Ap2a2, Dpep2, Ces1d, Upp1, Zg16, Adam12, Phf2, Phrf1, Slc43a2, Gm13977, Mfsd8, Cotl1, Efcab3, Commd4, Kantr, Rnf2 12, Ankmy2, Popdc3, Arpc1a, Npdc1, Sema6a, Etfb, D030040B21Rik, Ttc21b, Plekhg5, Nat8f1, Zscan18, 5330439K02Rik, Rbfa, Zfp408, Aadacl3, Oit1, Tmem120a, Traf5, Krt8, Cd200, Ostm1, Zc3h12d, Ammecr1, AI413582, Commd5, Prkdc, Tmprss2, Prkcsh, Wdr74, Nme1, Tex15, Zfp563, Gpn3, F5, Mib2, Gm4926, Sbspon, Nup155, Pbx4, Defb48, Ddx20, Itga4, Pcsk5, Dtna, Epcam, Gm28940, Gm44196, Eif3g, Polg2, Gm364, Mtch2, Htt, Ing5, Gm42945, Aknad1, Ehbp1
[Embryonic abnormality marker genes]
Dmd, Tyk2, Nyap2, Mettl22, Manea, Nudt18, Lair1, Slamf1, March3, Ulk2, AI429214, Zfp938, Wdr59, Spryd7, Grin2b, Fsd2, Ciao3, Zhx1, Grm1, Adm, Ugt2b38, T bc1d25, Lrrc61, Fam132b, Exoc6b, Reps2, Cdh24, Fech, Agpat1, Plcb3, A230056P14Rik, Spsb4, Dmrtc1b, Raver2, 4921531C22Rik, Tbc1d31, Nutm1, Nek11, Morc 2a, Trhr2, Aacs, Atp5o, Sirt5, Id3, Zfp507, AW551984, Lias, Usp20, Wfdc1, Tubd1, Zmym6, Klf10, Mfsd11, Nudt15, Nisch, Hipk3, Tatdn3, Tyw3, Ubl4a, Atp6ap1 l, Snx8, Gm12364, Ndufa7, Zfp341, Col4a1, Zfp595, G2e3, Slc15a2, Sgsm1, Parp4, Ciao1, Fam20b, Acvr2b, Ttc25, Mau2, Sybu, Kdm3b, AY512915, Gm13136, Trmt6
[10] The biomarker according to [9] above, characterized in that the gene is Cdk2ap1 or Dmd.
[11] The biomarker described in [9] or [10] above, characterized in that the polar body is a second polar body.
[12] A method for identifying genes for evaluation and/or selection of mammalian embryos, comprising the following steps (A) and (B):
(A) detecting a transcription product of a gene in a polar body derived from an in vitro fertilized mammalian zygote;
(B) when the level of the transcript of the gene detected in step (A) is higher than the level in a polar body derived from an embryo whose development does not progress to the blastocyst stage, the gene is evaluated as being a normal development embryo marker gene or is likely to be a normal development embryo marker gene;
evaluating, when the level of the transcript of the gene detected in step (A) is lower than the level in a polar body derived from an embryo whose development does not progress to the blastocyst stage, that the gene is an abnormal development embryo marker gene or is likely to be an abnormal development embryo marker gene;
[13] The method according to [12] above, characterized in that the polar body is a second polar body.
[14] The method according to [12] or [13] above, wherein in step (A), the transcription product of the gene is detected by a sequencing method using a next-generation sequencer.

In another embodiment of the present invention,
[15] The above step (a),
a step (b-1-1) of creating or collecting data for evaluating a mammalian embryo, the step comprising: a step of: creating or collecting data for evaluating that, when the transcription product of the gene detected in step (a) is a transcription product of a [group of normal development embryo marker genes], the fertilized egg is an embryo that will develop normally; or an embryo that is likely to develop normally, when the level of the transcription product of the gene detected in step (a) is higher than the level in a polar body derived from an embryo whose development does not progress to the blastocyst stage; and creating or collecting data for evaluating that, when the transcription product of the gene detected in step (a) is a transcription product of a [group of abnormal development embryo marker genes], the fertilized egg is an embryo that will develop normally; or an embryo that is likely to develop normally, when the level of the transcription product of the gene detected in step (a) is lower than the level in a polar body derived from an embryo whose development does not progress to the blastocyst stage.
[16] For use in evaluating and/or selecting mammalian embryos,
Examples of such a gene include a forward primer, a reverse primer, and/or a probe that hybridizes to a transcription product or cDNA of one or more genes selected from the above-mentioned [group of normal development embryo marker genes] and [group of abnormal development embryo marker genes] in a polar body derived from an in vitro fertilized mammalian fertilized egg.

According to the present invention, normally developing mammalian fertilized eggs (embryos) can be evaluated and selected relatively early after fertilization, which is expected to improve the conception rate (pregnancy rate) of mammals. In addition, the transcription products of the normal development embryo marker genes and abnormal development embryo marker genes used as indicators for the evaluation and selection are derived from the polar body, not from the embryo, so damage to the embryo itself can be kept relatively low, and high-quality embryos can be evaluated and selected in a minimally invasive manner.

In the present Example described later, a method for collecting the second polar body released from the embryo using a mouse fertilized egg and a method for culturing the embryo in vitro are specifically shown, but these techniques have been established in mammals such as humans, cows, and pigs, and the normal development embryo marker genes and abnormal development embryo marker genes have orthologs among mammals. Therefore, the present invention is useful not only for mice but also for mammals in general, and therefore contributes to the field of breeding (reproduction) technology, for example, the livestock industry that produces high-quality livestock (e.g., cows with marbled characteristics) or livestock that are difficult to breed, and the field of infertility treatment.

Fig. 1A shows microscopic images of the first polar body (shown in a dotted circle), a fertilized egg (shown in an arrow), and a second polar body (shown in a solid circle) released from the fertilized egg. Fig. 1B shows the results of an analysis of common transcripts between the transcripts of 16397 genes in a fertilized egg sample and the transcripts of 11107 genes in a second polar body sample. This figure shows the results of analyzing the levels of transcripts of six types of normal development embryo marker genes (Trappc12 [Fig. 2A], Kif13a [Fig. 2B], Redrum [Fig. 2C], Enpep [Fig. 2D], Gart [Fig. 2E], and Rogdi [Fig. 2F]) for second polar bodies derived from normal development embryos ("N" in the figure) and second polar bodies derived from abnormal development embryos ("A" in the figure). ● in the graph indicates each sample (same below). FIG. 3 shows the results of analyzing the levels of transcripts of six types of normal development embryo marker genes (Rab13 [FIG. 3A], Prdx3 [FIG. 3B], Spire2 [FIG. 3C], Sipa1 [FIG. 3D], Atxn7l1 [FIG. 3E], and Ube2m [FIG. 3F]) for second polar bodies derived from normal development embryos ("N" in the figure) and second polar bodies derived from abnormal development embryos ("A" in the figure). FIG. 4 shows the results of analyzing the transcript levels of six types of normal development embryo marker genes (Rrp1 [FIG. 4A], Otx1 [FIG. 4B], Phf2 [FIG. 4C], Phrf1 [FIG. 4D], Rnf212 [FIG. 4E], and Ankmy2 [FIG. 4F]) for second polar bodies derived from normal development embryos ("N" in the figure) and second polar bodies derived from abnormal development embryos ("A" in the figure). FIG. 5 shows the results of analyzing the transcript levels of six normal development embryo marker genes (Popdc3 [FIG. 5A], Zscan18 [FIG. 5B], Krt8 [FIG. 5C], Wdr74 [FIG. 5D], Nme1 [FIG. 5E], and Tex15 [FIG. 5F]) for second polar bodies derived from normal development embryos ("N" in the figure) and second polar bodies derived from abnormal development embryos ("A" in the figure). FIG. 6 shows the results of analyzing the transcript levels of six types of normal development embryo marker genes (Mib2 [FIG. 6A], Pbx4 [FIG. 6B], Ddx20 [FIG. 6C], Cdk2ap1 [FIG. 6D], Pcsk5 [FIG. 6E], and Ing5 [FIG. 6F]) for second polar bodies derived from normal development embryos ("N" in the figure) and second polar bodies derived from abnormal development embryos ("A" in the figure). FIG. 7 shows the results of analyzing the transcript levels of six types of abnormal development embryo marker genes (Tyk2 [FIG. 7A], Mettl22 [FIG. 7B], Ulk2 [FIG. 7C], Grin2b [FIG. 7D], Adm [FIG. 7E], and Tbc1d25 [FIG. 7F]) for second polar bodies derived from normally developed embryos ("N" in the figure) and second polar bodies derived from abnormally developed embryos ("A" in the figure). FIG. 8 shows the results of analyzing the transcript levels of six abnormal embryo marker genes (Fech [FIG. 8A], Plcb3 [FIG. 8B], Spsb4 [FIG. 8C], Raver2 [FIG. 8D], Nek11 [FIG. 8E], and Zfp507 [FIG. 8F]) for second polar bodies derived from normally developed embryos ("N" in the figure) and second polar bodies derived from abnormally developed embryos ("A" in the figure). FIG. 9 shows the results of analyzing the transcript levels of six abnormal development embryo marker genes (Lias [FIG. 9A], Tubd1 [FIG. 9B], Zmym6 [FIG. 9C], Mfsd11 [FIG. 9D], Nudt15 [FIG. 9E], and Slc15a2 [FIG. 9F]) for second polar bodies derived from normally developed embryos ("N" in the figure) and second polar bodies derived from abnormally developed embryos ("A" in the figure). FIG. 10 shows the results of analyzing the transcript levels of five abnormal development embryo marker genes (Parp4 [FIG. 10A], Fam20b [FIG. 10B], Mau2 [FIG. 10C], Kdm3b [FIG. 10D], and Dmd [FIG. 10E]) for second polar bodies derived from normally developed embryos ("N" in the figure) and second polar bodies derived from abnormally developed embryos ("A" in the figure). This figure shows the results (mean ± standard error) of an analysis of the proportion of second polar bodies in which the transcript of the abnormal embryo marker gene (Dmd) was detected for each of the second polar bodies derived from normally developed embryos ("N" in the figure) and the second polar bodies derived from abnormally developed embryos ("A" in the figure). "*" in the figure indicates that there is a statistically significant difference (p = 0.027). This figure shows the results (mean ± standard error) of analysis of the level of the transcript of a normal development embryo marker gene (Cdk2ap1 gene) for the second polar body derived from a normal development embryo ("N" in the figure) and the second polar body derived from an abnormal development embryo ("A" in the figure). "**" in the figure indicates that there is a statistically significant difference (p = 0.002).

The method for evaluating a mammalian embryo of the present invention includes the steps of:
A step (a) of detecting and, if necessary, quantifying the transcription products of one or more genes (hereinafter, sometimes referred to as "the embryo marker gene group") selected from the following [normal development embryo marker gene group] and [abnormal development embryo marker gene group] in a polar body (hereinafter, sometimes referred to as "detection target polar body") derived from a mammalian fertilized egg fertilized in vitro (for example, in vitro fertilization by insemination, intracytoplasmic sperm injection; the same applies below);
When the level of the transcript of the [group of normal development embryo marker genes] in the polar body to be detected is higher than the level in a polar body derived from an embryo whose development does not progress to the blastocyst stage (hereinafter sometimes referred to as a "control polar body"), the fertilized egg is evaluated as an embryo that develops normally; or an embryo that is likely to develop normally;
There are no particular limitations on the method (hereinafter sometimes referred to as "the present evaluation method") as long as it includes a step (b-1) of evaluating, when the level of the transcription product of the [abnormal development embryo marker gene group] in the polar body to be detected is lower than the corresponding level in the control polar body, that the fertilized egg is an embryo that will develop normally; or an embryo that is likely to develop normally; and the present evaluation method may further include a step (b-3) of selecting a fertilized egg that has been evaluated in the above step (b-1) as an embryo that will develop normally; or an embryo that is likely to develop normally.
[Normal embryo development marker genes]
Cdk2ap1, Trappc12, Kif13a, Redrum, Enpep, Gart, Rogdi, 2310003L06Rik, Rabl3, F730016J06Rik, Prdx3, Xpnpep1, Abcc5, 4933431E20Rik, Spire2, Mgme1, Ak9, Sipa1, Narf, Tmem147, Rimbp2, Dlgap1, Cd209f, Itpr3, Atxn7l1, T hap3, Dr1, Casq2, Dnah11, Nsun5, Tek, Sec1, Zc3h7a, Vgll1, Nfkbid, Rin2, Ube2m, Zfp954, Fkbp9, Rrp1, Otx1, Ap2a2, Dpep2, Ces1d, Upp1, Zg16, Adam12, Phf2, Phrf1, Slc43a2, Gm13977, Mfsd8, Cotl1, Efcab3, Commd4, Kantr, Rnf2 12, Ankmy2, Popdc3, Arpc1a, Npdc1, Sema6a, Etfb, D030040B21Rik, Ttc21b, Plekhg5, Nat8f1, Zscan18, 5330439K02Rik, Rbfa, Zfp408, Aadacl3, Oit1, Tmem120a, Traf5, Krt8, Cd200, Ostm1, Zc3h12d, Ammecr1, AI413582, Commd5, Prkdc, Tmprss2, Prkcsh, Wdr74, Nme1, Tex15, Zfp563, Gpn3, F5, Mib2, Gm4926, Sbspon, Nup155, Pbx4, Defb48, Ddx20, Itga4, Pcsk5, Dtna, Epcam, Gm28940, Gm44196, Eif3g, Polg2, Gm364, Mtch2, Htt, Ing5, Gm42945, Aknad1, Ehbp1
[Embryonic abnormality marker genes]
Dmd, Tyk2, Nyap2, Mettl22, Manea, Nudt18, Lair1, Slamf1, March3, Ulk2, AI429214, Zfp938, Wdr59, Spryd7, Grin2b, Fsd2, Ciao3, Zhx1, Grm1, Adm, Ugt2b38, T bc1d25, Lrrc61, Fam132b, Exoc6b, Reps2, Cdh24, Fech, Agpat1, Plcb3, A230056P14Rik, Spsb4, Dmrtc1b, Raver2, 4921531C22Rik, Tbc1d31, Nutm1, Nek11, Morc 2a, Trhr2, Aacs, Atp5o, Sirt5, Id3, Zfp507, AW551984, Lias, Usp20, Wfdc1, Tubd1, Zmym6, Klf10, Mfsd11, Nudt15, Nisch, Hipk3, Tatdn3, Tyw3, Ubl4a, Atp6ap1 l, Snx8, Gm12364, Ndufa7, Zfp341, Col4a1, Zfp595, G2e3, Slc15a2, Sgsm1, Parp4, Ciao1, Fam20b, Acvr2b, Ttc25, Mau2, Sybu, Kdm3b, AY512915, Gm13136, Trmt6

Furthermore, the method for selecting a mammalian embryo of the present invention is not particularly limited as long as it comprises the steps of: (a) detecting, in the polar body to be detected, the transcription products of one or more genes selected from the above-mentioned [group of normal development embryo marker genes] and [group of abnormal development embryo marker genes], and quantifying them as necessary; and (b-2) selecting a fertilized egg derived from the polar body to be detected when the level of the transcription products of the [group of normal development embryo marker genes] in the polar body to be detected is higher than the level in the control polar body, and selecting a fertilized egg derived from the polar body to be detected when the level of the transcription products of the [group of abnormal development embryo marker genes] in the polar body to be detected is lower than the level in the control polar body (hereinafter, may be referred to as "the present selection method").

The present evaluation method and each step in the present evaluation method are all performed in vitro and do not include in vivo steps (such as a step of collecting eggs from a woman's ovaries, or a step of implanting a fertilized egg or an embryo after its development into a woman's uterus (embryo transfer), which are so-called medical procedures performed by a doctor).

As used herein, "selecting fertilized eggs" means separating or isolating the fertilized eggs to be selected from a group of fertilized eggs in order to distinguish between the fertilized eggs to be selected and the fertilized eggs that are not to be selected, or removing the fertilized eggs that are not to be selected. The fertilized eggs to be selected may be separated or isolated one by one, or may be separated or isolated as a group of fertilized eggs to be selected.

The kit for evaluation and/or selection of mammalian embryos of the present invention is not particularly limited as long as it is a kit for evaluation and/or selection of mammalian embryos (hereinafter, sometimes referred to as the "evaluation/selection kit") that includes a reverse primer (hereinafter, sometimes referred to as the "reverse primer for RNA detection") and/or a probe (hereinafter, sometimes referred to as the "RNA detection probe") that hybridizes to the transcription product of the embryo marker gene group in the polar body to be detected, and/or a primer set consisting of a forward primer and a reverse primer (hereinafter, sometimes referred to as the "cDNA detection primer set") that hybridizes to the cDNA of the embryo marker gene group in the polar body to be detected, and/or a probe (hereinafter, sometimes referred to as the "cDNA detection probe") for use in the evaluation and/or selection of mammalian embryos. The evaluation/selection kit is an invention of use related to a kit for evaluation and/or selection of mammalian embryos, and these kits usually include components generally used in this type of detection kit, such as a cell lysis buffer, a carrier, a pH buffer, and a stabilizer, as well as an instruction manual, instructions for evaluation and/or selection of mammalian embryos, and other attached documents.

The biomarker for evaluation and/or selection of mammalian embryos of the present invention is not particularly limited as long as it is a biomarker for use in evaluation and/or selection of mammalian embryos, containing one or more genes selected from the above-mentioned [normal development embryo marker gene group] and [abnormal development embryo marker gene group], in which the level of the transcript of the [normal development embryo marker gene group] in the polar body to be detected is higher than that in the control polar body, and the level of the transcript of the [abnormal development embryo marker gene group] in the polar body to be detected is lower than that in the control polar body. Using the level of the transcript of the present embryo marker gene group in the polar body derived from an in vitro fertilized mammalian fertilized egg as an indicator, it is possible to accurately evaluate whether the mammalian fertilized egg is a mammalian embryo that develops normally, and to select a mammalian embryo that develops normally, so that the present embryo marker gene group can be a biomarker for use in evaluation and/or selection of mammalian embryos.

In addition, the method for identifying genes for evaluation and/or selection of mammalian embryos of the present invention includes the following steps:
A step (A) of detecting a transcription product of a gene in a polar body to be detected;
There are no particular limitations on the method (hereinafter, sometimes referred to as the present identification method) as long as it includes a step (B) of evaluating, when the level of the gene transcript in the polar body to be detected is higher than that in the control polar body, that the gene is a normal development embryo marker gene or is highly likely to be a normal development embryo marker gene, and, when the level of the gene transcript in the polar body to be detected is lower than that in the control polar body, that the gene is an abnormal development embryo marker gene or is highly likely to be an abnormal development embryo marker gene. The number of polar bodies used in the present identification method may be at least one (preferably 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) that is sufficient for evaluation, and is, for example, 200 or less (preferably 160, 130, 100, 90, 80, 70, 60, 50, or 40 or less) in consideration of cost-effectiveness.

As used herein, when "the level of the gene transcript is lower than the level in a polar body derived from an embryo that does not progress to the blastocyst stage (control polar body)," this also includes when the level of the gene transcript is not detectable (below the detection sensitivity).

Specific examples of the polar body include the second polar body (specifically, a polar body released from a mammalian egg [fertilized egg] after the second meiotic division is resumed when fertilization is initiated by the entry of a mammalian sperm into a mammalian egg [i.e., secondary oocyte] whose cell cycle has stopped in the middle of the second meiotic division [around metaphase]); and/or the first polar body (specifically, a polar body generated after the first meiotic division of a mammalian primary oocyte together with a mammalian secondary oocyte). Among these, the second polar body is a suitable example because its effect has been demonstrated in the present embodiment described below. When the second polar body is used as the polar body, the present evaluation method, the present selection method, and the present identification method (collectively referred to as "the present method") may further include a step of detecting a transcription product of the present embryo marker gene group in the first polar body and a step of detecting a mutation of the present embryo marker gene group in the genomic DNA in the second polar body, but the method does not include these steps because the purpose of the present method can be achieved without carrying out these steps.

In this specification, a "second polar body derived from an in vitro fertilized mammalian fertilized egg" can be said in other words as a "second polar body released from an in vitro fertilized mammalian fertilized egg", and a "second polar body derived from an embryo that does not progress to the blastocyst stage" can be said in other words as a "second polar body released from a mammalian embryo that does not progress to the blastocyst stage when the fertilized egg is a fertilized egg". Usually, one second polar body is released from one mammalian egg.

In this specification, the term "first polar body derived from an in vitro fertilized mammalian fertilized egg" can be rephrased as "a first polar body generated together with a mammalian secondary oocyte (the pre-differentiation state of a mammalian egg used for in vitro fertilization of a mammalian fertilized egg) by the first meiotic division of a mammalian primary oocyte," and the term "first polar body derived from an embryo that does not progress to the blastocyst stage" can be rephrased as "a first polar body generated together with a mammalian secondary oocyte (the pre-differentiation state of a mammalian egg used for in vitro fertilization of a fertilized egg, which is the pre-development state of a mammalian embryo that does not progress to the blastocyst stage) by the first meiotic division of a mammalian primary oocyte." Usually, one first polar body is generated from one mammalian primary oocyte.

In this specification, "embryo that develops normally" means, more specifically, "embryo that develops normally at least to the blastocyst stage". In addition, in this specification, "normal development embryo marker gene" means a gene whose transcript level is higher in the polar body derived from an embryo that develops normally than in the polar body derived from an embryo that does not develop to the blastocyst stage. In addition, in this specification, "abnormal development embryo marker gene" means a gene whose transcript level is higher in the polar body derived from an embryo that does not develop to the blastocyst stage than in the polar body derived from an embryo that develops normally. More specifically, these marker genes can be used to accurately evaluate whether a mammalian fertilized egg is a mammalian embryo that develops normally, and to select a mammalian embryo that develops normally, using the transcript level of the gene in the polar body derived from an in vitro fertilized mammalian fertilized egg as an indicator.

In this specification, "transcription product of a gene" means RNA synthesized (transcribed) by DNA-dependent RNA polymerase (also simply referred to as "RNA polymerase") using the antisense strand of double-stranded DNA encoding a gene present in genomic DNA as a template, and is usually a single-stranded polynucleotide. The RNA in question includes not only mRNA that is translated into protein, but also RNA that is not translated into protein (non-coding RNA [ncRNA]). The RNA also includes initial transcription products (immature mRNA or ncRNA), mature transcription products (mature mRNA or ncRNA) generated by post-transcriptional processing (splicing), and splicing variants thereof.

In this specification, "cDNA of a gene" means cDNA (complementary DNA) synthesized from the RNA of a gene by PCR using reverse transcriptase, and is usually a double-stranded polynucleotide.

Examples of mammals of the present invention include rodents such as mice, rats, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, horses, and sheep; carnivores such as dogs and cats; and primates such as humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees. Of these, preferred examples include humans, cows, and pigs.

The in vitro fertilized mammalian fertilized egg can be prepared, for example, by in vitro fertilization (so-called in vitro fertilization by insemination) in which at least one collected mammalian sperm and at least one collected mammalian egg (i.e., secondary oocyte) are naturally fertilized in a culture medium, or by a method in which mammalian sperm are injected into at least one collected mammalian egg by micromanipulation (i.e., intracytoplasmic sperm injection). The first polar body attached to the mammalian egg (i.e., secondary oocyte) before in vitro fertilization and the second polar body released from the mammalian fertilized egg after in vitro fertilization can each be recovered and isolated by micromanipulation.

In the above steps (a) and (A), the method of detecting the transcription product (i.e., RNA) of the gene in the polar body to be detected can be a method of directly detecting the RNA of the gene, or a method of (indirectly) detecting cDNA synthesized using the RNA of the gene as a template. Examples of the method of detecting the RNA of such a gene include the RT (Reverse Transcription)-PCR method using the present RNA detection reverse primer, etc.; the Northern blotting method, the microarray method, the ISH method, etc. using the present RNA detection probe, etc. In addition, examples of methods for detecting the cDNA of the above genes include the LAMP method using the present cDNA detection primer set, the PCR method (e.g., real-time PCR method [intercalator method, 5'-nuclease method, cycling probe method, etc.], ddPCR method); the LCR method; sequencing method using a next-generation sequencer; Southern hybridization method using the present cDNA detection probe, the microarray method, and the ISH method; among these, the sequencing method using a next-generation sequencer is a suitable example.

In this specification, the term "next-generation sequencer" is also referred to as a second-generation sequencer, and refers to a nucleotide sequence determination device that can simultaneously determine the nucleotide sequences of tens of millions of DNA fragments. Examples of the sequencing principle in a next-generation sequencer include a principle in which the DNA to be detected is amplified on a flow cell using a bridge PCR method and a sequencing-by-synthesis method, and a principle in which sequencing is performed using an emulsion PCR method and a pyrosequencing method that determines the sequence by measuring the amount of pyrophosphate released during DNA synthesis. More specifically, examples of next-generation sequencers include the MiniSeq, MiSeq, NextSeq, HiSeq, and HiSeq X series manufactured by Illumina, and the Roche 454 GS FLX sequencer manufactured by Roche.

In the present invention, the level (amount) of the transcription product of a gene in a polar body may be an absolute value (concentration [copy number]) or a relative value. When it is a relative value, for example, the transcription level of a housekeeping gene such as Gapdh or β-actin can be used as an internal standard.

In the present invention, the level of the gene transcription product in the polar body to be detected and the level of the gene transcription product in the control polar body correspond to each other. Therefore, when one is an absolute value or a relative value, the other is also an absolute value or a relative value, respectively. The level value of the gene transcription product in the control polar body may be measured each time the method of the present invention is carried out, but a value measured in advance may also be used. In addition, it is preferable that the polar bodies to be compared are prepared by substantially the same method. In addition, it is preferable that the method for detecting the gene transcription product is substantially the same for both polar bodies to be compared.

In step (b-1) of the present evaluation method, if the level of the transcripts of the [group of marker genes for normal development embryos] in the polar body to be detected is not higher than the corresponding level in the control polar body, the fertilized egg derived from the polar body to be detected is evaluated as an embryo that does not develop normally; or an embryo that is unlikely to develop normally; and if the level of the transcripts of the [group of marker genes for abnormal development embryos] in the polar body to be detected is not lower than the corresponding level in the control polar body, the fertilized egg derived from the polar body to be detected is evaluated as an embryo that does not develop normally; or an embryo that is unlikely to develop normally.

In addition, in step (b-2) of the present selection method, if the level of the transcription products of the [group of marker genes for normal development embryos] in the polar body to be detected is not higher than the corresponding level in the control polar body, the fertilized egg derived from the polar body to be detected is not selected, and if the level of the transcription products of the [group of marker genes for abnormal development embryos] in the polar body to be detected is not lower than the corresponding level in the control polar body, the fertilized egg derived from the polar body to be detected is not selected.

In addition, in step (B) of the present identification method, when the level of the gene transcript in the polar body to be detected is not higher than the level in the control polar body, the gene is evaluated as not being a normal development embryo marker gene or is unlikely to be a normal development embryo marker gene; and when the level of the gene transcript in the polar body to be detected is not lower than the level in the control polar body, the gene is evaluated as not being an abnormal development embryo marker gene or is unlikely to be an abnormal development embryo marker gene. In step (B) of the present identification method, when evaluating whether the level of the gene transcript in the polar body to be detected is higher or lower than the level in the control polar body, the level of the transcript of the present embryo marker gene group in a polar body derived from a normally developing embryo can be used as a positive control.

In order to evaluate (determine) whether the level of the gene transcript in the target polar body is higher or lower than the level of the gene transcript in the control polar body, any threshold (cutoff value) can be set, and examples of such thresholds include the average value of the gene transcript levels in the control polar body; the average value + standard deviation (SD); the average value + 2 SD; the average value + 3 SD; the median; the interquartile range, etc. In addition, the threshold can also be calculated using a ROC (Receiver Operating Characteristic) curve using statistical analysis software based on data on the gene transcript levels in the target polar body and data on the gene transcript levels in the control polar body, so as to increase sensitivity (the proportion of embryos that develop normally that can be correctly determined as positive) and specificity (the proportion of embryos that do not develop to the blastocyst stage that can be correctly determined as negative).

In the above [Group of marker genes for normal development embryos] and [Group of marker genes for abnormal development embryos], the gene names are written in accordance with the notation method for mouse genes for convenience, but in mammals other than mice, they correspond to their orthologues. For example, the above Cdk2ap1 corresponds to CDK2AP1 in humans, cows, and pigs, the above Dmd corresponds to DMD in humans, cows, and pigs, and the above AW551984 corresponds to VWA5A in humans and pigs.

Among the above-mentioned [group of normal development embryo marker genes], examples of normal development embryo marker genes derived from humans, cows, and pigs include RNAs containing a nucleotide sequence that has 80% or more sequence identity to the transcription products (RNAs) of the genes shown in Table 1 below. The nucleotide sequences of the RNAs of the genes in Table 1 can be understood from the contents described in the "NCBI Reference Sequence" section of Table 1. For example, in the case of Cdk2ap1, human (NM_001270433) is the mRNA of human-derived CDK2AP1 registered with the NCBI (National Center for Biotechnology Information) under the number NM_001270433 (specifically, the mRNA consisting of the nucleotide sequence of SEQ ID NO: 1), bovine (NM_001076369) is the mRNA of bovine-derived CDK2AP1 registered with the NCBI under the number NM_001076369 (specifically, the mRNA consisting of the nucleotide sequence of SEQ ID NO: 2), and porcine (NM_001190166) is the mRNA of porcine-derived CDK2AP1 registered with the NCBI under the number NM_001190166 (specifically, the mRNA consisting of the nucleotide sequence of SEQ ID NO: 3).

Among the above-mentioned [group of abnormal development embryo marker genes], examples of abnormal development embryo marker genes derived from humans, cows, and pigs include RNAs containing a nucleotide sequence that has 80% or more sequence identity to the transcription products (RNAs) of the genes shown in Table 2 below. The nucleotide sequences of the RNAs of the genes in Table 2 can be understood from the contents described in the "NCBI Reference Sequence" section of Table 2. For example, in the case of Dmd, human (NM_000109) is the mRNA of human-derived DMD registered in NCBI under the number NM_000109 (specifically, the mRNA consisting of the nucleotide sequence of SEQ ID NO: 4), bovine (XM_024988359) is the mRNA of bovine-derived DMD registered in NCBI under the number XM_024988359 (specifically, the mRNA consisting of the nucleotide sequence of SEQ ID NO: 5), and porcine (NM_001012408) is the mRNA of porcine-derived DMD registered in NCBI under the number NM_001012408 (specifically, the mRNA consisting of the nucleotide sequence of SEQ ID NO: 6). In humans, cattle, and pigs, there are many single nucleotide polymorphisms (SNPs) in genes, so there may be RNA that contains a nucleotide sequence that has 80% or more sequence identity with the specific nucleotide sequence. The nucleotide sequence registered with NCBI may be the latest sequence as of the filing date or priority date of this application, or may be a sequence updated after the filing date or priority date of this application. In this specification, the nucleotide sequence of mRNA is expressed as "u (uracil residue)" instead of "t (thymine residue)" in the nucleotide sequences of SEQ ID NOs: 1 to 6 in the sequence listing.

As used herein, "sequence identity of 80% or more" means sequence identity of at least 80%, preferably 85% or more, more preferably 88% or more, even more preferably 90% or more, even more preferably 92% or more, particularly preferably 94% or more, especially more preferably 96% or more, especially even more preferably 98% or more, even more especially preferably 99% or more, and most preferably 100% sequence identity. The identity of nucleotide sequences can be determined using a program called BLASTX based on the algorithm BLAST by Carlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, Proc Natl Acad Sci USA 90: 5873, 1993) or a program called BLASTN based on a program called BLASTP (Altschul SF, et al: J Mol Biol 215: 403, 1990). When analyzing nucleotide sequences using BLASTN, the parameters are, for example, score = 100 and wordlength = 12.

Cdk2ap1 and/or Dmd are suitable examples of the embryo marker gene group of the present invention, the effects of which have been demonstrated in more detail in the present Example described below.

The present reverse primer for detecting RNA specifically hybridizes to the transcription product (RNA) of the present embryo marker gene group, and is preferably a primer capable of generating an amplified product by RT-PCR, and corresponds to a portion of the antisense strand that codes for the present embryo marker gene group present in genomic DNA.

The RNA detection probe in question specifically hybridizes to the transcription product (RNA) of the embryo marker gene group in question, and is preferably a probe that can be detected by a method such as Northern blotting, microarray, or ISH, and corresponds to a portion of the antisense strand that codes for the embryo marker gene group in question present in genomic DNA.

The forward primer and reverse primer in the present cDNA detection primer set hybridize to the antisense strand and sense strand, respectively, of the cDNA of the present embryo marker gene group, and are preferably primers that can be detected by methods such as the LAMP method and PCR method, and correspond to a part of the sense strand and a part of the antisense strand, respectively, that code for the present embryo marker gene group present in the genomic DNA.

The length of the primer of the present invention is, for example, 10 nucleotides or more (preferably 13, 16, 20, 23, 26, or 30 nucleotides or more) and 100 nucleotides or less (preferably 90, 80, 70, 60, 50, or 40 nucleotides or less). The length of the probe of the present invention is, for example, 10 nucleotides or more (preferably 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides or more) and 1000 nucleotides or less (preferably 900, 800, 700, 600, 500, 400, 300, or 200 nucleotides or less).

In the present invention, the primers and probes may be DNA, RNA, or DNA/RNA chimeras, but are preferably DNA. In addition, in part or in whole, the nucleotides may be replaced by artificial nucleic acids such as PNA (polyamide nucleic acid, peptide nucleic acid), LNA (registered trademark, locked nucleic acid, bridged nucleic acid), ENA (registered trademark, 2'-O,4'-C-Ethylene-bridged nucleic acids), GNA (Glycerol nucleic acid), and TNA (Threonine nucleic acid).

In the present invention, the primers and probes can be labeled in order to visualize and/or quantify the transcription product or cDNA of the gene to be detected. Examples of the labeling substance in such a label include enzymes such as peroxidase (e.g., horseradish peroxidase), alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apo-glucose oxidase, urease, luciferase, and acetylcholinesterase; fluorescent substances (e.g., allophycocyanin [APC], phycoerythrin [PE], FITC [fluorescein isothiocyanate], Alexa Fluor 488, Alexa Fluor 647, AlexaFluor 700, PE-TexasRed, PE-Cy5, and PE-Cy7); green fluorescent protein (GFP), cyan fluorescent protein (CFP), blue fluorescent protein (Blue Fluorescence Protein), and the like. Examples of the fluorescent reporter include fluorescent proteins such as yellow fluorescent protein (BFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), and luciferase; radioisotopes such as ^3H , ^14C , ^125I , and ^131I ; biotin, avidin, or a chemiluminescent substance; and combinations of a reporter fluorescent substance and a quencher fluorescent substance or structure (for example, a combination of 5-FAM [5-Carboxyfluorescein] and 5-TAMRA [5-Carboxytetramethylrhodamine], or a combination of VIC and MGB [Minor Groove Binder]).

The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. In the following examples, mice were reared under light control (light period: 6:30 to 20:30, dark period: 20:30 to 6:30) and in an environment maintained at a room temperature of 26°C, and used in the experiments. The rearing of mice and their use in the experiments were performed in accordance with the guidelines for animal experiments established by the Kinki University Faculty of Biomedical Engineering Experimental Animal Subcommittee. In addition, the operation of transferring eggs, inseminated eggs (fertilized eggs), and second polar bodies was performed using a hematocrit capillary plain (Hirschmann) slightly thicker than the diameter of the eggs. The isolation of the second polar body was performed under a microscope (IX73, Olympus) using a micromanipulator (Narishige). Sperm were inseminated with a microchip using a pipette. Furthermore, the sperm pre-culture and insemination were carried out under conditions of 5% CO ₂ /20% O ₂ and 37°C.

Example 1: Analysis of gene transcription products in fertilized eggs and gene transcription products in the second polar body released from the fertilized eggs 1. Materials and methods 1-1 Culture medium Sperm pre-insemination culture (pre-culture) and insemination were performed in 200 μL drops of mHTF culture medium placed on a culture dish (manufactured by BD Falcon). In addition, embryo development of the fertilized eggs was performed in 10 μL drops of mKSOM culture medium placed on the culture dish. In addition, in order to prevent evaporation of the culture medium and sudden changes in temperature, the drops of culture medium were covered with mineral oil (manufactured by SIGMA ALDRICH).

1-2 In Vitro Fertilization (So-called In Vitro Fertilization by Insemination) and Embryonic Development In vitro fertilization and embryonic development were carried out according to the following procedures [1] to [6].
[1] Male DBA/2 mice (manufactured by Japan CLEA) were sacrificed by cervical dislocation, and the tail of the epididymis was collected. The duct of the tail of the epididymis was incised with small straight scissors, and the sperm mass was collected with a dissection needle. The collected sperm mass was transferred to a drop-shaped mHTF culture medium and pre-cultured for 1 hour or more.
[2] DBA/2 strain female mice (manufactured by Japan CLEA) were intraperitoneally administered 7.5 (IU) of pregnant mare serum gonadotropin (PMSG) (manufactured by Aska Pharmaceutical Co., Ltd.). After 48 hours, 7.5 (IU) of human chorionic gonadotropin (hCG) (manufactured by Aska Pharmaceutical Co., Ltd.) was intraperitoneally administered to induce superovulation.
[3] 15 hours after hCG administration, female mice were sacrificed by cervical dislocation, and the ampulla of the oviduct was collected. The collected ampulla of the oviduct was immersed in mineral oil in an insemination dish, and cumulus cell-oocyte complexes (COCs) were collected from the ampulla of the oviduct. The collected COCs were placed in a drop-shaped mHTF culture medium.
[4] The sperm mass pre-cultured in [1] above was transferred to the drop-shaped mHTF culture medium containing COC prepared in [3] above at a concentration of 200 sperm/μL, and insemination was performed.
[5] Two hours after insemination, 12 μL of 10 mg/mL hyaluronidase (Sigma Aldrich) was added to the drop-shaped mHTF culture medium to detach the cumulus cells. The oocytes were transferred to a drop-shaped 35 μL of mKSOM culture medium. This procedure was repeated until the cumulus cells and floating sperm were removed.
[6] Six hours after insemination, eggs in which both male and female pronuclei were clearly visible were considered fertilized eggs. Eggs in which no pronuclei were visible were considered unfertilized eggs, fertilized eggs in which three or more pronuclei were visible were considered polyspermic eggs, and eggs in which only one pronucleus was visible were considered parthenogenetic eggs.

1-3 Preparation and storage of second polar body samples and fertilized egg samples Second polar body samples and fertilized egg samples were prepared and stored according to the following procedures [1] to [3].
[1] The fertilized eggs obtained by the procedure [6] in "1-2" above were cultured for an additional hour. After confirming that the second polar body (see FIG. 1A) had been released from the fertilized eggs, the fertilized eggs and the second polar bodies were each isolated and recovered one by one using micromanipulation.
[2] Each recovered second polar body was washed with 1x PBS(-) containing 0.1% BSA (manufactured by SIGMA ALDRICH) (hereinafter referred to as "0.1% BSA-PBS"), and then a mixture of 1 μL of 10x Reaction Buffer (10x Lysis Buffer containing RNase inhibitor) from the SMART-seq v4 Ultra Low Input RNA Kit (manufactured by Takara) and 8.5 μL of UltraPure DNase/RNase-Free Distilled Water (manufactured by Invitrogen) (hereinafter referred to as "RNase inhibitor-containing lysis solution") was added to a PCR tube in advance, and 1 μL of 0.1% BSA-PBS containing each second polar body was added thereto to prepare a second polar body sample. In addition, each of the collected fertilized eggs was treated with acidic Tyrode's solution to dissolve the zona pellucida, and then an RNase inhibitor-containing dissolution solution was added to a PCR tube in advance, to which 1 μL of 0.1% BSA-PBS containing each fertilized egg was added to prepare a fertilized egg sample.
[3] The second polar body sample and the fertilized egg sample were flash frozen in liquid nitrogen and then stored in a freezer at -80°C.

1-4 Preparation of cDNA library and RNA sequence (RNA-seq) analysis 10.5 μL of the second polar body sample and fertilized egg sample prepared in "1-3" above were used to synthesize and amplify cDNA using SMART-seq v4 Ultra Low Input RNA Kit (manufactured by Takara). Considering that there is a large difference between the amount of maternal transcripts in the fertilized egg and the amount of maternal transcripts in the second polar body, 1 μL of 12 μM 3' SMART-Seq CDS Primer II A (manufactured by Takara) as a reverse transcription primer and 1 μL of UltraPure DNase/RNase-Free Distilled Water (manufactured by Invitrogen) were added to the second polar body sample, and 2 μL of the above 3' SMART-Seq CDS Primer II A was added to the fertilized egg sample, which was then treated at 72°C for 3 minutes and then left on ice for 2 minutes to perform denaturation of higher-order structures and primer annealing. In addition, to prepare a fertilized egg sample containing ERCC Spike-In RNA (manufactured by Thermo Fisher Scientific), 1 μL was removed from the fertilized egg sample prepared in "1-3" above, and 1 μL of diluted ERCC Spike-In RNA (calculated assuming that the total RNA amount of the fertilized egg sample is 350 pg. This was prepared by diluting the original ERCC Spike-In RNA 1/100,000-fold, and then mixing 7 μL of the diluted ERCC Spike-In RNA-containing solution with 3 μL of UltraPure DNase/RNase-Free Distilled Water) was added, and then denaturation of the higher-order structure and primer annealing were performed by the above-mentioned treatment. In addition, in order to prepare a second polar body sample containing ERCC Spike-In RNA, 1 μL of diluted ERCC Spike-In RNA (calculated assuming that the total RNA amount of the second polar body sample is 20 pg/μL. The stock solution of ERCC Spike-In RNA was diluted 1/1,000,000 times, and then 2 μL of the diluted ERCC Spike-In RNA-containing solution was mixed with 3 μL of UltraPureDNase/RNase-Free Distilled Water) and 1 μL of 12 μM 3' SMART-Seq CDS Primer II A were added to the second polar body sample prepared in the above "1-3", and denaturation of the higher-order structure and primer annealing were performed by the above treatment. Then, 7.5 μL of First Strand Mix was added to each sample, and the mixture was reacted at 42 ° C for 90 minutes and at 70 ° C for 10 minutes to synthesize single-stranded cDNA. Next, 30 μl of Second Strand Mix was added to each sample, and double-stranded cDNA was synthesized and amplified. The number of PCR cycles was 14-15 for the fertilized egg sample and 19-20 for the second polar body sample. After the amplification was completed, the cDNA was purified using AMPure XP beads (BECKMAN COULTER) and 80% ethanol. The cDNA sample was diluted to a concentration of 200 pg/μl and fragmented and tagged using Nextera DNA Sample Preparation Kit (Illumina). The amplification and fragmentation of the cDNA were confirmed using Bioanalyzer and High Sensitivity DNA kit (both manufactured by Agilent). Then, RNA-seq analysis was performed using NextSeq (Illmina).

2. Results Transcripts of 16,397 and 11,107 genes were identified from the fertilized egg and second polar body samples, respectively (see FIG. 1B). Furthermore, of the 11,107 gene transcripts from the second polar body samples, 10,690 gene transcripts (96.2%) were common to the transcripts of genes from the fertilized egg samples (i.e., maternal transcripts) (see FIG. 1B).
These results indicate that analysis of the transcripts of genes in the second polar body enables minimally invasive analysis of maternal transcripts in fertilized eggs.

Example 2: Identification of a group of marker genes (the present embryo marker genes) that can evaluate normally developed embryos Since it was found that the majority of the transcripts of genes in the second polar body are maternal transcripts of fertilized eggs, we attempted to identify maternal transcripts that can evaluate the quality of an embryo from the transcripts of genes in the second polar body. Specifically, the second polar body was collected from the fertilized egg obtained by the procedure [6] of "1-2" above by the procedure [1] of "1-3" above, and the fertilized egg after the second polar body collection was cultured for 4 to 5 days. The embryos that developed to the blastocyst stage were evaluated as normally developed embryos, and the embryos that did not develop to the blastocyst stage (for example, embryos in which the development was stopped before the blastocyst stage or embryos in which the development was delayed) were evaluated as abnormally developed embryos. Using the second polar bodies of each of the normally developed embryos (n = 5) and the abnormally developed embryos (n = 5), RNA-seq analysis was performed according to the method described in the above item "1-4", and then the transcripts (maternal transcripts) of genes that fluctuate between the second polar bodies derived from both embryos were identified by bioinformatics analysis. The bioinformatics analysis was performed in the following manner. First, excess sequences not used in the analysis, such as adapter sequences, were removed (trimmed) from the sequence reads obtained by NextSeq using software such as trim_galore or cutadapt. The trimmed sequence reads were then mapped to the mouse mm10 genome using STAR software. The expression levels of each gene were then counted using featureCounts, and the number of transcripts expressed in the fertilized eggs and second polar bodies was calculated. To identify genes that showed differential expression between normal and abnormally developed embryos, DESeq2 was used to calculate differentially expressed genes (FDR: False Discovery Rate ≦ 0.05).

In the second polar bodies derived from normally developed embryos, the transcripts of 9,705 types of genes were identified, and of these, the transcripts of 113 types of genes (i.e., normal development embryo marker genes) shown in Table 3 were identified as being significantly increased compared to the second polar bodies derived from abnormally developed embryos. Figures 2 to 6 show the transcripts of 30 types of normal development embryo marker genes shown in Table 3 (Trappc12 [number 1], Kif13A [number 2], Redrum [number 3], Enpep [number 4], Gart [number 5], Rogdi [number 6], Rabl3 [number 8], Prdx3 [number 10], Spire2 [number 14], Sipa1 [number 17], Atxn7l1 [number 24], Ube2m [number 36], Rrp1 [number 39], Otx1 [number 40], Phf2 [number 47], P HRFL [number 48], Rnf212 [number 56], Ankmy2 [number 57], Popdc3 [number 58], Zscan18 [number 67], Krt8 [number 75], Wdr74 [number 85], Nme1 [number 86], Tex15 [number 87], Mib2 [number 91], Pbx4 [number 95], Ddx20 [number 97], Cdk2ap1 [number 99], Pcsk5 [number 100], and Ing5 [number 110]) are shown in the graph plotting the levels of the transcripts. In addition, in the second polar body derived from abnormally developed embryos, the transcripts of 9411 kinds of genes were identified, and among them, the transcripts of 80 kinds of genes (i.e., abnormally developed embryo marker genes) shown in Table 4 were identified as being significantly increased compared to the second polar body derived from normally developed embryos. 7 to 10 show 23 types of abnormal embryo marker genes shown in Table 4 (Tyk2 [number 1], Mettl22 [number 3], Ulk2 [number 9], Grin2b [number 14], Adm [number 19], Tbc1d25 [number 21], Fech [number 27], Plcb3 [number 29], Spsb4 [number 31], Raver2 [number 33], Nek11 [number 37], Zfp50 7 [number 44], Lias [number 46], Tubd1 [number 49], Zmym6 [number 50], Mfsd11 [number 52], Nudt15 [number 53], Slc15a2 [number 67], Parp4 [number 69], Fam20b [number 71], Mau2 [number 74], Kdm3b [number 76], and Dmd [number 79]) are shown.

These results indicate that it is possible to evaluate whether a fertilized egg is an embryo that will develop normally by using as an indicator whether the level of the transcript of a normal development embryo marker gene in a polar body derived from a fertilized egg, such as the second polar body released from the fertilized egg, is higher than the corresponding level in a second polar body derived from an abnormal development embryo, or whether the level of the transcript of a normal development embryo marker gene is lower than the corresponding level in a second polar body derived from an abnormal development embryo.

表３中の「log2倍率」は、異常発生胚由来の第二極体における母性転写産物の量に対する正常発生胚由来の第二極体における母性転写産物の量を、２の指数で示したものである。例えば、表中の「log2倍率」が１の場合、異常発生胚由来の第二極体における母性転写産物の量に対して、正常発生胚由来の第二極体における母性転写産物の量は、２（＝２^１）倍であることを意味する。

The "log2 fold" in Table 3 indicates the amount of maternal transcript in the second polar body derived from a normally developed embryo relative to the amount of maternal transcript in the second polar body derived from an abnormally developed embryo, expressed as an exponent of 2. For example, when the "log2 fold" in the table is 1, it means that the amount of maternal transcript in the second polar body derived from a normally developed embryo is 2 (=2 ¹ ) times the amount of maternal transcript in the second polar body derived from an abnormally developed embryo.

表４中の「log2倍率」は、正常発生胚由来の第二極体における母性転写産物の量に対する異常発生胚由来の第二極体における母性転写産物の量を、２の指数で示したものである。例えば、表中の「log2倍率」が１の場合、正常発生胚由来の第二極体における母性転写産物の量に対して、異常発生胚由来の第二極体における母性転写産物の量は、２（＝２^１）倍であることを意味する。

The "log2 fold" in Table 4 indicates the amount of maternal transcript in the second polar body derived from an abnormally developed embryo relative to the amount of maternal transcript in the second polar body derived from a normally developed embryo, expressed as an exponent of 2. For example, when the "log2 fold" in the table is 1, it means that the amount of maternal transcript in the second polar body derived from an abnormally developed embryo is 2 (=2 ¹ ) times the amount of maternal transcript in the second polar body derived from a normally developed embryo.

Example 3. Reverse transcription (RT)-quantitative polymerase chain reaction (qPCR) analysis The difference in the amount of the transcription products of the present embryo marker gene group between the second polar body derived from a normally developed embryo and the second polar body derived from an abnormally developed embryo was analyzed by RT-qPCR described in the following items "1-1" to "1-2". The present embryo marker gene group to be detected is Dmd, which is one of the abnormally developed embryo marker genes, and Cdk2ap1, which is one of the normally developed embryo marker genes. In addition, for the analysis of the transcription products of Dmd, the number of second polar bodies derived from normally developed embryos and the second polar bodies derived from the abnormally developed embryos used in the analysis was increased to 37. In addition, for the analysis of the transcription products of Cdk2ap1, the number of second polar bodies derived from normally developed embryos and the second polar bodies derived from the abnormally developed embryos used in the analysis was increased to 96.

1. Method 1-1 Synthesis of cDNA from second polar body samples 10.5 μL of a second polar body sample derived from a normally developed embryo and a second polar body sample derived from an abnormally developed embryo were each divided into 8.0 μL for RT+ and 2.5 μL for RT-, and 4 μL of UltraPure DNase/RNase-Free Distilled Water was added to the RT- sample to make a total of 6.5 μL. In order to increase the specificity of the primers in RT-qPCR, 100 μM of the reverse primer for detecting Cdk2ap1 to be detected (see Table 5), 100 μM of the reverse primer for detecting Dmd to be detected (see Table 5), and 100 μM of the reverse primer for detecting Gapdh as an internal standard (see Table 5), and 100 μM of oligo (dT) 15 primer (Sigma) were mixed in amounts of 2 μL each, and a total of 840 μL of UltraPure DNase / RNase-Free Distilled Water was added to prepare Gene Specific Primer (GSP). 1 μL of GSP and 1 μL of 10 nM dNTP mixture were added to the second polar body sample, and the mixture was incubated at 65 ° C. for 5 minutes using a thermal cycler, then placed on ice and rapidly cooled for 1 minute or more. Then, 8.5 μL of RT Mixture (Invitrogen) was added to each sample, and 0.5 μL of SuperScriptIII Reverse Transcriptase (Thermo Fisher) was added only to the second polar body sample for RT+. Each sample was treated at 50° C. for 50 minutes and at 85° C. for 5 minutes using a thermal cycler to synthesize cDNA.

1-2 qPCR
A mixture of qPCR solution (SYBR Premix EX Taq [Takara]; ROX reference dye [Takara]; and a primer set consisting of a forward primer for detecting Gapdh and a reverse primer for detecting Gapdh [10 μM each; see Table 5]; a primer set consisting of a forward primer for detecting Cdk2ap1 and a reverse primer for detecting Cdk2ap1 [10 μM each; see Table 5]; or a primer set consisting of a forward primer for detecting Dmd and a reverse primer for detecting Dmd [10 μM each; see Table 5];) and 4 μL of a template 2-fold diluted synthetic cDNA were mixed, and RT-qPCR was performed using a 7300 real-time PCR system (Applied Biosystems). Three reaction solutions were prepared for each sample and the experiment was performed. The standard curve for qPCR was prepared by extracting RNA from the ovaries of DBA/2 female mice using an RNeasy Mini Kit (Qiagen), reverse transcribing the cDNA using oligo(dT)20 primer (Thermo Fisher), and diluting the resulting DNA 1000-fold as a template.

2. Results The results of analyzing the transcript of Dmd, one of the marker genes for abnormal embryo development, are shown in Figure 11. As shown in Figure 11, the percentage of second polar bodies in which the transcript of Dmd was detected was approximately 30% in the second polar bodies derived from normally developed embryos, whereas it was high, approximately 70%, in the second polar bodies derived from abnormal embryos.

This result shows that it is possible to evaluate whether a fertilized egg is an embryo that will develop normally by using the presence or absence of transcription products of abnormal development embryo marker genes in polar bodies derived from the fertilized egg, such as the second polar body released from the fertilized egg, and supports the results of Examples 2 and 3 above.

Figure 12 shows the results of an analysis of the transcript of Cdk2ap1, one of the marker genes for normal developmental embryos. As shown in Figure 12, the level of the Cdk2ap1 transcript was significantly higher, approximately two-fold, in second polar bodies derived from normal developmental embryos compared to second polar bodies derived from abnormal developmental embryos. Furthermore, a binomial logistic analysis was performed based on the data used to obtain the analysis results in Figure 12, and it was confirmed that normal developmental embryos could be identified with a specificity of 85%.

This result indicates that it is possible to evaluate whether a fertilized egg is a normally developing embryo by using as an indicator whether the level of the transcript of a normal development embryo marker gene in a polar body derived from a fertilized egg, such as a second polar body released from the fertilized egg, is higher than the corresponding level in a second polar body derived from an abnormally developed embryo, and supports the results of Example 3 above.

The present invention contributes to improving productivity in the livestock industry and infertility treatment.

Claims (9)

A method for evaluating a mammalian embryo, comprising the steps of:
(a) detecting the transcripts of all genes in second polar bodies derived from in vitro fertilized mammalian zygotes;
(b) selecting the following [normal development embryo marker gene group] and [abnormal development embryo marker gene group] from among all the detected gene transcription products;
[Normal embryo development marker genes]
Cdk2ap1, Trappc12, Kif13a, Redrum, Enpep, Gart, Rogdi, 2310003L06Rik, Rabl3, F730016J06Rik, Prdx3, Xpnpep1, Abcc5, 4933431E20Rik, Spire2, Mgme1, Ak9, Sipa1, Narf, Tmem147, Rimbp2, Dlgap1, Cd209f, Itpr3, Atxn7l1, T hap3, Dr1, Casq2, Dnah11, Nsun5, Tek, Sec1, Zc3h7a, Vgll1, Nfkbid, Rin2, Ube2m, Zfp954, Fkbp9, Rrp1, Otx1, Ap2a2, Dpep2, Ces1d, Upp1, Zg16, Adam12, Phf2, Phrf1, Slc43a2, Gm13977, Mfsd8, Cotl1, Efcab3, Commd4, Kantr, Rnf2 12, Ankmy2, Popdc3, Arpc1a, Npdc1, Sema6a, Etfb, D030040B21Rik, Ttc21b, Plekhg5, Nat8f1, Zscan18, 5330439K02Rik, Rbfa, Zfp408, Aadacl3, Oit1, Tmem120a, Traf5, Krt8, Cd200, Ostm1, Zc3h12d, Ammecr1, AI413582, Commd5, Prkdc, Tmprss2, Prkcsh, Wdr74, Nme1, Tex15, Zfp563, Gpn3, F5, Mib2, Gm4926, Sbspon, Nup155, Pbx4, Defb48, Ddx20, Itga4, Pcsk5, Dtna, Epcam, Gm28940, Gm44196, Eif3g, Polg2, Gm364, Mtch2, Htt, Ing5, Gm42945, Aknad1, Ehbp1
[Embryonic abnormality marker genes]
Dmd, Tyk2, Nyap2, Mettl22, Manea, Nudt18, Lair1, Slamf1, March3, Ulk2, AI429214, Zfp938, Wdr59, Spryd7, Grin2b, Fsd2, Ciao3, Zhx1, Grm1, Adm, Ugt2b38, T bc1d25, Lrrc61, Fam132b, Exoc6b, Reps2, Cdh24, Fech, Agpat1, Plcb3, A230056P14Rik, Spsb4, Dmrtc1b, Raver2, 4921531C22Rik, Tbc1d31, Nutm1, Nek11, Morc 2a, Trhr2, Aacs, Atp5o, Sirt5, Id3, Zfp507, AW551984, Lias, Usp20, Wfdc1, Tubd1, Zmym6, Klf10, Mfsd11, Nudt15, Nisch, Hipk3, Tatdn3, Tyw3, Ubl4a, Atp6ap1 l, Snx8, Gm12364, Ndufa7, Zfp341, Col4a1, Zfp595, G2e3, Slc15a2, Sgsm1, Parp4, Ciao1, Fam20b, Acvr2b, Ttc25, Mau2, Sybu, Kdm3b, AY512915, Gm13136, Trmt6
(c) quantifying the transcription products of one or more genes selected from the group of [normal development embryo marker genes] and [abnormal development embryo marker genes] in the second polar body in the test sample;
(d) when the transcription product of the gene quantified in step (c) is a transcription product of the [group of normal development embryo marker genes], when the level of the transcription product of the gene quantified in step (c) is higher than the corresponding level in a second polar body derived from an embryo whose development does not progress to the blastocyst stage, evaluating the fertilized egg as an embryo that will develop normally or an embryo that is likely to develop normally;
a step of evaluating, when the transcription product of the gene quantified in step (c) is a transcription product of the [group of marker genes for an abnormal developmental embryo], the fertilized egg being an embryo that will develop normally or is likely to develop normally when the level of the transcription product of the gene quantified in step (c) is lower than the level in a second polar body derived from an embryo whose development does not progress to the blastocyst stage; A method for selecting a mammalian embryo, comprising the steps of: (a) selecting a mammalian embryo from among the mammalian embryos;
(a) detecting the transcripts of all genes in second polar bodies derived from in vitro fertilized mammalian zygotes;
(b) selecting the following [normal development embryo marker gene group] and [abnormal development embryo marker gene group] from among all the detected gene transcription products;
[Normal embryo development marker genes]
Cdk2ap1, Trappc12, Kif13a, Redrum, Enpep, Gart, Rogdi, 2310003L06Rik, Rabl3, F730016J06Rik, Prdx3, Xpnpep1, Abcc5, 4933431E20Rik, Spire2, Mgme1, Ak9, Sipa1, Narf, Tmem147, Rimbp2, Dlgap1, Cd209f, Itpr3, Atxn7l1, T hap3, Dr1, Casq2, Dnah11, Nsun5, Tek, Sec1, Zc3h7a, Vgll1, Nfkbid, Rin2, Ube2m, Zfp954, Fkbp9, Rrp1, Otx1, Ap2a2, Dpep2, Ces1d, Upp1, Zg16, Adam12, Phf2, Phrf1, Slc43a2, Gm13977, Mfsd8, Cotl1, Efcab3, Commd4, Kantr, Rnf2 12, Ankmy2, Popdc3, Arpc1a, Npdc1, Sema6a, Etfb, D030040B21Rik, Ttc21b, Plekhg5, Nat8f1, Zscan18, 5330439K02Rik, Rbfa, Zfp408, Aadacl3, Oit1, Tmem120a, Traf5, Krt8, Cd200, Ostm1, Zc3h12d, Ammecr1, AI413582, Commd5, Prkdc, Tmprss2, Prkcsh, Wdr74, Nme1, Tex15, Zfp563, Gpn3, F5, Mib2, Gm4926, Sbspon, Nup155, Pbx4, Defb48, Ddx20, Itga4, Pcsk5, Dtna, Epcam, Gm28940, Gm44196, Eif3g, Polg2, Gm364, Mtch2, Htt, Ing5, Gm42945, Aknad1, Ehbp1
[Embryonic abnormality marker genes]
Dmd, Tyk2, Nyap2, Mettl22, Manea, Nudt18, Lair1, Slamf1, March3, Ulk2, AI429214, Zfp938, Wdr59, Spryd7, Grin2b, Fsd2, Ciao3, Zhx1, Grm1, Adm, Ugt2b38, T bc1d25, Lrrc61, Fam132b, Exoc6b, Reps2, Cdh24, Fech, Agpat1, Plcb3, A230056P14Rik, Spsb4, Dmrtc1b, Raver2, 4921531C22Rik, Tbc1d31, Nutm1, Nek11, Morc 2a, Trhr2, Aacs, Atp5o, Sirt5, Id3, Zfp507, AW551984, Lias, Usp20, Wfdc1, Tubd1, Zmym6, Klf10, Mfsd11, Nudt15, Nisch, Hipk3, Tatdn3, Tyw3, Ubl4a, Atp6ap1 l, Snx8, Gm12364, Ndufa7, Zfp341, Col4a1, Zfp595, G2e3, Slc15a2, Sgsm1, Parp4, Ciao1, Fam20b, Acvr2b, Ttc25, Mau2, Sybu, Kdm3b, AY512915, Gm13136, Trmt6
(c) quantifying the transcription products of one or more genes selected from the group of [normal development embryo marker genes] and [abnormal development embryo marker genes] in the second polar body in the test sample;
(d) when the transcription product of the gene quantified in step (c) is a transcription product of the [group of normally developing embryo marker genes], selecting the fertilized egg when the level of the transcription product of the gene quantified in step (c) is higher than the level in a second polar body derived from an embryo whose development does not progress to the blastocyst stage;
a step of selecting the fertilized egg when the level of the gene transcription product quantified in step (c) is lower than the level in a second polar body derived from an embryo whose development does not progress to the blastocyst stage, when the amount of the gene transcription product detected in step (c) is the group of [abnormal development embryo marker genes]; The method according to claim 1 or 2, characterized in that the gene is Cdk2ap1 or Dmd. The method according to any one of claims 1 to 3, characterized in that in step (a), the transcription products of all genes are detected by a sequencing method using a next-generation sequencer. A kit for evaluating and/or selecting mammalian embryos, comprising: detecting the transcription products of all genes in a second polar body derived from an in vitro fertilized mammalian fertilized egg; and selecting from the transcription products of all detected genes one or more genes selected from the following [group of marker genes for normal development embryos] and [group of marker genes for abnormal development embryos]; and/or a primer set and/or a probe consisting of a forward primer and a reverse primer that hybridize to the cDNA of the gene; and an attached document.
[Normal embryo development marker genes]
Cdk2ap1, Trappc12, Kif13a, Redrum, Enpep, Gart, Rogdi, 2310003L06Rik, Rabl3, F730016J06Rik, Prdx3, Xpnpep1, Abcc5, 4933431E20Rik, Spire2, Mgme1, Ak9, Sipa1, Narf, Tmem147, Rimbp2, Dlgap1, Cd209f, Itpr3, Atxn7l1, T hap3, Dr1, Casq2, Dnah11, Nsun5, Tek, Sec1, Zc3h7a, Vgll1, Nfkbid, Rin2, Ube2m, Zfp954, Fkbp9, Rrp1, Otx1, Ap2a2, Dpep2, Ces1d, Upp1, Zg16, Adam12, Phf2, Phrf1, Slc43a2, Gm13977, Mfsd8, Cotl1, Efcab3, Commd4, Kantr, Rnf2 12, Ankmy2, Popdc3, Arpc1a, Npdc1, Sema6a, Etfb, D030040B21Rik, Ttc21b, Plekhg5, Nat8f1, Zscan18, 5330439K02Rik, Rbfa, Zfp408, Aadacl3, Oit1, Tmem120a, Traf5, Krt8, Cd200, Ostm1, Zc3h12d, Ammecr1, AI413582, Commd5, Prkdc, Tmprss2, Prkcsh, Wdr74, Nme1, Tex15, Zfp563, Gpn3, F5, Mib2, Gm4926, Sbspon, Nup155, Pbx4, Defb48, Ddx20, Itga4, Pcsk5, Dtna, Epcam, Gm28940, Gm44196, Eif3g, Polg2, Gm364, Mtch2, Htt, Ing5, Gm42945, Aknad1, Ehbp1
[Embryonic abnormality marker genes]
Dmd, Tyk2, Nyap2, Mettl22, Manea, Nudt18, Lair1, Slamf1, March3, Ulk2, AI429214, Zfp938, Wdr59, Spryd7, Grin2b, Fsd2, Ciao3, Zhx1, Grm1, Adm, Ugt2b38, T bc1d25, Lrrc61, Fam132b, Exoc6b, Reps2, Cdh24, Fech, Agpat1, Plcb3, A230056P14Rik, Spsb4, Dmrtc1b, Raver2, 4921531C22Rik, Tbc1d31, Nutm1, Nek11, Morc 2a, Trhr2, Aacs, Atp5o, Sirt5, Id3, Zfp507, AW551984, Lias, Usp20, Wfdc1, Tubd1, Zmym6, Klf10, Mfsd11, Nudt15, Nisch, Hipk3, Tatdn3, Tyw3, Ubl4a, Atp6ap1 l, Snx8, Gm12364, Ndufa7, Zfp341, Col4a1, Zfp595, G2e3, Slc15a2, Sgsm1, Parp4, Ciao1, Fam20b, Acvr2b, Ttc25, Mau2, Sybu, Kdm3b, AY512915, Gm13136, Trmt6 The kit according to claim 5, characterized in that the gene is Cdk2ap1 or Dmd. 1. Use of a biomarker in the evaluation of a mammalian embryo, comprising the steps of: (a) detecting a mammalian embryo;
(a) detecting the transcripts of all genes in second polar bodies derived from in vitro fertilized mammalian zygotes;
(b) selecting the following [group of marker genes for normal development embryos] and [group of marker genes for abnormal development embryos] as biomarkers from among all the detected gene transcription products;
[Normal embryo development marker genes]
Cdk2ap1, Trappc12, Kif13a, Redrum, Enpep, Gart, Rogdi, 2310003L06Rik, Rabl3, F730016J06Rik, Prdx3, Xpnpep1, Abcc5, 4933431E20Rik, Spire2, Mgme1, Ak9, Sipa1, Narf, Tmem147, Rimbp2, Dlgap1, Cd209f, Itpr3, Atxn7l1, T hap3, Dr1, Casq2, Dnah11, Nsun5, Tek, Sec1, Zc3h7a, Vgll1, Nfkbid, Rin2, Ube2m, Zfp954, Fkbp9, Rrp1, Otx1, Ap2a2, Dpep2, Ces1d, Upp1, Zg16, Adam12, Phf2, Phrf1, Slc43a2, Gm13977, Mfsd8, Cotl1, Efcab3, Commd4, Kantr, Rnf2 12, Ankmy2, Popdc3, Arpc1a, Npdc1, Sema6a, Etfb, D030040B21Rik, Ttc21b, Plekhg5, Nat8f1, Zscan18, 5330439K02Rik, Rbfa, Zfp408, Aadacl3, Oit1, Tmem120a, Traf5, Krt8, Cd200, Ostm1, Zc3h12d, Ammecr1, AI413582, Commd5, Prkdc, Tmprss2, Prkcsh, Wdr74, Nme1, Tex15, Zfp563, Gpn3, F5, Mib2, Gm4926, Sbspon, Nup155, Pbx4, Defb48, Ddx20, Itga4, Pcsk5, Dtna, Epcam, Gm28940, Gm44196, Eif3g, Polg2, Gm364, Mtch2, Htt, Ing5, Gm42945, Aknad1, Ehbp1
[Embryonic abnormality marker genes]
Dmd, Tyk2, Nyap2, Mettl22, Manea, Nudt18, Lair1, Slamf1, March3, Ulk2, AI429214, Zfp938, Wdr59, Spryd7, Grin2b, Fsd2, Ciao3, Zhx1, Grm1, Adm, Ugt2b38, T bc1d25, Lrrc61, Fam132b, Exoc6b, Reps2, Cdh24, Fech, Agpat1, Plcb3, A230056P14Rik, Spsb4, Dmrtc1b, Raver2, 4921531C22Rik, Tbc1d31, Nutm1, Nek11, Morc 2a, Trhr2, Aacs, Atp5o, Sirt5, Id3, Zfp507, AW551984, Lias, Usp20, Wfdc1, Tubd1, Zmym6, Klf10, Mfsd11, Nudt15, Nisch, Hipk3, Tatdn3, Tyw3, Ubl4a, Atp6ap1 l, Snx8, Gm12364, Ndufa7, Zfp341, Col4a1, Zfp595, G2e3, Slc15a2, Sgsm1, Parp4, Ciao1, Fam20b, Acvr2b, Ttc25, Mau2, Sybu, Kdm3b, AY512915, Gm13136, Trmt6
(c) quantifying the transcription products of one or more genes selected from the group of [normal development embryo marker genes] and [abnormal development embryo marker genes] in the second polar body in the test sample;
(d) when the transcription product of the gene quantified in step (c) is a transcription product of the [group of normal development embryo marker genes], when the level of the transcription product of the gene quantified in step (c) is higher than the corresponding level in a second polar body derived from an embryo whose development does not progress to the blastocyst stage, evaluating the fertilized egg as an embryo that will develop normally or an embryo that is likely to develop normally;
a step of evaluating, when the transcription product of the gene quantified in step (c) is a transcription product of the [group of marker genes for an abnormal developmental embryo], the fertilized egg being an embryo that will develop normally or is likely to develop normally when the level of the transcription product of the gene quantified in step (c) is lower than the level in a second polar body derived from an embryo whose development does not progress to the blastocyst stage; A use of a biomarker in selecting a mammalian embryo, comprising the steps of: (a) selecting a mammalian embryo from the selected mammalian embryo;
(a) detecting the transcripts of all genes in second polar bodies derived from in vitro fertilized mammalian zygotes;
(b) selecting the following [normal development embryo marker gene group] and [abnormal development embryo marker gene group] as biomarkers from among all the detected gene transcription products;
[Normal embryo development marker genes]
Cdk2ap1, Trappc12, Kif13a, Redrum, Enpep, Gart, Rogdi, 2310003L06Rik, Rabl3, F730016J06Rik, Prdx3, Xpnpep1, Abcc5, 4933431E20Rik, Spire2, Mgme1, Ak9, Sipa1, Narf, Tmem147, Rimbp2, Dlgap1, Cd209f, Itpr3, Atxn7l1, T hap3, Dr1, Casq2, Dnah11, Nsun5, Tek, Sec1, Zc3h7a, Vgll1, Nfkbid, Rin2, Ube2m, Zfp954, Fkbp9, Rrp1, Otx1, Ap2a2, Dpep2, Ces1d, Upp1, Zg16, Adam12, Phf2, Phrf1, Slc43a2, Gm13977, Mfsd8, Cotl1, Efcab3, Commd4, Kantr, Rnf2 12, Ankmy2, Popdc3, Arpc1a, Npdc1, Sema6a, Etfb, D030040B21Rik, Ttc21b, Plekhg5, Nat8f1, Zscan18, 5330439K02Rik, Rbfa, Zfp408, Aadacl3, Oit1, Tmem120a, Traf5, Krt8, Cd200, Ostm1, Zc3h12d, Ammecr1, AI413582, Commd5, Prkdc, Tmprss2, Prkcsh, Wdr74, Nme1, Tex15, Zfp563, Gpn3, F5, Mib2, Gm4926, Sbspon, Nup155, Pbx4, Defb48, Ddx20, Itga4, Pcsk5, Dtna, Epcam, Gm28940, Gm44196, Eif3g, Polg2, Gm364, Mtch2, Htt, Ing5, Gm42945, Aknad1, Ehbp1
[Embryonic abnormality marker genes]
Dmd, Tyk2, Nyap2, Mettl22, Manea, Nudt18, Lair1, Slamf1, March3, Ulk2, AI429214, Zfp938, Wdr59, Spryd7, Grin2b, Fsd2, Ciao3, Zhx1, Grm1, Adm, Ugt2b38, T bc1d25, Lrrc61, Fam132b, Exoc6b, Reps2, Cdh24, Fech, Agpat1, Plcb3, A230056P14Rik, Spsb4, Dmrtc1b, Raver2, 4921531C22Rik, Tbc1d31, Nutm1, Nek11, Morc 2a, Trhr2, Aacs, Atp5o, Sirt5, Id3, Zfp507, AW551984, Lias, Usp20, Wfdc1, Tubd1, Zmym6, Klf10, Mfsd11, Nudt15, Nisch, Hipk3, Tatdn3, Tyw3, Ubl4a, Atp6ap1 l, Snx8, Gm12364, Ndufa7, Zfp341, Col4a1, Zfp595, G2e3, Slc15a2, Sgsm1, Parp4, Ciao1, Fam20b, Acvr2b, Ttc25, Mau2, Sybu, Kdm3b, AY512915, Gm13136, Trmt6
(c) quantifying the transcription products of one or more genes selected from the group of [normal development embryo marker genes] and [abnormal development embryo marker genes] in the second polar body in the test sample;
(d) when the transcription product of the gene quantified in step (c) is a transcription product of the [group of normally developing embryo marker genes], selecting the fertilized egg when the level of the transcription product of the gene quantified in step (c) is higher than the level in a second polar body derived from an embryo whose development does not progress to the blastocyst stage;
a step of selecting the fertilized egg when the level of the transcription product of the gene quantified in step (c) is a transcription product of the [group of abnormal development embryo marker genes], is lower than the level in the second polar body derived from an embryo whose development does not progress to the blastocyst stage; The use according to claim 7 or 8, characterized in that the gene is Cdk2ap1 or Dmd.