JP2021069348A - Mammal embryo selection method using biomarker - Google Patents

Abstract

To provide a method for evaluating and selecting a good-quality embryo by suppressing damage to an embryo at a relatively low level, and a method for identifying and using a biomarker for evaluating and selecting a good-quality embryo.SOLUTION: The level of a transcription product of an embryo marker gene in a polar body derived from a mammal fertilized egg which is fertilized in vitro is used as an index, whether the mammal fertilized egg is a normally occurring mammal embryo or not is evaluated properly, so that the normally occurring mammal embryo is selected.SELECTED DRAWING: None

Description

The present invention is a method for evaluating a mammalian embryo; a method for selecting a mammalian embryo; a kit for evaluating and / or selecting a mammalian embryo; a biomarker for evaluating and / or selecting a mammalian embryo; an evaluation of a mammalian embryo. And / or a method for identifying genes for selection; etc.

Currently, one in six couples in developed countries suffers from infertility. For this reason, in addition to general infertility treatment by artificial insemination (AI), implementation of advanced infertility treatment (ART) such as in vitro fertilization (IVF) and microinsemination (ICSI) is increasing, but in Japan The success rate is still low at 10% or less (Non-Patent Document 1). Recently, the decrease in conception rate has become a big problem in the livestock industry. In particular, cattle have a long gestation period, so a single miscarriage is an economically significant loss.

Many embryos that do not maintain pregnancy are thought to have underdeveloped during cleavage or premature miscarriage after implantation. As one of the main causes, it is assumed that the quality of gametes and embryos deteriorates due to late marriage and external environmental stress. The quality of embryos has traditionally been evaluated as good or bad based on differences in embryo morphology and division rate, but in many cases it depends on individual senses, and it is difficult to say that it is accurate, and there is little scientific basis.

In recent years, a method for detecting chromosomal abnormalities by performing detailed imaging of embryos in search of a more objective index has been developed (Non-Patent Document 2). However, such a method requires a process of synthesizing mRNA encoding a fluorescent protein in vitro and injecting it into a fertilized egg, and its application to human embryos for which nucleic acid injection is not approved is difficult. A method for evaluating the quality of embryos (fertilized eggs) at an early stage based on a less invasive and scientific basis has been desired.

On the other hand, a method for detecting the presence or absence of a maternally-derived etiologic gene in a fertilized egg by performing a genetic diagnosis on a polar body generated in the process of meiosis of an egg has been reported (Patent Document 1).

Japanese Unexamined Patent Publication No. 2-86800

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

An object of the present invention is to provide a method for evaluating / selecting a good quality embryo while suppressing damage to the embryo relatively low, a method for identifying and using a biomarker for evaluating / selecting a good quality embryo, and the like. There is.

In order to solve the above problems, the present inventors have analyzed the transcript of the gene in the fertilized egg and the transcript of the gene in the second polar body released from the fertilized egg. As a result, the present inventors have analyzed the gene in the second polar body. We found that the majority of the transcripts of the fertilized eggs were maternal transcripts of fertilized eggs. In addition, in order to identify a maternal transcript that can evaluate the quality of the embryo, we tried to identify it from the transcript of the gene in the second polar body. As a result, in the second polar body derived from the normal development embryo, it was derived from the abnormal development embryo. 113 types of genes (ie, normal developmental embryo marker genes) were identified as transcripts that were significantly increased compared to the second polar body of. In addition, 80 kinds of genes (that is, abnormally developed embryo marker genes) were identified as transcripts in which a significant increase was observed in the abnormally developed embryo-derived second polar body as compared with the normally developed embryo-derived second polar body. Was done. In addition, it was confirmed that normal developmental embryos and abnormal developmental embryos can be distinguished by using the levels of the transcripts of these normal developmental embryo marker genes and abnormal developmental embryo marker genes as indicators. The present invention has been completed based on these findings.

That is, the present invention is as follows.
[1] A method for evaluating a mammalian embryo, which comprises the following steps (a) and (b-1).
(A) Transcripts of one or more genes selected from the following [normal developmental embryo marker gene group] and [abnormal developmental embryonic marker gene group] in the polar body derived from in vitro fertilized mammalian fertilized eggs. Step to detect;
[Normal developmental embryo marker gene group]
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, Thap3, Dr1, Casq2, Dnah11, Nsun5, Tek, Sec1, Zc3h7a, Vgll1, Nfkbid, Rin2, Ube2m, Zfp954, Fkbp9, Rrp1, Otx1, Ap2a2, Rrp1, Otx1, Ap2a2 Gm13977, Mfsd8, Cotl1, Efcab3, Commd4, Kantr, Rnf212, 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, Tmprs2, Prkksh, Wdr74, Nme1, Tex15, Zfp563, Gpn3, F5, Mib2, Gmp49 Dtna, Epcam, Gm28940, Gm44196, Eif3g, Polg2, Gm364, Mtch2, Htt, Ing5, Gm42945, Aknad1, Ehbp1
[Abnormal developmental embryo marker gene group]
Dmd, Tyk2, Nyap2, Mettl22, Manea, Nudt18, Lair1, Slamf1, March3, Ulk2, AI429214, Zfp938, Wdr59, Spryd7, Grin2b, Fsd2, Ciab Reps2, Cdh24, Fech, Agpat1, Plcb3, A230056P14Rik, Spsb4, Dmrtc1b, Raver2, 4921531C22Rik, Tbc1d31, Nutm1, Nek11, Morc2a, Trhr2, Aacs Zmym6, Klf10, Mfsd11, Nudt15, Nitch, Hipk3, Tattn3, Tyw3, Ubl4a, Atp6ap1l, Snx8, Gm12364, Ndufa7, Zfp341, Col4a1, Zfp2m Sybu, Kdm3b, AY512915, Gm13136, Trmt6
(B-1) When the transcript of the gene detected in step (a) is a transcript of [normal development embryo marker gene group], the level of the transcript of the gene detected in step (a) is the scutellum. When it is higher than the level in the polar body derived from an embryo that does not progress to the blastocyst stage, the fertilized egg is evaluated as a normally developing embryo or an embryo likely to develop normally.
When the transcript of the gene detected in step (a) is a transcript of [abnormal development embryo marker gene group], the level of the transcript of the gene detected in step (a) develops until the blastocyst stage. A step of assessing that a fertilized egg is or is likely to develop normally when it is below that level in a polar body derived from a non-progressive embryo;
[2] A method for selecting mammalian embryos, which comprises the following steps (a) and (b-2).
(A) Transcripts of one or more genes selected from the following [normal developmental embryo marker gene group] and [abnormal developmental embryonic marker gene group] in the polar body derived from in vitro fertilized mammalian fertilized eggs. Step to detect;
[Normal developmental embryo marker gene group]
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, Thap3, Dr1, Casq2, Dnah11, Nsun5, Tek, Sec1, Zc3h7a, Vgll1, Nfkbid, Rin2, Ube2m, Zfp954, Fkbp9, Rrp1, Otx1, Ap2a2, Rrp1, Otx1, Ap2a2 Gm13977, Mfsd8, Cotl1, Efcab3, Commd4, Kantr, Rnf212, 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, Tmprs2, Prkksh, Wdr74, Nme1, Tex15, Zfp563, Gpn3, F5, Mib2, Gmp49 Dtna, Epcam, Gm28940, Gm44196, Eif3g, Polg2, Gm364, Mtch2, Htt, Ing5, Gm42945, Aknad1, Ehbp1
[Abnormal developmental embryo marker gene group]
Dmd, Tyk2, Nyap2, Mettl22, Manea, Nudt18, Lair1, Slamf1, March3, Ulk2, AI429214, Zfp938, Wdr59, Spryd7, Grin2b, Fsd2, Ciab Reps2, Cdh24, Fech, Agpat1, Plcb3, A230056P14Rik, Spsb4, Dmrtc1b, Raver2, 4921531C22Rik, Tbc1d31, Nutm1, Nek11, Morc2a, Trhr2, Aacs Zmym6, Klf10, Mfsd11, Nudt15, Nitch, Hipk3, Tattn3, Tyw3, Ubl4a, Atp6ap1l, Snx8, Gm12364, Ndufa7, Zfp341, Col4a1, Zfp2m Sybu, Kdm3b, AY512915, Gm13136, Trmt6
(B-2) When the transcript of the gene detected in step (a) is a transcript of [normal development embryo marker gene group], the level of the transcript of the gene detected in step (a) is the scutellum. When the level is higher than the relevant level in the polar body derived from an embryo that does not progress to the spore stage, the fertilized egg is selected.
When the transcript of the gene detected in step (a) is the amount of transcript of [abnormal development embryo marker gene group], the level of transcript of the gene detected in step (a) develops until the blastocyst stage. The step of sorting the fertilized egg when it is lower than the level in the polar body derived from the embryo that does not progress;
[3] The method according to the above [1] or [2], wherein the gene is Cdk2ap1 or Dmd.
[4] The method according to any one of the above [1] to [3], wherein the polar body is a second polar body.
[5] The method according to any one of the above [1] to [4], wherein in the step (a), the transcript of the gene is detected by a sequencing method using a next-generation sequencer.
[6] Transcripts of one or more genes selected from the following [normal developmental embryo marker gene group] and [abnormal developmental embryonic marker gene group] in the polar body derived from in vitro fertilized mammalian fertilized eggs. For evaluation of mammalian embryos and / or comprising a reverse primer and / or a probe that hybridizes to, and / or a primer set consisting of a forward primer and a reverse primer that hybridize to the cDNA of the gene and / or a probe. / Or a sorting kit.
[Normal developmental embryo marker gene group]
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, Thap3, Dr1, Casq2, Dnah11, Nsun5, Tek, Sec1, Zc3h7a, Vgll1, Nfkbid, Rin2, Ube2m, Zfp954, Fkbp9, Rrp1, Otx1, Ap2a2, Rrp1, Otx1, Ap2a2 Gm13977, Mfsd8, Cotl1, Efcab3, Commd4, Kantr, Rnf212, 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, Tmprs2, Prkksh, Wdr74, Nme1, Tex15, Zfp563, Gpn3, F5, Mib2, Gmp49 Dtna, Epcam, Gm28940, Gm44196, Eif3g, Polg2, Gm364, Mtch2, Htt, Ing5, Gm42945, Aknad1, Ehbp1
[Abnormal developmental embryo marker gene group]
Dmd, Tyk2, Nyap2, Mettl22, Manea, Nudt18, Lair1, Slamf1, March3, Ulk2, AI429214, Zfp938, Wdr59, Spryd7, Grin2b, Fsd2, Ciab Reps2, Cdh24, Fech, Agpat1, Plcb3, A230056P14Rik, Spsb4, Dmrtc1b, Raver2, 4921531C22Rik, Tbc1d31, Nutm1, Nek11, Morc2a, Trhr2, Aacs Zmym6, Klf10, Mfsd11, Nudt15, Nitch, Hipk3, Tattn3, Tyw3, Ubl4a, Atp6ap1l, Snx8, Gm12364, Ndufa7, Zfp341, Col4a1, Zfp2m Sybu, Kdm3b, AY512915, Gm13136, Trmt6
[7] The kit according to the above [6], wherein the gene is Cdk2ap1 or Dmd.
[8] The kit according to the above [6] or [7], wherein the polar body is a second polar body.
[9] A biomarker for evaluation and / or selection of mammalian embryos, which comprises one or more genes selected from the following [normal developmental embryo marker gene group] and [abnormal developmental embryonic marker gene group]. hand,
When the gene is a gene of [normal development embryo marker gene group], the level of the transcript of the gene in the polar body derived from the in vitro fertilized mammalian fertilized egg progresses to the blastocyst stage. Higher than that level in polar bodies derived from non-embryos,
When the gene is a gene of [abnormal development embryo marker gene group], the level of the transcript of the gene in the polar body derived from the in vitro fertilized mammalian fertilized egg progresses to the blastocyst stage. The biomarker, characterized in that it is lower than that level in a polar body derived from a non-embryo.
[Normal developmental embryo marker gene group]
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, Thap3, Dr1, Casq2, Dnah11, Nsun5, Tek, Sec1, Zc3h7a, Vgll1, Nfkbid, Rin2, Ube2m, Zfp954, Fkbp9, Rrp1, Otx1, Ap2a2, Rrp1, Otx1, Ap2a2 Gm13977, Mfsd8, Cotl1, Efcab3, Commd4, Kantr, Rnf212, 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, Tmprs2, Prkksh, Wdr74, Nme1, Tex15, Zfp563, Gpn3, F5, Mib2, Gmp49 Dtna, Epcam, Gm28940, Gm44196, Eif3g, Polg2, Gm364, Mtch2, Htt, Ing5, Gm42945, Aknad1, Ehbp1
[Abnormal developmental embryo marker gene group]
Dmd, Tyk2, Nyap2, Mettl22, Manea, Nudt18, Lair1, Slamf1, March3, Ulk2, AI429214, Zfp938, Wdr59, Spryd7, Grin2b, Fsd2, Ciab Reps2, Cdh24, Fech, Agpat1, Plcb3, A230056P14Rik, Spsb4, Dmrtc1b, Raver2, 4921531C22Rik, Tbc1d31, Nutm1, Nek11, Morc2a, Trhr2, Aacs Zmym6, Klf10, Mfsd11, Nudt15, Nitch, Hipk3, Tattn3, Tyw3, Ubl4a, Atp6ap1l, Snx8, Gm12364, Ndufa7, Zfp341, Col4a1, Zfp2m Sybu, Kdm3b, AY512915, Gm13136, Trmt6
[10] The biomarker according to the above [9], wherein the gene is Cdk2ap1 or Dmd.
[11] The biomarker according to the above [9] or [10], wherein the polar body is a second polar body.
[12] A method for identifying a gene for evaluation and / or selection of a mammalian embryo, which comprises the following steps (A) and (B).
(A) A step of detecting a transcript of a gene in a polar body derived from an in vitro fertilized mammalian fertilized egg;
(B) When the level of the transcript of the gene detected in step (A) is higher than the level in the polar body derived from an embryo whose development does not progress to the blastocyst stage, the gene is a normally developing embryo marker gene. It is evaluated that it is likely to be a normal developmental embryo marker gene.
When the level of the transcript of the gene detected in step (A) is lower than that level in the polar body derived from an embryo that does not progress to the blastocyst stage, the gene is an abnormally developing embryo marker gene or Step to evaluate that it is likely to be an abnormally developed embryo marker gene;
[13] The method according to the above [12], wherein the polar body is a second polar body.
[14] The method according to [12] or [13] above, wherein in the step (A), the transcript of the gene is detected by a sequencing method using a next-generation sequencer.

Further, as another embodiment of the present invention,
[15] In the above step (a),
When the transcript of the gene detected in step (a) is a transcript of [normally developing embryo marker gene group], the level of the transcript of the gene detected in step (a) develops until the embryonic vesicle stage. Create or collect data to evaluate that the fertilized egg is an embryo that develops normally; or an embryo that is likely to develop normally when it is higher than that level in a polar body derived from a non-progressive embryo. Then, when the transcript of the gene detected in step (a) is a transcript of [abnormal development embryo marker gene group], the level of the transcript of the gene detected in step (a) is up to the vesicle stage. Create data to evaluate that the fertilized egg is an embryo that develops normally; or an embryo that is likely to develop normally when it is lower than the level in the polar body derived from an embryo that does not progress development. Alternatively, a method for creating or collecting data for evaluating a mammalian embryo, including a step of collecting (b-1-1), and a method for collecting or collecting data.
[16] For use in the evaluation and / or selection of mammalian embryos,
Hybrid to transcripts or cDNAs of one or more genes selected from the above [normal developmental embryo marker gene group] and [abnormal developmental embryonic marker gene group] in the polar body derived from in vitro fertilized mammalian fertilized eggs. Examples include forward primers, reverse primers, and / or probes for soaking.

According to the present invention, a normally developing mammalian fertilized egg (embryo) can be evaluated and selected relatively early after fertilization, so that the conception rate (pregnancy rate) of the mammal is expected to be improved. In addition, since the transcripts of the normally-developing embryo marker gene and the abnormally-developing embryo marker gene, which are used as indicators for evaluation and selection, are not derived from the embryo but from the polar body, the damage to the embryo itself is kept relatively low. It is possible to evaluate and select good quality embryos in a minimally invasive manner.

In this example, which will be described later, a method of collecting the second polar body released from the embryo and a method of culturing the embryo in vitro using a fertilized mouse egg are specifically shown. It has been established in mammals such as humans, cows, and pigs, and the normal development embryo marker gene and the abnormal development embryo marker gene have orthologs among mammals. Therefore, since the present invention is useful not only in mice but also in mammals in general, the field of breeding (reproductive) technology, for example, high-quality livestock (for example, cattle exhibiting marbling traits) or livestock difficult to breed. It contributes to the livestock industry that produces cattle and the field of fertility treatment.

FIG. 1A shows the first polar body (circled by the dotted line in the figure), the fertilized egg (arrow in the figure), and the second polar body released from the fertilized egg (the solid circle in the figure). It is a microscope image of the enclosed part). FIG. 1B is a diagram showing the results of analyzing the transcripts common to the transcripts of 16397 kinds of genes in the fertilized egg sample and the transcripts of 11107 kinds of genes in the second polar body sample. Six types of normally developing embryo marker genes (Trappc12 [Trappc12]] are used for each of the second polar body derived from a normally developing embryo (“N” in the figure) and the second polar body derived from an abnormally developing embryo (“A” in the figure). FIG. 2A shows the results of analyzing the levels of transcripts of Kif13a [FIG. 2B], Embryogenesis [FIG. 2C], Embryo [FIG. 2D], Gate [FIG. 2E], and Rogdi [FIG. 2F]. ● in the graph indicates each sample (the same applies hereinafter). Six types of normally developing embryo marker genes (Labl3 [Rabl3 [] for each of the second polar body derived from a normally developing embryo (“N” in the figure) and the second polar body derived from an abnormally developing embryo (“A” in the figure). 3A], Prdx3 [FIG. 3B], Polar2 [FIG. 3C], Shipa1 [FIG. 3D], Atxn7l1 [FIG. 3E], and Ube2m [FIG. 3F]) are shown. Six types of normally developing embryo marker genes (Rrp1 [Rrp1 [] for each of the second polar body derived from a normally developing embryo (“N” in the figure) and the second polar body derived from an abnormally developing embryo (“A” in the figure). FIG. 4A shows the results of analyzing the levels of transcripts of Otx1 [FIG. 4B], Phf2 [FIG. 4C], Phrf1 [FIG. 4D], Rnf212 [FIG. 4E], and Ankmy2 [FIG. 4F]. Six types of normally developing embryo marker genes (Popdc3 [Popdc3 [] for each of the second polar body derived from a normally developing embryo (“N” in the figure) and the second polar body derived from an abnormally developing embryo (“A” in the figure). 5A], Zscan18 [FIG. 5B], Krt8 [FIG. 5C], Wdr74 [FIG. 5D], Nme1 [FIG. 5E], and Tex15 [FIG. 5F]) are shown. Six types of normally developing embryo marker genes (Mib2 [Mib2 [] for each of the second polar body derived from a normally developing embryo (“N” in the figure) and the second polar body derived from an abnormally developing embryo (“A” in the figure). FIG. 6A shows the results of analyzing the levels of transcripts of Pbx4 [FIG. 6B], Ddx20 [FIG. 6C], Cdk2ap1 [FIG. 6D], Pcsk5 [FIG. 6E], and Ing5 [FIG. 6F]. Six types of abnormally developing embryo marker genes (Tyk2 [Tyk2 [] for each of the second polar body derived from a normally developing embryo (“N” in the figure) and the second polar body derived from an abnormally developing embryo (“A” in the figure). FIG. 7A shows the results of analyzing the levels of transcripts of Embryo 22 [FIG. 7B], Ulk2 [FIG. 7C], Grin2b [FIG. 7D], Adm [FIG. 7E], and Tbc1d25 [FIG. 7F]. Six types of abnormally developing embryo marker genes (Fech [] for each of the second polar body derived from a normally developing embryo (“N” in the figure) and the second polar body derived from an abnormally developing embryo (“A” in the figure). FIG. 8A shows the results of analyzing the levels of transcripts of Plcb3 [FIG. 8B], Spsb4 [FIG. 8C], River2 [FIG. 8D], Nek11 [FIG. 8E], and Zfp507 [FIG. 8F]. Six types of abnormally developing embryo marker genes (Lias [] for each of the second polar body derived from a normally developing embryo (“N” in the figure) and the second polar body derived from an abnormally developing embryo (“A” in the figure). 9A], Tubd1 [FIG. 9B], Zmym6 [FIG. 9C], Mfsd11 [FIG. 9D], Nutt15 [FIG. 9E], and Slc15a2 [FIG. 9F]) are shown. Five types of abnormally developing embryo marker genes (Parp4 [] for each of the second polar body derived from a normally developing embryo (“N” in the figure) and the second polar body derived from an abnormally developing embryo (“A” in the figure). 10A], Fam20b [FIG. 10B], Mau2 [FIG. 10C], Kdm3b [FIG. 10D], and Dmd [FIG. 10E]) are shown. A transcript of the abnormally developing embryo marker gene (Dmd) for each of the second polar body derived from a normally developing embryo (“N” in the figure) and the second polar body derived from an abnormally developing embryo (“A” in the figure). It is a figure which shows the result (mean value ± standard error) of the analysis of the ratio of the 2nd polar body which was detected. “*” In the figure indicates that there is a statistically significant difference (p = 0.027). Transcription of the normal developmental embryo marker gene (Cdk2ap1 gene) for each of the normal developmental embryo-derived second polar body (“N” in the figure) and the abnormally developing embryo-derived second polar body (“A” in the figure). It is a figure which shows the result (mean value ± standard error) which analyzed the level of a product. “**” in the figure indicates that there is a statistically significant difference (p = 0.002).

As a method for evaluating a mammalian embryo of the present invention,
In vitro fertilized (for example, in vitro fertilization by in vitro fertilization, microinsemination; the same applies hereinafter) in a polar body derived from a fertilized mammalian egg (hereinafter, may be referred to as a "detection target polar body"), the following [normally developing embryos] Detects transcripts of one or more genes (hereinafter sometimes referred to as the "embryo marker gene group") selected from [marker gene group] and [abnormal development embryo marker gene group], and quantifies as necessary. Step (a) and
The level of the transcript of the [normally developing embryo marker gene group] in the polar body to be detected is the relevant polar body derived from an embryo whose development does not progress to the embryonic vesicle stage (hereinafter, may be referred to as "control polar body"). When above the level, the fertilized egg is evaluated as a normally developing embryo; or an embryo that is likely to develop normally;
When the level of the transcript of the [abnormal developmental embryo marker gene group] in the polar body to be detected is lower than the level in the control polar body, the fertilized egg is a normally developing embryo; or can develop normally. The method is not particularly limited as long as it is a method including the step (b-1) of evaluating the embryo as having high sex (hereinafter, may be referred to as "the evaluation method"), and the evaluation method includes the above step (the present evaluation method). In b-1), the step (b-3) of selecting a fertilized egg evaluated as an embryo that develops normally; or an embryo that is likely to develop normally; may be further included. ..
[Normal developmental embryo marker gene group]
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, Thap3, Dr1, Casq2, Dnah11, Nsun5, Tek, Sec1, Zc3h7a, Vgll1, Nfkbid, Rin2, Ube2m, Zfp954, Fkbp9, Rrp1, Otx1, Ap2a2, Rrp1, Otx1, Ap2a2 Gm13977, Mfsd8, Cotl1, Efcab3, Commd4, Kantr, Rnf212, 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, Tmprs2, Prkksh, Wdr74, Nme1, Tex15, Zfp563, Gpn3, F5, Mib2, Gmp49 Dtna, Epcam, Gm28940, Gm44196, Eif3g, Polg2, Gm364, Mtch2, Htt, Ing5, Gm42945, Aknad1, Ehbp1
[Abnormal developmental embryo marker gene group]
Dmd, Tyk2, Nyap2, Mettl22, Manea, Nudt18, Lair1, Slamf1, March3, Ulk2, AI429214, Zfp938, Wdr59, Spryd7, Grin2b, Fsd2, Ciab Reps2, Cdh24, Fech, Agpat1, Plcb3, A230056P14Rik, Spsb4, Dmrtc1b, Raver2, 4921531C22Rik, Tbc1d31, Nutm1, Nek11, Morc2a, Trhr2, Aacs Zmym6, Klf10, Mfsd11, Nudt15, Nitch, Hipk3, Tattn3, Tyw3, Ubl4a, Atp6ap1l, Snx8, Gm12364, Ndufa7, Zfp341, Col4a1, Zfp2m Sybu, Kdm3b, AY512915, Gm13136, Trmt6

Further, as a method for selecting a mammalian embryo of the present invention, one or two or more genes selected from the above-mentioned [normal developmental embryo marker gene group] and [abnormal developmental embryo marker gene group] in the polar body to be detected are used. The step (a) of detecting the transcript and quantifying it as needed; and when the level of the transcript of the [normally developing embryonic marker genes] in the polar body to be detected is higher than the level in the control polar body. Fertilized eggs derived from the polar body to be detected are selected, and when the level of the transcript of [abnormal development embryo marker gene group] in the polar body to be detected is lower than the level in the control polar body, fertilization derived from the polar body to be detected is performed. The method is not particularly limited as long as it is a method including the step of selecting eggs (b-2); (hereinafter, may be referred to as “the selection method”).

The evaluation method and each step in the evaluation method are all performed in vitro, and the steps in vivo (for example, the step of collecting an egg from the ovary of a woman; the fertilized egg or the embryo after its development is transferred to the uterus of a woman. It does not include the process of implanting the uterus (embryo transfer); so-called medical practice by a doctor).

In the present specification, "selecting fertilized eggs" means to separate fertilized eggs to be selected from a group of fertilized eggs in order to distinguish between fertilized eggs to be selected and fertilized eggs not to be selected. Or it means to isolate or remove fertilized eggs that are not to be sorted. The fertilized eggs to be sorted may be separated or isolated one by one, or may be separated or isolated as a group of fertilized eggs to be sorted.

The kit for evaluation and / or selection of mammalian embryos of the present invention is a reverse primer that hybridizes to the transcript of the embryo marker gene group in the polar body to be detected (hereinafter referred to as "reverse primer for RNA detection"). (Sometimes) and / or probe (hereinafter sometimes referred to as "probe for RNA detection"), and / or forward primer and reverse primer that hybridize to the cDNA of the embryo marker gene group in the polar body to be detected. Evaluation and / or evaluation of mammalian embryos comprising a primer set consisting of (hereinafter, sometimes referred to as "the present cDNA detection primer set") and / or a probe (hereinafter, sometimes referred to as "the present cDNA detection probe"). The kit is not particularly limited as long as it is a kit for use in sorting (hereinafter, may be referred to as "the evaluation / sorting kit"), and the evaluation / sorting kit evaluates and / or sorts mammalian embryos. These kits contain components commonly used in this type of detection kit, such as cell lysis buffers, carriers, pH buffers, stabilizers, as well as instruction manuals and mammalian embryos. Attachments such as instructions for evaluation and / or sorting are usually included.

Further, as the biomarker for evaluation and / or selection of the mammalian embryo of the present invention, one or more genes selected from the above-mentioned [normal developmental embryo marker gene group] and [abnormal developmental embryonic marker gene group] can be used. A biomarker for use in the evaluation and / or selection of mammalian embryos, including, the level of transcript of [normally developing embryonic marker genes] in the polar body to be detected is higher than that level in the control polar body. , The biomarker in which the level of the transcript of the [abnormal developmental embryonic marker gene group] in the polar body to be detected is lower than the level in the control polar body is not particularly limited. Using the level of the transcript of the Embryo Marker Genes in the polar body derived from the in vitro fertilized mammalian fertilized egg as an index, it is possible to accurately determine whether or not the mammalian fertilized egg is a normally developing embryonic embryo. Since the embryonic embryos that can be evaluated and normally developed can be selected, the Embryo Marker Gene Group can be a biomarker for use in the evaluation and / or selection of mammalian embryos.

Further, as a method for identifying a gene for evaluation and / or selection of a mammalian embryo of the present invention,
The step (A) of detecting a transcript of a gene in the polar body to be detected, and
When the level of the transcript of the gene in the pole to be detected is higher than that in the control pole, the gene is a normal developmental embryo marker gene; or likely to be a normal developmental embryo marker gene; When the level of transcript of the gene in the polar body to be evaluated and detected is lower than that level in the control pole, the gene is an abnormally developing embryo marker gene; or may be an abnormally developing embryo marker gene. It is not particularly limited as long as it is a method including the step (B) evaluated as high; (hereinafter, may be referred to as “the present identification method”). The number of polar bodies used in the present identification method may be a sufficient number for evaluation, and is at least one (preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more). In consideration of cost effectiveness, for example, it is 200 or less (preferably 160, 130, 100, 90, 80, 70, 60, 50, or 40 or less).

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

The polar body is specifically a second polar body (specifically, a mammalian oocyte whose cell cycle is stopped in the middle of the second meiosis [around the middle of division] [that is, a secondary oocyte]. The polar body is released from the mammalian oocyte [fertilized egg] after the fertilization is started by the entry of the mammalian sperm into the body and the second meiosis is resumed); and / or the first polar body (specifically). Can be mentioned as a polar body generated after the first meiosis of the mammalian primary oocyte together with the mammalian secondary oocyte), and among these, the effect has been demonstrated in this example described later. Therefore, the second polar body can be preferably exemplified. The evaluation method, the selection method, and the identification method (collectively, the present method) are referred to as the first polar body when the second polar body is used as the above polar body. It may include a step of detecting a transcript of the present embryo marker gene group in the 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 these steps are carried out. Since the object of the present method can be achieved without this method, it is preferable that the method does not include these steps.

In the present specification, the "second polar body derived from the in vitro fertilized mammalian fertilized egg" can be, in other words, the "second polar body released from the in vitro fertilized mammalian fertilized egg". In other words, the "second polar body derived from an embryo that does not progress to the vesicle stage" is released from the fertilized egg when the mammalian embryo that does not progress to the vesicle stage is a fertilized egg. It can be called "the second polar body". Usually one mammalian egg releases one second polar body.

In the present specification, the "primary polar body derived from an in vitro fertilized mammalian fertilized egg" is, in other words, "a mammalian secondary oocyte due to the first meiotic division of the mammalian primary oocyte. It can be said to be the "first polar body that occurred with the predifferential state of the mammalian oocyte used for in vitro fertilization of the mammalian fertilized egg", and "the first polar body derived from an embryo that does not progress to the embryonic vesicle stage". In other words, the "polar body" is the "pre-developmental state of the mammalian secondary oocyte (the state before development of the mammalian embryo that does not progress to the embryonic stage) due to the first meiosis of the mammalian primary oocyte. It can be said that the first polar body is produced together with the pre-differentiation state of the mammalian oocyte used for in vitro fertilization of the fertilized egg. Usually one mammalian primary oocyte yields one primary polar body.

As used herein, the term "normally developing embryo" means, more specifically, "an embryo that normally develops at least until the blastocyst stage". Further, in the present specification, the "normally developing embryo marker gene" is used in a polar body derived from a normally developing embryo rather than in a polar body derived from an embryo whose development does not progress until the blastocyst stage. , Means a gene with a high level of transcript. Further, in the present specification, the "abnormally developing embryo marker gene" is used in a polar body derived from an embryo that does not progress to the blastocyst stage, rather than in a polar body derived from a normally developing embryo. , Means a gene with a high level of transcript. More specifically, these marker genes use the level of the transcript of the gene in the polar body derived from the in vitro fertilized mammalian fertilized egg as an index, and the mammal in which the fertilized mammalian egg normally develops. It is possible to accurately evaluate whether or not it is an embryo and select a mammalian embryo that develops normally.

In the present specification, the "gene transcript" is a DNA-dependent RNA polymerase (simply, "RNA polymerase") using an antisense strand as a template among double-stranded DNA encoding a gene existing in genomic DNA. It means RNA synthesized (transcribed) by (also referred to as), and is usually a single-stranded polynucleotide. The RNA includes mRNA that is translated into protein as well as RNA that is not translated into protein (non-coding RNA [ncRNA]). The RNA also includes an early transcript (immature mRNA or ncRNA), a mature transcript produced by post-transcriptional processing (splicing) (mature mRNA or ncRNA), and splicing variants thereof.

As used herein, the term "gene cDNA" means a cDNA (complementary DNA) synthesized from the RNA of a gene by a PCR method using reverse transcriptase, and is usually a double-stranded polynucleotide. ..

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, and cats such as dogs and cats. Primates such as humans, monkeys, red-tailed monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees can be exemplified, and among them, humans, cows, and pigs can be preferably exemplified.

In the in vitro fertilized mammalian fertilized egg, for example, at least one collected mammalian sperm and at least one collected mammalian egg (that is, a secondary egg mother cell) are naturally mixed in a culture medium. Prepared by in vitro fertilization to be fertilized (so-called in vitro fertilization by in vitro fertilization) or by injecting mammalian sperm into at least one collected mammalian egg by micromanipulation (that is, microinsemination). Can be done. The first polar body attached to the mammalian egg (that is, the secondary oocyte) before in vitro fertilization and the second polar body released from the fertilized mammalian egg after in vitro fertilization are recovered by micromanipulation, respectively. -Can be isolated.

In the above steps (a) and (A), as a method for detecting a transcript (that is, RNA) of a gene in a polar body to be detected, a method for directly detecting RNA of a gene and a method using RNA of a gene as a template are used. A method of (indirectly) detecting the synthesized cDNA can be mentioned. Examples of the method for detecting RNA of such a gene include RT (Reverse Transcription) -PCR method using the RNA detection reverse primer and the like; Northern blotting method, microarray method and ISH method using the RNA detection probe and the like. Etc. can be mentioned. Further, as a method for detecting the cDNA of the above gene, for example, a LAMP method or a PCR method using the Primer Set for cDNA Detection of the present case (for example, a real-time PCR method [intercalator method, 5'-nuclease method, cycling probe method). Etc.], ddPCR method); LCR method; sequencing method using next-generation sequencer; Southern hybridization method using the probe for cDNA detection, microarray method, ISH method; etc. , A sequencing method using a next-generation sequencer can be preferably exemplified.

As used herein, the term "next generation sequencer" is also referred to as a second generation sequencer, and means a nucleotide sequencing device capable of simultaneously and simultaneously determining the nucleotide sequences of tens of millions of DNA fragments. As the sequencing principle in the next-generation sequencer, for example, the bridge PCR method and the Sequencing-by-synthesis method are used to amplify the DNA to be detected on the flow cell and perform sequencing while synthesizing it, or the emulsion PCR method and DNA. A principle can be mentioned in which sequencing is performed by a pyrosequencing method in which sequencing is performed by measuring the amount of pyrophosphate released during synthesis. More specific examples of the next-generation sequencer include Illumina's MiniSeq, MiSeq, NextSeq, HiSeq and HiSeq X series, and Roche's Roche 454 GS FLX sequencer.

In the present invention, the level (amount) of the transcript of the gene in the polar body may be an absolute value (concentration [number of copies]) or a relative value, and when it is a relative value, for example, Gapdh or Relative values using the transcription level of housekeeping genes such as β-actin as an internal standard can be mentioned.

In the present invention, the level of the transcript of the gene in the polar body to be detected and the level of the transcript of the gene 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 and a relative value, respectively. The level value of the transcript of the gene in the control polar body may be measured each time the method is carried out, or a value measured in advance may be used. Further, the polar body is preferably prepared by substantially the same method for both comparisons. Further, the method for detecting the transcript of the gene is preferably substantially the same for both comparisons.

In the step (b-1) of the evaluation method, when the level of the transcript of [normal development embryo marker gene group] in the polar body to be detected is not higher than the level in the polar body to be detected, it is derived from the polar body to be detected. The fertilized egg is evaluated as an embryo that does not develop normally; or an embryo that is unlikely to develop normally; and the level of the transcript of [abnormal development embryo marker gene group] in the polar body to be detected is controlled. When not below that level in the polar body, the fertilized egg 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 the step (b-2) of the selection method, when the level of the transcript of [normally developing embryo marker gene group] in the polar body to be detected is not higher than the level in the polar body to be detected, the polar body to be detected is detected. When the level of the transcript of [abnormal developmental embryo marker gene group] in the polar body to be detected is not lower than the level in the polar body to be detected without selecting the fertilized egg of the origin, the fertilized egg derived from the polar body to be detected is selected. Do not sort.

Further, in the step (B) of the identification method, when the level of the transcript of the gene in the electrode to be detected is not higher than the level in the control electrode, the gene is not a normally developing embryo marker gene; It is unlikely that it is a normally developing embryo marker gene; and when the level of transcript of the gene in the pole to be detected is not lower than that level in the control pole, the gene is an abnormally developing embryo marker gene. Not; or unlikely to be an abnormally developing embryo marker gene; In step (B) of the identification method, when evaluating whether the level of the transcript of the gene in the polar body to be detected is higher or lower than the level in the control polar body, it is derived from an embryo that develops normally. The level of the transcript of the Embryo Marker Genes in the polar body can be used as a positive control.

A threshold (cutoff value) to evaluate (determine) whether the level of the gene transcript in the pole to be detected is higher or lower than the level of the gene transcript in the control pole. Can be set to any value, and such thresholds include, for example, the mean value of the gene transcript level in the control electrode; mean value + standard deviation (SD); mean value + 2SD; mean value + 3SD; median. Value; Interquartile range, etc. can be mentioned. In addition, the threshold is set so that the sensitivity (the rate at which normally developing embryos can be correctly judged as positive) and the specificity (the rate at which embryos that do not progress to the blastocyst stage can be correctly judged as negative) are high. It can also be calculated using the ROC (Receiver Operating Characteristic) curve using statistical analysis software based on the data on the level of the transcript of the gene in the polar body to be detected and the data on the level of the transcript of the gene in the control polar body. it can.

In the above [normal development embryo marker gene group] and [abnormal development embryo marker gene group], the notation of the gene name follows the notation method of the gene name in the mouse for convenience, but in mammals other than the mouse, the ortholog is used. Equivalent to. For example, Cdk2ap1 corresponds to CDK2AP1 in humans, cattle, and pigs, Dmd corresponds to DMD in humans, cattle, and pigs, and AW551984 corresponds to VWA5A in humans and pigs.

Among the above [normal development embryo marker gene group], the normal development embryo marker gene derived from human, bovine, and pig is, for example, 80% or more sequence identical to the transcript (RNA) of the gene shown in Table 1 below. RNA containing a nucleotide sequence having sex can be mentioned. The RNA nucleotide sequence of the gene in Table 1 can be understood from the contents described in the item of "NCBI Reference Sequence" in Table 1. For example, in the case of Cdk2ap1, the human (NM_001270433) is NCBI (National Center for). Biotechnology Information) is the mRNA of human-derived CDK2AP1 registered with the number NM_001270433 (specifically, the mRNA consisting of the nucleotide sequence of SEQ ID NO: 1), and the bovine (NM_001076369) is registered with NCBI with the number NM_001076369. The bovine-derived CDK2AP1 mRNA (specifically, the mRNA consisting of the nucleotide sequence of SEQ ID NO: 2), and the pig (NM_001190166) is the pig-derived CDK2AP1 mRNA registered in NCBI with the number NM_001190166 (specifically). Is an mRNA consisting of the nucleotide sequence of SEQ ID NO: 3).

Among the above [abnormal development embryo marker gene group], the abnormal development embryo marker gene derived from human, bovine, and pig is, for example, 80% or more sequence identical to the transcript (RNA) of the gene shown in Table 2 below. RNA containing a nucleotide sequence having sex can be mentioned. The nucleotide sequence of the RNA of the gene in Table 2 can be understood from the contents described in the item of "NCBI Reference Sequence" in Table 2. For example, in the case of Dmd, the human (NM_000109) is numbered NM_000109 in NCBI. The human-derived DMD mRNA registered in (specifically, the mRNA consisting of the nucleotide sequence of SEQ ID NO: 4), and the bovine (XM_024988359) is the bovine-derived DMD mRNA registered in NCBI with the number XM_024988359. (Specifically, mRNA consisting of the nucleotide sequence of SEQ ID NO: 5), and pig (NM_001012408) is RNA of pig-derived DMD registered in NCBI with the number NM_001012408 (specifically, mRNA of SEQ ID NO: 6). It is an mRNA consisting of a nucleotide sequence. Since there are many single nucleotide polymorphisms (SNPs) of genes in humans, bovines, and pigs, RNA containing a nucleotide sequence having 80% or more sequence identity with the above-mentioned specific nucleotide sequence. Can exist. In addition, the nucleotide sequence registered in NCBI may be the latest sequence as of the filing date or priority date of the present application, or may be an updated sequence after the filing date or priority date of the present application. .. In the present specification, as the nucleotide sequence of mRNA, "t (thymine residue)" in the nucleotide sequence of SEQ ID NOs: 1 to 6 in the sequence listing is applied as "u (uracil residue)".

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

As the embryo marker gene group, Cdk2ap1 and / or Dmd can be preferably exemplified because the effect has been demonstrated in more detail in this example described later.

The reverse primer for RNA detection is a primer that specifically hybridizes to the transcript (RNA) of the embryo marker gene group and is preferably capable of producing an amplification product by RT-PCR, and is present in genomic DNA. Corresponds to a part of the antisense strand encoding the embryonic marker gene group.

The RNA detection probe specifically hybridizes to the transcript (RNA) of the embryonic marker gene group, and is preferably a probe capable of detection by a method such as Northern blotting method, microarray method, or ISH method. It corresponds to a part of the antisense strand encoding the Embryonic Marker Genes present in the genomic DNA.

The forward primer and reverse primer in the primer set for cDNA detection hybridize with the antisense strand and the sense strand of the cDNA of the embryo marker gene group, respectively, and can be detected by a method such as the LAMP method or the PCR method, respectively. It is a primer and corresponds to a part of the sense strand and a part of the antisense strand that encode the embryo marker gene group present in the genomic DNA, respectively.

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 primer or probe may be DNA, RNA, or a DNA / RNA chimera, but is preferably DNA. In addition, in some or all of them, PNA (polyamide nucleic acid, peptide nucleic acid), LNA (registered trademark, locked nucleic acid, Bridged Nucleic Acid, crosslinked nucleic acid), ENA (registered trademark, 2'-O, 4'-C- The nucleotides may be substituted with artificial nucleic acids such as Ethylene-bridged nucleic acids), GNA (Glycerol nucleic acid, glycerol nucleic acid), and TNA (Threosenucleic acid, Treose nucleic acid).

In the present invention, primers and probes can use labeled substances for visualizing and / or quantifying transcripts or cDNAs of genes to be detected. Labeling substances in such labeled substances include peroxidase (eg, horseradishperoxidase), alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucos-6-phosphate dehydrogenase, alcohol dehydrogenase, malic acid dehydrogenase, penicillinase, etc. Enzymes such as catalase, apoglucose oxidase, urease, luciferase or acetylcholine esterase; fluorescent substances (eg, allophicocyanin [APC], phycoerythrin [PE], FITC [fluorescein isothiocyanate], Alexa Fluor 488, Alexa Fluor 647, Alexa Fluor 700, Alexa Fluor -TexasRed, PE-Cy5, PE-Cy7); Green Fluorescence Protein (GFP), Cyan Fluorescence Protein (CFP), Blue Fluorescence Protein (BFP), Yellow Fluorescence Protein ; YFP), red fluorescent protein (red fluorescence protein; RFP), luciferase (luciferase) fluorescence of such ^proteins; 3 ^H, 14 ^C, radioisotopes such as ¹²⁵ I or ¹³¹ I; biotin, avidin or chemiluminescent substances; A combination 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], a combination of VIC and MGB [Minor Groove Binder]). Can be mentioned.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these examples. In the following examples, the mice are light-controlled (light period; 6:30 to 20:30, dark period; 20:30 to 6:30), and in an environment maintained at room temperature of 26 ° C. It was bred and tested in an experiment. The breeding of mice and the use of experiments were carried out in accordance with the guidelines for animal experiments established by the Experimental Animal Subcommittee of the Faculty of Biophysical Engineering, Kinki University. The operation of transferring the egg, the fertilized egg (fertilized egg), and the second polar body was performed using a hematocrit capillary plain (manufactured by Hirschmann) slightly thicker than the diameter of the egg. Isolation of the second polar body was carried out under a microscope (IX73, manufactured by Olympus) using a micromanipulator (manufactured by Naricige). Sperm were inoculated with a microchip using a Pipetman. In addition, sperm preculture and insemination were carried out under the conditions of _{5% CO 2} /20% O _{2 and 37 ° C.}

Example 1: Analysis of the transcript of the gene in the fertilized egg and the transcript of the gene in the second polar body released from the fertilized egg 1. Materials and Methods 1-1 Culture Solution The sperm culture before insemination (preculture) and insemination are carried out in a 200 μL drop-shaped mHTF culture solution placed on a culture dish (manufactured by BD Falcon). I went there. In addition, embryonic development of fertilized eggs was carried out in a 10 μL drop-shaped mKSOM culture medium placed on the culture dish. The drop-shaped culture solution was covered with mineral oil (manufactured by SIGMA ALDRICH) in order to suppress evaporation of the culture solution and sudden changes in temperature.

1-2 In vitro fertilization (so-called in vitro fertilization by fertilization) and embryogenesis In vitro fertilization and embryogenesis were performed according to the following procedures [1] to [6].
[1] DBA / 2 strain male mice (manufactured by Claire Japan) were sacrificed by cervical dislocation, and then the epididymal tail was collected. The tube at the tail of the epididymis was incised with a small straight scissors, and the sperm mass was collected with an anatomical 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 Claire Japan) were intraperitoneally administered with equine chorionic gonadotropin (PMSG) (manufactured by Aska Pharmaceutical Co., Ltd.) 7.5 (IU). .. After 48 hours, human chorionic gonadotropin (hCG) (manufactured by Aska Pharmaceutical Co., Ltd.) was intraperitoneally administered 7.5 (IU) to induce excessive ovulation.
[3] Fifteen hours after administration of hCG, female mice were sacrificed by cervical dislocation, and the ampulla of uterine tube was collected. The collected ampulla of uterine tube was immersed in mineral oil of a dish for insemination, and a cumulus cell-egg complex (COC) was collected from the ampulla of uterine tube. The removed COC was placed in a drop-shaped mHTF culture medium.
[4] The sperm mass precultured in the above [1] was transferred to a drop-shaped mHTF culture solution containing COC prepared in the above [3] so as to have 200 sperms / μL, and insemination was performed. ..
[5] Two hours after insemination, 12 μL of 10 mg / mL hyaluronidase (manufactured by SIGMA ALDRICH) was added to the drop-shaped mHTF culture solution, and the cumulus cells were exfoliated. Eggs were transferred into 35 μL drop-shaped mKSOM culture medium. This operation was performed until the cumulus cells and floating sperm were removed.
[6] Six hours after insemination, an egg in which both male and female pronuclei were clearly observed was regarded as a fertilized egg. Eggs for which no pronucleus can be confirmed are regarded as unfertilized eggs, fertilized eggs for which three or more pronuclei can be confirmed are regarded as polysperm fertilized eggs, and eggs for which only one pronucleus can be confirmed are regarded as parthenogenetic eggs. ..

1-3 Preparation and storage of the second polar body sample and the fertilized egg sample The second polar body sample and the fertilized egg sample were prepared and stored according to the following procedures [1] to [3].
[1] The fertilized egg obtained by the procedure [6] of the above "1-2" was further cultured for 1 hour, and it was confirmed that the second polar body (see FIG. 1A) was released from the fertilized egg. After that, the fertilized egg and the second polar body were isolated and recovered one by one by micromanipulation.
[2] After washing each recovered second electrode with 1 × PBS (-) containing 0.1% BSA (manufactured by SIGMA ALDRICH) (hereinafter referred to as “0.1% BSA-PBS”). , 1 μL of 10 × Reaction Buffer (10 × Lysis Buffer containing RNase inhibitor) in SMART-seq v4 Ultra Low Input RNA Kit (TaKaRa) and 8.5 μL of UltraPure DNase / RNase-Free Distilled Water (Invitrogen) A mixed solution with (hereinafter referred to as “RNase inhibitor-containing solution”) is added in advance into a PCR tube, and 1 μL of 0.1% BSA-PBS containing each secondary electrode is added thereto. A bipolar sample was prepared. In addition, each collected fertilized egg is treated with an acidic tie load solution to dissolve the zona pellucida, and then an RNase inhibitor-containing solution is added in advance in a PCR tube, and each fertilized egg is contained therein. .1 μL of 1% BSA-PBS was added to prepare a fertilized egg sample.
[3] The second polar body sample and the fertilized egg sample were instantly frozen in liquid nitrogen and then stored in a freezer at −80 ° C.

1-4 Preparation of cDNA library and analysis of RNA sequence (RNA-seq) SMART-seq v4 Ultra Low Input RNA Kit (SMART-seq v4 Ultra Low Input RNA Kit) CDNA was synthesized and amplified using TaKaRa). Considering that there is a large difference between the amount of maternal transcript contained in the fertilized egg and the amount of maternal transcript contained in the secondary polar body, in the secondary polar body sample, 12 μM 3'SMART-Seq, which is a reverse transcription primer, is considered. Add 1 μL of CDS Primer II A (TaKaRa) and 1 μL of UltraPure DNase / RNase-FreeDistilled Water (Invitrogen), and in the fertilized egg sample, add 2 μL of the above 3'SMART-Seq CDS Primer II A and at 72 ° C. After treatment for 3 minutes, the mixture was allowed to stand on ice for 2 minutes to modify the higher-order structure and perform primer annealing. Further, in order 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 diluted ERCC Spike-In. RNA (calculated assuming that the total RNA amount of the fertilized egg sample is 350 pg. After diluting the stock solution of ERCC Spike-In RNA 1 / 100,000 times, 7 μL of diluted ERCC Spike-In RNA-containing solution and 3 μL of UltraPure DNase / RNase- After adding 1 μL (prepared by mixing with Free Distilled Water), the higher-order structure was modified and primer annealing was performed by the above treatment. Further, in order to prepare a secondary polar body sample containing ERCC Spike-In RNA, 1 μL of diluted ERCC Spike-In RNA (of the secondary polar body sample) was added to the secondary polar body sample prepared in the above “1-3”. Calculated assuming that the total amount of RNA is 20 pg / μL. After diluting the stock solution of ERCC Spike-In RNA 1/1,000,000 times, 2 μL of diluted ERCC Spike-In RNA-containing solution and 3 μL of UltraPure DNase / RNase-Free (Produced by mixing Distilled Water) and 12 μM 3'SMART-Seq CDS Primer II A 1 μL were added, and higher-order structure modification and primer annealing were performed by the above treatment. Then, 7.5 μL of First strand mix was added to each sample and 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 to synthesize and amplify double-stranded cDNA. The number of PCR cycles was 14 to 15 cycles for the fertilized egg sample and 19 to 20 cycles for the second polar body sample. After completion of amplification, cDNA was purified using AM Pure XP beads (manufactured by BECKMAN COULTER) and 80% ethanol. A cDNA sample diluted to a concentration of 200 pg / μl was fragmented and a tag sequence was added using the Nextera DNA Sample Preparation Kit (manufactured by Illumina). Amplification and fragmentation of cDNA was confirmed by Bioanalyzer and High Sensitivity DNA kit (both manufactured by Agilent). Then, RNA-seq analysis was performed using NextSeq (manufactured by Illmina).

2. Results Transcripts of 16397 and 11107 genes were identified from the fertilized egg sample and the second polar body sample, respectively (see FIG. 1B). Further, among the transcripts of 11107 genes derived from the second polar body sample, the transcripts of 10690 genes corresponding to 96.2% are the transcripts of the genes derived from the fertilized egg sample (that is, maternal transcripts). ) (See Fig. 1B).
This result indicates that the maternal transcript of a fertilized egg can be analyzed minimally invasively by analyzing the transcript of the gene in the second polar body.

Example 2: Identification of a group of marker genes (embryo marker genes) capable of evaluating normally developing embryos Since most of the transcripts of genes in the second polar body were found to be maternal transcripts of fertilized eggs, embryos We attempted to identify maternal transcripts that could assess the quality of the gene from the transcripts of the genes in the second polar body. Specifically, the second polar body is recovered from the fertilized egg obtained by the procedure [6] of the above "1-2" by the procedure [1] of the above "1-3", and after the recovery of the second polar body. Fertilized eggs were cultured for 4 to 5 days, embryos that developed to the vesicle stage embryo were evaluated as normal developmental embryos, and embryos that did not develop to the vesicle stage embryo (for example, development before the blastocyst stage) Embryogens with arrest or delayed development are evaluated as abnormally developed embryos, and the second polar bodies of normal-developed embryos (n = 5) and abnormally-developed embryos (n = 5) are used. After performing RNA-seq analysis according to the method described in the above item "1-4", the transcript of the gene that fluctuates between the secondary polar bodies derived from both embryos (maternal transcription) by bioinformatic analysis. Product) was identified. The bioinformatics analysis was carried out according to the following flow. First, from the sequence read obtained by NextSeq, extra sequences not used for analysis such as adapter sequences were removed (trimmed) using trim_galore or cutadapt software. The trimmed sequence reads were then mapped to the mouse mm10 genome using STAR software. The expression level of each gene was then counted using featureCounts to calculate the number of transcripts expressed in the fertilized egg and the second polar body. Then, in order to identify the gene showing the expression variation between the normal development embryo and the abnormal development embryo, the expression variation gene was calculated using DESeq2 (FDR: False Discovery Rate ≤ 0.05).

Transcripts of 9705 types of genes were identified in the polar body derived from normally developing embryos, and among them, a significant increase was observed as compared with the secondary polar body derived from abnormally developing embryos, as shown in Table 3. Transcripts of 113 genes (ie, normal developmental embryonic marker genes) were identified. In FIGS. 2 to 6, 30 types of normally developing embryonic marker genes shown in Table 3 (Trappc12 [No. 1], Kif13A [No. 2], Redrum [No. 3], Empep [No. 4], Gart [No. 5], Rogdi [No. 6], Labl3 [No. 8], Prdx3 [No. 10], Spire2 [No. 14], Shipa1 [No. 17], Atxn7l1 [No. 24], Ube2m [No. 36], Rrp1 [No. 39], Otx1 [ No. 40], Phf2 [No. 47], Phrf1 [No. 48], Rnf212 [No. 56], Ankmy2 [No. 57], Popdc3 [No. 58], Zscan18 [No. 67], Krt8 [No. 75], Wdr74 [No. 85] ], Nme1 [No. 86], Tex15 [No. 87], Mib2 [No. 91], Pbx4 [No. 95], Ddx20 [No. 97], Cdk2ap1 [No. 99], Pcsk5 [No. 100], and Ing5 [No. 110]. ) Is shown in a graph plotting the level of the transcript. In addition, in the bipolar body derived from abnormally developed embryos, transcripts of 9411 kinds of genes were identified, and among them, a significant increase was observed as compared with the secondary polar body derived from normally developed embryos, Table 4 Transcripts of 80 types of genes (ie, abnormally developed embryonic marker genes) shown in the above were identified. In FIGS. 7 to 10, 23 types of abnormally developing embryo marker genes shown in Table 4 (Tyk2 [No. 1], Mettl22 [No. 3], Ulk2 [No. 9], Grin2b [No. 14], Adm [No. 19], Tbc1d25 [number 21], Fech [number 27], Plcb3 [number 29], Spsb4 [number 31], River2 [number 33], Nek11 [number 37], Zfp507 [number 44], Lias [number 46], Tubd1 [ No. 49], Zmym6 [No. 50], Mfsd11 [No. 52], Nudt15 [No. 53], Slc15a2 [No. 67], Parp4 [No. 69], Fam20b [No. 71], Mau2 [No. 74], Kdm3b [No. 76] ], And the graph plotting the level of the transcript of Dmd [No. 79]) is shown.

These results show that in the polar body derived from fertilized eggs such as the second polar body released from the fertilized egg, the level of the transcript of the normally developing embryo marker gene is higher than the level in the second polar body derived from the abnormally developing embryo. The fertilized egg is an embryo in which the fertilized egg normally develops based on whether or not the embryo is high and the level of the transcript of the abnormally developed embryo marker gene is lower than the level in the second polar body derived from the abnormally developed embryo. It shows that it can be evaluated whether or not.

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 magnification" in Table 3 indicates the amount of maternal transcripts in the second polar body derived from normal embryos with respect to the amount of maternal transcripts in the second polar body derived from abnormally developed embryos, as an index of 2. Is. For example, when the "log2 magnification" in the table is 1, the amount of maternal transcript in the second polar body derived from normally developing embryos is greater than the amount of maternal transcripts in the second polar body derived from abnormally developing embryos. It means that it is 2 (= 2 ^{1) times.}

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 magnification" in Table 4 indicates the amount of maternal transcript in the second polar body derived from abnormally developed embryos as an index of 2 with respect to the amount of maternal transcripts in the second polar body derived from normal embryos. Is. For example, when the "log2 magnification" in the table is 1, the amount of maternal transcript in the second polar body derived from abnormally developing embryos is greater than the amount of maternal transcripts in the second polar body derived from abnormally developing embryos. It means that it is 2 (= 2 ^{1) times.}

Example 3. Reverse transcription (RT) -quantitative polymerase chain reaction (qPCR) analysis Differences in the amount of transcripts of the Embryogenic Marker Genes between the second polar body derived from normally developing embryos and the second polar body derived from abnormally developing embryos , 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 developing embryo marker genes, and Cdk2ap1, which is one of the normally developing embryo marker genes. In the analysis of the transcript of Dmd, the number of the second polar body derived from the normal development embryo and the second polar body derived from the abnormal development embryo used in the analysis was increased to 37. In addition, in the analysis of the transcript of Cdk2ap1, the number of the second polar body derived from the normal development embryo and the second polar body derived from the abnormal development embryo used in the analysis was increased to 96.

1. 1. Method 1-1 Synthesis of cDNA from 2nd polar body Samples of 10.5 μL of 2nd polar body sample derived from normal developmental embryo and 2nd polar body sample derived from abnormally developing embryo were prepared in 8.0 μL for RT + and RT-, respectively. For RT-, 4 μL of UltraPure DNase / RNase-Free Distilled Water was added to make a total of 6.5 μL. In order to increase the specificity of the primer in RT-qPCR, the Cdk2ap1 detection reverse primer (see Table 5) 100 μM to be detected, the Dmd detection reverse primer (see Table 5) 100 μM to be detected, and the internal standard Gapdh 100 μM of reverse primer for detection (see Table 5) and 100 μM of oligo (dT) 15 primer (manufactured by Sigma) were mixed by 2 μL each, and UltraPureDNase / RNase-Free Distilled Water was added so as to make a total of 840 μL. Specific Primer (GSP) was prepared. To the second polar body sample, 1 μL of GSP and 1 μL of a 10 nM dNTP mixture were added, incubated at 65 ° C. for 5 minutes using a thermal cycler, placed on ice, and rapidly cooled for 1 minute or more. Then, 8.5 μL of RT Mixture (manufactured by Invitrogen) was added to each sample, and then 0.5 μL of SuperScript III Reverse Transcriptase (manufactured by Thermo Fisher) was added only to the second polar body sample for RT +. Each sample was treated with a thermal cycler at 50 ° C. for 50 minutes and at 85 ° C. for 5 minutes to synthesize cDNA.

1-2 qPCR
A primer set consisting of a mixed solution for qPCR (SYBR Premix EX Taq [Takara]; ROX reference dye [Takara]; and a forward primer for Gapdh detection and a reverse primer for Gapdh detection [10 μM each; see Table 5]; And a primer set consisting of a forward primer for Cdk2ap1 detection and a reverse primer for Cdk2ap1 detection [10 μM each; see Table 5]; or a primer set consisting of a forward primer for Dmd detection and a reverse primer for Dmd detection [10 μM each; see Table 5]. , And 4 μL of synthetic cDNA diluted 2-fold as a template were mixed, and RT-qPCR was performed using a 7300 real-time PCR system (manufactured by Applied Biosystems). Experiments were conducted by preparing three reaction solutions for each sample. The calibration curve in qPCR was reversed using an oligo (dT) 20 primer (manufactured by Thermo Fisher) after RNA was extracted from the ovary of a DBA / 2 strain female mouse using the RNeasy Mini Kit (manufactured by Qiagen). The copied cDNA was prepared by diluting it 1000 times using a template.

2. Results FIG. 11 shows the results of analysis of a transcript of Dmd, which is one of the abnormally developed embryo marker genes. As shown in FIG. 11, the proportion of the second polar body in which the transcript of Dmd was detected was about 30% in the second polar body derived from the normally developing embryo, whereas it was derived from the abnormally developing embryo. In the second polar body, it was as high as about 70%.

This result shows whether the fertilized egg normally develops in the polar body derived from the fertilized egg such as the second polar body released from the fertilized egg, using the presence or absence of the transcript of the abnormally developing embryo marker gene as an index. It shows that it can be evaluated, and supports the results of Examples 2 and 3 above.

FIG. 12 shows the results of analysis of the transcript of Cdk2ap1, which is one of the normally developing embryonic marker genes. As shown in FIG. 12, the level of the transcript of Cdk2ap1 was significantly higher in the second polar body derived from the normally developing embryo than in the second polar body derived from the abnormally developing embryo. Furthermore, when a binomial logistic analysis was performed based on the data used to obtain the analysis result of FIG. 12, it was confirmed that a normally developing embryo could be determined with a specificity of 85%.

This result shows that in the polar body derived from the fertilized egg such as the second polar body released from the fertilized egg, the level of the transcript of the normally developing embryo marker gene is higher than that in the second polar body derived from the abnormally developing embryo. It is shown that it is possible to evaluate whether or not the fertilized egg is an embryo that normally develops by using whether or not it is high, which supports the result of Example 3 above.

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

Claims (14)

A method for evaluating a mammalian embryo, which comprises the following steps (a) and (b-1).
(A) Transcripts of one or more genes selected from the following [normal developmental embryo marker gene group] and [abnormal developmental embryonic marker gene group] in the polar body derived from in vitro fertilized mammalian fertilized eggs. Step to detect;
[Normal developmental embryo marker gene group]
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, Thap3, Dr1, Casq2, Dnah11, Nsun5, Tek, Sec1, Zc3h7a, Vgll1, Nfkbid, Rin2, Ube2m, Zfp954, Fkbp9, Rrp1, Otx1, Ap2a2, Rrp1, Otx1, Ap2a2 Gm13977, Mfsd8, Cotl1, Efcab3, Commd4, Kantr, Rnf212, 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, Tmprs2, Prkksh, Wdr74, Nme1, Tex15, Zfp563, Gpn3, F5, Mib2, Gmp49 Dtna, Epcam, Gm28940, Gm44196, Eif3g, Polg2, Gm364, Mtch2, Htt, Ing5, Gm42945, Aknad1, Ehbp1
[Abnormal developmental embryo marker gene group]
Dmd, Tyk2, Nyap2, Mettl22, Manea, Nudt18, Lair1, Slamf1, March3, Ulk2, AI429214, Zfp938, Wdr59, Spryd7, Grin2b, Fsd2, Ciab Reps2, Cdh24, Fech, Agpat1, Plcb3, A230056P14Rik, Spsb4, Dmrtc1b, Raver2, 4921531C22Rik, Tbc1d31, Nutm1, Nek11, Morc2a, Trhr2, Aacs Zmym6, Klf10, Mfsd11, Nudt15, Nitch, Hipk3, Tattn3, Tyw3, Ubl4a, Atp6ap1l, Snx8, Gm12364, Ndufa7, Zfp341, Col4a1, Zfp2m Sybu, Kdm3b, AY512915, Gm13136, Trmt6
(B-1) When the transcript of the gene detected in step (a) is a transcript of [normal development embryo marker gene group], the level of the transcript of the gene detected in step (a) is the scutellum. When it is higher than the level in the polar body derived from an embryo that does not progress to the blastocyst stage, the fertilized egg is evaluated as a normally developing embryo or an embryo likely to develop normally.
When the transcript of the gene detected in step (a) is a transcript of [abnormal development embryo marker gene group], the level of the transcript of the gene detected in step (a) develops until the blastocyst stage. A step of assessing that a fertilized egg is or is likely to develop normally when it is below that level in a polar body derived from a non-progressive embryo; A method for selecting mammalian embryos, which comprises the following steps (a) and (b-2).
(A) Transcripts of one or more genes selected from the following [normal developmental embryo marker gene group] and [abnormal developmental embryonic marker gene group] in the polar body derived from in vitro fertilized mammalian fertilized eggs. Step to detect;
[Normal developmental embryo marker gene group]
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, Thap3, Dr1, Casq2, Dnah11, Nsun5, Tek, Sec1, Zc3h7a, Vgll1, Nfkbid, Rin2, Ube2m, Zfp954, Fkbp9, Rrp1, Otx1, Ap2a2, Rrp1, Otx1, Ap2a2 Gm13977, Mfsd8, Cotl1, Efcab3, Commd4, Kantr, Rnf212, 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, Tmprs2, Prkksh, Wdr74, Nme1, Tex15, Zfp563, Gpn3, F5, Mib2, Gmp49 Dtna, Epcam, Gm28940, Gm44196, Eif3g, Polg2, Gm364, Mtch2, Htt, Ing5, Gm42945, Aknad1, Ehbp1
[Abnormal developmental embryo marker gene group]
Dmd, Tyk2, Nyap2, Mettl22, Manea, Nudt18, Lair1, Slamf1, March3, Ulk2, AI429214, Zfp938, Wdr59, Spryd7, Grin2b, Fsd2, Ciab Reps2, Cdh24, Fech, Agpat1, Plcb3, A230056P14Rik, Spsb4, Dmrtc1b, Raver2, 4921531C22Rik, Tbc1d31, Nutm1, Nek11, Morc2a, Trhr2, Aacs Zmym6, Klf10, Mfsd11, Nudt15, Nitch, Hipk3, Tattn3, Tyw3, Ubl4a, Atp6ap1l, Snx8, Gm12364, Ndufa7, Zfp341, Col4a1, Zfp2m Sybu, Kdm3b, AY512915, Gm13136, Trmt6
(B-2) When the transcript of the gene detected in step (a) is a transcript of [normal development embryo marker gene group], the level of the transcript of the gene detected in step (a) is the scutellum. When the level is higher than the relevant level in the polar body derived from an embryo that does not progress to the spore stage, the fertilized egg is selected.
When the transcript of the gene detected in step (a) is the amount of transcript of [abnormal development embryo marker gene group], the level of transcript of the gene detected in step (a) develops until the blastocyst stage. The step of sorting the fertilized egg when it is lower than the level in the polar body derived from the embryo that does not progress; The method according to claim 1 or 2, wherein the gene is Cdk2ap1 or Dmd. The method according to any one of claims 1 to 3, wherein the polar body is a second polar body. The method according to any one of claims 1 to 4, wherein in the step (a), the transcript of the gene is detected by a sequencing method using a next-generation sequencer. Hybridizes to transcripts of one or more genes selected from the following [normal developmental embryo marker genes] and [abnormal developmental embryonic marker genes] in the polar body derived from in vitro fertilized mammalian fertilized eggs. For evaluation and / or selection of mammalian embryos comprising a reverse primer and / or a probe and / or a primer set consisting of a forward primer and a reverse primer that hybridize to the cDNA of the gene and / or a probe. Kit for.
[Normal developmental embryo marker gene group]
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, Thap3, Dr1, Casq2, Dnah11, Nsun5, Tek, Sec1, Zc3h7a, Vgll1, Nfkbid, Rin2, Ube2m, Zfp954, Fkbp9, Rrp1, Otx1, Ap2a2, Rrp1, Otx1, Ap2a2 Gm13977, Mfsd8, Cotl1, Efcab3, Commd4, Kantr, Rnf212, 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, Tmprs2, Prkksh, Wdr74, Nme1, Tex15, Zfp563, Gpn3, F5, Mib2, Gmp49 Dtna, Epcam, Gm28940, Gm44196, Eif3g, Polg2, Gm364, Mtch2, Htt, Ing5, Gm42945, Aknad1, Ehbp1
[Abnormal developmental embryo marker gene group]
Dmd, Tyk2, Nyap2, Mettl22, Manea, Nudt18, Lair1, Slamf1, March3, Ulk2, AI429214, Zfp938, Wdr59, Spryd7, Grin2b, Fsd2, Ciab Reps2, Cdh24, Fech, Agpat1, Plcb3, A230056P14Rik, Spsb4, Dmrtc1b, Raver2, 4921531C22Rik, Tbc1d31, Nutm1, Nek11, Morc2a, Trhr2, Aacs Zmym6, Klf10, Mfsd11, Nudt15, Nitch, Hipk3, Tattn3, Tyw3, Ubl4a, Atp6ap1l, Snx8, Gm12364, Ndufa7, Zfp341, Col4a1, Zfp2m Sybu, Kdm3b, AY512915, Gm13136, Trmt6 The kit according to claim 6, wherein the gene is Cdk2ap1 or Dmd. The kit according to claim 6 or 7, wherein the polar body is a second polar body. A biomarker for evaluation and / or selection of mammalian embryos, which comprises one or more genes selected from the following [normal developmental embryo marker gene group] and [abnormal developmental embryonic marker gene group].
When the gene is a gene of [normal development embryo marker gene group], the level of the transcript of the gene in the polar body derived from the in vitro fertilized mammalian fertilized egg progresses to the blastocyst stage. Higher than that level in polar bodies derived from non-embryos,
When the gene is a gene of [abnormal development embryo marker gene group], the level of the transcript of the gene in the polar body derived from the in vitro fertilized mammalian fertilized egg progresses to the blastocyst stage. The biomarker, characterized in that it is lower than that level in a polar body derived from a non-embryo.
[Normal developmental embryo marker gene group]
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, Thap3, Dr1, Casq2, Dnah11, Nsun5, Tek, Sec1, Zc3h7a, Vgll1, Nfkbid, Rin2, Ube2m, Zfp954, Fkbp9, Rrp1, Otx1, Ap2a2, Rrp1, Otx1, Ap2a2 Gm13977, Mfsd8, Cotl1, Efcab3, Commd4, Kantr, Rnf212, 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, Tmprs2, Prkksh, Wdr74, Nme1, Tex15, Zfp563, Gpn3, F5, Mib2, Gmp49 Dtna, Epcam, Gm28940, Gm44196, Eif3g, Polg2, Gm364, Mtch2, Htt, Ing5, Gm42945, Aknad1, Ehbp1
[Abnormal developmental embryo marker gene group]
Dmd, Tyk2, Nyap2, Mettl22, Manea, Nudt18, Lair1, Slamf1, March3, Ulk2, AI429214, Zfp938, Wdr59, Spryd7, Grin2b, Fsd2, Ciab Reps2, Cdh24, Fech, Agpat1, Plcb3, A230056P14Rik, Spsb4, Dmrtc1b, Raver2, 4921531C22Rik, Tbc1d31, Nutm1, Nek11, Morc2a, Trhr2, Aacs Zmym6, Klf10, Mfsd11, Nudt15, Nitch, Hipk3, Tattn3, Tyw3, Ubl4a, Atp6ap1l, Snx8, Gm12364, Ndufa7, Zfp341, Col4a1, Zfp2m Sybu, Kdm3b, AY512915, Gm13136, Trmt6 The biomarker according to claim 9, wherein the gene is Cdk2ap1 or Dmd. The biomarker according to claim 9 or 10, wherein the polar body is a second polar body. A method for identifying a gene for evaluation and / or selection of a mammalian embryo, which comprises the following steps (A) and (B).
(A) A step of detecting a transcript of a gene in a polar body derived from an in vitro fertilized mammalian fertilized egg;
(B) When the level of the transcript of the gene detected in step (A) is higher than the level in the polar body derived from an embryo whose development does not progress to the blastocyst stage, the gene is a normally developing embryo marker gene. It is evaluated that it is likely to be a normal developmental embryo marker gene.
When the level of the transcript of the gene detected in step (A) is lower than that level in the polar body derived from an embryo that does not progress to the blastocyst stage, the gene is an abnormally developing embryo marker gene or Step to evaluate that it is likely to be an abnormally developed embryo marker gene; The method according to claim 12, wherein the polar body is a second polar body. The method according to claim 12 or 13, wherein in the step (A), the transcript of the gene is detected by a sequencing method using a next-generation sequencer.

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