CN107419029A - A kind of kit of screening high-quality blastaea - Google Patents
A kind of kit of screening high-quality blastaea Download PDFInfo
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- CN107419029A CN107419029A CN201710840325.4A CN201710840325A CN107419029A CN 107419029 A CN107419029 A CN 107419029A CN 201710840325 A CN201710840325 A CN 201710840325A CN 107419029 A CN107419029 A CN 107419029A
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Abstract
The invention discloses a kind of kit of screening high-quality blastaea.Kit disclosed in this invention is to carry out being born before the implantation of people's supplementary reproduction blastaea the kit of safety prediction, including for detecting the product of the wall trophocyte of people's supplementary reproduction blastaea and the expression quantity of 398 genes shown in table 2 in polar trophoblast cell.If in 398 genes of the wall trophocyte of people's supplementary reproduction blastaea (blastaea before the implantation of external artificial insemination the 5th day) expression quantity of each gene be polar trophoblast cell 398 genes in more than twice of expression quantity of same gene or half times it is following, indicate people's supplementary reproduction blastaea by normal development and pregnant woman's normal labor.For the present invention for science judgment transplantation strategies and decision-making, reducing pregnant woman's perinatal period complication and the possibility of affected children ranges in supplementary reproduction has important application value.
Description
Technical field
The present invention relates to biomedical sector, and in particular to a kind of kit of screening high-quality blastaea.
Background technology
Embryonic limb bud cell turns into still unsolved hot issue in reproductive medicine research field.Embryonic limb bud cell is mammal
Key link in reproductive process, after it refers to that blastocyst runs to uterus, the process with endometrium interaction, mainly include
The positioning of blastocyst, adhere to and penetrate, this process is considerably complicated, is that embryo and endometrium both sides' collective effect are completed, is located at
Embryo periphery trophocyte played in embryo's invasion procedure key effect, it by secrete array of cytokines and
Hormone exercises pregnant recognition reaction, also by secreting these factors, functions of hormones make embryo endometrium is occurred adhesion and
The effect of intrusion.
Research in terms of Embryonic limb bud cell and ectopic pregnancy shows, the gene and molecule to be played a crucial role to Embryonic limb bud cell
It may not be to derive from parent, and be derived from embryo, Maternal-fetal interface tissue is only to provide the carrier of embryonic development.Strengthen embryo
On the basis of the research of implantation stage Cytobiology and molecular biology, crucial special point of the endogenous of regulation and control Embryonic limb bud cell is found and determined
Son, it is favorably improved Embryonic limb bud cell success rate.Molecule mechanism at present on Embryonic limb bud cell is not also fully aware of, the research of the past
More using embryonic feeder confluent monolayer cells as an entirety, gene expression time consequent succession is studied, lacks cellular gene expression space
Research, it is also difficult to find people's natural pregnancy physiological blastaea as research.
Auxiliary procreation technology solves the problems, such as a large amount of reproductive systems, helps Infertile couples to obtain filial generation, to society and family's band
Come happy.However, the natural pregnancy that compares, epidemiology and clinical studies show, excluding infertile disease factor influences, auxiliary life
Plantation technology has uncertain hidden danger in itself, auxiliary procreation technology pregnancy outcome risk and natus health (childhood and into
Term) risk increase, brings great risk to social economy and family and bears for a long time.In this area, high-quality blastaea have compared with
Good developmental potentiality, has an implantation normal endometrium potentiality, and future development is into normal fetus.High-quality blastaea has security,
Without perinatal period complication and Averse pregnancy outcomes.Therefore, how in the preceding high-quality blastaea of progress of people's supplementary reproduction blastaea implantation
Screening, there is important application value.
The content of the invention
The technical problems to be solved by the invention be how people's supplementary reproduction blastaea implantation before carry out be born security it is pre-
Survey, the screening high-quality blastaea specially before the implantation of people's supplementary reproduction blastaea, high-quality blastaea is by normal development.
In order to solve the above technical problems, the invention provides one kind to carry out birth safety before the implantation of people's supplementary reproduction blastaea
Property prediction kit.
A kind of kit that birth safety prediction is carried out before the implantation of people's supplementary reproduction blastaea provided by the present invention, can
Including the production for detecting the wall trophocyte of people's supplementary reproduction blastaea and the expression quantity of 398 genes in polar trophoblast cell
Product；398 genes are following genes of the people shown in table 2：SCARB1 genes；PPP3CA genes；EVL genes；CAPG bases
Cause；PLCG2 genes；DIP2C genes；INA genes；HAUS8 genes；BPHL genes；IFT74 genes；FANCG genes；PSKH1 bases
Cause；PDE8A genes；DLX3 genes；SLC35E3 genes；FBXO36 genes；ZNF7 genes；TNFAIP1 genes；TP53INP1 bases
Cause；PKMYT1 genes；DTX4 genes；NFYA genes；VEPH1 genes；TPCN2 genes；ANKRD6 genes；GCLC genes；ABCD4
Gene；AP1G2 genes；MFSD10 genes；EIF5A2 genes；ASB8 genes；SLC35F5 genes；CGRRF1 genes；HES6 bases
Cause；PIGU genes；TMTC4 genes；LMF2 genes；MGAT2 genes；C1orf112 genes；LYRM1 genes；TAOK2 genes；
TFPT genes；MAP3K2 genes；FAM193A genes；FUCA2 genes；DPF2 genes；CAMK2D genes；YIPF1 genes；
C14orf109 genes；TMEM5 genes；EED genes；CDK16 genes；LRP1 genes；TUBB6 genes；CDK5 genes；THOP1 bases
Cause；RNF139 genes；BRP44L genes；HSD17B10 genes；C5orf22 genes；GYG1 genes；SLC35C1 genes；ZBED1
Gene；SNX29 genes；ANXA6 genes；CBLL1 genes；C11orf82 genes；TRMT5 genes；ATP6V0D1 genes；BARD1
Gene；ARL6IP4 genes；ELOVL6 genes；INTS7 genes；CHMP1B genes；GNAI2 genes；AKT1 genes；HSDL2 bases
Cause；C19orf10 genes；TCOF1 genes；RBX1 genes；EID1 genes；IREB2 genes；C14orf2 genes；NDUFA7 genes；
PFDN2 genes；SNRPA genes；RPS19 genes；TMEM208 genes；SSNA1 genes；RPS23 genes；KHSRP genes；EIF1B
Gene；COX5B genes；PFDN4 genes；PDCD5 genes；ARFGEF2 genes；COX8A genes；RPL26L1 genes；SYF2 bases
Cause；RPL32 genes；CPSF2 genes；RPLP1 genes；IPO4 genes；NDUFB6 genes；PREP genes；ANAPC7 genes；SLU7
Gene；PDGFA genes；RPS24 genes；RPL28 genes；RTF1 genes；RBM12 genes；NAA38 genes；CTCF genes；
POLR2F genes；RBM27 genes；TXNDC17 genes；SMARCA4 genes；DAAM1 genes；SMC2 genes；EIF4G3 genes；
NDUFA13 genes；BLOC1S1 genes；TIMM10 genes；RPL22 genes；AGPS genes；RPL27A genes；TIMM8B genes；
TOMM5 genes；MRPS28 genes；BOLA2 genes；RBMX2 genes；AGFG1 genes；NDUFS5 genes；HS6ST1 genes；
SLC35E1 genes；LARP4 genes；SNRPD2 genes；S100A13 genes；RPL23A genes；MRP63 genes；ABCF1 genes；
FKBP2 genes；GABARAPL2 genes；LEPR genes；POLR2L genes；DDX6 genes；WHSC1 genes；ROCK1 genes；
MORF4L2 genes；TFAM genes；SUPT7L genes；SERTAD2 genes；CCDC59 genes；LMO4 genes；COMMD9 genes；
RBM19 genes；KRR1 genes；RPS12 genes；NDUFA6 genes；NDUFA2 genes；FUS genes；HEATR5A genes；ILKAP
Gene；NTAN1 genes；S100A6 genes；Sdhd gene；NOP10 genes；MGST2 genes；MCRS1 genes；SETD2 genes；
TARBP2 genes；LRRC57 genes；RPL27 genes；WBSCR22 genes；RNMT genes；RPL30 genes；PBRM1 genes；
EARS2 genes；PRELP genes；PPP1R14A genes；CSNK1G1 genes；KRTCAP2 genes；MTOR genes；COX6B1 genes；
RBM42 genes；TFE3 genes；RPL23 genes；KLF3 genes；RC3H1 genes；C11orf10 genes；SNRPE genes；COX7C
Gene；XIAP genes；MFSD5 genes；TRMT11 genes；RPS15A genes；BOLA3 genes；RPS25 genes；SNRNP200 bases
Cause；C12orf57 genes；WDR26 genes；RPL22L1 genes；TAF1D genes；DGCR2 genes；SPAG7 genes；OSBP genes；
PDAP1 genes；GRHL1 genes；CHCHD7 genes；NDUFC1 genes；KIAA1919 genes；GON4L genes；RPS15 genes；
TMEM69 genes；SLC6A8 genes；TRIM59 genes；PPP6R1 genes；PDE6A genes；TCEB2 genes；USP25 genes；
UQCRB genes；QRSL1 genes；RAB33B genes；AP4E1 genes；POLR2I genes；COX6C genes；MRPL33 genes；
RPL34 genes；KCTD5 genes；NDUFB4 genes；UBN2 genes；ATP5D genes；RPL36AL genes；COX7A2 genes；
METTL13 genes；C15orf63 genes；RPL35A genes；NDUFA1 genes；RNF145 genes；OR7E37P genes；CRIP1 bases
Cause；PDRG1 genes；SNRPG genes；IQSEC1 genes；NDUFB2 genes；IRF1 genes；ATP2B4 genes；USP47 genes；
SHFM1 genes；TMEM56 genes；NIF3L1 genes；UQCR11 genes；MED10 genes；NDUFB3 genes；CMIP genes；
TMEM41B genes；TATDN3 genes；NUFIP1 genes；KLRG2 genes；ACAD8 genes；RPLP2 genes；MRPL52 genes；
ROMO1 genes；RPL31 genes；TAF10 genes；RPS26 genes；CEPT1 genes；OSTM1 genes；SMARCAD1 genes；
COMMD6 genes；UQCR10 genes；RPL36 genes；CARD6 genes；COX7B genes；ZNF766 genes；RPL35 genes；
POLRMT genes；MRPS21 genes；TMF1 genes；RNF125 genes；IMPDH1 genes；UGT8 genes；ZRANB1 genes；MTRR
Gene；RPL41 genes；MUL1 genes；CHURC1 genes；C17orf61 genes；CCNT2 genes；RREB1 genes；FAM8A1 bases
Cause；ATP5J2 genes；MYLIP genes；UQCC genes；UQCRQ genes；MRPL24 genes；UBL5 genes；ATOX1 genes；
ATP5E genes；RPS28 genes；COX17 genes；TMEM128 genes；USP32 genes；YIPF5 genes；CHCHD10 genes；
SPATA5 genes；DDX51 genes；SEC61G genes；MTX2 genes；SLC20A2 genes；SRXN1 genes；PSTPIP2 genes；
SNRPF genes；DNAL1 genes；RICTOR genes；APOC1 genes；CXXC1 genes；NOM1 genes；DGAT1 genes；RPL38 bases
Cause；RPL37A genes；C1RL genes；TMEM168 genes；SMUG1 genes；CAMK1D genes；RPS21 genes；SMURF2 genes；
OST4 genes；TLR7 genes；FLJ13197 genes；KIAA1522 genes；TAB1 genes；FLJ39653 genes；CC2D1A genes；
SGTB genes；MDM2 genes；FAM165B genes；GRIN2D genes；PWWP2A genes；TGFBR1 genes；MYEOV2 genes；
ZNF493 genes；SYNJ2 genes；ELOVL7 genes；TADA1 genes；SETD4 genes；ANKS4B genes；LARP6 genes；
C17orf89 genes；RIPK4 genes；PIN4 genes；NR1H3 genes；ZSCAN12 genes；DPM3 genes；NAV2 genes；
TRIOBP genes；C10orf47 genes；SERAC1 genes；SLC46A1 genes；FBRSL1 genes；PTPN23 genes；PIF1 bases
Cause；SPTB genes；ARHGAP26 genes；ABI2 genes；HMGN5 genes；C12orf56 genes；RIC8B genes；TDRKH genes；
SLC9A6 genes；ANKRD13D genes；ZNF426 genes；SMYD4 genes；CEP97 genes；ACTR5 genes；ZBTB1 genes；
VPS18 genes；GBAS genes；ZMYND19 genes；GALNT7 genes；LYRM5 genes；ANKS6 genes；SS18L1 genes；
P4HA1 genes；MYPOP genes；ZNF99 genes；SCARA3 genes；MID1 genes；PPM1N genes；ZNF700 genes；ZFP30
Gene；TMEM159 genes；ZNF333 genes；BMP8B genes；TECPR2 genes；UPRT genes；CIDEB genes.
The ID_REF of 398 genes is as shown in arranging table 2 the 1st.
In mentioned reagent box, the expression quantity of 398 genes can be to be carried out using Illumina HiSeq2000 systems
It is unicellular that obtained gene expression amount is sequenced.
In mentioned reagent box, the kit may also include the carrier for being described below content：Detect people's supplementary reproduction blastaea
The expression quantity of 398 genes in wall trophocyte and polar trophoblast cell, according to the wall trophocyte and the polar trophoblast
The difference of the expression quantity of 398 genes described in cell carries out birth safety prediction before being implanted into people's supplementary reproduction blastaea.
In mentioned reagent box, the carrier can record following content：If people's supplementary reproduction blastaea is (external artificial
Be fertilized the blastaea before implantation in the 5th day) wall trophocyte 398 genes in the expression quantity of each gene be that the people is auxiliary
Help more than twice of the expression quantity of same gene in the polar trophoblast cell of reproduction blastaea or half times is following (is specially
The expression quantity of each gene is in the polar trophoblast cell of people's supplementary reproduction blastaea in preceding 78 genes during table 2 the 1st arranges
More than twice of the expression quantity of same gene, the expression quantity of each gene is described to table 2 the 1st in rear 320 genes in arranging
The half of the expression quantity of same gene times is following in the polar trophoblast cell of people's supplementary reproduction blastaea), indicate the people
Supplementary reproduction blastaea is by normal development.
In mentioned reagent box, in the wall trophocyte and polar trophoblast cell for detecting people's supplementary reproduction blastaea
The product of the expression quantity of 398 genes can be the product of the unicellular sequencing of progress, such as Illumina HiSeq2000 systems.
In mentioned reagent box, the supplementary reproduction blastaea can be the blastaea before external artificial insemination implantation in the 5th day.
The table of 398 genes in the wall trophocyte and polar trophoblast cell for detecting people's supplementary reproduction blastaea
Product up to amount carries out the application being born in the kit of safety prediction before being implanted into described in preparing in people's supplementary reproduction blastaea
Fall within protection scope of the present invention；398 genes are specially 398 genes described in the row of table 2 the 1st.
In above-mentioned application, the supplementary reproduction blastaea can be the blastaea before external artificial insemination implantation in the 5th day.
The table of 398 genes in the wall trophocyte and polar trophoblast cell for detecting people's supplementary reproduction blastaea
The reagent that birth safety prediction is carried out before the implantation of people's supplementary reproduction blastaea is being prepared up to the product of amount and the carrier
Application in box falls within protection scope of the present invention；398 genes are specially 398 genes described in the row of table 2 the 1st.
In above-mentioned application, the supplementary reproduction blastaea can be the blastaea before external artificial insemination implantation in the 5th day.
Influence of people's auxiliary procreation technology to placenta development is specified, mainly finds the wall nourishing in people's supplementary reproduction blastaea
The expression difference of 398 genes in confluent monolayer cells and polar trophoblast cell.If people's supplementary reproduction blastaea (external artificial insemination
Blastaea before implantation in 5 days) wall trophocyte 398 genes in the expression quantity of each gene be people's supplementary reproduction
In 398 genes of the polar trophoblast cell of blastaea more than twice of the expression quantity of same gene or half times it is following,
Indicate people's supplementary reproduction blastaea by normal development and pregnant woman's normal labor；Otherwise people's supplementary reproduction blastaea will be abnormal
Development, influence children's health and perinatal period complication occur；This possibility is with 398 genes in people's supplementary reproduction blastaea
Difference degree increases and risk increase.The main contributions of the present invention are science judgment transplantation strategies and decision-making, reduce supplementary reproduction
The possibility of middle pregnant woman's perinatal period complication and affected children ranges.
Brief description of the drawings
Fig. 1 is blastaea aspect graph.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, it is conventional method unless otherwise specified.Test material used in following embodiments, it is from often unless otherwise specified
Rule biochemical reagents shop is commercially available.Quantitative test in following examples, it is respectively provided with and repeats to test three times, results averaged.
The approval that research contents and specimen collection obtain scientific research Ethics Committee of Beijing Jishuitan Hospital (cures human relations to examine：
2014075)。
Illumina HiSeq2000 systems are the product of Shanghai Ren Ke bio tech ltd.1×Expand High
Fidelity PCR buffer solutions be Roche Holding Ag product, catalog number 11732641001.
Full cell lysis buffer solution：Containing 0.1% (0.1mg/100mL) SDS, 150mmol/L NaCl and 1.0mmol/L
EDTA pH7.5,50mmol/L Tris-HCl cushioning liquid.
First, using conventional method (i.e. Gardner points-scoring systems) screening high-quality blastaea
High-quality blastaea refers to preferable developmental potentiality, with implantation normal endometrium potentiality, future development into just
Normal embryo and without perinatal period complication and the blastaea of Averse pregnancy outcomes.
The present inventor (is scored) screening high-quality blastaea using Gardner points-scoring systems by discernable by eye,
Comprise the following steps that：
1st, according to the superovulated scheme of routine of Beijing Jishuitan Hospital's reproductive center, ovum is obtained.
2nd, ovum is taken, nature insemination is carried out according to patient profiles.
3rd, the 24h of ovum is taken, observes fertilization situation.
4th, the 72h of ovum is taken, using Gardner points-scoring system screening high-quality blastaeas.High-quality blastaea scores in Gardner
Scoring in system is 5AA.
Gardner points-scoring systems are as follows：(1) carried out by stages according to blister cavities degrees of expansion：The 50% of blister cavities deficiency embryo is 1
Phase, what blister cavities exceeded embryo 50% was 2 phases, and it was 3 phases that blister cavities, which takes whole embryo, and it was 4 phases that it is thinning, which to expand oolemma, for blastaea, blastaea
It was 5 phases to start hatching, and blastaea was hatched as 6 phases completely；(2) inner cell mass scoring is carried out to the blastaea of 4~6 phases in step (1)：A
For cell number is more, arrangement is close, B is that cell number is less, arrangement is loose, and C is that cell number is seldom；(3) in step (1)
The blastaea of 4~6 phases carries out Trophectoderm cells scoring：A is cell number is more, blister cavities surrounding has cell, arranges close, B
For cell number it is less, arrangement it is more loose, C be cell number it is seldom.
5th, after completing step 4, the high-quality blastaea in part is transplanted respectively.
20 supplementary reproduction women for being implanted into high-quality blastaea are carried out to keep track return visit, 20 women normally divide
Childbirth, return visit is kept track to child to 1 years old half, child's indices are normal.
6th, after completing step 4, the 20 high-quality blastaeas do not transplanted are taken, continue culture to the 5d, Ran Hou of fertilization
Micro- Microscopic observation form, 20 high-quality blastaeas (being respectively provided with smooth trophectoderm and well-developed blastocoele) are carried out
Freezen protective.
High-quality blastaea is shown in Fig. 1 in the 5d of fertilization form (PTE is polar trophoblast cell, and MTE is wall trophocyte).
The donations Mr. and Mrs of 20 research samples (i.e. 20 high-quality blastaeas) sign Written informed consent, informed consent form
Confirm patient's voluntary donations/contributions blastaea be used for human embryo developmental mechanism study, and be apprised of donations blastaea may bring dive
In risk.The age of mother of 20 research samples is and infertile caused by Tubal factor between -35 years old 25 years old, and 20
The seminal fluid of the father of individual research sample detects normally.
2nd, the expression quantity of each gene in high-quality blastaea is detected
1st, trophocyte separates
It is by (the production of Japanese Narishige companies of hard glass microelectrode blank to separate micro pipette and draw micro pipette
Product) draw what is formed, diameter is respectively 1-2 μm and 10-20 μm.
Take 20 of step 1 freezen protective to study sample, cell is first found under 20 times of object lens × 10 times eyepieces, then
Using the mechanically decoupled polar trophoblast cell of separation micro pipette or wall trophocyte under 60 times of object lens × 10 times eyepieces, and it is sent into
Draw in micro pipette, be then transferred in the Eppendorf pipes containing 1 × Expand High Fidelity PCR buffer solutions.
From this 20 research samples, 8 wall trophocytes and 9 polar trophoblast cells have been isolated.
2nd, unicellular sequencing and the acquisition of gene expression amount
The 8 wall trophocytes and 9 polar trophoblast cells that step 1 separates are taken respectively, using Illumina
HiSeq2000 systems carry out unicellular sequencing, obtain the expression quantity of each gene in the complete genome DNA of each cell.Specifically
Step is as follows：
(1) unicellular cDNA is prepared
Unicellular cDNA is prepared using progressively unicellular RNA-Seq methods：By micro pipette select it is unicellular be transferred to it is complete
In cell lysis buffer solution, first poly (A) tail is added in the first chain cDNA 3' ends using terminal deoxynucleotidyl transferase
And using it as template, performing PCR is entered with the universal primer UPM (universal primer, UPM) containing Partial joints sequence and expanded
Increase reaction (totally 30 circulations), obtain unicellular cDNA.
(2) RNA-Seq libraries are prepared
The unicellular cDNA of 200ng are taken, cut into 150bp-350bp fragment, are then prepared according to TrueSeq DNA libraries
The operating procedure of kit (product of Illumina companies), prepares sequencing library.
(3) it is sequenced
Sequencing library prepared by step (2) is sequenced.
Each trophocyte obtains ten thousand 100bp of 20-60 reading.The reading of genetic fragment, from UCSC Genome
Browser(http://genome.ucsc.edu) download hg19RefSeq, using Burrows-Wheeler Aligner [5,
6] (BWA, Version 0.5.9-r16) is contrasted the data of screening with hg19RefSeq, obtains known (UCSC
hg19,http://genome.ucsc.edu) expression quantity.
3rd, gene annotation
Annotation of gene function uses DAVID (http with function enrichment://david.abcc.ncifcrf.gov/) and biology
(the http of molecular function annotation system MAS 3.0://bioinfo.capitalbio.com/mas3/) carry out.
4th, statistical method
Statistical procedures are carried out with the statistical softwares of SPSS 16.0, data are represented with mean ± standard deviation, Student's, t
Each experimental data of check analysis, p<0.05 is that difference is statistically significant.
The expression quantity of 398 genes of 8 wall trophocytes and 9 polar trophoblast cells the results are shown in Table 1 (m1-m8
For 8 wall trophocytes, p1-p9 is 9 polar trophoblast cells).398 bases in wall trophocyte and polar trophoblast cell
Because the average and standard deviation of expression quantity are shown in Table 2, (M standard deviations are the standard deviation of wall trophocyte, and P standard deviations are that polar trophoblast is thin
The standard deviation of born of the same parents).The ratio of expression quantity of 398 genes in wall trophocyte and polar trophoblast cell is shown in Table 3.
Table 1
Table 2
Table 3
As a result show, in blastaea (blastaea before the 5d that is fertilized in auxiliary procreation technology, implantation), 398 genes are in wall
Expression quantity in trophocyte is that more than twice of the expression quantity in polar trophoblast cell or half times are following, its
In expression quantity of 78 genes in wall trophocyte be more than twice of the expression quantity in polar trophoblast cell, 320
Expression quantity of the individual gene in wall trophocyte is that the half times of the expression quantity in polar trophoblast cell is following.
Claims (8)
1. a kind of kit that birth safety prediction is carried out before the implantation of people's supplementary reproduction blastaea, including for detecting people's auxiliary
The product of the expression quantity of 398 genes in the wall trophocyte of reproduction blastaea and polar trophoblast cell；398 genes are
Following genes of people：SCARB1 genes；PPP3CA genes；EVL genes；CAPG genes；PLCG2 genes；DIP2C genes；INA bases
Cause；HAUS8 genes；BPHL genes；IFT74 genes；FANCG genes；PSKH1 genes；PDE8A genes；DLX3 genes；
SLC35E3 genes；FBXO36 genes；ZNF7 genes；TNFAIP1 genes；TP53INP1 genes；PKMYT1 genes；DTX4 genes；
NFYA genes；VEPH1 genes；TPCN2 genes；ANKRD6 genes；GCLC genes；ABCD4 genes；AP1G2 genes；MFSD10 bases
Cause；EIF5A2 genes；ASB8 genes；SLC35F5 genes；CGRRF1 genes；HES6 genes；PIGU genes；TMTC4 genes；
LMF2 genes；MGAT2 genes；C1orf112 genes；LYRM1 genes；TAOK2 genes；TFPT genes；MAP3K2 genes；
FAM193A genes；FUCA2 genes；DPF2 genes；CAMK2D genes；YIPF1 genes；C14orf109 genes；TMEM5 genes；
EED genes；CDK16 genes；LRP1 genes；TUBB6 genes；CDK5 genes；THOP1 genes；RNF139 genes；BRP44L bases
Cause；HSD17B10 genes；C5orf22 genes；GYG1 genes；SLC35C1 genes；ZBED1 genes；SNX29 genes；ANXA6 bases
Cause；CBLL1 genes；C11orf82 genes；TRMT5 genes；ATP6V0D1 genes；BARD1 genes；ARL6IP4 genes；ELOVL6
Gene；INTS7 genes；CHMP1B genes；GNAI2 genes；AKT1 genes；HSDL2 genes；C19orf10 genes；TCOF1 bases
Cause；RBX1 genes；EID1 genes；IREB2 genes；C14orf2 genes；NDUFA7 genes；PFDN2 genes；SNRPA genes；
RPS19 genes；TMEM208 genes；SSNA1 genes；RPS23 genes；KHSRP genes；EIF1B genes；COX5B genes；PFDN4
Gene；PDCD5 genes；ARFGEF2 genes；COX8A genes；RPL26L1 genes；SYF2 genes；RPL32 genes；CPSF2 bases
Cause；RPLP1 genes；IPO4 genes；NDUFB6 genes；PREP genes；ANAPC7 genes；SLU7 genes；PDGFA genes；RPS24
Gene；RPL28 genes；RTF1 genes；RBM12 genes；NAA38 genes；CTCF genes；POLR2F genes；RBM27 genes；
TXNDC17 genes；SMARCA4 genes；DAAM1 genes；SMC2 genes；EIF4G3 genes；NDUFA13 genes；BLOC1S1 bases
Cause；TIMM10 genes；RPL22 genes；AGPS genes；RPL27A genes；TIMM8B genes；TOMM5 genes；MRPS28 genes；
BOLA2 genes；RBMX2 genes；AGFG1 genes；NDUFS5 genes；HS6ST1 genes；SLC35E1 genes；LARP4 genes；
SNRPD2 genes；S100A13 genes；RPL23A genes；MRP63 genes；ABCF1 genes；FKBP2 genes；GABARAPL2 bases
Cause；LEPR genes；POLR2L genes；DDX6 genes；WHSC1 genes；ROCK1 genes；MORF4L2 genes；TFAM genes；
SUPT7L genes；SERTAD2 genes；CCDC59 genes；LMO4 genes；COMMD9 genes；RBM19 genes；KRR1 genes；
RPS12 genes；NDUFA6 genes；NDUFA2 genes；FUS genes；HEATR5A genes；ILKAP genes；NTAN1 genes；
S100A6 genes；Sdhd gene；NOP10 genes；MGST2 genes；MCRS1 genes；SETD2 genes；TARBP2 genes；LRRC57
Gene；RPL27 genes；WBSCR22 genes；RNMT genes；RPL30 genes；PBRM1 genes；EARS2 genes；PRELP genes；
PPP1R14A genes；CSNK1G1 genes；KRTCAP2 genes；MTOR genes；COX6B1 genes；RBM42 genes；TFE3 genes；
RPL23 genes；KLF3 genes；RC3H1 genes；C11orf10 genes；SNRPE genes；COX7C genes；XIAP genes；MFSD5
Gene；TRMT11 genes；RPS15A genes；BOLA3 genes；RPS25 genes；SNRNP200 genes；C12orf57 genes；
WDR26 genes；RPL22L1 genes；TAF1D genes；DGCR2 genes；SPAG7 genes；OSBP genes；PDAP1 genes；GRHL1
Gene；CHCHD7 genes；NDUFC1 genes；KIAA1919 genes；GON4L genes；RPS15 genes；TMEM69 genes；SLC6A8
Gene；TRIM59 genes；PPP6R1 genes；PDE6A genes；TCEB2 genes；USP25 genes；UQCRB genes；QRSL1 genes；
RAB33B genes；AP4E1 genes；POLR2I genes；COX6C genes；MRPL33 genes；RPL34 genes；KCTD5 genes；
NDUFB4 genes；UBN2 genes；ATP5D genes；RPL36AL genes；COX7A2 genes；METTL13 genes；C15orf63 bases
Cause；RPL35A genes；NDUFA1 genes；RNF145 genes；OR7E37P genes；CRIP1 genes；PDRG1 genes；SNRPG bases
Cause；IQSEC1 genes；NDUFB2 genes；IRF1 genes；ATP2B4 genes；USP47 genes；SHFM1 genes；TMEM56 genes；
NIF3L1 genes；UQCR11 genes；MED10 genes；NDUFB3 genes；CMIP genes；TMEM41B genes；TATDN3 genes；
NUFIP1 genes；KLRG2 genes；ACAD8 genes；RPLP2 genes；MRPL52 genes；ROMO1 genes；RPL31 genes；TAF10
Gene；RPS26 genes；CEPT1 genes；OSTM1 genes；SMARCAD1 genes；COMMD6 genes；UQCR10 genes；RPL36 bases
Cause；CARD6 genes；COX7B genes；ZNF766 genes；RPL35 genes；POLRMT genes；MRPS21 genes；TMF1 genes；
RNF125 genes；IMPDH1 genes；UGT8 genes；ZRANB1 genes；MTRR genes；RPL41 genes；MUL1 genes；CHURC1
Gene；C17orf61 genes；CCNT2 genes；RREB1 genes；FAM8A1 genes；ATP5J2 genes；MYLIP genes；UQCC bases
Cause；UQCRQ genes；MRPL24 genes；UBL5 genes；ATOX1 genes；ATP5E genes；RPS28 genes；COX17 genes；
TMEM128 genes；USP32 genes；YIPF5 genes；CHCHD10 genes；SPATA5 genes；DDX51 genes；SEC61G genes；
MTX2 genes；SLC20A2 genes；SRXN1 genes；PSTPIP2 genes；SNRPF genes；DNAL1 genes；RICTOR genes；
APOC1 genes；CXXC1 genes；NOM1 genes；DGAT1 genes；RPL38 genes；RPL37A genes；C1RL genes；TMEM168
Gene；SMUG1 genes；CAMK1D genes；RPS21 genes；SMURF2 genes；OST4 genes；TLR7 genes；FLJ13197 bases
Cause；KIAA1522 genes；TAB1 genes；FLJ39653 genes；CC2D1A genes；SGTB genes；MDM2 genes；FAM165B bases
Cause；GRIN2D genes；PWWP2A genes；TGFBR1 genes；MYEOV2 genes；ZNF493 genes；SYNJ2 genes；ELOVL7 bases
Cause；TADA1 genes；SETD4 genes；ANKS4B genes；LARP6 genes；C17orf89 genes；RIPK4 genes；PIN4 genes；
NR1H3 genes；ZSCAN12 genes；DPM3 genes；NAV2 genes；TRIOBP genes；C10orf47 genes；SERAC1 genes；
SLC46A1 genes；FBRSL1 genes；PTPN23 genes；PIF1 genes；SPTB genes；ARHGAP26 genes；ABI2 genes；
HMGN5 genes；C12orf56 genes；RIC8B genes；TDRKH genes；SLC9A6 genes；ANKRD13D genes；ZNF426 bases
Cause；SMYD4 genes；CEP97 genes；ACTR5 genes；ZBTB1 genes；VPS18 genes；GBAS genes；ZMYND19 genes；
GALNT7 genes；LYRM5 genes；ANKS6 genes；SS18L1 genes；P4HA1 genes；MYPOP genes；ZNF99 genes；
SCARA3 genes；MID1 genes；PPM1N genes；ZNF700 genes；ZFP30 genes；TMEM159 genes；ZNF333 genes；
BMP8B genes；TECPR2 genes；UPRT genes；CIDEB genes.
2. kit as claimed in claim 1, it is characterised in that：The kit includes the carrier for being described below content：Inspection
The wall trophocyte of people's supplementary reproduction blastaea and the expression quantity of 398 genes in polar trophoblast cell are surveyed, is grown according to the wall
The difference for supporting the expression quantity of 398 genes described in confluent monolayer cells and the polar trophoblast cell is planted to people's supplementary reproduction blastaea
Birth safety prediction is carried out before entering.
3. kit as claimed in claim 1 or 2, it is characterised in that：The carrier records following content：If people aids in
The expression quantity of each gene is the pole of people's supplementary reproduction blastaea in 398 genes of the wall trophocyte of reproduction blastaea
In trophocyte more than twice of the expression quantity of same gene or half times it is following, indicate people's supplementary reproduction capsule
Embryo is by normal development.
4. the kit as described in claims 1 to 3 is any, it is characterised in that：The supplementary reproduction blastaea for it is external manually by
Blastaea before essence implantation in the 5th day.
5. for detecting the wall trophocyte of people's supplementary reproduction blastaea and the expression quantity of 398 genes in polar trophoblast cell
Application of the product in any kit of Claims 1-4 is prepared；398 genes are described in claim 1
398 genes.
6. application as claimed in claim 5, it is characterised in that：The supplementary reproduction blastaea is that external artificial insemination is planted on the 5th day
Blastaea before entering.
7. for detecting the wall trophocyte of people's supplementary reproduction blastaea and the expression quantity of 398 genes in polar trophoblast cell
Application of the carrier in any kit of Claims 1-4 is prepared described in product and Claims 2 or 3；It is described
398 genes are 398 genes described in claim 1.
8. application as claimed in claim 7, it is characterised in that：The supplementary reproduction blastaea is that external artificial insemination is planted on the 5th day
Blastaea before entering.
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| CN201710840325.4A CN107419029A (en) | 2017-09-18 | 2017-09-18 | A kind of kit of screening high-quality blastaea |
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| CN201710840325.4A CN107419029A (en) | 2017-09-18 | 2017-09-18 | A kind of kit of screening high-quality blastaea |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182561A (en) * | 2018-12-03 | 2019-01-11 | 吉林大学 | Application of the ARHGAP26 as ox superfecundation trait molecular marker |
| CN109988845A (en) * | 2019-01-30 | 2019-07-09 | 江苏万成生物医学研究院有限公司 | A kind of application of new stomach cancer marker circ-EIF4G3 |
| CN110055328A (en) * | 2019-03-28 | 2019-07-26 | 昆明医科大学第一附属医院 | A kind of adenocarcinoma of lung diagnosis marker based on metabolic gene spectrum |
| CN111157745A (en) * | 2020-01-17 | 2020-05-15 | 上海交通大学 | Application of human SNRPA protein in lung cancer recurrence or metastasis monitoring |
| CN112899360A (en) * | 2021-02-02 | 2021-06-04 | 北京航空航天大学 | Application method of composition for detecting occurrence probability of Terchester-Coriolis syndrome |
| CN117721222A (en) * | 2024-02-07 | 2024-03-19 | 北京大学第三医院（北京大学第三临床医学院） | Method for predicting embryo implantation by single cell transcriptome and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100323442A1 (en) * | 2006-10-17 | 2010-12-23 | Emmanuel Edward Baetge | Modulation of the phosphatidylinositol-3-kinase pathway in the differentiation of human embryonic stem cells |
| CN104561309A (en) * | 2015-01-04 | 2015-04-29 | 北京大学第三医院 | Kit for predicting birth safety before people-assisted reproduction blastosphere implantation |
| CN105861658A (en) * | 2016-04-12 | 2016-08-17 | 中国科学院北京基因组研究所 | Noninvasive detection method for screening healthily grown blastulas |
-
2017
- 2017-09-18 CN CN201710840325.4A patent/CN107419029A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100323442A1 (en) * | 2006-10-17 | 2010-12-23 | Emmanuel Edward Baetge | Modulation of the phosphatidylinositol-3-kinase pathway in the differentiation of human embryonic stem cells |
| CN104561309A (en) * | 2015-01-04 | 2015-04-29 | 北京大学第三医院 | Kit for predicting birth safety before people-assisted reproduction blastosphere implantation |
| CN105861658A (en) * | 2016-04-12 | 2016-08-17 | 中国科学院北京基因组研究所 | Noninvasive detection method for screening healthily grown blastulas |
Non-Patent Citations (1)
| Title |
|---|
| LIYING YAN等: "Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells", 《NATURE STRUCTURAL & MOLECULAR BIOLOGY》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182561A (en) * | 2018-12-03 | 2019-01-11 | 吉林大学 | Application of the ARHGAP26 as ox superfecundation trait molecular marker |
| CN109182561B (en) * | 2018-12-03 | 2022-03-04 | 吉林大学 | Application of ARHGAP26 as bovine superovulation trait molecular marker |
| CN109988845A (en) * | 2019-01-30 | 2019-07-09 | 江苏万成生物医学研究院有限公司 | A kind of application of new stomach cancer marker circ-EIF4G3 |
| CN110055328A (en) * | 2019-03-28 | 2019-07-26 | 昆明医科大学第一附属医院 | A kind of adenocarcinoma of lung diagnosis marker based on metabolic gene spectrum |
| CN111157745A (en) * | 2020-01-17 | 2020-05-15 | 上海交通大学 | Application of human SNRPA protein in lung cancer recurrence or metastasis monitoring |
| CN112899360A (en) * | 2021-02-02 | 2021-06-04 | 北京航空航天大学 | Application method of composition for detecting occurrence probability of Terchester-Coriolis syndrome |
| CN117721222A (en) * | 2024-02-07 | 2024-03-19 | 北京大学第三医院（北京大学第三临床医学院） | Method for predicting embryo implantation by single cell transcriptome and application |
| CN117721222B (en) * | 2024-02-07 | 2024-05-10 | 北京大学第三医院（北京大学第三临床医学院） | Method for predicting embryo implantation by single cell transcriptome and application |
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