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WO2004055217A1 - Methodes de determination de la validite d'un embryon - Google Patents

Methodes de determination de la validite d'un embryon Download PDF

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Publication number
WO2004055217A1
WO2004055217A1 PCT/US2003/039450 US0339450W WO2004055217A1 WO 2004055217 A1 WO2004055217 A1 WO 2004055217A1 US 0339450 W US0339450 W US 0339450W WO 2004055217 A1 WO2004055217 A1 WO 2004055217A1
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Prior art keywords
gene
embryo
cell
expression
protein
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English (en)
Inventor
Douglas Powers
Shungping Wang
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EMBRYOMICS Inc
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EMBRYOMICS Inc
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Priority to AU2003293517A priority Critical patent/AU2003293517A1/en
Publication of WO2004055217A1 publication Critical patent/WO2004055217A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/367—Infertility, e.g. sperm disorder, ovulatory dysfunction

Definitions

  • ART Assisted Reproductive Technologies
  • in vitro fertilization consists of pharmaceutical stimulation of the female's ovaries to produce a large number of follicles. Eggs surgically harvested from these follicles are then mixed in the laboratory with the male's sperm. If fertilization is successful, the embryos are incubated for a short time and then transferred back to the female. If one of these embryos implants in the uterine wall, a successful pregnancy may follow. There are several modifications of this basic technique.
  • intracytoplasmic sperm injection can be used for cases of low sperm count or cases where the sperm has difficulty fertilizing the egg.
  • Another in vitro fertilization modification is Assisted Hatching (AH), a procedure in which the zona pellucida (the outer wall of the embryo) is mechanically cut or chemically etched, thereby partially exposing the embryo.
  • AH Assisted Hatching
  • in vitro fertilization procedures can also incorporate donor tissues, including sperm, ova and embryos, for those individuals who cannot produce their own.
  • in vitro fertilization is now a widely accepted treatment for unexplained infertility (RCOG 1998). Approximately 50,000 human in vitro fertilization procedures are performed in the United States annually. Despite its great successes, in vitro fertilization has several problems. First and foremost, the procedure is unpredictable with estimated live birth rates per cycle varying between 13% and 28%. hi addition, due to the implantation of several embryos, in vitro fertilization may result in a pregnancy with multiple embryos. Moreover, although the costs vary widely depending on drugs, testing and other laboratory fees, typical in vitro fertilization charges are on the order of $10,000 per procedure, not counting travel, lost work time, and emotional costs for the patients.
  • the present invention is based, in part, on the discovery that by obtaining a cell from an embryo, analysis of nucleic acid, e.g., cDNA, expression of two or more genes at preselected levels within the cell can be used to evaluate the likelihood of the embryo implanting into a uterus and/or developing to term. Such information can be useful, e.g., for selecting an embryo (or embryos) for implantation during in vitro fertilization procedures. Despite the success of current in vitro fertilization procedures, one of the problems of currently available procedures is that the likelihood of an embryo developing to term is low and somewhat unpredictable with estimated live birth rates per cycle varying between 13% and 28%.
  • the present invention can be used to select embryos with an increased likelihood of surviving to term, thereby increasing the rates of live births obtained by in vitro fertilization procedures. Additionally, such information can be useful, e.g., in selecting non- human mammalian embryos produced by cloning and transgenic technologies for implantation. Again, the rates of live births from cloning and transgenic technologies are fairly low, and the ability to select embryos with an increased likelihood of surviving to term would be beneficial in both the cloning and transgenic animal fields. This information is also useful for evaluating embryos that serve as a source of stem cells. Currently, there are few methods of evaluating a source of stem cells to determine if it is a viable source.
  • the present invention provides a method of evaluating an embryo to determine if the embryo is likely to develop to term, thereby -. providing a way of evaluating whether the embryo should be used as a source of stems cells. Such methods can provide, e.g., a way for various parties such as regulatory bodies (e.g., the FDA) to evaluate the quality of a source of stem cells. Accordingly, in one aspect, the invention features a method of evaluating an embryo, e.g., evaluating the likelihood of an embryo to implant into a uterus and/or develop to term or evaluating the embryo as a source of stem cells. The method includes: removing a cell from an embryo; and evaluating the expression of at least two, and preferably at least three, preselected genes in the cell, to thereby evaluate the embryo.
  • the method can further include selecting the embryo for implantation. In some embodiments, the method can also include implanting the embryo into a female recipient. hi other embodiments, the method can further include selecting the embryo as a stem cell donor. In some embodiments, the methods can also include selecting a stem cell from the embryo.
  • the method includes evaluating to determine whether or not one or more, and preferably all of the genes are expressed or non- expressed. In another embodiment, the method includes evaluating to determine if the at least two, preferably three, preselected genes are expressed at a preselected level.
  • the preselected level can be below a reference level, e.g., it is at or below a level which correlates to no expression or the preselected level can be any level of expression above a reference level, e.g., it is above a level which correlates to no expression.
  • the method includes evaluating a gene or genes having a defined expression pattern (i.e. a reference expression profile) during normal early embryonic development and determining if at the stage of embryo development during which the cell is obtained, the gene or genes are being expressed at a level consistent with the reference expression profile. Expression levels can be evaluated at one of the following stages of embryonic development: 4-cell stage, 8-cell stage, compacted embryo stage, morulae stage, and/or blastocyst stage. In one embodiment, the method includes comparing the level of expression of the gene or genes to a reference expression profile.
  • the reference expression profile can provide a profile of changes in expression of the gene over early embryonic development in a normal embryo, for example, normal gene expression at the unfertilized egg, (fertilized) 1- cell, 2-cell, 4-cell, 8-cell, compacted embryo, morulae, and/or blastocyst stage of embryogenesis.
  • Examples of products encoded by genes that normally have a reference profiles during embryogenesis that show a constant increase in expression levels from the egg or from the (fertilized) 1-cell stage, through the 8-cell stage include: mitochondrial ribosomal protein L27; aminolevulinate, delta-, dehydratase; eukaryotic translation initiation factor 4E binding protein 1; GTP binding protein 1; mitochondrial ribosomal protein L17; cyclin-dependent kinase inhibitor 1 A (P21); NADH dehydrogenase (ubiquinone) flavoprotein 1; proteasome (prosome, macropain) 26S subunit, ATPase 3; platelet-activating factor acetylhydrolase, isoform lb, alphal subunit; RIKEN cDNA 2210402A09 gene product; RuvB-like protein 2; peroxiredoxin 6; RIKEN cDNA 0610027F08 gene product; acid beta glucosidase; peptidylproly
  • cerevisiae homolog EH-domain containing 1 protein; ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c (subunit 9), isoform 1; actin, beta, cytoplasmic; mitochondrial ribosomal protein L30; and actin related protein 2/3 complex, subunit 5.
  • Examples of products encoded by genes that normally have a reference expression profile during embryogenesis that shows a net decline in expression from the egg or 1-cell stage, through the 4-cell stage, followed by an increase in expression by the 8-cell stage (, e.g., such that expression at the 8-cell stage is greater than or equal to expression levels at the egg or 1-cell stage) include: stearoyl-Coenzyme A desaturase 2; methylenetetrahydrofolate dehydrogenase (NADP+ dependent)/ methenyltetrahydrofolate cyclohydrolase/ formyltetrahydrofolate synthase; UDP- GahbetaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1; ATPase, H+ transporting, NO subunit B; leucyl-tR ⁇ A synthetase; beta-1,4 mannosyltransferase; ligase I, D ⁇ A, ATP-
  • RIKEN cDNA 1810073M23 gene product RIKEN cDNA 1810073M23 gene product
  • KDEL Lis-Asp-Glu-Leu
  • endoplasmic reticulum protein retention receptor 2 differential display and activated by p53 gene product
  • solute carrier family 12, member 7; RAB5C, member RAS oncogene family solute carrier family 12, member 7; RAB5C, member RAS oncogene family
  • flightless I Drosophila
  • wild-type p53-induced gene 1 product wild-type p53-induced gene 1 product
  • inositol polyphosphate-5-phosphatase inositol polyphosphate-5-phosphatase.
  • Examples of products encoded by genes that are normally expressed at low levels at the egg stage, followed by an increase of expression, then a subsequent decrease in expression, followed by an increase of expression by the 8-cell stage include: hydroxysteroid (17-beta) dehydrogenase 4; defender against cell death 1; topoisomerase (DNA) III beta; low-density lipoprotein receptor-related protein 10; zinc finger, DHHC domain containing 3 protein; FK506 binding protein 9; ATP- binding cassette, sub-family D (ALD), member 3; H2-K region expressed gene 4 gene product; cysteine-rich hydrophobic domain 2; adaptor-related protein complex 3, beta 1 subunit; solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5; RIKEN cDNA 3010027G13 gene product; guanine nucleotide binding protein, beta 1 ; and LanC (bacterial lantibiotic synthetase component C)-like gene product.
  • Examples of products encoded by genes that are normally expressed at low levels from the egg stage through the 2-cell embryonic stage and subsequently demonstrate an increase in expression by the 8-cell stage include: dolichyl-di- phosphooligosaccharide-protein glycotransferase; copropo hyrinogen oxidase; ATPase, H+ transporting, VI subunit F; mitochondrial ribosomal protein LI 1 ; eukaryotic translation initiation factor 2, subunit 2 (beta); cysteine rich protein 2; platelet derived growth factor, alpha; cyclin-dependent kinase 4; CD40 ligand- activated specific transcript 3; cellular repressor of ElA-stimulated genes; glutathione S-transferase omega 1; RTKEN cDNA 2210402 A09 gene product; connective tissue growth factor; protein tyrosine phosphatase, receptor-type, F interacting protein, binding protein 2; Tcfcp2-related transcriptional repress
  • 2210408E11 gene product glucosidase, alpha, acid; cathepsin D; hexosaminidase A; isocitrate dehydrogenase 2 (NADP+), mitochondrial; translocase of inner mitochondrial membrane 9 (yeast) homolog; solute carrier family 19 (thiamine transproter), member 2; transmembrane 9 superfamily member 1; expressed sequence AI173274; capping protein (actin filament) muscle Z-line, beta; dynactin 1; solute carrier family 1, member 1; RIKEN cDNA 1810011E08 gene product; shroom; and jumonji.
  • Examples of products encoded by genes that normally demonstrate a fairly constant level of gene expression from the egg to the 8-cell stage include: RIKEN cDNA 0610038K03 gene; StAR-related lipid transfer (START) domain containing 4; Hermansky-Pudlak syndrome 1 homolog (human); protein kinase, cAMP dependent regulatory, type I beta; 3-hydroxybutyrate dehydrogenase (heart, mitochondrial); secreted frizzled-related sequence protein 1 ; and X (inactive)-specific transcript, antisense
  • Examples of products encoded by genes that normally demonstrate a rise in expression after the fertilized egg stage, followed by a subsequent decline in expression by the 8-cell stage include: integrin beta 1 (fibronectin receptor beta) and acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl- Coenzyme A thiola
  • Examples of products encoded by genes that normally demonstrate little change in expression from the egg stage to the 1-cell stage, followed by an increase in expression by the 2-cell stage to a level that changes relatively little into the 4-cell stage, and whose expression level subsequently rises by the 8-cell stage, include: solute carrier family 25 (mitochondrial carrier; dicarboxylate transporter), member 10 and adaptor protein complex AP-2, mul .
  • the method includes evaluating expression of the genes by evaluating the level of a nucleic acid, e.g., RNA or cDNA, from the cell.
  • the method further includes obtaining nucleic acid, e.g., RNA, from the cell.
  • the nucleic acid, e.g., RNA can be obtained from the cell within at least 5, 10, 15, 20, 25, 30 minutes, 1 hour, 2, 3, 4, 5 hours from the time the cell is removed from the embryo.
  • the method can further include obtaining cDNA from the RNA, e.g., using reverse transcription, e.g., the cDNA is obtained within at least 5, 10, 15, 20, 25, 30 minutes, 1 hour, 2, 3, 4, 5 hours from the time the RNA is obtained.
  • the cell can be maintained under conditions that do not significantly alter the expression of the genes prior to obtaining the nucleic acid, e.g.-, RNA.
  • the cell can be cryopreserved.
  • the method includes evaluating the expression within at least 5, 10, 15, 20, 25, 30 minutes, 1 hour, 2, 3, 4, 5 hours from the time the cell is removed from the embryo.
  • the method includes evaluating expression of the genes by: providing an array of a plurality of capture probes, e.g., nucleic acid sequences which can hybridize to specific nucleic acids from the cell, wherein each of the capture probes is positionally distinguishable from other capture probes of the plurality on the array; and hybridizing nucleic acid, e.g., cDNA, from the cell with the array of capture probes, to thereby analyze for expression of the specified nucleic acids from the cell.
  • the array is an array described herein.
  • the method further includes evaluating a selected gene, preferably a gene other than the genes evaluated for expression, to determine if a preselected form of the selected gene is present or expressed in the cell.
  • the selected gene can be evaluated by hybridizing a nucleic acid, e.g., RNA or cDNA, from the selected gene to a probe, wherein the probe is able to differentially hybridize to the nucleic acid, e.g., RNA or cDNA, from a first, e.g., a non-mutant, and a second, e.g., a mutant, form of the selected gene.
  • the selected gene can also be evaluated by, e.g., hybridizing RNA or cDNA from the selected gene to a probe, wherein the probe is able to differentially hybridize RNA or cDNA from alternatively spliced RNA forms.
  • evaluating the selected gene includes hybridizing genomic DNA from the selected gene to a probe or primer, wherein the probe or primer is able to distinguish between a first, e.g., a non-mutant, and a second, e.g., a mutant, form of the selected gene, e.g., a gene associated with a disorder.
  • the selected gene can be, e.g.: the gene encoding cystic fibrosis transmembrane conductase regulator (CFTR), the gene encoding dystrophin, the Beta Thalassemia gene, the gene encoding Factor VIII, the gene encoding Factor LX, Tay-Sachs gene, survival motor neuron (SMN) gene, and/or the HD gene.
  • the selected gene is evaluated to detect the absence or presence of the second mutant form of the selected gene, e.g., to evaluate whether the embryo carries a mutation associated with a disorder.
  • disorders for which the embryo can be evaluated include but are not limited to: Adenoleukodystrophy, Amyotrophic Lateral Sclereosis (ALS), Becker Muscular Dystrophy, Beta Thalassemia, Cerebellar Ataxia, Charcot-Marie-Tooth Disease, Chondrodysplasia Aganglionic Megacolon, Conradi- Hunnerman Syndrome, Cystic Fibrosis, Duchenne Muscular Dystrophy, Hemophilia A or B, Huntington's Disease, Fragile X Syndrome, Glycogen Storage Disease, Hirschsprung Disease, Icthyosis, Lesch Nyhan, Myopathies, Polycystic Ovary Syndrome, Restinosis Pigmentosa, Sickle Cell Anemia, Tay-Sachs Disease, and Von Willebran Disease.
  • the expression of the genes can be evaluated by: providing an array of a plurality of capture probes, wherein the array includes a plurality of capture probes, e.g., nucleic acid sequences which can hybridize to specific nucleic acids from the cell, namely nucleic acids from the preselected genes, and further includes a probe which differentially hybridizes to a nucleic acid, e.g., RNA or cDNA, from a first and second form of the selected gene.
  • Arrays which can be used include, e.g., the arrays described herein.
  • the method further includes evaluating a selected gene or cluster of genes, preferably other than the preselected gene evaluated for expression, to determine if the embryo is aneuploid, i.e., an embryo has too many or too few copies of a chromosome.
  • the selected gene or cluster of genes can be evaluated by hybridizing a nucleic acid, e.g., RNA or DNA, from the selected gene or cluster of genes to a probe, and determining the number of copies of the gene or the cluster of genes that hybridize to the probe. The presence of more than two copies of a gene or cluster of genes is indicative that the embryo has more then two copies of the chromosome associated with that gene or genes.
  • the presence of one copy of the gene or cluster of genes is indicative that the embryo has one copy of the chromosome associated with that gene or genes. Having an extra chromosome is referred to as “trisomy”, missing a chromosome is referred to as “monosomy”.
  • trisomy Having an extra chromosome is referred to as "trisomy”
  • missing a chromosome is referred to as “monosomy”.
  • the absence or presence of one or more chromosomes, e.g. one or more of chromosomes 1-22, above or below the normal number of chromosomes indicates that the embryo is unlikely to give rise to a healthy embryo.
  • the embryo is evaluated for one or more of: more than two of chromosome 21 in the embryo which is indicative of a risk for Down's Syndrome; more than two of chromosome 13 in the embryo which is indicative of a risk for Patau syndrome; more than two of chromosome 18 in the embryo which is indicative of a risk for Edwards syndrome; more than two of chromosome 16 in the embryo which is indicative of a risk for abnormal fetal development and/or miscarriage; more than two of chromosome 22 in the embryo ' which is indicative of a risk for abnormal fetal development and/or miscarriage; absence or presence of only one copy of chromosome 5 in the embryo which is indicative of a risk for Cri du Chat; more than two X chromosomes in the embryo which is indicative of a risk for Klinefelter's syndrome; more than one X chromosome in the embryo which is indicative of a risk for Triple X syndrome; and the absence of chromosome X in the embryo which is indicative of a risk for
  • the copy number of the genes or genes can be evaluated by providing an array of a plurality of capture probes, wherein the array includes a plurality of capture probes, e.g., nucleic acid sequences which can hybridize to specific nucleic acids from the cell, namely nucleic acids from the preselected genes, and further includes a probe or probes which hybridizes to all or a portion of a gene or genes used to determine if the embryo is an aneuploid for one or more chromosomes.
  • the array includes a plurality of capture probes, e.g., nucleic acid sequences which can hybridize to specific nucleic acids from the cell, namely nucleic acids from the preselected genes, and further includes a probe or probes which hybridizes to all or a portion of a gene or genes used to determine if the embryo is an aneuploid for one or more chromosomes.
  • the nucleic acids from the cell can be labeled.
  • the method can further include comparing the hybridization pattern of the labeled nucleic acids from the cell to the hybridization pattern of a control cell, e.g., nucleic acids from a cell which gave rise to a healthy embryo.
  • two arrays are used, one for each evaluation, and the nucleic acids from the cell of the embryo being evaluated for aneuploidy and the nucleic acid from the control cell are labeled, e.g., with detectable different labels.
  • the probe for evaluating aneuploid can be a bacterial artificial chromosome (BAC), PI or a cosmid clone.
  • BAC bacterial artificial chromosome
  • PI bacterial artificial chromosome
  • cosmid clone a cosmid clone.
  • Arrays which can be used include, e.g., the arrays described herein.
  • the method further includes evaluating the absence or presence of a selected gene (or genes) associated with a sex chromosome, e.g., the Y chromosome, to thereby evaluate the sex of the embryo.
  • a selected gene or genes associated with a sex chromosome, e.g., the Y chromosome
  • the nucleic acid, e.g., RNA or cDNA, from the cell can be evaluated for the absence or presence of a gene specifically expressed on the Y chromosome but not the X chromosome, the presence of the gene being indicative of a male embryo.
  • the gene associated with the Y chromosome can be, e.g., the sry gene.
  • the expression of the genes can be evaluated by:- providing an array of a plurality of capture probes, wherein the array includes a plurality of capture probes, e.g., nucleic acid sequences which can hybridize to specific nucleic acids from the cell, namely nucleic acids from the preselected genes, and further includes a probe which hybridizes to a nucleic acid associated with one sex chromosome but not the other, e.g., it is associated with the Y chromosome but not the X chromosome.
  • Arrays which can be used include, e.g., the arrays described herein.
  • the method can include at least three of the following steps: evaluating the expression of atleast two, and preferably, at least three preselected genes to evaluate the likelihood of an embryo to implant into the uterus and/or survive to term; evaluating the presence of a first, e.g., non-mutant, and a second, e.g., mutant, form of a gene; evaluating a selected gene or cluster of genes to determine the absence or presence of one or more chromosomes above or below the normal number of chromosomes; and evaluating a selected gene to determine the sex of the embryo.
  • the method further includes determining if a preselected factor is present for one or both parents of the embryo.
  • the method can further include entering a value for the determination of the preselected factor into a database, e.g., a database described herein.
  • the database further includes one or both of data on the expression of the preselected genes and data on whether the selected embryo developed to term.
  • the factor can be, e.g., the presence of a treatment (e.g., the administration of a compound, e.g., a drug (e.g., a hormone), vitamin, food, or dietary supplement); the presence of an environmental factor (e.g., the presence of a substance in the environment, residence in a preselected geographic area, performing a preselected occupation); and/or the presence of a genetic factor or physical factor such as age.
  • the method can further include determining if a preselected condition was used to produce the embryo.
  • the method can further include entering a value for the determination of the preselected condition into a database e.g., a database described herein.
  • the database further includes one or both of data on the expression of the preselected genes and data on whether the selected embryo developed to term.
  • the preselected condition can be, e.g., any condition used for in vitro fertilization.
  • the preselected condition can be a fertilization technique, a germ cell (e.g., oocyte or sperm) retrieval technique, and/or the presence or absence of one or more factors in culture media.
  • the method includes evaluating a pre-implantation embryo wherein the pre-implantation embryo is in the two to thirty-two cell stages, preferably the four to sixteen cell stages, the 8-cell stage, of embryogenesis.
  • the method further includes evaluating the expression of at least one control gene.
  • the control gene can be, e.g., selected from the group, consisting of the beta actin gene, the beta-2-microglobulin gene, the glyceraldehyde-3 -phosphate dehydrogenase gene, the hypoxanthine phosphoribosyl- transferase 1 gene, the ribosomal protein L13a, subunit A gene of the succinate dehydrogenase complex, the TATA binding protein gene, and the ubiquitin C gene.
  • the method includes evaluating expression of the preselected genes, wherein expression of at least five, ten, fifteen, twenty, twenty- five, thirty, thirty-five, forty, forty-five, fifty or more preselected genes are evaluated.
  • at least one of the preselected genes for which expression is evaluated can be a gene whose product is involved in biosynthesis.
  • the evaluated gene can encode any one of the following biosynthetic gene products or human homologs thereof: mitochondrial ribosomal protein L27; stearoyl-Coenzyme A desaturase 2; methylenetetrahydrofolate dehydrogenase (NADP+ dependent)/ methenyltetrahydrofolate cyclohydrolase/ formyltetrahydrofolate synthase; dolichyl- di-phosphooligosaccharide-protein glycotransferase; hydroxysteroid (17-beta) dehydrogenase 4; UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1; coproporphyrinogen oxidase; ATPase, H+ transporting, Nl subunit F; aminolevulinate, delta-, dehydratase; RIKE ⁇ cD ⁇ A 0610038K03 gene 1; StAR- related lipid transfer
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in cell proliferation.
  • gene products involved in cell proliferation include: ligase I, D ⁇ A, ATP-dependent; protein kinase, cAMP dependent regulatory, type I beta; cysteine rich protein 2; platelet derived growth factor, alpha; cyclin-dependent kinase 4; cyclin-dependent kinase inhibitor 1A (P21); CD40 ligand-activated specific transcript 3; protein kinase, cAMP dependent regulatory, type I, alpha; topoisomerase (D ⁇ A) III beta; or protein phosphatase 2a, catalytic subunit, alpha isoform; and human homologs thereof.
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in metabolism.
  • gene products involved in metabolism include: mitochondrial ribosomal protein L27; cellular repressor of El A-stimulated genes; ⁇ ADH dehydrogenase (ubiquinone) flavoprotein 1 ; thioredoxin 1 ; proteasome (prosome, macropain) 26S subunit, ATPase 3; platelet-activating factor acetylhydrolase, isoform lb, alphal subunit; glutathione S-transferase omega 1; RIKEN cDNA 2210402 A09 gene product; low-density lipoprotein receptor-related protein 10; connective tissue growth factor; S-adenosylhomocysteine hydrolase; protein tyrosine phosphatase, receptor- type, F interacting protein, binding protein 2; POU domain, class 5, transcription factor 1 ; Tcf
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in cell growth and/or cell maintenance.
  • Examples gene products involved in cell growth and/or cell maintenance include: cell division cycle 42 (S. cerevisiae) homolog; EH-domain containing 1 protein; ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c (subunit 9), isoform 1; SEC 13 (S.
  • solute carrier family 25 mitochondrial ribosomal protein L30; capping protein (actin filament) muscle Z-line, beta; RAB5C, member RAS on
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in development.
  • gene products involved in development include: secreted frizzled-related sequence protein 1; shroom; jumonji; X (inactive)-specific transcript, antisense; and human homologs thereof.
  • at least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in signal transduction. Examples of gene products involved in signal transduction include: guanine nucleotide binding protein, beta 1; inositol polyphosphate-5-phosphatase;
  • the embryo is an in vitro fertilized mammalian embryo, e.g., a human embryo.
  • the embryo is a non-human mammalian embryo, e.g., a non-human mammalian embryo selected from the group consisting of a mouse embryo, a goat embryo, a cow embryo, a sheep embryo, a pig embryo, a horse embryo, a canine embryo, a feline embryo, and a rabbit embryo.
  • the non-human mammalian embryo can be, e.g., a transgenic embryo or a cloned embryo.
  • the method further includes providing a result of the evaluation to a third party, e.g., a hospital, clinic, government entity, reimbursing party or insurance company.
  • a third party e.g., a hospital, clinic, government entity, reimbursing party or insurance company.
  • payment for a medical procedure e.g., an in vitro fertilization service, or payment by a reimbursing party, e.g., a government entity or insurance company, is conditional on a selected outcome of the evaluation method.
  • the invention features a method of evaluating a cloned or transgenic non-human animal, e.g., evaluating the likelihood of the cloned or transgenic animal to develop to term.
  • the method includes: removing a cell from an embryo; and evaluating the expression of at least two, and preferably at least three, preselected genes in the cell, to thereby evaluate the embryo.
  • the non-human animal can be, e.g., a mammal, e.g., a goat, cow, sheep, pig, horse, dog, cat, rabbit, or mouse.
  • the method can further include selecting the embryo for implantation, hi some embodiments, the method can also include implanting the embryo into a female recipient.
  • the method includes evaluating to determine whether or not one or more, and preferably all of the genes are expressed or non- expressed. In another embodiment, the method includes evaluating to determine if the at least two, preferably three, preselected genes are expressed at a preselected level. For example, the preselected level can be below a reference level, e.g., it is at or below a level which correlates to no expression or the preselected level can be any level of expression above a reference level, e.g., it is above a level which correlates to no expression. In some embodiments, the method includes evaluating expression of the genes by evaluating the level of a nucleic acid, e.g., RNA or cDNA, from the cell.
  • a nucleic acid e.g., RNA or cDNA
  • the method further includes obtaining nucleic acid, e.g., RNA, from the cell.
  • the nucleic acid, e.g., RNA can be obtained from the cell within at least 5, 10, 15, 20, 25, 30 minutes, 1 hour, 2, 3, 4, 5 hours from the time the cell is removed from the embryo.
  • the method can further include obtaining cDNA from the RNA, e.g., using reverse transcription, e.g., the cDNA is obtained within at least 5, 10, 15, 20, 25, 30 minutes, 1 hour, 2, 3, 4, 5 hours from the time the RNA is obtained.
  • the cell can be maintained under conditions which do not significantly alter the expression of the genes prior to obtaining the nucleic acid, e.g., RNA.
  • the cell can be cryopreserved.
  • the method includes evaluating the expression within at least 5, 10, 15, 20, 25, 30 minutes, 1 hour, 2, 3, 4, 5 hours from the time the cell is removed from the embryo.
  • the method includes evaluating expression of the genes by: providing an array of a plurality of capture probes, e.g., nucleic acid sequences which can hybridize to specific nucleic acids from the cell, wherein each of the capture probes is positionally distinguishable from other capture probes of the plurality on the array; and hybridizing nucleic acid, e.g., cDNA, from the cell with the array of capture probes, to thereby analyze for expression of the specified nucleic acids from the cell.
  • the array is an array described herein.
  • the method further includes evaluating the absence or presence of a selected gene (or genes) associated with a sex chromosome, e.g., the Y chromosome, to thereby evaluate the sex of the embryo.
  • a selected gene or genes associated with a sex chromosome, e.g., the Y chromosome
  • the nucleic acid, e.g., RNA or cDNA, from the cell can be evaluated for the absence or presence of a gene specifically expressed on the Y chromosome but not the X chromosome, the presence of the gene being indicative of a male embryo.
  • the expression of the genes can be evaluated by: providing an array of a plurality of capture probes, wherein the array includes a plurality of capture probes, e.g., nucleic acid sequences which can hybridize to specific nucleic acids from the cell, namely nucleic acids from the preselected genes, and further includes a probe which hybridizes to a nucleic acid associated with one sex chromosome but not the other, e.g., it is associated with the Y chromosome but not the X chromosome.
  • Arrays which can be used include, e.g., the arrays described herein.
  • the method further includes determining if a preselected factor is present for one or both parents of the embryo.
  • the method can further include entering a value for the determination of the preselected factor into a database, e.g., a database described herein.
  • the database further includes one or both of data on the expression of the preselected genes and data on whether the selected embryo developed to term.
  • the factor can be, e.g., the presence of a treatment (e.g., the administration of a compound, e.g., a drug (e.g., a hormone), vitamin, food, or dietary supplement); the presence of an environmental factor (e.g., the presence of a substance in the environment); and/or the presence of a genetic factor or physical factor such as age.
  • a treatment e.g., the administration of a compound, e.g., a drug (e.g., a hormone), vitamin, food, or dietary supplement)
  • an environmental factor e.g., the presence of a substance in the environment
  • a genetic factor or physical factor such as age.
  • the method can further include determining if a preselected condition was used to produce the embryo.
  • the method can further include entering a value for the determination of the preselected condition into a database e.g., a database described herein.
  • the database further includes one or both of data on the expression of the preselected genes and data on whether the selected embryo developed to term.
  • the preselected condition can be, e.g., any condition used for microinjection and cloning techniques.
  • the preselected condition can be a fertilization technique, a germ cell (e.g., oocyte or sperm) retrieval technique, and/or the presence or absence of one or more factors in culture media.
  • the method includes evaluating a pre-implantation embryo wherein the pre-implantation embryo is in the two to thirty-two cell stages, preferably the four to sixteen cell stages, eight cell stage, of embryogenesis. In some embodiments, the method further includes evaluating the expression of at least one control gene.
  • control gene can be, e.g., selected from the group consisting of the beta actin gene, the beta-2 -micro globulin gene, the glyceraldehyde-3-phosphate dehydrogenase gene, the hypoxanthine phosphoribosyl- transferase 1 gene, the ribosomal protein L13a, subunit A gene of the succinate dehydrogenase complex, the TATA binding protein gene, and the ubiquitin C gene.
  • the invention features a database comprising a plurality of records.
  • Each record includes at least one, two or preferably all of the following: data on the expression of at least two, preferably at least three preselected genes obtained from a cell of an embryo; data on whether the embryo was implanted and/or carried to term; and data on a preselected factor present in one or both parents of the embryo.
  • the preselected factor can be one or more of: the presence of a treatment (e.g., the administration of a compound e.g., a drug (e.g., a hormone), vitamin, food or dietary supplement); the presence of an environmental factor (e.g., the presence of a substance in the environment); the presence of a genetic factor or physical factor such as age.
  • the database includes at least two records, and the preselected factor in each of the records differs from the other record.
  • the preselected factor can be administration of a compound and in one record, the preselected factor includes administration of the compound and in the other record, the compound is not administered, is administered at a different dose and/or a different compound is administered.
  • the preselected factor can be an environmental factor and in one record the factor is present and in the other record the environmental factor is not present or is present at a different level.
  • the preselected factor can be a physical factor such as age and the age in one record varies from the age in the other record, e.g., a difference in age of at least 5, 10, 15, 20 years or more.
  • each record of the database includes data on at least two preselected factors present in one or both parents of the embryo.
  • the database includes at least two records, and at least one preselected factor in each of the records differs from the other record.
  • the database includes at least two records and at least one preselected factor in the records differ and at least one of the other preselected factors is the same.
  • the database can include at least two records and each record includes at least one preselected factor and at least one preselected condition.
  • the database can be any kind of storage system capable of storing the various data for each of the records as described herein.
  • the database may be a flat file, a relational database, a table in a database, an object in a computer readable volatile or non- volatile memory, data accessible by computer program, such as data stored in a resource fork of an application program file on a computer readable storage medium.
  • the database is in a computer readable medium (e.g., a computer memory or storage device).
  • each record of the database includes data on whether at least two, preferably at least three preselected genes obtained from a cell of an embryo are expressed or non-expressed. In other embodiments, each record of the database includes data on whether at least two, preferably at least three of the preselected genes are expressed at a preselected level.
  • the data can indicate, e.g., whether the preselected level is below a reference level, e.g., it is at or below a level which correlates to no expression, or whether the preselected level is any level of expression above a reference level, e.g., it is above a level which correlates to no expression.
  • the data on whether at least two and preferably at least three preselected genes is obtained by a method described herein.
  • each record includes data on the expression of at least five, ten, fifteen, twenty, twenty-five, thirty, thirty-five, forty, forty-five, fifty or more preselected genes.
  • each record can further include data on the expression of at least one control gene.
  • control genes include: beta actin gene, the beta-2 -micro globulin gene, the glyceraldehyde-3 -phosphate dehydrogenase gene, the hypoxanthine phosphoribosyl-transferase 1 gene, the ribosomal protein LI 3a, subunit A gene of the succinate dehydrogenase complex, the TATA binding protein gene, and the ubiquithrC gene.
  • the invention features a method of evaluating the effect of a preselected factor, in one or both parents of an embryo, on the likelihood of an embryo implanting into a uterus and or developing to term using e.g., a database as described herein.
  • the preselected factor can be one or more of: the presence of a treatment (e.g., the administration of a compound, e.g., a drug (e.g., a hormone), vitamin, food, or dietary supplement); the presence of an environmental factor (e.g., the presence of a substance in the environment, residence in a preselected geographic area, performing a preselected occupation); the presence of a genetic factor or physical factor such as age.
  • a treatment e.g., the administration of a compound, e.g., a drug (e.g., a hormone), vitamin, food, or dietary supplement
  • an environmental factor e.g., the presence of a substance in the environment, residence in a preselected geographic area, performing
  • the invention features a database comprising a plurality of records.
  • Each record includes at least one, two or preferably all of the following: data on the expression of at least two, preferably at least three preselected genes obtained from a cell of an embryo; data on whether the embryo was implanted into the uterus and/or carried to term; and data on a preselected condition used to produce the embryo.
  • the preselected condition can be any condition used for in vitro fertilization.
  • the preselected condition can be any of: a fertilization technique, a germ cell (e.g., sperm or oocyte) retrieval technique, the presence or absence of one or more factors in culture media.
  • the database includes at least two records, and the preselected condition in each of the records differs from the other record.
  • the preselected condition can be a culture component and in one record the preselected condition includes the presence of the component and in the other record the component is not present or is present at a different amount.
  • each record of the database includes data on at least two preselected conditions.
  • the database includes at least two records, and at least one preselected condition in each of the records differs from the other record.
  • the database includes at least two records and at least one preselected condition in the records differ and at least one of the other preselected conditions is the same.
  • the database can be any kind of storage system capable of storing the various data for each of the records as described herein.
  • the database may be a flat file, a relational database, a table in a database, an object in a computer readable volatile or non- volatile memory, data accessible by computer program, such as data stored in a resource fork of an application program file on a computer readable storage medium.
  • the database is in a computer readable medium (e.g., a computer memory or storage device).
  • each record of the database includes data on whether at least two, preferably at least three preselected genes obtained from a cell of an embryo are expressed or non-expressed.
  • each record of the database includes data on whether at least two, preferably at least three of the preselected genes are expressed at a preselected level.
  • the data can indicate, e.g., whether the preselected level is below a reference level, e.g., it is at or below a level which correlates to no expression, or whether the preselected level is any level of expression above a reference level, e.g., it is above a level which correlates to no expression.
  • the data on whether at least two and preferably at least three preselected genes is obtained by a method described herein.
  • each record in the database includes data on the expression of at least one preselected gene whose product is involved in biosynthesis.
  • gene products involved in biosynthesis include: mitochondrial ribosomal protein L27; stearoyl-Co enzyme A desaturase 2; methylenetetrahydrofolate dehydrogenase (NADP+ dependent)/ methenyltetrahydrofolate cyclohydrolase/ formyltetrahydrofolate synthase; dolichyl-di-phosphooligosaccharide-protein glycotransferase; hydroxysteroid (17-beta) dehydrogenase 4; UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1; coproporphyrinogen oxidase; ATPase, H+ transporting, Nl subunit F; aminolevulinate, delta-, dehydratase; RD E ⁇ cD ⁇ A 06
  • each record in the database includes data on the expression of at least one preselected gene encoding a product that is involved in cell proliferation.
  • gene products involved in cell proliferation include: ligase I, D ⁇ A, ATP-dependent; protein kinase, cAMP dependent regulatory, type I beta; cysteine rich protein 2; platelet derived growth factor, alpha; cyclin-dependent kinase 4; cyclin-dependent kinase inhibitor 1 A (P21); CD40 ligand-activated specific transcript 3; protein kinase, cAMP dependent regulatory, type I, alpha; topoisomerase (D ⁇ A) III beta; protein phosphatase 2a, catalytic subunit, alpha isoform; or human homologs thereof.
  • each record in the database includes data on the expression of at least one preselected gene that encodes a product involved in metabolism.
  • gene products involved in metabolism include: mitochondrial ribosomal protein L27; cellular repressor of El A-stimulated genes; ⁇ ADH dehydrogenase (ubiquinone) flavoprotein 1; thioredoxin 1; proteasome (prosome, macropain) 26S subunit, ATPase 3; platelet-activating factor acetylhydrolase, isoform lb, alphal subunit; glutathione S-transferase omega 1; RIKEN cDNA 2210402 A09 gene product; low-density lipoprotein receptor-related protein 10; connective tissue growth factor; S-adenosylhomocysteine hydrolase; protein tyrosine phosphatase, receptor-type, F interacting protein, binding protein 2; POU domain, class 5, transcription factor 1; Tcfcp2
  • each record in the database includes data on at least one preselected gene that encodes a product involved in cell growth and or cell maintenance.
  • gene products involved in cell growth and/or cell maintenance include: cell division cycle 42 (S. cerevisiae) homolog; EH-domain containing 1 protein; ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c (subunit 9), isoform 1; EC 13 (S. cerevisiae) homolog; EH-domain containing 1 protein; ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c (subunit 9), isoform 1; EC 13 (S.
  • solute carrier family 25 mitochondrial ribosomal protein L30; capping protein (actin filament) muscle Z-line, beta; RAB5C, member RAS on
  • each record in the database includes at data on at least one preselected gene that encodes a product involved in development.
  • gene products involved in development include: secreted frizzled-related sequence protein 1; shroom; jumonji; X (inactive)-specific transcript, antisense; or human homologues thereof.
  • each record in the database includes data on at least one preselected gene that encodes a gene product involved in signal transduction.
  • gene products involved in signal transduction include: guanine nucleotide binding protein, beta 1; inositol polyphosphate-5-phosphatase; LanC
  • each record includes data on the expression of at least five, ten, fifteen, twenty, twenty-five, thirty, thirty-five, forty, forty-five, fifty or more preselected genes.
  • at least one of the preselected genes for which expression is evaluated can be a gene whose product is involved in biosynthesis.
  • the evaluated gene can encode any one of the following biosynthetic gene products or human homologs thereof: mitochondrial ribosomal protein L27; stearoyl-Coenzyme A desaturase 2; methylenetetrahydrofolate dehydrogenase (NADP+ dependent)/ methenyltetrahydrofolate cyclohydrolase/ formyltetrahydrofolate synthase; dolichyl-di-phosphooligosaccharide-protein glycotransferase; hydroxysteroid (17-beta) dehydrogenase 4; UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1; copropo ⁇ hyrinogen oxidase; ATPase, H+ transporting, VI subunit F; aminolevulinate, delta-, dehydratase; RTKEN cDNA 0610038K03 gene 1; StAR-related lipid transfer (
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in cell proliferation.
  • gene products involved in cell proliferation include: ligase I, D ⁇ A, ATP-dependent; protein kinase, cAMP dependent regulatory, type I beta; cysteine rich protein 2; platelet derived growth factor, alpha; cyclin-dependent kinase 4; cyclin-dependent kinase inhibitor 1 A (P21); CD40 ligand-activated specific transcript 3; protein kinase, cAMP dependent regulatory, type I, alpha; topoisomerase (D ⁇ A) III beta; or protein phosphatase 2a, catalytic subunit, alpha isoform; and human homologs thereof.
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in metabolism.
  • gene products involved in metabolism include: mitochondrial ribosomal protein L27; cellular repressor of El A-stimulated genes; ⁇ ADH dehydrogenase (ubiquinone) flavoprotein 1; thioredoxin 1; proteasome (prosome, macropain) 26S subunit, ATPase 3; platelet-activating factor acetylhydrolase, isoform lb, alphal subunit; glutathione S-transferase omega 1; RIKE ⁇ cD ⁇ A 2210402A09 gene product; low-density lipoprotein receptor-related protein 10; connective tissue growth factor; S-adenosylhomocysteine hydrolase; protein tyrosine phosphatase, receptor- type, F interacting protein, binding protein 2; POU domain, class 5, transcription factor 1 ; Tcf
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in cell growth and/or cell maintenance.
  • Examples gene products involved in cell growth and/or cell maintenance include: cell division cycle 42 (S. cerevisiae) homolog; EH-domain containing 1 protein; ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c (subunit 9), isoform 1; SEC 13 (S.
  • solute carrier family 25 mitochondrial ribosomal protein L30; capping protein (actin filament) muscle Z-line, beta; RAB5C, member RAS on
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in development.
  • gene products involved in development include: secreted frizzled-related sequence protein 1; shroom; jumonji; X (inactive)-specific transcript, antisense; and human homologs thereof.
  • at least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in signal transduction.
  • gene products involved in signal transduction include: guanine nucleotide binding protein, beta 1; inositol polyphosphate-5-phosphatase; LanC (bacterial lantibiotic synthetase component C)-like; talin; and human homologs thereof.
  • each record can further include data on the expression of at least one control gene.
  • control genes include beta actin gene, the beta-2-microglobulin gene, the glyceraldehyde-3-phosphate dehydrogenase gene, the hypoxanthine phosphoribosyl-transferase 1 gene, the ribosomal protein LI 3a, subunit A gene of the succinate dehydrogenase complex, the TATA binding protein gene, and the ubiquitin C gene.
  • the database can include data on one or more preselected condition(s) and one or more preselected factor(s) as described herein.
  • the invention features a method of evaluating the effect of a preselected condition on the likelihood of an embryo implanting into a uterus and/or developing to term using e.g., a database as described herein.
  • the preselected condition can be one or more of: any condition used for in vitro fertilization.
  • the preselected condition can be any of: a fertilization technique, a germ cell retrieval technique, the presence or absence of one or more factors in culture media.
  • the invention features an array of a plurality of capture probes, e.g., nucleic acid sequences which can hybridize to specific nucleic acids from a cell of an embryo.
  • Each of the capture probes is positionally distinguishable from other capture probes of the plurality on the array, and at least one probe, preferably at least two or three probes, hybridizes specifically with a gene the expression of which is predictive of the likelihood that an embryo which expresses the gene at a predetermined level, e.g., positive expression or no expression, will implant into the uterus and/or progress to term and at least one probe which can specifically hybridize to a nucleic acid encoding or expressed by a specific allele of a selected gene, e.g., it can distinguish between a first, e.g., a mutant, and a second, e.g., a non-mutant, form of a gene or between alternatively expressed forms such as splice variants.
  • the array can further include at least one probe that hybridizes to a nucleic acid associated with one sex chromosome but not the other, e.g., a nucleic acid associated with the Y chromosome but not the X chromosome.
  • the array includes less than 200, 150, 100, 75, 50 or 25 capture probes.
  • the capture probes are single stranded probes in the array. In other embodiments, the capture probes have a structure comprising a double stranded portion and a single stranded portion in the array.
  • hybridization to the array is detected by mass spectrophotometry, e.g., by MALDI-TOF mass spectrophotometry.
  • the array can be a high medium or low density array.
  • Examples of low density arrays include 96 well microtiter plates.
  • the array can be made by light directed synthesis, or deposited by a movable liquid dispensing head.
  • the array further includes at least one capture probe which can hybridize to a specific control gene.
  • at least one or the probes can hybridize to a control gene selected, e.g., from the group consisting of the beta actin gene, the beta-2-microglobulin gene, the glyceraldehyde-3 -phosphate dehydrogenase gene, the hypoxanthine phosphoribosyl-transferase 1 gene, the ribosomal protein LI 3 a, subunit A gene of the succinate dehydrogenase complex, the TATA binding protein gene, and the ubiquitin C gene.
  • the array includes at least five, ten, fifteen, twenty, twenty-five, thirty, thirty-five, forty, forty- five, fifty or more probes which hybridize specifically with genes the expression of which are predictive of the likelihood that an embryo which expresses the genes at a predetermined level, e.g., positive expression or no expression, will implant into a uterus and/or progress to term.
  • the array can include one or more probes that specifically hybridize to a gene whose product is involved in biosynthesis.
  • the evaluated gene can encode any one of the following biosynthetic gene products or human homologs thereof: mitochondrial ribosomal protein L27; stearoyl-Coenzyme A desaturase 2; methylenetetrahydrofolate dehydrogenase (NADP+ dependent)/ methenyltetrahydrofolate cyclohydrolase/ formyltetrahydrofolate synthase; dolichyl- di-phosphooligosaccharide-protein glycotransferase; hydroxysteroid (17-beta) dehydrogenase 4; UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1; copropo ⁇ hyrinogen oxidase; ATPase, H+ transporting, VI subunit F; aminolevulinate, delta-, dehydratase; RTKEN cDNA 0610038K03 gene 1; StAR- related lipid transfer (STAR)
  • the array can include one or more probes that specifically hybridize to a gene encoding a product involved in cell proliferation.
  • gene products involved in cell proliferation include: ligase I, D ⁇ A, ATP- dependent; protein kinase, cAMP dependent regulatory, type I beta; cysteine rich protein 2; platelet derived growth factor, alpha; cyclin-dependent kinase 4; cyclin- dependent kinase inhibitor 1 A (P21); CD40 ligand-activated specific transcript 3; protein kinase, cAMP dependent regulatory, type I, alpha; topoisomerase (D ⁇ A) III beta; or protein phosphatase 2a, catalytic subunit, alpha isoform; and human homologs thereof.
  • the array can include one or more probes that specifically hybridize to a gene encoding a product involved in metabolism.
  • gene products involved in metabolism include: mitochondrial ribosomal protein L27; cellular repressor of El A-stimulated genes; NADH dehydrogenase (ubiquinone) flavoprotein 1; thioredoxin 1; proteasome (prosome, macropain) 26S subunit, ATPase 3; platelet-activating factor acetylhydrolase, isoform lb, alphal subunit; glutathione S-transferase omega 1; RTKEN cDNA 2210402 A09 gene product; low-density lipoprotein receptor-related protein 10; connective tissue growth factor; S-adenosylhornocysteine hydrolase; protein tyrosine phosphatase, receptor- type, F interacting protein, binding protein 2; POU domain, class 5, transcription factor 1; Tcfcp2-related transcriptional re
  • the array can include one or more probes that specifically hybridize to a gene encoding a product involved in cell growth and or cell maintenance.
  • gene products involved in cell growth and/or cell maintenance include: cell division cycle 42 (S. cerevisiae) homolog; EH-domain containing 1 protein; ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c (subunit 9), isoform 1; SEC 13 (S.
  • solute carrier family 25 mitochondrial ribosomal protein L30; capping protein (actin filament) muscle Z-line, beta; RAB5C, member RAS on
  • the array can include one or more probes that specifically hybridize to a gene encoding a product involved in development.
  • Examples of gene products involved in development include: secreted frizzled-related sequence protein 1; shroom; jumonji; X (inactive)-specific transcript, antisense; and human homologs thereof.
  • at least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in signal transduction.
  • Examples of gene products involved in signal transduction include: guanine nucleotide binding protein, beta 1; inositol polyphosphate-5-phosphatase; LanC (bacterial lantibiotic synthetase component C)-like; talin; and human homologs thereof.
  • the array includes at least one, two, three, four, five, six, seven, eight, nine, ten or more probes which can specifically hybridize to a nucleic acid encoding or expressed by a specific allele of a selected gene, e.g., the probe or probes can distinguish between a first, e.g., a non-mutant, and a second, e.g., a mutant, form of the selected gene, or between alternatively expressed forms such as splice variants.
  • the probe or probes specifically hybridize to a selected gene associated with a disorder.
  • the selected gene can be, e.g.: the gene encoding cystic fibrosis transmembrane conductase regulator (CFTR), the gene encoding dystrophin, the Beta Thalassemia gene, the gene encoding Factor NIII, the gene encoding Factor IX, Tay-Sachs gene, survival motor neuron (SM ⁇ ) gene, the HD gene.
  • the probe or probes specifically hybridizes to a selected gene to detect the absence or presence of the second mutant form of the selected gene, e.g., to detect whether the embryo carries a mutation associated with a disorder.
  • disorders for which the embryo can be evaluated include but are not limited to: Adenoleukodystrophy, Amyotrophic Lateral Sclereosis (ALS), Becker Muscular Dystrophy, Beta Thalassemia, Cerebellar Ataxia, Charcot-Marie- Tooth Disease, Chondrodysplasia Aganglionic Megacolon, Conradi-Hunnerman Syndrome, Cystic Fibrosis, Duchenne Muscular Dystrophy, Hemophilia A or B,
  • Huntington's Disease Fragile X Syndrome, Glycogen Storage Disease, Hirschsprung Disease, Icthyosis, Lesch ⁇ yhan, Myopathies, Polycystic Ovary Syndrome, Restenosis Pigmentosa, Sickle Cell Anemia, Tay-Sachs Disease, and Non Willebran Disease.
  • the invention features a method of providing information.
  • the method includes: (1) providing an embryo; (2) removing a cell from the embryo; (3) providing nucleic acid, e.g., R ⁇ A or cD ⁇ A, from the cell; (4) evaluating the expression of at least two, and preferably at least three genes in the cell; (5) thereby evaluating the embryo, e.g., for the likelihood of implanting into a uterus and/or developing to term or for its suitability as a source of stem cells, wherein one or more of steps 1, 2, and 3 are performed by a first party, e.g., a fertility clinic, and at least one step, e.g., step 4, is performed by a second party, e.g., a service provider, and said second party transmits the evaluation, e.g., of step 4 and/or step 5 to the first party.
  • a first party e.g., a fertility clinic
  • a second party e.g., a service provider
  • the method further includes a step (2a) of freezing the cell, wherein the first party performs step (2a). In some embodiments, the method further includes a step (2a) of maintaining the cell under conditions which do not significantly alter the expression of the genes prior to obtaining the nucleic acid from the cell.
  • the method further includes a step (6) of selecting an embryo based upon the evaluation.
  • the first party performs step (6).
  • the second party performs step (6).
  • the method further includes a step (7) of implanting the embryo into a recipient female, wherein step (7) is performed by the first party.
  • the evaluation step of the method includes determining whether or not one or more, and preferably all of the genes are expressed or non- expressed.
  • the method includes evaluating to determine if the at least two, preferably three, preselected genes are expressed at a preselected level.
  • the preselected level can be below a reference level, e.g., it is at or below a level which correlates to no expression or the preselected level can be any level of expression above a reference level, e.g., it is above a level which correlates to no expression.
  • the evaluation step of the method includes evaluating a gene or genes having a defined expression pattern (i.e. a reference expression profile) during normal early embryonic development and determining if at the stage of embryo development during which the cell is obtained, the gene or genes are being expressed at a level consistent with the reference expression profile. Expression levels can be evaluated at one of the following stages of embryonic development: unfertilized egg stage, (fertilized) 1-cell stage, 2-cell stage, 4-cell stage, 8-cell stage, compacted embryo stage, morulae stage, and/or blastocyst stage. In one embodiment, the evaluation step of the method includes comparing the level of expression of the gene or genes to a reference expression profile.
  • a defined expression pattern i.e. a reference expression profile
  • the reference expression profile can provide a profile of changes in expression of the gene over early embryonic development in a normal embryo, for example, normal gene expression at the unfertilized egg, (fertilized) 1-cell, 2-cell, 4-cell, 8-cell, compacted embryo, morulae, and/or blastocyst stage of embryogenesis.
  • Examples of products encoded by genes that normally have a reference profiles during embryogenesis that show a constant increase in expression levels from the egg or from the (fertilized) 1-cell stage, through the 8-cell stage include: mitochondrial ribosomal protein L27; aminolevulinate, delta-, dehydratase; eukaryotic translation initiation factor 4E binding protein 1 ; GTP binding protein 1 ; mitochondrial ribosomal protein L17; cyclin-dependent kinase inhibitor 1 A (P21); NADH dehydrogenase (ubiquinone) flavoprotein 1 ; proteasome (prosome, macropain) 26S subunit, ATPase 3; platelet-activating factor acetylhydrolase, isoform lb, alphal subunit; RTKEN cDNA 2210402A09 gene product; RuvB-like protein 2; peroxiredoxin 6; RTKEN cDNA 0610027F08 gene product; acid beta glucosidase; peptidylpro
  • Examples of products encoded by genes that normally have a reference expression profile during embryogenesis that shows a net decline in expression from the egg or 1-cell stage, through the 4-cell stage, followed by an increase in expression by the 8-cell stage (, e.g., such that expression at the 8-cell stage is greater than or equal to expression levels at the egg or 1-cell stage) include: stearoyl-Coenzyme A desaturase 2; methylenetetrahydrofolate dehydrogenase (NADP+ dependent)/ methenyltetrahydrofolate cyclohydrolase/ formyltetrahydrofolate synthase; UDP- GahbetaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1; ATPase, H+ transporting, NO subunit B; leucyl-tR ⁇ A synthetase; beta-1,4 mannosyltransferase; ligase I, D ⁇ A, ATP-
  • Examples of products encoded by genes that are normally expressed at low levels at the egg stage, followed by an increase of expression, then a subsequent decrease in expression, followed by an increase of expression by the 8-cell stage include: hydroxysteroid (17-beta) dehydrogenase 4; defender against cell death 1; topoisomerase (DNA) III beta; low-density lipoprotein receptor-related protein 10; zinc finger, DHHC domain containing 3 protein; FK506 binding protein 9; ATP- binding cassette, sub-family D (ALD), member 3; H2-K region expressed gene 4 gene product; cysteine-rich hydrophobic domain 2; adaptor-related protein complex 3, beta 1 subunit; solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5; RTKEN cDNA 3010027G13 gene product; guanine nucleotide binding protein, beta 1 ; LanC (bacterial lantibiotic synthetase component C)-like gene product; and human homologs
  • Examples of products encoded by genes that are normally expressed at low levels from the egg stage through the 2-cell embryonic stage and subsequently demonstrate an increase in expression by the 8-cell stage include: dolichyl-di- phosphooligosaccharide-protein glycotransferase; copropo ⁇ hyrinogen oxidase; ATPase, H+ transporting, Nl subunit F; mitochondrial ribosomal protein LI 1; eukaryotic translation initiation factor 2, subunit 2 (beta); cysteine rich protem 2; platelet derived growth factor, alpha; cyclin-dependent kinase 4; CD40 ligand- activated specific transcript 3; cellular repressor of ElA-stimulated genes; glutathione S-transferase omega 1; RTKEN cDNA 2210402A09 gene product; connective tissue growth factor; protein tyrosine phosphatase, receptor-type, F interacting protein, binding protein 2; Tcfcp2-related transcriptional re
  • 2210408E11 gene product glucosidase, alpha, acid; cathepsin D; hexosaminidase A; isocitrate dehydrogenase 2 (NADP+), mitochondrial; translocase of inner mitochondrial membrane 9 (yeast) homolog; solute carrier family 19 (thiamine transproter), member 2; transmembrane 9 superfamily member 1; expressed sequence All 73274; capping protein (actin filament) muscle Z-line, beta; dynactin 1; solute carrier family 1, member 1; RTKEN cDNA 1810011E08 gene product; shroom; jumonji; and human homologs thereof.
  • Examples of products encoded by genes that normally demonstrate a fairly constant level of gene expression from the egg to the 8-cell stage include: RTKEN cDNA 0610038K03 gene; StAR-related lipid transfer (START) domain containing 4; Hermansky-Pudlak syndrome 1 homolog (human); protem kinase, cAMP dependent regulatory, type I beta; 3-hydroxybutyrate dehydrogenase (heart, mitochondrial); secreted frizzled-related sequence protein 1 ; X (inactive)-specific transcript, antisense; and human homologs thereof.
  • Examples of products encoded by genes that normally demonstrate a rise in expression after the fertilized egg stage, followed by a subsequent decline in expression by the 8-cell stage include: integrin beta 1 (fibronectin receptor beta) acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl- Coenzyme A thiolase); and human homologs thereof.
  • An example of a gene that normally demonstrates a constant decrease in expression from the egg stage through the 8-cell stage is the gene coding for heat shock factor binding protein 1, and a human homolog thereof.
  • Examples of products encoded by genes that normally demonstrate little change in expression from the egg stage to the 1-cell stage, followed by an increase in expression by the 2-cell stage to a level that changes relatively little into the 4-cell stage, and whose expression level subsequently rises by the 8-cell stage, include: solute carrier family 25 (mitochondrial carrier; dicarboxylate transporter), member 10 and adaptor protein complex AP-2, mul ; and human homologs thereof.
  • step (3) includes evaluating the expression of at least five, ten, fifteen, twenty, twenty five, thirty, thirty-five, forty, forty-five, fifty or more preselected genes.
  • at least one of the preselected genes for which expression is evaluated can be a gene whose product is involved in biosynthesis.
  • the evaluated gene can encode any one of the following biosynthetic gene products or human homologs thereof: mitochondrial ribosomal protein L27; stearoyl-Co enzyme A desaturase 2; methylenetetrahydrofolate dehydrogenase (NADP+ dependent)/ methenyltetrahydrofolate cyclohydrolase/ formyltetrahydrofolate synthase; dolichyl-di-phosphooligosaccharide-protein glycotransferase; hydroxysteroid (17-beta) dehydrogenase 4; UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1; copropo ⁇ hyrinogen oxidase; ATPase, H+ transporting, VI subunit F; aminolevulinate, delta-, dehydratase; RTKEN cDNA 0610038K03 gene 1; StAR-related lipid transfer (START
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in cell proliferation.
  • gene products involved in cell proliferation include: ligase I, D ⁇ A, ATP-dependent; protein kinase, cAMP dependent regulatory, type I beta; cysteine rich protein 2; platelet derived growth factor, alpha; cyclin-dependent kinase 4; cyclin-dependent kinase inhibitor 1 A (P21); CD40 ligand-activated specific transcript 3; protein kinase, cAMP dependent regulatory, type I, alpha; topoisomerase (D ⁇ A) III beta; or protein phosphatase 2a, catalytic subunit, alpha isoform; and human homologs thereof.
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in metabolism.
  • gene products involved in metabolism include: mitochondrial ribosomal protein L27; cellular repressor of ElA-stimulated genes; NADH dehydrogenase (ubiquinone) flavoprotein 1; thioredoxin 1; proteasome (prosome, macropain) 26S subunit, ATPase 3; platelet-activating factor acetylhydrolase, isoform lb, alphal subunit; glutathione S-transferase omega 1; RTKEN cDNA 2210402 A09 gene product; low-density lipoprotein receptor-related protein 10; connective tissue growth factor; S-adenosylhomocysteine hydrolase; protein tyrosine phosphatase, receptor- type, F interacting protein, binding protein 2; POU domain, class 5, transcription factor 1 ; Tcfcp2 -
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in cell growth and/or cell maintenance.
  • Examples gene products involved in cell growth and/or cell maintenance include: cell division cycle 42 (S. cerevisiae) homolog; EH-domain containing 1 protein; ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c (subunit 9), isoform 1; SEC 13 (S.
  • solute carrier family 25 mitochondrial ribosomal protein L30; capping protein (actin filament) muscle Z-line, beta; RAB5C, member RAS onc
  • RTKEN cDNA 1810011E08 gene product RTKEN cDNA 3010027G13 gene product; or human homologs thereof.
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in development.
  • gene products involved in development include: secreted frizzled-related sequence protein 1; shroom; jumonji; X (inactive)-specific transcript, antisense; and human homologs thereof.
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in signal transduction.
  • gene products involved in signal transduction include: guanine nucleotide binding protein, beta 1; inositol polyphosphate-5-phosphatase;
  • LanC bacterial lantibiotic synthetase component C-like; talin; and human homologs thereof.
  • step (3) further includes evaluating the expression of at least one control gene.
  • the control gene can be, e.g., the beta actin gene, the beta-2- micro globulin gene, the glyceraldehyde-3 -phosphate dehydrogenase gene, the hypoxanthine phosphoribosyl-transferase 1 gene, the ribosomal protein LI 3 a, subunit A gene of the succinate dehydrogenase complex, the TATA binding protein gene, and the ubiquitin C gene.
  • the method further includes a step (8) of providing a result of the evaluation to a third party, e.g., a hospital, clinic, government entity, reimbursing party or insurance company.
  • a third party e.g., a hospital, clinic, government entity, reimbursing party or insurance company.
  • the expression of the genes is evaluated by: providing an array of a plurality of capture probes, e.g., nucleic acid sequences which can hybridize to specific nucleic acids from the cell, wherein each of the capture probes is positionally distinguishable from other capture probes of the plurality on the array; and hybridizing nucleic acid, e.g., cDNA, from the cell with the array of capture probes, to thereby analyze expression of the specified nucleic acids from the cell.
  • the array can be, e.g., an array described herein.
  • the method includes a step (3a) of evaluating a selected gene, preferably a gene other than the genes evaluated for expression, to determine if a preselected form of the selected gene is present in the cell, wherein step (3 a) is performed by the second party.
  • Evaluation of the selected gene can include hybridizing a nucleic acid, e.g., RNA or cDNA, from the selected gene to a probe or primer, wherein the probe or primer is able to differentially hybridize to the nucleic acid, e.g., RNA or cDNA, from a first, e.g., a non-mutant, and a second, e.g., a mutant, form of the selected gene.
  • Step (3a) can be performed, e.g., using an array of a plurality of capture probes, e.g., nucleic acids which can hybridize to specific cDNAs from the cell, wherein each of the capture probes is positionally distinguishable from other capture probes of the plurality on the array, and which at least one of the probes which can specifically hybridize to a nucleic acid encoding or expressed by a specific allele of a selected gene, e.g., it can distinguish between a first, e.g., a mutant, and a second, e.g., a non-mutant, form of a gene or between alternatively expressed forms such as splice variants.
  • a first e.g., a mutant
  • a second e.g., a non-mutant
  • the array can be, e.g., an array described herein.
  • the selected gene is a gene associated with a disorder.
  • the selected gene can be: the gene encoding systrophin, the Beta Thalassemia gene, the gene encoding factor NIII, the gene encoding factor IX, Tay Sachs gene, survival motor neuron gene and for the HD gene.
  • step 3 a includes evaluating the absence or presence of the second mutant form of the selected gene, e.g., to detect whether the embryo carries a mutation associated with a disorder.
  • disorders for which the embryo can be evaluated include but are not limited to: Adenoleukodystrophy, Amyotrophic Lateral Sclereosis (ALS), Becker Muscular Dystrophy, Beta Thalassemia, Cerebellar Ataxia, Charcot-Marie-Tooth Disease, Chondrodysplasia Aganglionic Megacolon, Conradi-Hunnerman Syndrome, Cystic Fibrosis, Duchenne Muscular Dystrophy, Hemophilia A or B, Huntington's Disease, Fragile X Syndrome, Glycogen Storage Disease, Hirschsprung Disease, Icthyosis, Lesch Nyhan, Myopathies, Polycystic Ovary Syndrome, Restenosis Pigmentosa, Sickle Cell Anemia, Tay-Sachs Disease, and Non Willebran Disease.
  • the method further includes a step (3b) of evaluating a selected gene or cluster of genes, preferably other than the preselected gene evaluated for expression, to determine if the embryo is an aneuploid, i.e., the embryo has too few or too many copies of a chromosome.
  • Step (3b) can be performed by either the first party or the second party.
  • step (3b) can be performed in addition to or instead of step (3a).
  • the selected gene or cluster of genes can be evaluated by hybridizing a nucleic acid, e.g., an R ⁇ A or D ⁇ A, from a the selected gene or cluster of genes to a probe, and determining the number of copies of the gene or cluster of genes the hybridize to the probe.
  • the presence of more than two copies of a gene or cluster of genes is indicative that the embryo has more then two copies of the chromosome associated with that gene or genes.
  • the presence of one copy of the gene or cluster of genes is indicative that the embryo has one copy of the chromosome associated with that gene or genes. Having an extra chromosome is referred to as "trisomy”, missing a chromosome is referred to as "monosomy”.
  • the absence or presence of one or more chromosomes, e.g. one or more of chromosomes 1-22, above or below the normal number of chromosomes indicates that the embryo is unlikely to give rise to a healthy embryo.
  • the embryo is evaluated for one or more of: more than two of chromosome 21 in the embryo which is indicative of a risk for Down's Syndrome; more than two of chromosome 13 in the embryo which is indicative of a risk for Patau syndrome; more than two of chromosome 18 in the embryo which is indicative of a risk for Edwards syndrome; more than two of chromosome 16 in the embryo which is indicative of a risk for abnormal fetal development and/or miscarriage; more than two of chromosome 22 in the embryo which is indicative of a risk for abnormal fetal development and/or miscarriage; absence or presence of only one copy of chromosome 5 in the embryo which is indicative of a risk for Cri du Chat; more than one X chromosome in the embryo which is indicative of a risk for Klinefelter's syndrome; more than two X chromosomes in the embryo which is indicative of a risk for Triple X syndrome; and the absence of chromosome X in the embryo which is indicative of a risk
  • the method further includes step (3 c) of evaluating the presence or expression of at least one selected gene associated with a sex chromosome, e.g., associated with one sex chromosome but not the other, to determine if the sex chromosome is absent or present, thereby evaluating the sex of the embryo.
  • the selected gene is associated with the Y chromosome, e.g., the gene is the sry gene.
  • Step (3c) is performed by the second party.
  • the method can include performing two or more of: step (3a), step (3b) and step (3 c).
  • the invention features a method of providing information which includes: (1) providing an embryo; (2) removing a cell from the embryo; (3) providing RNA from the cell; (4) obtaining cDNA from the RNA; (5) evaluating the cDNA to determine expression of at least two, and preferably at least three genes in the cell, to (6) thereby evaluate the embryo, e.g., for the likelihood of implanting into a uterus and/or developing to term, wherein steps 1, 2, 3 and 4 are performed by a first party, e.g., a fertility clinic, and step 5 is performed by a second party, e.g., a service provider, and said second party transmits the evaluation of step 5 and/or step 6 to the first party.
  • steps 1, 2, 3 and 4 are performed by a first party, e.g., a fertility clinic
  • step 5 is performed by a second party, e.g., a service provider, and said second party transmits the evaluation of step 5 and/or step 6 to the first party.
  • the method further includes a step (2a) of freezing the cell, wherein the first party performs step (2a). In some embodiments, the method further includes a step (7) of selecting an embryo based upon the evaluation. In one embodiment, the first party performs step (7). In another embodiment, the second party performs step (7). In some embodiments, the method further includes a step (8) of implanting the embryo into a recipient female, wherein step (8) is performed by the first party.
  • the evaluation step of the method includes determining whether or not one or more, and preferably all of the genes are expressed or non- expressed.
  • the method includes evaluating to determine if the at least two, preferably three, preselected genes are expressed at a preselected level.
  • the preselected level can be below a reference level, e.g., it is at or below a level which correlates to no expression or the preselected level can be any level of expression above a reference level, e.g., it is above a level which correlates to no expression.
  • the evaluation step of the method includes evaluating a gene or genes having a defined expression pattern (i.e. a reference expression profile) during normal early embryonic development and determining if at the stage of embryo development during which the cell is obtained, the gene or genes are being expressed at a level consistent with the reference expression profile.
  • Expression levels can be evaluated at one of the following stages of embryonic development: unfertilized egg stage, (fertilized) 1-cell stage, 2-cell stage, 4-cell stage, 8-cell stage, compacted embryo stage, morulae stage, and or blastocyst stage.
  • the evaluation step of the method includes comparing the level of expression of the gene or genes to a reference expression profile.
  • the reference expression profile can provide a profile of changes in expression of the gene over early embryonic development in a normal embryo, for example, normal gene expression at the unfertilized egg, (fertilized) 1-cell, 2-cell, 4-cell, 8-cell, compacted embryo, morulae, and/or blastocyst stage of embryogenesis.
  • Examples of products encoded by genes that normally have a reference profiles during embryogenesis that show a constant increase in expression levels from the egg or from the (fertilized) 1-cell stage, through the 8-cell stage include: mitochondrial ribosomal protein L27; aminolevulinate, delta-, dehydratase; eukaryotic translation initiation factor 4E binding protein 1; GTP binding protein 1; mitochondrial ribosomal protein L17; cyclin-dependent kinase inliibitor 1A (P21); NADH dehydrogenase (ubiquinone) flavoprotein 1; proteasome (prosome, macropain) 26S subunit, ATPase 3; platelet-activating factor acetylhydrolase, isoform lb, alphal subunit; RTKEN cDNA 2210402A09 gene product; RuvB-like protein 2; peroxiredoxin 6; RTKEN cDNA 0610027F08 gene product; acid beta glucosidase; peptidy
  • Examples of products encoded by genes that normally have a reference expression profile during embryogenesis that shows a net decline in expression from the egg or 1-cell stage, through the 4-cell stage, followed by an increase in expression by the 8-cell stage (, e.g., such that expression at the 8-cell stage is greater than or equal to expression levels at the egg or 1-cell stage) include: stearoyl-Coenzyme A desaturase 2; methylenetetrahydrofolate dehydrogenase (NADP+ dependent)/ methenyltetrahydrofolate cyclohydrolase/ formyltetrahydrofolate synthase; UDP- GahbetaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1; ATPase, H+ transporting, NO subunit B; leucyl-tR ⁇ A synthetase; beta- 1,4 mannosyltransferase; ligase I, D ⁇ A, ATP-
  • Examples of products encoded by genes that are normally expressed at low levels at the egg stage, followed by an increase of expression, then a subsequent decrease in expression, followed by an increase of expression by the 8-cell stage include: hydroxysteroid (17-beta) dehydrogenase 4; defender against cell death 1; topoisomerase (DNA) III beta; low-density lipoprotein receptor-related protein 10; zinc finger, DHHC domain containing 3 protein; FK506 binding protein 9; ATP- binding cassette, sub-family D (ALD), member 3; H2-K region expressed gene 4 gene product; cysteine-rich hydrophobic domain 2; adaptor-related protein complex 3, beta 1 subunit; solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5; RIKEN cDNA 3010027G13 gene product; guanine nucleotide binding protein, beta 1; LanC (bacterial lantibiotic synthetase component C)-like gene product; and human homologs
  • Examples of products encoded by genes that are normally expressed at low levels from the egg stage through the 2-cell embryonic stage and subsequently demonstrate an increase in expression by the 8-cell stage include: dolichyl-di- phosphooligosaccharide-protein glycotransferase; copropo ⁇ hyrinogen oxidase; ATPase, H+ transporting, Nl subunit F; mitochondrial ribosomal protein LI 1 ; eukaryotic translation initiation factor 2, subunit 2 (beta); cysteine rich protem 2; platelet derived growth factor, alpha; cyclin-dependent kinase 4; CD40 ligand- activated specific transcript 3; cellular repressor of El A-stimulated genes; glutathione S-transferase omega 1; RIKE ⁇ cD ⁇ A 2210402 A09 gene product; connective tissue growth factor; protein tyrosine phosphatase, receptor-type, F interacting protein, binding protein 2; Tcfcp2-related
  • Examples of products encoded by genes that normally demonstrate a fairly constant level of gene expression from the egg to the 8-cell stage include: RTKEN cDNA 0610038K03 gene; StAR-related lipid transfer (START) domain containing 4; Hermansky-Pudlak syndrome 1 homolog (human); protein kinase, cAMP dependent regulatory, type I beta; 3-hydroxybutyrate dehydrogenase (heart, mitochondrial); secreted frizzled-related sequence protein 1 ; X (inactive)-specific transcript, antisense; and human homologs thereof.
  • Examples of products encoded by genes that normally demonstrate a rise in expression after the fertilized egg stage, followed by a subsequent decline in expression by the 8-cell stage include: integrin beta 1 (fibronectin receptor beta) acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl- Coenzyme A thiolase); and human homologs thereof.
  • An example of a gene that normally demonstrates a constant decrease in expression from the egg stage through the 8-cell stage is the gene coding for heat shock factor binding protein 1, and a human homolog thereof.
  • step (5) includes evaluating the expression of at least five, ten, fifteen, twenty, twenty five, thirty, thirty-five, forty, forty- five, fifty or more preselected genes.
  • at least one of the preselected genes for which expression is evaluated can be a gene whose product is involved in biosynthesis.
  • the evaluated gene can encode any one of the following biosynthetic gene products or human homologs thereof: mitochondrial ribosomal protein L27; stearoyl-Coenzyme A desaturase 2; methylenetetrahydrofolate dehydrogenase (NADP+ dependent)/ methenyltetrahydrofolate cyclohydrolase/ formyltetrahydrofolate synthase; dolichyl-di-phosphooligosaccharide-protein glycotransferase; hydroxysteroid (17-beta) dehydrogenase 4; UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1; copropo ⁇ hyrinogen oxidase; ATPase, H+ transporting, VI subunit F; aminolevulinate, delta-, dehydratase; RTKEN cDNA 0610038K03 gene 1; StAR-related lipid transfer (
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in cell proliferation.
  • gene products involved in cell proliferation include: ligase I, D ⁇ A, ATP-dependent; protein kinase, cAMP dependent regulatory, type I beta; cysteme rich protein 2; platelet derived growth factor, alpha; cyclin-dependent kinase 4; cyclin-dependent kinase inhibitor 1 A (P21); CD40 ligand-activated specific transcript 3; protein kinase, cAMP dependent regulatory, type I, alpha; topoisomerase (D ⁇ A) III beta; or protein phosphatase 2a, catalytic subunit, alpha isoform; and human homologs thereof.
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in metabolism.
  • gene products involved in metabolism include: mitochondrial ribosomal protein L27; cellular repressor of ElA-stimulated genes; ⁇ ADH dehydrogenase (ubiquinone) flavoprotein 1; thioredoxin 1; proteasome (prosome, macropain) 26S subunit, ATPase 3; platelet-activating factor acetylhydrolase, isoform lb, alphal subunit; glutathione S-transferase omega 1; RIKE ⁇ cD ⁇ A 2210402 A09 gene product; low-density lipoprotein receptor-related protein 10; connective tissue growth factor; S-adenosylhomocysteine hydrolase; protein tyrosine phosphatase, receptor- type, F interacting protein, binding protein 2; POU domain, class 5, transcription factor 1; Tcfc
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in cell growth and/or cell maintenance.
  • Examples gene products involved in cell growth and/or cell maintenance include: cell division cycle 42 (S. cerevisiae) homolog; EH-domain containing 1 protem; ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c (subunit 9), isoform 1; SEC 13 (S.
  • solute carrier family 25 mitochondrial ribosomal protein L30; capping protein (actin filament) muscle Z-line, beta; RAB5C, member RAS on
  • RTKEN cDNA 1810011E08 gene product RTKEN cDNA 3010027G13 gene product; or human homologs thereof.
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in development.
  • gene products involved in development include: secreted frizzled-related sequence protein 1; shroom; jumonji; X (inactive)-specific transcript, antisense; and human homologs thereof.
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in signal transduction.
  • gene products involved in signal transduction include: guanine nucleotide binding protein, beta 1; inositol polyphosphate-5 -phosphatase;
  • LanC bacterial lantibiotic synthetase component C-like; talin; and human homologs thereof.
  • the expression of the genes is evaluated by: providing an array of a plurality of capture probes, e.g., nucleic acid sequences which can hybridize to specific cDNAs from the cell, wherein each of the capture probes is positionally distinguishable from other capture probes of the plurality on the array; and hybridizing cDNA from the cell with the array of capture probes, to thereby analyze expression of the specified nucleic acids from the cell.
  • the array can be, e.g., an array described herein.
  • the method includes a step (5a) of evaluating a selected gene, preferably a gene other than the genes evaluated for expression, to determine if a preselected form of the selected gene is present in the cell, wherein step (5a) is performed by the second party.
  • Evaluation of the selected gene can include hybridizing RNA or cDNA from the selected gene to a probe or primer, wherein the probe or primer is able to differentially hybridize to RNA or cDNA from a first, e.g., a non-mutant, and a second, e.g., a mutant, form of the selected gene.
  • Step (5a) can be performed, e.g., using an array of a plurality of capture probes, e.g., nucleic acids which can hybridize to specific cDNAs from the cell, wherein each of the capture probes is positionally distinguishable from other capture probes of the plurality on the array, and which at least one of the probes which can specifically hybridize to a nucleic acid encoding or expressed by a specific allele of a selected gene, e.g., it can distinguish between a first, e.g., a mutant, and a second, e.g., a non-mutant, form of a gene or between alternatively expressed forms such as splice variants.
  • a first e.g., a mutant
  • a second e.g., a non-mutant
  • the array can be, e.g., an array described herein.
  • the selected gene is a gene associated with a disorder.
  • the selected gene can be: the gene encoding systrophin, the Beta Thalassemia gene, the gene encoding factor NIII, the gene encoding factor IX, Tay Sachs gene, survival motor neuron gene and for the HD gene.
  • step 3 a includes evaluating the absence or presence of the second mutant form of the selected gene, e.g., to detect whether the embryo carries a mutation associated with a disorder.
  • disorders for which the embryo can be evaluated include but are not limited to: Adenoleukodystrophy, Amyotrophic Lateral Sclereosis (ALS), Becker Muscular Dystrophy, Beta Thalassemia, Cerebellar Ataxia, Charcot-Marie-Tooth Disease, Chondrodysplasia Aganglionic Megacolon, Conradi-Hunnerman Syndrome, Cystic Fibrosis, Duchenne Muscular Dystrophy, Hemophilia A or B, Huntington's Disease, Fragile X Syndrome, Glycogen Storage Disease, Hirschsprung Disease, Icthyosis, Lesch ⁇ yhan, Myopathies, Polycystic Ovary Syndrome, Restenosis Pigmentosa, Sickle Cell Anemia, Tay-Sachs Disease, and Non Willebran Disease.
  • ALS Amyotrophic Lateral Sclereosis
  • Beta Thalassemia Cerebellar Ataxia
  • Charcot-Marie-Tooth Disease Chondrodysplasi
  • the method further includes a step (5b) of evaluating a selected gene or cluster of genes, preferably other than the preselected gene evaluated for expression, to determine if the embryo is an aneuploid, i.e., the embryo has too few or too many copies of a chromosome.
  • Step (5b) can be performed by either the first party or the second party.
  • step (5b) can be performed in addition to or instead of step (5 a).
  • the selected gene or cluster of genes can be evaluated by hybridizing a nucleic acid, e.g., an R A or D ⁇ A, from a the selected gene or cluster of genes to a probe, and determining the number of copies of the gene or cluster of genes the hybridize to the probe.
  • the presence of more than two copies of a gene or cluster of genes is indicative that the embryo has more then two copies of the chromosome associated with that gene or genes.
  • the presence of one copy of the gene or cluster of genes is indicative that the embryo has one copy of the chromosome associated with that gene or genes. Having an extra chromosome is referred to as "trisomy”, missing a chromosome is referred to as "monosomy”.
  • the absence or presence of one or more chromosomes, e.g. one or more of chromosomes 1-22, above or below the normal number of chromosomes indicates that the embryo is unlikely to give rise to a healthy embryo.
  • the embryo is evaluated for one or more of: more than two of chromosome 21 in the embryo which is indicative of a risk for Down's Syndrome; more than two of chromosome 13 in the embryo which is indicative of a risk for Patau syndrome; more than two of chromosome 18 in the embryo which is indicative of a risk for Edwards syndrome; more than two of chromosome 16 in the embryo which is indicative of a risk for abnormal fetal development and/or miscarriage; more than two of chromosome 22 in the embryo which is indicative of a risk for abnormal fetal development and/or miscarriage; absence or presence of only one copy of chromosome 5 in the embryo which is indicative of a risk for Cri du Chat; more than one X chromosome in the embryo which is indicative of a risk for Klinefelter's syndrome; more than two X chromosome in the embryo which is indicative of a risk for Triple X syndrome; and the absence of chromosome X in the embryo which is indicative of a risk for
  • the method further includes step (5c) of evaluating the presence or expression of at least one selected gene associated with a sex chromosome, e.g., associated with one sex chromosome but not the other, to determine if the sex chromosome is absent or present, thereby evaluating the sex of the embryo.
  • the selected gene is associated with the Y chromosome, e.g., the gene is the sry gene.
  • Step (5c) is performed by the second party.
  • the method can include performing two or more of: step (5a), step (5b) and step (5 c).
  • step (5) further includes evaluating the expression of at least one control gene.
  • the control gene can be, e.g., the beta actin gene, beta actin gene, the beta-2 -micro globulin gene, the glyceraldehyde-3 -phosphate dehydrogenase gene, the hypoxanthine phosphoribosyl-transferase 1 gene, the ribosomal protein L13a, subunit A gene of the succinate dehydrogenase complex, the TATA binding protein gene, and the ubiquitin C gene.
  • the beta actin gene e.g., the beta actin gene, beta actin gene, the beta-2 -micro globulin gene, the glyceraldehyde-3 -phosphate dehydrogenase gene, the hypoxanthine phosphoribosyl-transferase 1 gene, the ribosomal protein L13a, subunit A gene of the succinate dehydrogenase complex, the TATA binding protein gene, and the ubiquitin C gene.
  • the method further includes a step (9) of providing a result of the evaluation to a third party, e.g., a hospital, clinic, government entity, reimbursing party or insurance company.
  • a third party e.g., a hospital, clinic, government entity, reimbursing party or insurance company.
  • the invention features a method of providing information which includes: (1) providing an embryo; (2) removing a cell from the embryo; (3) providing nucleic acid, e.g., RNA or cDNA, from the cell; (4) evaluating the expression of at least two, and preferably at least three genes in the cell; (5) thereby evaluating the embryo, e.g., for the likelihood of implanting into a uterus and/or developing to term, wherein steps 1, and 2 are performed by a first party, e.g., a fertility clinic, and steps 3 and 4 are performed by a second party, e.g., a service provider, and said second party transmits the evaluation of step 4 and/or step 5 to the first party.
  • a first party e.g., a fertility clinic
  • steps 3 and 4 are performed by a second party, e.g., a service provider
  • the method further includes a step (2a) of freezing the cell, wherein the first party performs step (2a)
  • the nucleic acid e.g., RNA or cDNA is obtained from the cell within at least 5, 10, 15, 20, 25, 30 minutes, 1 hour, 2, 3, 4, 5, hours form the time the cell is removed.
  • the method further includes step (3 a) of obtaining cDNA form the RNA, e.g., using reverse transcription e.g., the cDNA is obtained within at least 5, 10, 15, 20, 25, 30 minutes, 1 hour, 2, 3, 4, 5, hours form the time the RNA is obtained.
  • the method further includes a step (6) of selecting an embryo based upon the evaluation.
  • the first party performs step (6).
  • the second party performs step (6).
  • the method further includes a step (7) of implanting the embryo into a recipient female, wherein step (7) is performed by the first party.
  • the evaluation step of the method includes determining whether or not one or more, and preferably all of the genes are expressed or non- expressed.
  • the method includes evaluating to determine if the at least two, preferably three, preselected genes are expressed at a preselected level.
  • the preselected level can be below a reference level, e.g., it is at or below a level which correlates to no expression or the preselected level can be any level of expression above a reference level, e.g., it is above a level which correlates to no expression.
  • the evaluation step of the method includes evaluating a gene or genes having a defined expression pattern (i.e.
  • the evaluation step of the method includes comparing the level of expression of the gene or genes to a reference expression profile.
  • the reference expression profile can provide a profile of changes in expression of the gene over early embryonic development in a normal embryo, for example, normal gene expression at the unfertilized egg, (fertilized) 1-cell, 2-cell, 4-cell, 8-cell, compacted embryo, morulae, and/or blastocyst stage of embryogenesis.
  • Examples of products encoded by genes that normally have a reference profiles during embryogenesis that show a constant increase in expression levels from the egg or from the (fertilized) 1-cell stage, through the 8-cell stage include: mitochondrial ribosomal protein L27; aminolevulinate, delta-, dehydratase; eukaryotic translation initiation factor 4E binding protein 1; GTP binding protein 1; mitochondrial ribosomal protein L17; cyclin-dependent kinase inhibitor 1A (P21); NADH dehydrogenase (ubiquinone) flavoprotein 1 ; proteasome (prosome, macropain) 26S subunit, ATPase 3; platelet-activating factor acetylhydrolase, isoform lb, alphal subunit; RTKEN cDNA 2210402A09 gene product; RuvB-like protein 2; peroxiredoxin 6; RTKEN cDNA 0610027F08 gene product; acid beta glucosidase; peptidylprolyl
  • Examples of products encoded by genes that normally have a reference expression profile during embryogenesis that shows a net decline in expression from the egg or 1-cell stage, through the 4-cell stage, followed by an increase in expression by the 8-cell stage (, e.g., such that expression at the 8-cell stage is greater than or equal to expression levels at the egg or 1-cell stage) include: stearoyl-Coenzyme A desaturase 2; methylenetetrahydrofolate dehydrogenase (NADP+ dependent)/ methenyltetrahydrofolate cyclohydrolase/ formyltetrahydrofolate synthase; UDP- GahbetaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1; ATPase, H+ transporting, NO subunit B; leucyl-tR ⁇ A synthetase; beta-l,4 mannosyltransferase; ligase I, D ⁇ A, ATP
  • proteasome (prosome, macropain) subunit alpha type 7; protein disulfide isomerase-related; isocitrate dehydrogenase 3 (NAD+) alpha; nuclear factor of activated T-cells, cytoplasmic 2 interacting protein; mitogen activated protein kinase 12; RTKEN cDNA 2900026G05 gene product; proteasome (prosome, macropain) subunit, beta type 6; DNA methyltransferase 3B; nudix (nucleoside diphosphate linked moiety X)-type motif 1; glutathione S-transferase, pi 2; acid phosphatase 1, soluble; ATPase, Na+/K+ transporting, alpha 1 polypeptide; small fragment nuclease; proteasome (prosome, macropain) subunit, beta type 3; SEC 13 related (S.
  • Examples of products encoded by genes that are normally expressed at low levels at the egg stage, followed by an increase of expression, then a subsequent decrease in expression, followed by an increase of expression by the 8-cell stage include: hydroxysteroid (17-beta) dehydrogenase 4; defender against cell death 1; topoisomerase (DNA) III beta; low-density lipoprotein receptor-related protein 10; zinc finger, DHHC domain containing 3 protein; FK506 binding protein 9; ATP- binding cassette, sub-family D (ALD), member 3; H2-K region expressed gene 4 gene product; cysteine-rich hydrophobic domain 2; adaptor-related protein complex 3, beta 1 subunit; solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5; RTKEN cDNA 3010027G13 gene product; guanine nucleotide binding protein, beta 1 ; LanC (bacterial lantibiotic synthetase component C)-like gene product; and human homologs
  • Examples of products encoded by genes that are normally expressed at low levels from the egg stage through the 2-cell embryonic stage and subsequently demonstrate an increase in expression by the 8-cell stage include: dolichyl-di- phosphooligosaccharide-protein glycotransferase; copropo ⁇ hyrinogen oxidase; ATPase, H+ transporting, VI subunit F; mitochondrial ribosomal protein LI 1 ; eukaryotic translation initiation factor 2, subunit 2 (beta); cysteine rich protein 2; platelet derived growth factor, alpha; cyclin-dependent kinase 4; CD40 ligand- activated specific transcript 3; cellular repressor -of El A-stimulated genes; glutathione S-transferase omega 1; RTKEN cDNA 2210402A09 g ⁇ ene product; connective tissue growth factor; protein tyrosine phosphatase, receptor-type, F interacting protein, binding protein 2; Tcfc
  • Examples of products encoded by genes that normally demonstrate a fairly constant level of gene expression from the egg to the 8-cell stage include: RTKEN cDNA 0610038K03 gene; StAR-related lipid transfer (START) domain containing 4; Hermansky-Pudlak syndrome 1 homolog (human); protein kinase, cAMP dependent regulatory, type I beta; 3-hydroxybutyrate dehydrogenase (heart, mitochondrial); secreted frizzled-related sequence protein 1 ; X (inactive)-specific transcript, antisense; and human homologs thereof.
  • Examples of products encoded by genes that normally demonstrate a rise in expression after the fertilized egg stage, followed by a subsequent decline in expression by the 8-cell stage include: integrin beta 1 (fibronectin receptor beta) acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl- Coenzyme A thiolase); and human homologs thereof.
  • integrin beta 1 fibronectin receptor beta
  • acetyl-Coenzyme A acyltransferase 2 mitochondrial 3-oxoacyl- Coenzyme A thiolase
  • human homologs thereof An example of a gene that normally demonstrates a constant decrease in expression from the egg stage through the 8-cell stage is the gene coding for heat shock factor binding protein 1, and a human homolog thereof.
  • Examples of products encoded by genes that normally demonstrate little change in expression from the egg stage to the 1-cell stage, followed by an increase in expression by the 2-cell stage to a level that changes relatively little into the 4-cell stage, and whose expression level subsequently rises by the 8-cell stage, include: solute carrier family 25 (mitochondrial carrier; dicarboxylate transporter), member 10 and adaptor protein complex AP-2, mul; and human homologs thereof.
  • step (4) includes evaluating the expression of at least five, ten, fifteen, twenty, twenty five, thirty, thirty-five, forty, forty-five, fifty or more preselected genes.
  • at least one of the preselected genes for which expression is evaluated can be a gene whose product is involved in biosynthesis.
  • the evaluated gene can encode any one of the following biosynthetic gene products or human homologs thereof: mitochondrial ribosomal protem L27; stearoyl-Coenzyme A desaturase 2; methylenetetrahydrofolate dehydrogenase (NADP+ dependent)/ methenyltetrahydrofolate cyclohydrolase/ formyltetrahydrofolate synthase; dolichyl-di-phosphooligosaccharide-protein glycotransferase; hydroxysteroid (17-beta) dehydrogenase 4; UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1; copropo ⁇ hyrinogen oxidase; ATPase, H+ transporting, VI subunit F; aminolevulinate, delta-, dehydratase; RTKEN cDNA 0610038K03 gene 1; StAR-related lipid transfer
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in cell proliferation.
  • gene products involved in cell proliferation include: ligase I, D ⁇ A, ATP-dependent; protein kinase, cAMP dependent regulatory, type I beta; cysteine rich protein 2; platelet derived growth factor, alpha; cyclin-dependent kinase 4; cyclin-dependent kinase inhibitor 1A (P21); CD40 ligand-activated specific transcript 3; protein kinase, cAMP dependent regulatory, type I, alpha; topoisomerase (D ⁇ A) III beta; or protein phosphatase 2a, catalytic subunit, alpha isoform; and human homologs thereof.
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in metabolism.
  • gene products involved in metabolism include: mitochondrial ribosomal protein L27; cellular repressor of El A-stimulated genes; ⁇ ADH dehydrogenase (ubiquinone) flavoprotein 1 ; thioredoxin 1 ; proteasome (prosome, macropain) 26S subunit, ATPase 3; platelet-activating factor acetylhydrolase, isoform lb, alphal subunit; glutathione S-transferase omega 1 ; RTKEN cDNA 2210402 A09 gene product; low-density lipoprotein receptor-related protein 10; connective tissue growth factor; S-adenosylhomocysteine hydrolase; protein tyrosine phosphatase, receptor- type, F interacting protein, binding protein 2; POU domain, class 5, transcription factor 1; Tcfc
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in cell growth and/or cell maintenance.
  • Examples gene products involved in cell growth and/or cell maintenance include: cell division cycle 42 (S. cerevisiae) homolog; EH-domain containing 1 protein; ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c (subunit 9), isoform 1; SEC 13 (S.
  • solute carrier family 25 mitochondrial ribosomal protein L30; capping protein (actin filament) muscle Z-line, beta; RAB5C, member RAS on
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in development.
  • gene products involved in development include: secreted frizzled-related sequence protein 1; shroom; jumonji; X (inactive)-specific transcript, antisense; and human homologs thereof.
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in signal transduction.
  • gene products involved in signal transduction include: guanine nucleotide binding protein, beta 1; inositol polyphosphate-5-phosphatase; LanC (bacterial lantibiotic synthetase component C)-like; talin; and human homologs thereof.
  • the expression of the genes is evaluated by: providing an array of a plurality of capture probes, e.g., nucleic acid sequences which can hybridize to specific nucleic acids from the cell, wherein each of the capture probes is positionally distinguishable from other capture probes of the plurality on the array; and hybridizing nucleic acid, e.g., cDNA, from the cell with the array of capture probes, to thereby analyze expression of the specified nucleic acids from the cell.
  • the array can be, e.g., an array described herein.
  • the method includes a step (4a) of evaluating a selected gene, preferably a gene other than the genes evaluated for expression, to determine if a preselected form of the selected gene is present in the cell, wherein step (4a) is performed by the second party.
  • Evaluation of the selected gene can include hybridizing a nucleic acid, e.g., RNA or cDNA, from the selected gene to a probe or primer, wherein the probe or primer is able to differentially hybridize to the nucleic acid, e.g., RNA or cDNA, from a first, e.g., a non-mutant, and a second, e.g., a mutant, form of the selected gene.
  • Step (4a) can be performed, e.g., using an array of a plurality of capture probes, e.g., nucleic acids which can hybridize to specific cDNAs from the cell, wherein each of the capture probes is positionally distinguishable from other capture probes of the plurality on the array, and which at least one of the probes which can specifically hybridize to a nucleic acid encoding or expressed by a specific allele of a selected gene, e.g., it can distinguish between a first, e.g., a mutant, and a second, e.g., a non-mutant, form of a gene or between alternatively expressed forms such as splice variants.
  • a first e.g., a mutant
  • a second e.g., a non-mutant
  • the array can be, e.g., an array described herein. .
  • the selected gene is a gene associated with a disorder.
  • the selected gene can be: the gene encoding systrophin, the Beta Thalassemia gene, the gene encoding factor NIII, the gene encoding factor IX, Tay Sachs gene, survival motor neuron gene and for the HD gene.
  • step 3 a includes evaluating the absence or presence of the second mutant form of the selected gene, e.g., to detect whether the embryo carries a mutation associated with a disorder.
  • disorders for which the embryo can be evaluated include but are not limited to: Adenoleukodystrophy, Amyotrophic Lateral Sclereosis (ALS), Becker Muscular Dystrophy, Beta Thalassemia, Cerebellar Ataxia, Charcot-Marie-Tooth Disease, Chondrodysplasia Aganglionic Megacolon, Conradi-Hunnerman Syndrome, Cystic Fibrosis, Duchenne Muscular Dystrophy, Hemophilia A or B, Huntington's Disease, Fragile X Syndrome, Glycogen Storage Disease, Hirschsprung Disease, Icthyosis, Lesch ⁇ yhan, Myopathies, Polycystic Ovary Syndrome', Restenosis Pigmentosa, Sickle Cell Anemia, Tay-Sachs Disease, and Non Willebran Disease.
  • the method further includes a step (4b) of evaluating a selected gene or cluster of genes, preferably other than the preselected gene evaluated for expression, to determine if the embryo is an aneuploid, i.e., the embryo has too few or too many copies of a chromosome.
  • Step (4b) can be performed by either the first party or the second party.
  • step (4b) can be performed in addition to or instead of step (4a).
  • the selected gene or cluster of genes can be evaluated by hybridizing a nucleic acid, e.g., an R ⁇ A or D ⁇ A, from a the selected gene or cluster of genes to a probe, and determining the number of copies of the gene or cluster of genes the hybridize to the probe.
  • the presence of more than two copies of a gene or cluster of genes is indicative that the embryo has more then two copies of the chromosome associated with that gene or genes.
  • the presence of one copy of the gene or cluster of genes is indicative that the embryo has one copy of the chromosome associated with that gene or genes. Having an extra chromosome is referred to as "trisomy”, missing a chromosome is referred to as "monosomy”.
  • the absence or presence of one or more chromosomes, e.g. one or more of chromosomes 1-22, above or below the normal number of chromosomes indicates that the embryo is unlikely to give rise to a healthy embryo.
  • the embryo is evaluated for one or more of: more than two of chromosome 21 in the embryo which is indicative of a risk for Down's Syndrome; more than two of chromosome 13 in the embryo which is indicative of a risk for Patau syndrome; more than two of chromosome 18 in the embryo which is indicative of a risk for Edwards syndrome; more than two of chromosome 16 in the embryo which is indicative of a risk for abnormal fetal development and/or miscarriage; more than two of chromosome 22 in the embryo which is indicative of a risk for abnormal fetal development and/or miscarriage; absence or presence of only one copy of chromosome 5 in the embryo which is indicative of a risk for Cri du Chat; more than two X chromosome in the embryo which is indicative of a risk for Klinefelter's syndrome; more than one X chromosome in the embryo which is indicative of a risk for Triple X syndrome; and the absence of chromosome X in the embryo which is indicative of a risk for
  • the method further includes step (4c) of evaluating the presence or expression of at least one selected gene associated with a sex chromosome, e.g., associated with one sex chromosome but not the other, to determine if the sex chromosome is absent or present, thereby evaluating the sex of the embryo.
  • the selected gene is associated with the Y chromosome, e.g., the gene is the sry gene.
  • Step (4c) is performed by the second party.
  • the method can include performing two or more of: step (4a), step (4b) and step (4c).
  • step (4) further includes evaluating the expression of at least one control gene.
  • the control gene can be, e.g., the beta actin gene, beta actin gene, the beta-2-micro globulin gene, the glyceraldehyde-3 -phosphate dehydrogenase gene, the hypoxanthine phosphoribosyl-transferase 1 gene, the ribosomal protein LI 3a, subunit A gene of the succinate dehydrogenase complex, the TATA binding protein gene, and the ubiquitin C gene.
  • the method further includes a step (8) of providing a result of the evaluation to a third party, e.g., a hospital, clinic, government entity, reimbursing party or insurance company.
  • the invention features a method of providing information which includes: (1) providing an embryo; (2) removing a cell from the embryo; (3) providing RNA from the cell; (4) obtaining cDNA from the RNA; (5) evaluating the cDNA to determine expression of at least two, and preferably at least three genes in the cell, to (6) thereby evaluate the embryo, e.g., for the likelihood of implanting into a uterus and/or developing to term, wherein steps 1, 2, 3 and 4 are performed by a first party, e.g., a fertility clinic, and step 5 is performed by a second party, e.g., a service provider, and said second party transmits the evaluation of step 5 and/or step 6 to the first party.
  • steps 1, 2, 3 and 4 are performed by a first party, e.g., a fertility clinic
  • step 5 is performed by a second party, e.g., a service provider, and said second party transmits the evaluation of step 5 and/or step 6 to the first party.
  • the method further includes a step (2a) of freezing the cell, wherein the first party performs step (2a).
  • the method further includes a step (7) of selecting an embryo based upon the evaluation.
  • the first party performs step (7).
  • the second party performs step (7).
  • the method further includes a step (8) of implanting the embryo into a recipient female, wherein step (8) is performed by the first party.
  • the evaluation step of the method includes determining whether or not one or more, and preferably all of the genes are expressed or non- expressed.
  • the method includes evaluating to determine if the at least two, preferably three, preselected genes are expressed at a preselected level.
  • the preselected level can be below a reference level, e.g., it is at or below a level which correlates to no expression or the preselected level can be any level of expression above a reference level, e.g., it is above a level which correlates to no expression.
  • the evaluation step of the method includes evaluating a gene or genes having a defined expression pattern (i.e. a reference expression profile) during normal early embryonic development and determining if at the stage of embryo development during which the cell is obtained, the gene or genes are being expressed at a level consistent with the reference expression profile. Expression levels can be evaluated at one of the following stages of embryonic development: unfertilized egg stage, (fertilized) 1-cell stage, 2-cell stage, 4-cell stage, 8-cell stage, compacted embryo stage, morulae stage, and/or blastocyst stage. In one embodiment, the evaluation step of the method includes comparing the level of expression of the gene or genes to a reference expression profile.
  • a defined expression pattern i.e. a reference expression profile
  • the reference expression profile can provide a profile of changes in expression of the gene over early embryonic development in a normal embryo, for example, normal gene expression at the unfertilized egg, (fertilized) 1-cell, 2-cell, 4-cell, 8-cell, compacted embryo, morulae, and/or blastocyst stage of embryogenesis.
  • Examples of products encoded by genes that normally have a reference profiles during embryogenesis that show a constant increase in expression levels from the egg or from the (fertilized) 1-cell stage, through the 8-cell stage include: mitochondrial ribosomal protein L27; aminolevulinate, delta-, dehydratase; eukaryotic translation initiation factor 4E binding protein 1 ; GTP binding protein 1 ; mitochondrial ribosomal protein L17; cyclin-dependent kinase inhibitor 1 A (P21); NADH dehydrogenase (ubiquinone) flavoprotein 1 ; proteasome (prosome, macropain) 26S subunit, ATPase 3; platelet-activating factor acetylhydrolase, isoform lb, alphal subunit; RTKEN cDNA 2210402 A09 gene product; RuvB-like protein 2; peroxiredoxin 6; RTKEN cDNA 0610027F08 gene product; acid beta glucosidase; peptidylpro
  • EH-domain containing 1 protein EH-domain containing 1 protein
  • ATP synthase H+ transporting, mitochondrial F0 complex, subunit c (subunit 9), isoform 1
  • actin, beta cytoplasmic
  • mitochondrial ribosomal protein L30 actin related protem 2/3 complex, subunit 5; and human homologs thereof.
  • Examples of products encoded by genes that normally have a reference expression profile during embryogenesis that shows a net decline in expression from the egg or 1-cell stage, through the 4-cell stage, followed by an increase in expression by the 8-cell stage (, e.g., such that expression at the 8-cell stage is greater than or equal to expression levels at the egg or 1-cell stage) include: stearoyl-Coenzyme A desaturase 2; methylenetetrahydrofolate dehydrogenase (NADP+ dependent)/
  • Examples of products encoded by genes that are normally expressed at low levels at the egg stage, followed by an increase of expression, then a subsequent decrease in expression, followed by an increase of expression by the 8-cell stage include: hydroxysteroid (17-beta) dehydrogenase 4; defender against cell death 1; topoisomerase (DNA) III beta; low-density lipoprotein receptor-related protein 10; zinc finger, DHHC domain containing 3 protein; FK506 binding protein 9; ATP- binding cassette, sub-family D (ALD), member 3; H2-K region expressed gene 4 gene product; cysteine-rich hydrophobic domain 2; adaptor-related protein complex 3, beta 1 subunit; solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5; RTKEN cDNA 3010027G13 gene product; guanine nucleotide binding protein, beta 1 ; LanC (bacterial lantibiotic synthetase component C)-like gene product; and human homologs
  • Examples of products encoded by genes that are normally expressed at low levels from the egg stage through the 2-cell embryonic stage and subsequently demonstrate an increase in expression by the 8-cell stage include: dolichyl-di- phosphooligosaccharide-protein glycotransferase; copropo ⁇ hyrinogen oxidase; ATPase, H+ transporting, Nl subunit F; mitochondrial ribosomal protein LI 1; eukaryotic translation initiation factor 2, subunit 2 (beta); cysteine rich protein 2; platelet derived growth factor, alpha; cyclin-dependent kinase 4; CD40 ligand- activated specific transcript 3; cellular repressor of ElA-stimulated genes; glutathione S-transferase omega 1; RTKE ⁇ cD ⁇ A 2210402A09 gene product; connective tissue growth factor; protein tyrosine phosphatase, receptor-type, F interacting protein, binding protein 2; Tcfcp2 -related
  • Examples of products encoded by genes that normally demonstrate a fairly constant level of gene expression from the egg to the 8-cell stage include: RTKEN cDNA 0610038K03 gene; StAR-related lipid transfer (START) domain containing 4; Hermansky-Pudlak syndrome 1 homolog (human); protein kinase, cAMP dependent regulatory, type I beta; 3-hydroxybutyrate dehydrogenase (heart, mitochondrial); secreted frizzled-related sequence protein 1 ; X (inactive)-specific transcript, antisense; and human homologs thereof.
  • Examples of products encoded by genes that normally demonstrate a rise in expression after the fertilized egg stage, followed by a subsequent decline in expression by the 8-cell stage include: integrin beta 1 (fibronectin receptor beta) acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl- Coenzyme A thiolase); and human homologs thereof.
  • An example of a gene that normally demonstrates a constant decrease in expression from the egg stage through the 8-cell stage is the gene coding for heat shock factor binding protein 1, and a human homolog thereof.
  • Examples of products encoded by genes that normally demonstrate little change in expression from the egg stage to the 1-cell stage, followed by an increase in expression by the 2-cell stage to a level that changes relatively little into the 4-cell stage, and whose expression level subsequently rises by the 8-cell stage, include: solute carrier family 25 (mitochondrial carrier; dicarboxylate transporter), member 10 and adaptor protein complex AP-2, mul; and human homologs thereof.
  • step (5) includes evaluating the expression of at least five, ten, fifteen, twenty, twenty five, thirty, thirty-five, forty, forty-five, fifty or more preselected genes.
  • at least one of the preselected genes for which expression is evaluated can be a gene whose product is involved in biosynthesis.
  • the evaluated gene can encode any one of the following biosynthetic gene products or human homologs thereof: mitochondrial ribosomal protein L27; stearoyl-Coenzyme A desaturase 2; methylenetetrahydrofolate dehydrogenase (NADP+ dependent)/ methenyltetrahydrofolate cyclohydrolase/ formyltetrahydrofolate synthase; dolichyl-di-phosphooligosaccharide-protein glycotransferase; hydroxysteroid (17-beta) dehydrogenase 4; UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1; copropo ⁇ hyrinogen oxidase; ATPase, H+ transporting, VI subunit F; aminolevulinate, delta-, dehydratase; RTKEN cDNA 0610038K03 gene 1; StAR-related lipid transfer (
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in cell proliferation.
  • gene products involved in cell proliferation include: ligase I, DNA, ATP-dependent; protein kinase, cAMP dependent regulatory, type I beta; cysteine rich protein 2; platelet derived growth factor, alpha; cyclin-dependent kinase 4; cyclin-dependent kinase inhibitor 1A (P21); CD40 ligand-activated specific transcript 3; protein kinase, cAMP dependent regulatory, type I, alpha; topoisomerase (DNA) III beta; or protein phosphatase 2a, catalytic subunit, alpha isoform; and human homologs thereof.
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in metabolism.
  • gene products involved in metabolism include: mitochondrial ribosomal protein L27; cellular repressor of ElA-stimulated genes; NADH dehydrogenase (ubiquinone) flavoprotein 1; thioredoxin 1; proteasome (prosome, macropain) 26S subunit, ATPase 3; platelet-activating factor acetylhydrolase, isoform lb, alphal subunit; glutathione S-transferase omega 1; RTKEN cDNA 2210402 A09 gene product; low-density lipoprotein receptor-related protein 10; connective tissue growth factor; S-adenosylhomocysteine hydrolase; protein tyrosine phosphatase, receptor- type, F interacting protein, binding protem 2; POU domain, class 5, transcription factor T; Tcfcp2-related transcription
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in cell growth and/or cell maintenance.
  • Examples gene products involved in cell growth and/or cell maintenance include: cell division cycle 42 (S. cerevisiae) homolog; EH-domain containing 1 protein; ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c (subunit 9), isoform 1; SEC 13 (S.
  • solute carrier family 25 mitochondrial ribosomal protein L30; capping protein (actin filament) muscle Z-line, beta; RAB5C, member RAS
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in development.
  • gene products involved in development include: secreted frizzled-related sequence protein 1 ; shroom; jumonji; X (inactive)-specific transcript, antisense; and human homologs thereof.
  • at least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in signal transduction.
  • gene products involved in signal transduction include: guanine nucleotide binding protein, beta 1 ; inositol polyphosphate-5 -phosphatase; LanC (bacterial lantibiotic synthetase component C)-like; talin; and human homologs thereof.
  • the expression of the genes is evaluated by: providing an array of a plurality of capture probes, e.g., nucleic acid sequences which can hybridize to specific cDNAs from the cell, wherein each of the capture probes is positionally distinguishable from other capture probes of the plurality on the array; and hybridizing cDNA from the cell with the anay of capture probes, to thereby analyze expression of the specified nucleic acids from the cell.
  • the array can be, e.g., an array described herein.
  • the method includes a step (5 a) of evaluating a selected gene, preferably a gene other than the genes evaluated for expression, to determine if a preselected form of the selected gene is present in the cell, wherein step (5a) is performed by the second party.
  • Evaluation of the selected gene can include hybridizing RNA or cDNA from the selected gene to a probe or primer, wherein the probe or primer is able to differentially hybridize to RNA or cDNA from a first, e.g., a non-mutant, and a second, e.g., a mutant, form of the selected gene.
  • Step (5a) can be performed, e.g., using an array of a plurality of capture probes, e.g., nucleic acids which can hybridize to specific cDNAs from the cell, wherein each of the capture probes is positionally distinguishable from other capture probes of the plurality on the array, and which at least one of the probes which can specifically hybridize to a nucleic acid encoding or expressed by a specific allele of a selected gene, e.g., it can distinguish between a first, e.g., a mutant, and a second, e.g., a non-mutant, form of a gene or between alternatively expressed forms such as splice variants.
  • a first e.g., a mutant
  • a second e.g., a non-mutant
  • the array can be, e.g., an array described herein.
  • the selected gene is a gene associated with a disorder.
  • the selected gene can be: the gene encoding systrophin, the Beta Thalassemia gene, the gene encoding factor NTH, the gene encoding factor IX, Tay Sachs gene, survival motor neuron gene and for the HD gene.
  • step (3 a) includes evaluating the absence or presence of the second mutant form of the selected gene, e.g., to detect whether the embryo carries a mutation associated with a disorder.
  • disorders for which the embryo can be evaluated include but are not limited to: Adenoleukodystrophy, Amyotrophic Lateral Sclereosis (ALS), Becker Muscular Dystrophy, Beta Thalassemia, Cerebellar Ataxia, Charcot-Marie-Tooth Disease, Chondrodysplasia Aganglionic Megacolon, Conradi-Hunnerman Syndrome, Cystic Fibrosis, Duchenne Muscular Dystrophy, Hemophilia A or B, Huntington's Disease, Fragile X Syndrome, Glycogen Storage Disease, Hirschsprung Disease, Icthyosis, Lesch Nyhan, Myopathies, Polycystic Ovary Syndrome, Restenosis Pigmentosa, Sickle Cell Anemia, Tay-Sachs Disease, and Von Willebran Disease.
  • the method further includes a step (5b) of evaluating a selected gene or cluster of genes, preferably other than the preselected gene evaluated for expression, to determine if the embryo is an aneuploid, i.e., the embryo has too few or too many copies of a chromosome.
  • Step (5b) can be performed by either the first party or the second party.
  • step (5b) can be performed in addition to or instead of step (5a).
  • the selected gene or cluster of genes can be evaluated by hybridizing a nucleic acid, e.g., an RNA or DNA, from a the selected gene or cluster of genes to a probe, and determining the number of copies of the gene or cluster of genes the hybridize to the probe.
  • the presence of more than two copies of a gene or cluster of genes is indicative that the embryo has more then two copies of the chromosome associated with that gene or genes.
  • the presence of one copy of the gene or cluster of genes is indicative that the embryo has one copy of the chromosome associated with that gene or genes. Having an extra chromosome is refened to as "trisomy”, missing a chromosome is refened to as "monosomy”.
  • the absence or presence of one or more chromosomes, e.g. one or more of chromosomes 1-22, above or below the normal number of chromosomes indicates that the embryo is unlikely to give rise to a healthy embryo.
  • the embryo is evaluated for one or more of: more than two of chromosome 21 in the embryo which is indicative of a risk for Down's Syndrome; more than two of chromosome 13 in the embryo which is indicative of a risk for Patau syndrome; more than two of chromosome 18 in the embryo which is indicative of a risk for Edwards syndrome; more than two of chromosome 16 in the embryo which is indicative of a risk for abnormal fetal development and/or miscarriage; more than two of chromosome 22 in the embryo which is indicative of a risk for abnormal fetal development and/or miscarriage; absence or presence of only one copy of chromosome 5 in the embryo which is indicative of a risk for Cri du Chat; more than two X chromosome in the embryo which is indicative of a risk for Klinefelter's syndrome; more than one X chromosome in the embryo which is indicative of a risk for Triple X syndrome; and the absence of chromosome X in the embryo which is indicative of a risk for
  • the method further includes step (5c) of evaluating the presence or expression of at least one selected gene associated with a sex chromosome, e.g., associated with one sex chromosome but not the other, to determine if the sex chromosome is absent or present, thereby evaluating the sex of the embryo.
  • the selected gene is associated with the Y chromosome, e.g., the gene is the sry gene.
  • Step (5c) is performed by the second party.
  • the method can include performing two or more of: step (5a), step (5b) and step (5 c).
  • step (5) further includes evaluating the expression of at least one control gene.
  • the control gene can be, e.g., the beta actin gene, beta actin gene, the beta-2-microglobulin gene, the glyceraldehyde-3-phosphate dehydrogenase gene, the hypoxanthine phosphoribosyl-transferase 1 gene, the ribosomal protein L13a, subunit A gene of the succinate dehydrogenase complex, the TATA binding protein gene, and the ubiquitin C gene.
  • the method further includes a step (9) of providing a result of the evaluation to a third party, e.g., a hospital, clinic, government entity, reimbursing party or insurance company.
  • a third party e.g., a hospital, clinic, government entity, reimbursing party or insurance company.
  • the invention features a method of evaluating a factor for its effect on the implantation of the embryo into a uterus and/or the viability of an embryo.
  • the method includes: providing an embryo having at least one parent in which the factor is present; removing a cell from the embryo; and evaluating the expression of at least two, and preferably at least three genes in the cell, to thereby determine the effect of the factor on viability of the embryo.
  • the factor is the presence of a treatment, e.g., the administration of a compound, e.g., a drug, vitamin, food, or dietary supplement.
  • the factor can be the presence of an environmental factor (e.g., the presence of a substance in the environment, residence with a preselected geographic area, performing a preselected occupation) and/or the presence of a genetic factor or a physical factor such as age.
  • the method further includes: providing a second embryo having at least one parent, preferably both parents in which the factor is absent; removing a cell from the second embryo; evaluating the expression of at least two, and preferably at least three genes in the cell of the second embryo, to thereby determine the effect of the absence of the factor on viability of the second embryo; and comparing the evaluation of the embryo and the second embryo to evaluate the effect of the presence or absence of the factor on embryo viability.
  • the method further includes: providing a second embryo having at least one parent in which the factor is present; removing a cell from the second embryo; evaluating the expression of at least two, and preferably at least three genes in the cell of the second embryo, to thereby determine the effect of the factor on viability of the second embryo; and comparing the' evaluation of the embryo and the second embryo to determine a conelation between the presence of the factor and embryo viability.
  • the method further includes: providing a third embryo having at least one parent, preferably both parents, in which the factor is absent; removing a cell from the third embryo; evaluating expression of at least two, preferably three genes, in the cell of the third embryo to determine the effect of the absence of the factor on the viability of the embryo; and comparing the evaluation of the first, second and third embryos to evaluate the effect the absence or presence of the factor has on embryo viability.
  • the method further includes evaluating if the embryo (or embryos) develops to term.
  • the method includes evaluating to determine whether or not one or more, and preferably all of the genes are expressed or non- expressed. In another embodiment, the method includes evaluating to determine if the at least two, preferably three, preselected genes are expressed at a preselected level.
  • the preselected level can be below a reference level, e.g., it is at or below a level which conelates to no expression or the preselected level can be any level of expression above a reference level, e.g., it is above a level which correlates to no expression.
  • the method includes evaluating expression of the genes by evaluating the level of a nucleic acid, e.g., RNA or cDNA, from the cell.
  • a nucleic acid e.g., RNA or cDNA
  • the method further includes evaluating the expression of at least one control gene.
  • the control gene can be, e.g., selected from the group consisting of the beta actin gene, the beta-2-micro globulin gene, the glyceraldehyde-3-phosphate dehydrogenase gene, the hypoxanthine phosphoribosyl- transferase 1 gene, the ribosomal protein LI 3 a, subunit A gene of the succinate dehydrogenase complex, the TATA binding protein gene, and the ubiquitin C gene.
  • the evaluation step of the method includes evaluating a gene or genes having a defined expression pattern (i.e. a reference expression profile) during normal early embryonic development and determining if at the stage of embryo development during which the cell is obtained, the gene or genes are being expressed at a level consistent with the reference expression profile. Expression levels can be evaluated at one of the following stages of embryonic development: unfertilized egg stage, (fertilized) 1-cell stage, 2-cell stage, 4-cell stage, 8-cell stage, compacted embryo stage, morulae stage, and/or blastocyst stage. In one embodiment, the evaluation step of the method includes comparing the level of expression of the gene or genes to a reference expression profile.
  • a defined expression pattern i.e. a reference expression profile
  • the reference expression profile can provide a profile of changes in expression of the gene over early embryonic development in a normal embryo, for example, normal gene expression at the unfertilized egg, (fertilized) 1-cell, 2-cell, 4-cell, 8-cell, compacted embryo, morulae, and/or blastocyst stage of embryogenesis.
  • Examples of products encoded by genes that normally have a reference profiles during embryogenesis that show a constant increase in expression levels from the egg or from the (fertilized) 1-cell stage, through the 8-cell stage include: mitochondrial ribosomal protein L27; aminolevulinate, delta-, dehydratase; eukaryotic translation initiation factor 4E binding protein 1 ; GTP binding protein 1 ; mitochondrial ribosomal protein L17; cyclin-dependent kinase inhibitor 1A (P21); NADH dehydrogenase (ubiquinone) flavoprotein 1 ; proteasome (prosome, macropain) 26S subunit, ATPase 3; platelet-activating factor acetylhydrolase, isoform lb, alphal subunit; RTKEN cDNA 2210402A09 gene product; RuvB-like protein 2; peroxiredoxin 6; RTKEN cDNA 0610027F08 gene product; acid beta glucosidase; peptidylpro
  • Examples of products encoded by genes that normally have a reference expression profile during embryogenesis that shows a net decline in expression from the egg or 1-cell stage, through the 4-cell stage, followed by an increase in expression by the 8-cell stage (, e.g., such that expression at the 8-cell stage is greater than or equal to expression levels at the egg or 1-cell stage) include: stearoyl-Coenzyme A desaturase 2; methylenetetrahydrofolate dehydrogenase (NADP+ dependent)/ methenyltetrahydrofolate cyclohydrolase/ formyltetrahydrofolate synthase; UDP- • GahbetaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1; ATPase, H+ transporting, NO subunit B; leucyl-tR ⁇ A synthetase; beta- 1,4 mannosyltransferase; ligase I, D ⁇ A, ATP
  • Examples of products encoded by genes that are normally expressed at low levels at the egg stage, followed by an increase of expression, then a subsequent decrease in expression, followed by an increase of expression by the 8-cell stage include: hydroxysteroid (17-beta) dehydrogenase 4; defender against cell death 1; topoisomerase (DNA) III beta; low-density lipoprotein receptor-related protein 10; zinc finger, DHHC domain containing 3 protein; FK506 binding protein 9; ATP- binding cassette, sub-family D (ALD), member 3; H2-K region expressed gene 4 gene product; cysteine-rich hydrophobic domain 2; adaptor-related protein complex 3, beta 1 subunit; solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5; RTKEN cDNA 3010027G13 gene product; guanine nucleotide binding protein, beta 1; LanC (bacterial lantibiotic synthetase component C)-like gene product; and human homologs thereof
  • Examples of products encoded by genes that are normally expressed at low levels from the egg stage through the 2-cell embryonic stage and subsequently demonstrate an increase in expression by the 8-cell stage include: dolichyl-di- phosphooligosaccharide-protein glycotransferase; copropo ⁇ hyrinogen oxidase; ATPase, H+ transporting, VI subunit F; mitochondrial ribosomal protein LI 1; eukaryotic translation initiation factor 2, subunit 2 (beta); cysteine rich protein 2; platelet derived growth factor, alpha; cyclin-dependent kinase 4; CD40 ligand- activated specific transcript 3; cellular repressor of ElA-stimulated genes; glutathione S-transferase omega 1; RTKEN cDNA 2210402A09 gene product; connective tissue growth factor; protein tyrosine phosphatase, receptor-type, F interacting protein, binding protein 2; Tcfcp2-related transcriptional repressor
  • Examples of products encoded by genes that normally demonstrate a fairly constant level of gene expression from the egg to the 8-cell stage include: RTKEN cDNA 0610038K03 gene; StAR-related lipid transfer (START) domain containing 4; Hermansky-Pudlak syndrome 1 homolog (human); protein kinase, cAMP dependent regulatory, type I beta; 3-hydroxybutyrate dehydrogenase (heart, mitochondrial); secreted frizzled-related sequence protein 1; X (inactive)-specific transcript, antisense; and human homologs thereof.
  • Examples of products encoded by genes that normally demonstrate a rise in expression after the fertilized egg stage, followed by a subsequent decline in expression by the 8-cell stage include: integrin beta 1 (fibronectin receptor beta) acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl- Coenzyme A thiolase); and human homologs thereof.
  • genes that normally demonstrates a constant decrease in expression from the egg stage through the 8-cell stage are the gene coding for heat shock factor binding protein 1, and a human homolog thereof.
  • Examples of products encoded by genes that normally demonstrate little change in expression from the egg stage to the 1-cell stage, followed by an increase in expression by the 2-cell stage to a level that changes relatively little into the 4-cell stage, and whose expression level subsequently rises by the 8-cell stage include: solute carrier family 25 (mitochondrial carrier; dicarboxylate transporter), member 10 and adaptor protein complex AP-2, mul ; and human homologs thereof.
  • the method includes evaluating the expression of at least five, ten, fifteen, twenty, twenty five, thirty, thirty-five, forty, forty- five, fifty or more preselected genes.
  • at least one of the preselected genes for which expression is evaluated can be a gene whose product is involved in biosynthesis.
  • the evaluated gene can encode any one of the following biosynthetic gene products or human homologs thereof: mitochondrial ribosomal protein L27; stearoyl-Coenzyme A desaturase 2; methylenetetrahydrofolate dehydrogenase (NADP+ dependent)/ methenyltetrahydrofolate cyclohydrolase/ formyltetrahydrofolate synthase; dolichyl-di-phosphooligosaccharide-protein glycotransferase; hydroxysteroid (17-beta) dehydrogenase 4; UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1; copropo ⁇ hyrinogen oxidase; ATPase, H+ transporting, Nl subunit F; aminolevulinate, delta-, dehydratase; RTKE ⁇ cD ⁇ A 0610038K03 gene 1; StAR-related
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in cell proliferation.
  • gene products involved in cell proliferation include: ligase I, D ⁇ A, ATP-dependent; protein kinase, cAMP dependent regulatory, type I beta; cysteine rich protein 2; platelet derived growth factor, alpha; cyclin-dependent kinase 4; cyclin-dependent kinase inhibitor 1A (P21); CD40 ligand-activated specific transcript 3; protein kinase, cAMP dependent regulatory, type I, alpha; topoisomerase (D ⁇ A) III beta; or protein phosphatase 2a, catalytic subunit, alpha isoform; and human homologs thereof.
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in metabolism.
  • gene products involved in metabolism include: mitochondrial ribosomal protein L27; cellular repressor of El A-stimulated genes; ⁇ ADH dehydrogenase (ubiquinone) flavoprotein 1; thioredoxin 1; proteasome (prosome, macropain) 26S subunit, ATPase 3; platelet-activating factor acetylhydrolase, isoform lb, alphal subunit; glutathione S-transferase omega 1; RTKEN cDNA 2210402 A09 gene product; low-density lipoprotein receptor-related protein 10; connective tissue growth factor; S-adenosylhomocysteine hydrolase; protein tyrosine phosphatase, receptor- type, F interacting protein, binding protein 2; POU domain, class 5, transcription factor 1; Tcfcp2-related
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in cell growth and/or cell maintenance.
  • Examples gene products involved in cell growth and/or cell maintenance include: cell division cycle 42 (S. cerevisiae) homolog; EH-domain containing 1 protein; ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c (subunit 9), isoform 1; SEC13 (S.
  • solute carrier family 25 mitochondrial ribosomal protein L30; capping protein (actin filament) muscle Z-line, beta; RAB5C, member RAS onc
  • RTKEN cDNA 1810011E08 gene product RTKEN cDNA 3010027G13 gene product; or human homologs thereof.
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in development.
  • gene products involved in development include: secreted frizzled-related sequence protein 1 ; shroom; jumonji; X (inactive)-specific transcript, antisense; and human homologs thereof.
  • At least one of the preselected genes for which expression is evaluated can be a gene encoding a product involved in signal transduction.
  • gene products involved in signal transduction include: guanine nucleotide binding protein, beta 1; inositol polyphosphate-5 -phosphatase;
  • LanC bacterial lantibiotic synthetase component C-like; talin; and human homologs thereof.
  • evaluating the expression of the genes is performed, e.g., using an array described herein.
  • the method is performed using, e.g., a database described herein.
  • the preselected factor is administration of a compound
  • the method further includes preparing or providing instructions, e.g., dosages, warnings, precautions, treatment groups, etc., wherein the instructions are based upon information obtained using the methods described herein.
  • instructions e.g., dosages, warnings, precautions, treatment groups, etc.
  • the invention features a method of evaluating a condition for its effect on the implantation of the embryo into a uterus and/or the viability of an embryo.
  • the method includes: providing an embryo produced using a preselected condition; removing a cell from the embryo; and evaluating the expression of at least two, and preferably at least three genes in the cell, to thereby determine the effect of the condition on viability of the embryo.
  • the preselected condition can be, e.g., any condition used for in vitro fertilization.
  • the preselected condition can be fertilization techniques, a germ cell (e.g., sperm or oocyte) retrieval technique, and/or the absence or presence of one or more factors in culture media.
  • the method further includes: providing a second embryo produced with the condition absent or the condition is different; removing a cell from the second embryo; evaluating the expression of at least two, and preferably at least three genes in the cell of the second embryo, to thereby determine the effect of the absence of the condition or change in the condition has on viability of the second embryo; and comparing the evaluation of the embryo and the second embryo to evaluate the effect of the presence or absence of the condition on embryo viability.
  • the method further includes: providing a second embryo produced with the condition present; removing a cell from the second embryo; evaluating the expression of at least two, and preferably at least three genes in the cell of the second embryo, to thereby determine the effect of the condition on viability of the second embryo; and comparing the evaluation of the embryo and the second embryo to determine a correlation between the presence of the condition and embryo viability, h a prefened embodiment, the method further includes: providing a third embryo produced with the condition absent; removing a cell from the third embryo; evaluating expression of at least two, preferably three genes, in the cell of the third embryo to determine the effect of the absence of the condition or difference in the condition on the viability of the embryo; and comparing the evaluation of the first, second and third embryos to evaluate the effect the absence or presence of the condition has on embryo viability.
  • the method further includes evaluating if the embryo (or embryos) develop to term.
  • the method includes evaluating to determine whether or not one or more, and preferably all of the genes are expressed or non- expressed. In another embodiment, the method includes evaluating to determine if the at least two, preferably three, preselected genes are expressed at a preselected level.
  • the preselected level can be below a reference level, e.g., it is at or below a level which correlates to no expression or the preselected level can be any level of expression above a reference level, e.g., it is above a level which conelates to no expression.
  • the method includes evaluating expression of the genes by evaluating the level of a nucleic acid, e.g., RNA or cDNA, from the cell. h some embodiments, the method further includes evaluating the expression of at least one control gene.
  • the control gene can be, e.g., selected from the group consisting of the beta actin gene, the beta-2-micro globulin gene, the glyceraldehyde-3 -phosphate dehydrogenase gene, the hypoxanthine phosphoribosyl- transferase 1 gene, the ribosomal protein L13a, subunit A gene of the succinate dehydrogenase complex, the TATA binding protein gene, and the ubiquitin C gene.
  • the method includes evaluating expression of the preselected genes, wherein expression of at least five, ten, fifteen, twenty, twenty- five, thirty, thirty-five, forty, forty- five, fifty or more preselected genes are evaluated.
  • at least one of the preselected genes for which expression is evaluated can be, e.g., any of the genes described herein.
  • evaluating the expression of the genes is performed, e.g., using an array described herein.
  • the method is performed using, e.g., a database described herein.
  • the method further includes preparing or providing instructions, e.g., regarding in vitro fertilization techniques or conditions, e.g., warnings or recommendations, wherein the instructions are based upon information obtained using the methods described herein.
  • the invention features a method of evaluating whether a reimbursing party, e.g., a government entity or insurance company will make a payment, e.g., to a patient, health care provider, hospital, clinic or other government entity or other insurance company, for e.g., payment of an in vitro fertilization service.
  • the method includes: obtaining an evaluation has preselected value, e.g., it is positive, making or not making the payment. In a prefened embodiment, the payment is made.
  • the invention features a method of reducing the likelihood of multiple births obtained from an in vitro fertilization procedure.
  • the method includes: providing a plurality of embryos; removing a cell from each of the embryos; evaluating the expression of at least two, preferably three genes from each of the cells; selecting an embryo or embryos expressing the two, preferably three genes at a preselected level, e.g., expression or no expression; and implanting the embryo or embryos, thereby reducing the likelihood of multiple births resulting from the implantation.
  • the invention provides methods of evaluating the likelihood of an embryo to develop to term.
  • This information can aid in selecting an embryo, e.g., an in vitro produced embryo, e.g., a human or non-human embryo, for implantation.
  • This has application in both in vitro fertilization procedures as well as cloning and transgenic technologies, by increasing the likelihood that an implanted embryo will develop to term.
  • this information can aid in selecting an embryo as a source of stem cells. By being able to determine which embryos are not likely to survive to term, those embryos can be eliminated as a source of stem cells.
  • the invention includes methods for evaluating an embryo, e.g., evaluating the likelihood of an embryo developing to term or evaluating the embryo as a source of stem cells.
  • the methods include providing an embryo, e.g., a human or non-human embryo.
  • the embryo can be produced in vivo or in vitro.
  • the embryo can be produced in vitro.
  • the methods and tools described herein can be used to evaluate the expression of a preselected gene by comparing the level of expression to a reference expression profile derived from expression of the gene obtained from the cell of a healthy embryo at various stages of embryogenesis.
  • Various expression profiles include: i) a constant increase in expression levels from the egg or from the (fertilized) I-cell stage, through the eight cells stage; ii) a net decline in expression from the egg or 1-cell stage through the 4-cell stage, followed by an increase in expression by the 8-cell
  • a cell can be obtained from an embryo at any one of the following stages: the egg (unfertilized) stage, the 1-cell (fertilized) stage, the 2-cell stage, the 4 cell stage, the 8-cell stage, the compacted embryo stage, the morula stage, or the blastocyst stage.
  • the expression level of two, three or more genes can be evaluated. The expression level from each gene can then be compared to the appropriate expression
  • the evaluated expression level is , compared to the value given in the profile above for the same gene, for the stage of embryonic development during which the evaluated cell was removed from an embryo.
  • Other genes which can be evaluated include other genes.
  • the gene can be an apoptosis related gene (e.g., a gene from the Bcl-2 family and/or a gene from the IAP family).
  • the apoptosis related gene can be from the Bcl-2 family and encode a protein having anti-apoptoic activity (e.g., Bcl-2, Bcl-Xl and Bcl-w) and/or the apoptosis related gene can be from the Bcl-2 family and encode a protein having pro-apoptoic activity (e.g., Bad, Box, Bak, and Bcl-X).
  • a protein having anti-apoptoic activity e.g., Bcl-2, Bcl-Xl and Bcl-w
  • pro-apoptoic activity e.g., Bad, Box, Bak, and Bcl-X
  • At least one of the preselected genes for which expression is evaluated can be: a heat shock protein gene (e.g., hsp60 and hsp70); a pattern formation gene (e.g., NFkB, rkB, IL-1, IL-1 receptor, serine protease, bone morphogenic protein gene (e.g., BMP-2, BMP-4), BRK, Chordin, TGF (e.g., TGF-alpha), En-1 , En@, sonic hedgehog, a wnt gene (e.g., Wntl, Wnt2), B-catenin); a cell cycle regulatory protein gene (e.g., CAK, Wee 1 kinase, Cdc 25 kinase, p27, p21, pi 6, SCF, APC, Cdc20, Hct 11, E2F and p53); a gene encoding a regulatory protein involved in polarization (e.g.,
  • the methods are useful in evaluating the likelihood of an in vitro produced human embryo to develop to term. Such methods can allow for an in vitro fertilized embryo to be selected for implantation such that the likelihood of the embryo resulting in a live birth is greater than, e.g., at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more, as compared to an embryo implanted without such an evaluation. Methods of providing an in vitro fertilized embryo are known in the art.
  • Ovarian stimulation Various methods are known for inducing ovulation to obtain oocytes for human in vitro fertilization procedures. Any method of inducing oocyte stimulation can be used to provide an in vitro produced embryo. Some of the more widely used protocols are described below.
  • Pituitary down-regulation with GnRH agonist GnRH agonist
  • pituitary down-regulation involves daily injections of a GnRH agonist, Lupron ® .
  • the Lupron ® is administered until "down-regulation" of pituitary GnRH receptors has occuned and pituitary FSH and LH release is reduced to a minimum.
  • the GnRH agonist is administered for a period of about 10-15 days before down-regulation occurs.
  • Down-regulation can be confirmed by the onset of menses and a serum estradiol level that is less than 50 pg/ml.
  • One of the quicker ways to achieve down-regulation is to start the GnRH agonist in the mid-luteal phase. It can also be started in the early follicular phase with the onset of menses.
  • the dose of the GnRH agonist can be reduced and the ovulation induction can be initiated with a daily FSH injection.
  • Microdose-Lupron protocol This protocol can be used for women who are poor responders or who have evidence of reduced ovarian reserve. Briefly, this protocol involves the administration of oral contraceptives for a period of 3 weeks. After the 3 -week course of the oral contraceptives has been completed, then microdoses of Lupron ® and FSH are administered twice daily. When Lupron ® is administered in this fashion, it acts as a stimulatory agent by inducing the release of FSH and LH.
  • Another protocol utilizes a GnRH antagonist, which suppresses a LH surge. In contrast to the GnRH agonist, the pituitary suppression achieved by GnRH antagonist is fairly rapid.
  • gonadotropins are initiated on cycle day 2. When the lead follicle reaches a diameter of 14 mm, the GnRH antagonist is administered (with the gonadotropins) on a daily basis.
  • a mature follicle as used herein refers to one that is at least 15-17 mm in diameter. Once this is achieved, the FSH and other medications are discontinued. The woman can then take a single injection of hCG, which further matures the egg to allow them to become fertilized. If it is judged that the response is insufficient, the cycle is cancelled and the treatment plan is reassessed.
  • the egg retrieval can be performed under vaginal ultrasound guidance. After the vaginal ultrasound is placed in the vagina and the ovarian follicles are located, a needle can be directed through the back wall of the vagina and directly into the ovarian follicles. The fluid is aspirated and then examined by an embryologist to see if an egg has been retrieved. All follicles within both ovaries are aspirated. Once the eggs are retrieved, they are placed in culture plates with nutrient media and then placed in the incubators. The procedure is performed under a light anesthesia and takes less than 5-10 minutes to perform. Progesterone is started the evening after the egg retrieval. Other methods and techniques for retrieving an oocyte can also be used.
  • sperm insemination On the day of egg retrieval, a sperm sample can be obtained and prepared to select out the most motile sperm.
  • the sperm sample can also be obtained in advance of egg retrieval and maintained under conditions, e.g., cyropreserved, such that the sperm remain viable.
  • Motile sperm are placed next to the egg in a culture dish and the culture dish is placed in an incubator. The next day the eggs can be examined to determine whether fertilization has occuned.
  • ICSI Intracytoplasmic sperm, injection
  • the methods provided herein are also useful in evaluating the likelihood of a cloned or transgenic non-human embryo to develop to term. Such methods can allow for the embryo to be selected for implantation such that the likelihood of the embryo resulting in a live birth is greater, e.g., at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%) or more, than an embryo implanted without such an evaluation.
  • Methods for generating non-human cloned and transgenic mammals are known in the art. Methods of providing transgenic mammals can involve introducing nucleic acid constructs into the germ line of a mammal to make a transgenic mammal.
  • one or several copies of the construct may be incorporated into the genome of a mammalian embryo by standard transgenic techniques.
  • non-human transgenic mammals can be produced using a somatic cell as a donor cell.
  • the genome of the somatic cell can then be inserted into an oocyte and the oocyte can be fused and activated to form a reconstructed embryo.
  • methods of producing cloned and transgenic animals using a somatic cell are described in PCT Publication WO 97/07669; Baguisi et al.
  • Transfected Cell Lines Genetically engineered cell lines can be used to produce a transgenic animal using cloning techniques.
  • a genetically engineered construct can be introduced into a cell via conventional transformation or transfection techniques.
  • the terms "transfection” and "transformation” include a variety of techniques for introducing a transgenic sequence into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextrane-mediated transfection, lipofection, or electroporation.
  • biological vectors e.g., viral vectors can be used as described below.
  • Suitable methods for transforming or transfecting host cells can be found in Sambrook et al., Molecular Cloning: A Laboratory Manuel, 2 nd ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other suitable laboratory manuals.
  • the DNA construct can be stably introduced into a donor cell line by electroporation using the following protocol: somatic cells, e.g., fibrob lasts, e.g., embryonic fibroblasts, are resuspended in PBS at about 4 x 10 6 cells/ml. Fifty micrograms of linearized DNA is added to the 0.5 ml cell suspension, and the suspension is placed in a 0.4 cm electrode gap cuvette (Biorad). Electroporation is performed using a Biorad Gene Pulser electroporator with a 330 volt pulse at 25 mA, 1000 microFarad and infinite resistance. If the DNA construct contains a neomyocin resistance gene for selection, neomyocin resistant clones are selected following • incubation with 350 microgram/ml of G418 (GibcoBRL) for 15 days.
  • somatic cells e.g., fibrob lasts, e.g., embryonic fibroblasts
  • PBS
  • the DNA construct can be stably introduced into a donor somatic cell line by lipofection using a protocol such as the following: about 2 x 10 5 cells are plated into a 3.5 cmiameter well and transfected with 2 micrograms of linearized DNA using
  • LipfectAMTNETM (GibcoBRL). Forty-eight hours after transfection, the cells are split 1:1000 and 1:5000 and, if the DNA construct contains a neomyosin resistance gene for selection, G418 is added to a final concentration of 0.35 mg/ml. Neomyocin resistant clones are isolated and expanded for cryopreservation as well as nuclear transfer.
  • Oocytes for use in producing a cloned or transgenic animal can be obtained at various times during an animal's reproductive cycle. Oocytes at various stages of the cell cycle can be obtained and then induced in vitro to enter a particular stage of meiosis. For example, oocytes cultured on serum-starved medium become anested in metaphase. In addition, anested oocytes can be induced to enter telophase by serum activation.
  • Oocytes can be matured in vitro before they are used to form a cloned embryo or a transgenic embryo, e.g., a transgenic embryo produced by microinjection or using a somatic cell donor.
  • the process of oocyte retrieval usually requires collecting immature oocytes from mammalian ovaries and maturing the oocyte in a medium prior to enucleation or fertilization until the oocyte reaches the desired meiotic stage, e.g., metaphase or telophase.
  • oocytes that have been matured in vivo can be used to form a cloned or transgenic embryos. Oocytes can be collected from a female mammal during superovulation.
  • oocytes can be recovered surgically by flushing the oocytes from the oviduct of the female donor. Methods of inducing superovulation in various mammals and the collection of oocytes from those mammals are known.
  • the methods further include removing a cell from the embryo.
  • Methods have been developed for preimplantation genetic diagnosis which allow the removal of one or more cells from an embryo without affecting the continued development of the embryo. Such methods can be used to obtain a cell for evaluation in the present methods.
  • the method is performed as follows. The procedure is performed on day 3 of embryo development, when the embryo is around 6-8 cell stage. Tyrodes solution is used to make a hole in the zona pellucida or non-contact laser has been used. Once the hole is made, cells (blastomeres) can be aspirated from the embryo.
  • the methods include evaluating the expression of at least two, preferably three preselected genes from within the cell to thereby evaluate the likelihood of the embryo developing to term.
  • the methods can further include evaluating expression of at least five, ten, fifteen, twenty, twenty- five, thirty, thirty-five, forty, forty- five, fifty or more genes from the cell.
  • the methods include evaluating the expression of the genes by evaluating the level of RNA or cDNA from the cell.
  • the RNA can be obtained from the cell at various times after the cell is removed from the embryo. For example, the RNA can be obtained from the cell within at least 5, 10, 15, 20, 25, 30, 35 minutes, 1 hour, 2, 3, 4, 5 hours from the time the cell is removed from the embryo.
  • the method can further include obtaining cDNA from the RNA using, e.g., reverse transcription.
  • RNA is isolated from the single cell biopsied from the embryo to be evaluated.
  • the rnRNA can be reverse transcribed to cDNA, during which process fluorescent dyes can be incorporated.
  • the labeled cDNAs can then be hybridized, e.g., to an array and, after washing, the fluorescent signals can be detected by a laser scanner.
  • RNA from a single cell isolated from embryo is extra with lysis buffer containing 1 st strand buffer and NP-40. The mixture is incubated at 65°C for 1 minute and 30 seconds, then cooled down to room temperature for 5 to 10 minutes. Diluted Superscript II is added and incubated at 37 °C for 30 minutes, followed by 10 minutes at 70°C to inactivate the enzyme. Terminal transferase mix containing terminal transferase is added to each reaction and incubated at 37C for 15 minutes, followed by 10 minutes at 65°C to inactivate the enzyme. PCR mixture is added to each reaction, heated at 95°C for 40 seconds, and then followed by /annealing/extension cycles.
  • the PCR products are purified using a PCR purification kit. 100 ng purified SC-PCR products is used as template, and the PCR is repeated. Components remain the same except using pdT-Cy3 primer instead of pd (T) 24-V primers followed by denature/annealing/extension cycles.
  • the PCR products are purified using Qiagen PCR purification kit, the O.D. measured and the product is, e.g., used for chip hybridization. ,
  • Expression of the preselected genes can be evaluated by determining whether one or more, preferably, all of the genes are expressed or non-expressed. This evaluation can include determining if the gene is expressed at a preselected level.
  • a "preselected level” as used herein is an expression level which can be compared to a reference standard to determine the absence or presence of expression. Levels at or below the reference standard can be considered non-expressed, whereas levels above the reference standard can be considered expressed.
  • the expression of the gene or genes can be evaluated by hybridizing the obtained nucleic acids, e.g., RNA or cDNA, from the cell with a probe or probes which hybridize to the specific nucleic acids from the cell.
  • RNA or cDNA e.g., RNA or cDNA
  • hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions describes conditions for hybridization and washing.
  • Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used.
  • Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by two washes in 0.2X SSC, 0.1%) SDS at least at 50°C (the temperature of the washes can be increased to 55°C for low stringency conditions); 2) medium stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C; 3) high stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C. Very high stringency conditions (4) are the prefened conditions and the ones that should be used unless otherwise specified.
  • one method of evaluating the expression of the preselected genes includes using an anay.
  • Such an array includes a substrate having a plurality of capture probes. At least two, preferably three, of the plurality of capture probes include a capture probe that specifically hybrizes to the nucleic acids of the preselected genes obtained from the cell.
  • the anay can have a density of at least than 10, 50, 100, 200, 500, 1,000, 2,000, or 10,000 or more addresses/cm 2 , and ranges between, hi a prefened embodiment, the plurality of capture probes is less than 500, 400, 300, 200, 100, 75, 50, 25 or less capture probes.
  • the array can include at least five, ten, fifteen, twenty, twenty- five, thirty, thirty-five, forty, forty-five, fifty of more preselected genes for which expression can be used to evaluate the likelihood of an embryo developing to term.
  • the capture probes can be single stranded probes in an anay or the capture probes can have a structure comprising a double stranded portion and a single stranded portion in an array.
  • the anay can further include at least one capture probe which hybridizes to a specific control gene.
  • control genes which the capture probe can hybridize include: the beta actin gene, the beta-2-microglobulin gene, the glyceraldehyde-3 -phosphate dehydrogenase gene, the hypoxanthine phosphoribosyl- transferase 1 gene, the ribosomal protein L13a, subunit A gene of the succinate dehydrogenase complex, the TATA binding protein gene, and the ubiquitin C gene.
  • the nucleic acid sequences of these genes are known in the art.
  • the substrate can be a two-dimensional substrate such as a glass slide, a wafer (e.g., silica or plastic), a mass spectroscopy plate, or a three-dimensional substrate such as a gel pad. Addresses in addition to address of the plurality can be disposed on the array.
  • An array can be generated by various methods, e.g., by photolithographic methods (see, e.g., U.S. Patent Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow methods as described in U.S. Patent No. 5,384,261), pin-based methods (e.g., as described in U.S. Pat. No.
  • Binding e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid.
  • hybridization to the array is detected by mass spectrophotometry, e.g., by MALDI-TOF mass spectrophotometry.
  • the array can also include at least one probe which can specifically hybridize to a nucleic acid encoding or expressed by a specific allele of a selected gene, e.g., it can distinguish between a first, e.g., a mutant, and a second, e.g., a non-mutant, form of a gene or between alternatively expressed forms such as splice variants.
  • the expression of the genes can be evaluated by: providing an array of a plurality of capture probes, e.g., nucleic acid sequence which can hybridize to specific nucleic acids from the cell.
  • the probe or probes specifically hybridize to a selected gene associated with a disorder.
  • the selected gene can be, e.g.: the gene encoding cystic fibrosis transmembrane conductase regulator (CFTR), the gene encoding dystrophin, the Beta Thalassemia gene, the gene encoding Factor NIII, the gene encoding Factor IX, Tay-Sachs gene, survival motor neuron (SM ⁇ ) gene, the HD gene.
  • the probe or probes specifically hybridizes to a selected gene to detect the absence or presence of the second mutant form of the selected gene, e.g., to detect whether the embryo carries a mutation associated with a disorder.
  • disorders for which the embryo can be evaluated include but are not limited to: Adenoleukodystrophy, Amyotrophic Lateral Sclereosis (ALS), Becker Muscular Dystrophy, Beta Thalassemia, Cerebellar Ataxia, Charcot-Marie-Tooth Disease, Chondrodysplasia Aganglionic Megacolon, Conradi- Hunnerman Syndrome, Cystic Fibrosis, Duchenne Muscular Dystrophy, Hemophilia A or B, Huntington's Disease, Fragile X Syndrome, Glycogen Storage Disease, Hirschsprung Disease, Icthyosis, Lesch ⁇ yhan, Myopathies, Polycystic Ovary Syndrome, Restenosis Pigmentosa, Sickle Cell Anemia, Tay-Sachs Disease, and Non Willebran Disease.
  • ALS Amyotrophic Lateral Sclereosis
  • Beta Thalassemia Cerebellar Ataxia
  • Charcot-Marie-Tooth Disease Chondrodysplasi
  • each address of a subset of the capture probes can be unique, overlapping, and complementary to a different variant of the selected gene.
  • the arrays described herein can further include at least one probe that hybridizes to a nucleic acid associated with one sex chromosome but not the other, e.g., a nucleic acid associated with the Y chromsome but not the X chromsome.
  • a probe which hybridizes to all or a portion of the sry gene can be used to determine the presence of a Y chromosome in the embryo, thereby indicating that the embryo is male. The absence of this gene indicates that the embryo is female.
  • Various arrays can also be used to that can ascertain differential expression patterns or copy numbers of one or more genes, clusters of genes or chromosomes in normal embryonic cells and cells obtained from the embryo to be evaluated.
  • anays of genomic bacterial artificial chromosomes (BAC), PI or cosmid clones can be used to measure differences in chromosomal structure or number between DNA from the embryo being evaluated and control DNA, e.g., DNA obtained from a healthy embryo. Since the clones on the array contain sequence tags, their positions on the array are accurately known relative to the genomic sequence.
  • the invention also features methods of analyzing the absence or presence of vaulting the presence of too few or too many chromosomes in an embryo.
  • the method includes providing an anay as described above; contacting the anay with a sample, e.g., a portion of genomic DNA that includes at least a portion of human chromosome to be evaluated for having too few or too many copies, and detecting binding of a nucleic acid from the sample to the anay.
  • the method further includes amplifying nucleic acid from the sample, e.g., genomic DNA that includes a portion of the human chromosome of interest, prior or during contact with the anay.
  • the method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality having a unique nucleic acid capture probe, contacting the array with a first sample from a cell of the embryo being evaluated; contacting the array with a second sample from a cell of a healthy embryo; and comparing the binding of the first sample with the binding of the second sample. Binding, e.g., in the case of a nucleic acid hybridization, with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid.
  • CGH arrays as described herein to evaluate: i) aneuploidy of embryos, ii) potential genetic disease, and iii) the sex of the embryo, e.g., using a combination of degenerate oligonucleotide-primed polymerase chain reaction (DOP- PCR) and CGH arrays.
  • DOP-PCR degenerate oligonucleotide-primed polymerase chain reaction
  • Whole genome can be amplified by DOP-PCR. Briefly, DNA from single cells from embryo biopsy is amplified in two phases. In the first phase, 10 ⁇ l of PCR buffer containing 2 ⁇ l of sample DNA, 250 ⁇ M dNTP, 3mM MgC12, 1.5 ⁇ M universal primer (5-CCGACTCGAGNNNNNNATGTGG-3') and 0.5 ⁇ l of ExTaq polymerase is subjected to initial denaturation at 94°C for 10 minutes, followed by five cycles of 94°C for 1 minute, 25°C for 1 minute, ramp from 25°C to 74°C in 3 minutes, and 74°C for 2 minutes in a thermal cycler.
  • PCR buffer containing 2 ⁇ l of sample DNA, 250 ⁇ M dNTP, 3mM MgC12, 1.5 ⁇ M universal primer (5-CCGACTCGAGNNNNNNATGTGG-3') and 0.5 ⁇ l of ExTaq polymerase is subjected to initial denaturation at 94°C
  • PCR buffer containing the first PCR product (lO ⁇ l), 200 ⁇ M dNTP, 3mM MgCl, 1.0 ⁇ M universal primer, and 1.0 ⁇ l of ExTaq polymerase is subjected to initial denaturation at 94°C for 5 minutes, 20 cycles of 94°C for 1 minute, 56°C for 1 minute, and 72°C for 2 minutes, and a final extension at 72°C for 10 minutes.
  • Probe DNA labeling, CGH, and digital image analysis can be performed as follows: DOP-PCR amplified DAN from single blastomere and normal DNAs are labeled with fluorescein-12-dUTP and tetramethylrhodamine-5-dUTP, respectively, by nick translation. Gains and losses in DNA copy number are defined by green to red ratios (G/R) greater than 1.2 and less than 0.8, respectively. High-level gains (amplifications) are defined by a G/R of 1.5 or greater.
  • G/R green to red ratios
  • High-level gains (amplifications) are defined by a G/R of 1.5 or greater.
  • Statistical analysis can be performed, e.g., using a Fisher's exact test and/or the Student t-test. Fisher's exact test is used to examine differences in the frequencies of chromosomal copy-number changes between two groups. The Student t-test is used to compare the difference in the mean numbers of copy-number abenations between groups. Difference at
  • the methods and tools described herein can be used not only to determine the likelihood of an embryo to develop to term but also to screen the nucleic acid, e.g., RNA or cDNA, from the cell to determine if the embryo carries a mutation in a selected gene, e.g., a gene associated with risk of a disorder.
  • the methods and tools described herein can also be used to detect genetic alterations in a selected gene, e.g., a gene known to be associated with a disorder, to thereby determining if an embryo is at risk for the disorder.
  • the methods include detecting, from the nucleic acid sample obtained from the cell, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding the selected gene, or the mis-expression of the selected gene.
  • such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleo tides from a gene; 2) an addition of one or more nucleotides to a gene; 3) a substitution of one or more nucleotides of a gene, 4) a chromosomal reanangement of a gene; 5) an alteration in the level of a messenger RNA transcript of a gene, 6) the presence of a non- wild type splicing pattern of a messenger RNA transcript of a gene, and 7) allelic loss of a gene.
  • genetic mutations in a gene can be detecting using, e.g., an array, e.g., an array described herein, which also includes at least one probe which can specifically hybridize to a nucleic acid encoding or expressed by a specific allele of a selected gene, e.g., it can distinguish between a first, e.g., a mutant, and a second, e.g., a non-mutant, form of a gene or between alternatively expressed forms such as splice variants.
  • Such anays can include a probe which is complementary to a region of a nucleic acid or a putative variant (e.g., allelic variant) thereof.
  • a hybridization array that allows the characterization of specific mutations by using smaller, specialized probe anays complementary to all variants or mutations detected can be used.
  • Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • PGD preimplantation genetic diagnosis
  • PGD has been used to identify the presence of the deltaF508 mutation, which accounts for 75% of identified cystic fibrosis mutations. In the majority of cases in which a couple faces a 25% to 50% risk for an affected child, one or both of them would cany the deltaF508 mutation.
  • genes which can be screened include: the gene encoding dystrophin, the Beta Thalassemia gene, the gene encoding Factor VIII, the gene encoding Factor IX, Tay-Sachs gene, and the HD gene.
  • disorders for which the embryo can be evaluated include but are not limited to: Adenoleukodystrophy, Amyotrophic Lateral Sclereosis (ALS), Becker Muscular Dystrophy, Beta Thalassemia, Cerebellar Ataxia, Charcot-Marie-Tooth Disease, Chondrodysplasia Aganglionic Megacolon, Conradi-Hunnerman Syndrome, Cystic Fibrosis, Duchenne Muscular Dystrophy, Hemophilia A or B, Huntington's Disease, Fragile X Syndrome, Glycogen Storage Disease, Hirschsprung Disease, Icthyosis, Lesch Nyhan, Myopathies, Polycystic Ovary Syndrome, Restenosis Pigmentosa, Sickle Cell Anemia, Tay-Sachs
  • the methods and tools described herein can be used to further evaluate the absence or presence of chromsomal abnormalities associated with a disorder, e.g., the absence or presence of too few or too many copies of a chromsome or chromosomes. This allows for selection of embryos for implantation that are not at risk for various disorders associated with the presence of too few or too many copies of a chromosome.
  • chromosome 21 in the embryo which is indicative of a risk for Down's Syndrome
  • the presence of more than two of chromosome 13 in the embryo which is indicative of a risk for Patau syndrome the presence of more than two of chromosome 18 in the embryo which is indicative of a risk for Edwards syndrome
  • the absence or presence of only one copy of chromosome 5 in the embryo which is indicative of a risk for Cri du Chat the presence of more than two X chromosome in the embryo which is indicative of a risk for Klinefelter's syndrome
  • the presence of more than two X chromosome in the embryo which is indicative of a risk for Triple X syndrome the absence of chromosome X in the embryo which is indicative of a risk for Turner's syndrome.
  • the methods of the invention include selecting an embryo based upon its evaluation and implanting that embryo into a female recipient.
  • Various methods of implanting human in vitro produced embryos and non-human cloned and transgenic animals are know. Several of those methods are described below.
  • the embryo transfer is performed usually 72 hours after the egg retrieval.
  • the catheter is passed through the cervix to approximately 1 cm below the uterine anatomy.
  • the depth of transfer may be judged from either a previous trial transfer, by measuring its length from external markings or by gently touching the uterine fundus and withdrawing 1 cm.
  • Embryos are loaded in the distal end of the transfer catheter and are gently injected using a small tuberculin syringe. After transfer the catheter is checked for retained embryos under a dissecting microscope.
  • a reconstructed embryo can be transfened to a recipient doe and allowed to develop into a cloned or transgenic mammal.
  • the reconstructed embryo can be transfened via the fimbria into the oviductal lumen of each recipient doe.
  • methods of transferring an embryo to a recipient mammal are known in the art and described, for example, in Ebert et al. (1994) Bio/Technology 12:699.
  • the information obtained by evaluating the likelihood of an embryo implanting into a uterus and/or developing to term can also be used to evaluate the effects that various factors and conditions, e.g., in vitro fertilization conditions, can have on embryo development.
  • the information can be stored in a database.
  • the invention includes a database having a plurality of records.
  • Each record includes at least one, two, preferably, all of the following information: data on the expression of at least two, preferably at least three preselected genes obtained from a cell of an embryo; data on whether the embryo implanted into the uterus and/or developed to term; and data on a preselected factor present in one or both of the parent of the embryo and/or data on a preselected condition, e.g., a condition used in an in vitro fertilization procedure.
  • the database can be any kind of storage system capable of storing various data for each of the records as described herein.
  • the database is a computer medium having a plurality of digitally encoded data records.
  • the data record can be structured as a table, e.g., a table that is part of a database such as a relational database (e.g., a SQL database of the Oracle or Sybase database environments).
  • machine-readable media refers to any medium that can be read and accessed directly by a machine, e.g., a digital computer or analogue computer.
  • a computer include a desktop PC, laptop, mainframe, server (e.g., a web server, network server, or server farm), handheld digital assistant, pager, mobile telephone, and the like.
  • the computer can be standalone or connected to a communications network, e.g., a local area network (such as a VPN or intranet), a wide area network (e.g., an Extranet or the Internet), or a telephone network (e.g., a wireless, DSL, or ISDN network).
  • a communications network e.g., a local area network (such as a VPN or intranet), a wide area network (e.g., an Extranet or the Internet), or a telephone network (e.g., a wireless, DSL, or ISDN network).
  • Machine-readable media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD- ROM; electrical storage media such as RAM, ROM, EPROM, EEPROM, flash memory, and the like; and hybrids of these categories such as magnetic/optical storage media.
  • a variety of data storage structures are available to a skilled artisan for creating a machine-readable medium having recorded thereon the data described herein.
  • the choice of the data storage structure will generally be based on the means chosen to access the stored information.
  • a variety of data processor programs and formats can be used to store the information of the present invention on computer readable medium.

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Abstract

L'invention concerne des méthodes et des outils permettant d'apprécier les chances qu'a un embryon de s'implanter dans l'utérus et/ou de se développer jusqu'à terme.
PCT/US2003/039450 2002-12-12 2003-12-12 Methodes de determination de la validite d'un embryon Ceased WO2004055217A1 (fr)

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CN108982867A (zh) * 2018-05-31 2018-12-11 西北妇女儿童医院 一种利用蛋白芯片对囊胚质量判断的方法
RU2795482C1 (ru) * 2022-10-25 2023-05-04 Публичное акционерное общество "Центр Генетики и Репродуктивной Медицины "ГЕНЕТИКО" Способ преимплантационного генетического тестирования ахондроплазии

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1960531A4 (fr) * 2005-12-06 2009-11-11 Copperman Alan Protocole pour detecter une proteine dans une culture contenant un embryon
CN108300802A (zh) * 2018-04-27 2018-07-20 江西省农业科学院水稻研究所 一种检测抗稻瘟病复等位基因Pi-2/gm/zt的通用分子标记及应用
CN108982867A (zh) * 2018-05-31 2018-12-11 西北妇女儿童医院 一种利用蛋白芯片对囊胚质量判断的方法
RU2795482C1 (ru) * 2022-10-25 2023-05-04 Публичное акционерное общество "Центр Генетики и Репродуктивной Медицины "ГЕНЕТИКО" Способ преимплантационного генетического тестирования ахондроплазии

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