EP1155320A1 - Isolement d'une fraction cytoplasmique sans alteration de la viabilite des cellules ovocytes et embryonnaires - Google Patents

Isolement d'une fraction cytoplasmique sans alteration de la viabilite des cellules ovocytes et embryonnaires

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Publication number
EP1155320A1
EP1155320A1 EP00906062A EP00906062A EP1155320A1 EP 1155320 A1 EP1155320 A1 EP 1155320A1 EP 00906062 A EP00906062 A EP 00906062A EP 00906062 A EP00906062 A EP 00906062A EP 1155320 A1 EP1155320 A1 EP 1155320A1
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EP
European Patent Office
Prior art keywords
oocyte
nucleotide sequence
mutation
polymorphism
fraction
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP00906062A
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German (de)
English (en)
Inventor
Robert Jansen
Kylie De Boer
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Sydney IVF Ltd
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Sydney IVF Ltd
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Filing date
Publication date
Application filed by Sydney IVF Ltd filed Critical Sydney IVF Ltd
Publication of EP1155320A1 publication Critical patent/EP1155320A1/fr
Withdrawn legal-status Critical Current

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    • 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
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a method of isolating a cytoplasmic fraction from an oocyte which does not impair the capacity of the oocyte to be fertilized.
  • the invention also relates to a method of isolating a cytoplasmic fraction from an embryonic cell which does not impair the developmental potential of the cell.
  • the present invention seeks to address these needs and in a first aspect provides a method of isolating a cytoplasmic fraction from an oocyte which does not impair the capacity of the oocyte to be fertilized, the method including the step of releasing a cytoplasmic fraction from the oocyte.
  • the volume of cytoplasmic fraction which is released from the oocyte is about 5% of the volume of the oocyte.
  • the volume is about 2% of the volume of the oocyte.
  • the method includes the following steps: a) inserting releasing means into the oocyte; b) drawing a cytoplasmic fraction which is about 5% of the volume of the oocyte into the releasing means ; and c) withdrawing the releasing means from the oocyte so that the fraction is isolated in the releasing means .
  • the volume of the cytoplasmic fraction drawn into the releasing means is typically less than 10 pL and preferably 8 pL.
  • the releasing means includes at least an ICSI pipette and preferably the cytoplasmic fraction is drawn approximately lOO ⁇ m into the pipette.
  • the drawing of the cytoplasmic fraction from the oocyte into the releasing means typically forms an extrusion of cytoplasmic contents between the releasing means and the oocyte.
  • the cytoplasmic fraction is isolated from the oocyte by gently stretching or shearing the extrusion so as to separate the extrusion.
  • the cytoplasmic fraction is isolated by stretching the extrusion.
  • the cytoplasmic fraction contains cytoplasmic organelles, and includes or consists of a sample of the oocyte 's mitochondria and mitochondrial products .
  • Mitochondria located in an oocyte are a template from which all mitochondria in the progeny descended from the fertilized oocyte are derived.
  • the present inventors recognised that a method for isolating a cytoplasmic fraction from an oocyte, without impairing the capacity of the oocyte to be fertilized, would allow the study of the relationship between the integrity of the mitochondrial genome and the function of both the oocyte and the progeny descended from the fertilized oocyte.
  • the invention relates to a method of analysing the mitochondrial genome of mitochondria located in an oocyte which does not impair the capacity of the oocyte to be fertilized, the method including the following steps: a) isolating a cytoplasmic fraction which includes mitochondria from the oocyte according to the method of the first aspect of the invention; b) analysing the mitochondrial genome of the mitochondria in the fraction.
  • Mutation of the mitochondrial genome causes, or at least is associated with, dysfunction or disease.
  • the nucleotide sequence deletion from nucleotide position number 8470 to 13,446 of the mitochondrial genome is understood to be associated with Kearns-Sayre syndrome (KSS) and chronic progressive external opthalmoplegia (CPEO) .
  • Other disease causing deletions which may or may not be observed with the common deletion, include a 7.4 kb deletion and 10.4 kb deletion/insertion in the mitochondrial genome of brain and heart,, as well as various point mutations .
  • the deletion is also observed in human tissue including skeletal muscle, heart, brain, oocytes, leukocytes, retina and ovaries.
  • Over 40 pathogenic point mutations of the mitochondrial genome can be associated with a broad spectrum of degenerative diseases involving the central nervous system, heart, muscle, endocrine system, kidney and liver.
  • Diseases associated with point mutations include Leigh Syndrome, MELAS (mitochonrdial encephalomyopathy, lactic acidosis and stroke like episodes), MERRF (myoclonus epilepsy with ragged-red fibres) , NARP (neruopathy, ataxia and retinitis pigmentosa) and LHON (Leber hereditary optic neuropathy) .
  • a method of isolating a cytoplasmic fraction from an oocyte without impairing the capacity of the oocyte to be fertilized is useful for detecting nucleotide sequence mutations in the mitochondrial genome, enabling the study of a nucleotide sequence, polymorphism or mutation in the context of the functional integrity of both the oocyte and the progeny descended from the fertilized oocyte.
  • the invention provides a method of detecting a nucleotide sequence, polymorphism or mutation in the mitochondrial genome of mitochondria located in an oocyte which does not impair the capacity of the oocyte to be fertilized, the method including the following steps: a) isolating a cytoplasmic fraction which includes mitochondria from the oocyte according to the method of the first aspect of the invention; and b) analysing the nucleotide sequence of the mitochondrial genome of the mitochondria in the cytoplasmic fraction for the presence of a nucleotide sequence, polymorphism or mutation in the mitochondrial genome.
  • a method of isolating a cytoplasmic fraction without impairing the capacity of the oocyte to be fertilized is useful for predicting whether the progeny descended from a fertilized oocyte will or will not contain a nucleotide sequence, polymorphism or mutation of the mitochondrial genome which causes, or is suspected of causing, or is associated with, disease or dysfunction.
  • the invention provides a method for predicting whether the progeny descended from a fertilized oocyte will contain a nucleotide sequence, polymorphism or mutation in a mitochondrial genome which causes, or is suspected of causing, or is associated with, a disease or dysfunction, wherein the method does not impair the capacity of the oocyte to be fertilized, the method including the following steps: a) isolating a cytoplasmic fraction which includes mitochondria from the oocyte according to the method of the first aspect of the invention; and b) analysing the mitochondrial genome of the mitochondria in the fraction for the presence of the nucleotide sequence, polymorphism or mutation in the mitochondrial genome; wherein the presence of the nucleotide sequence, polymorphism or mutation indicates a likelihood that the progeny descended from the fertilized oocyte will contain the nucleotide sequence, polymorphism or mutation.
  • nucleotide sequence, polymorphism or mutation in the mitochondrial genome of mitochondria in an oocyte which is known to be associated with, or cause, or is suspected of causing, disease or dysfunction, may not be sufficient to mediate the disease or dysfunction in the oocyte itself, or the progeny descended from the fertilized oocyte. Indeed, there appears to be at least an additional factor which contributes to the likelihood of, and/or the severity of the disease or dysfunction in the oocyte containing the nucleotide sequence, polymorphism or mutation, or the progeny descended from the fertilized oocyte.
  • the actual proportion, or "threshold level” of mitochondria which contain the particular nucleotide sequence, polymorphism or mutation in the mitochondrial genome in the oocyte will contribute to the likelihood and/or severity of disease or dysfunction in the oocyte, or progeny descended from the fertilized oocyte.
  • threshold level the level of mitochondria which contain the particular nucleotide sequence, polymorphism or mutation in the mitochondrial genome in the oocyte.
  • “Heteroplasmy” is observed when an oocyte contains more than one species of mitochondrial genome.
  • nucleotide sequences, polymorphisms and mutations of the mitochondrial genome which cause, or are suspected of causing, or are associated with, a disease or dysfunction, it is generally recognised that when a particular level of heteroplasmy is surpassed, the manifestations of the disease or dysfunction are sooner or later observed.
  • a method for determining the degree of heteroplasmy in a human oocyte is typically applied at specific stages of meiosis.
  • a method of isolating cytoplasmic fractions which does not impair the capacity of the oocyte to be fertilized is useful for determining the level of heteroplasmy in the oocyte, and for predicting the average level or likely range of heteroplasmy in tissues of the progeny descended from the fertilized oocyte.
  • the invention provides a method of determining the level of heteroplasmy of mitochondrial genomes in an oocyte which does not impair the capacity of the oocyte to be fertilized, the method including the following steps: a) isolating a cytoplasmic fraction which includes mitochondria from the oocyte according to the method of the first aspect of the invention; b) comparing the number of mitochondrial genomes in the fraction with a nucleotide sequence, polymorphism or mutation, with the number of genomes without the nucleotide sequence, polymorphism or mutation in the fraction.
  • the method of determining the level of heteroplasmy of mitochondrial genomes in an oocyte is useful for predicting whether the progeny descended from the fertilized oocyte are likely to suffer from a disease or dysfunction which is caused by, or associated with, the particular nucleotide sequence, polymorphism or mutation, and/or the severity of the disease or dysfunction.
  • the invention provides a method of determining whether the progeny descended from a fertilized oocyte are likely to suffer from a disease or dysfunction caused by, or suspected of being caused by, or associated with, a nucleotide sequence, polymorphism or mutation in a mitochondrial genome, wherein the method does not impair the capacity of the oocyte to be fertilized and includes the following steps: a) isolating a cytoplasmic fraction which includes mitochondria from the oocyte according to the method of the first aspect of the invention; b) analysing the mitochondrial genome of the mitochondria in the fraction for the presence of the nucleotide sequence, polymorphism or mutation; and determining that the progeny are likely to suffer from the disease or dysfunction where the analysis of the mitochondrial genome of mitochondria in the fraction demonstrates that the level of heteroplasmy of mitochondrial genomes with respect to the nucleotide sequence, polymorphism or mutation in the oocyte is at least the same as the level of heteroplasmy which is known to be associated with
  • cytoplasmic fractions from an oocyte involve the destruction of the oocyte. Although these methods reveal useful information about the oocyte that is thereby destroyed, the value of this information is confined to an extrapolation to other oocytes which are not evaluated. As a population of oocytes from the same individual are typically heterogenous with respect to ooplasmic content, there is some question as to the extent to which the information derived from the oocyte which is destroyed can be accurately or reliably extrapolated to other oocytes.
  • an oocyte is found to have either no nucleotide sequence, polymorphism or mutation in the mitochondrial genome which causes, or is associated with, disease or dysfunction in the oocyte, or the progeny of the fertilized oocyte, or a low degree of heteroplasmy in relation to the nucleotide sequence, polymorphsim or mutation, that other oocytes derived from the same individual will have the same mitochondrial genotype.
  • oocytes derived from a patient may contain a nucleotide sequence, polymorphism or mutation which causes, or is suspected of causing, or is associated with, disease or dysfunction in the progeny descended from the fertilized oocyte.
  • the method of isolating a cytoplasmic fraction from an oocyte which does not impair the capacity of the oocyte to be fertilized is particularly useful for screening oocytes for the presence of a nucleotide sequence, polymorphism or mutation which causes, or is suspected of causing, or is associated with, disease or dysfunction in the progeny descended from the fertilized oocyte, prior to fertilization.
  • the invention provides a method of screening an oocyte for the presence of a nucleotide sequence, polymorphism or mutation in the mitochondrial genome in the oocyte which causes, or is suspected of causing, or is associated with, disease or dysfunction in the progeny descended from the fertilized oocyte, wherein the method does not impair the capacity of the oocyte to be fertilized, and includes the following steps : a) isolating a cytoplasmic fraction which includes mitochondria from the oocyte according to the method of the first aspect of the invention; b) analysing the mitochondrial genome of the mitochondria in the fraction for the presence of the sequence, polymorphism or mutation.
  • oocytes Patients who present for in vi tro fertilization treatment frequently have few oocytes available for selection for fertilization, and of the available oocytes, it is anticipated that some of these will contain a nucleotide sequence, polymorphism or mutation in a mitochondrial genome which causes or is associated with a disease or dysfunction in progeny descended from the fertilized oocyte.
  • Other methods for isolating a cytoplasmic fraction from an oocyte are particularly unsuitable for selecting oocytes for fertilization, and could potentially result in the destruction of oocytes which do not contain a nucleotide sequence, polymorphism or mutation in a mitochondrial genome which causes or is associated with a disease or dysfunction, or oocytes which have a low level of heteroplasmy with respect to that mutation.
  • the method of isolating a cytoplasmic fraction from an oocyte which does not impair the capacity of the oocyte to be fertilized is particularly useful for selecting oocytes for fertilization which do not contain a nucleotide sequence, polymorphism or mutation which causes, or is suspected of causing, or is associated with, disease or dysfunction in the progeny descended from the fertilized oocyte, prior to fertilization.
  • the invention relates to a method of selecting an oocyte for fertilization which either : (i) does not contain a nucleotide sequence, polymorphism or mutation in the mitochondrial genome of mitochondria in the oocyte which causes, or is suspected of causing, or is associated with, a disease or dysfunction in the progeny descended from the fertilized oocyte; or
  • (ii) has a level of heteroplasmy of mitochondrial genomes with respect to the nucleotide sequence, polymorphism or mutation which is less than the level of heteroplasmy which is known to be associated with the manifestation of the disease or dysfunction; wherein the method does not impair the capacity of the oocyte to be fertilized, and includes the following steps: a) isolating a cytoplasmic fraction which includes mitochondria from the oocyte according to the method of the first aspect of the invention; b) analysing the mitochondrial genome of the mitochondria in the fraction; and c) selecting the oocyte for fertilization, provided that at least the degree of heteroplasmy of mitochondrial genomes with respect to the nucleotide sequence, polymorphism or mutation in the mitochondrial genome of the oocyte, is less than the degree of heteroplasmy which is known to be associated with the manifestation of the disease or dysfunction.
  • the oocytes which are screened or selected in accordance with the seventh or eighth aspect of the invention, respectively, may be fertilized by intra cytoplasmic sperm injection (ICSI) , or by in vi tro fertilization.
  • ICSI intra cytoplasmic sperm injection
  • the oocytes are fertilized by ICSI.
  • ICSI intra cytoplasmic sperm injection
  • the invention provides a method of fertilizing an oocyte, the method including the following steps : a) isolating a cytoplasmic fraction which includes mitochondria from the oocyte according to the method of the first aspect of the invention; b) analysing the mitochondrial genome of the mitochondria in the fraction for the presence of a nucleotide sequence, polymorphism or mutation which causes, or is suspected of causing, or is associated with, a disease or dysfunction in progeny descended from the fertilized oocyte; and c) fertilizing the oocyte, provided that the degree of heteroplasmy of mitochondrial genomes with respect to the nucleotide sequence, polymorphism or mutation in the oocyte, is less than the degree of heteroplasmy which is known to be associated with the manifestation of the disease or dysfunction.
  • the mitochondrial genome of mitochondria which are in the cytoplasmic fraction isolated according to the method of the first aspect of the invention can be analysed according to standard techniques. These techniques are exemplified further herein and include the polymerase chain reaction (PCR) , restriction fragment length polymorphism (RFLP) analysis, genomic hybridisation, nucleotide sequencing and gene function detection and/or measurement .
  • PCR polymerase chain reaction
  • RFLP restriction fragment length polymorphism
  • a representative fraction of genomes can be obtained when mitochondria are randomly distributed in the cytoplasm.
  • a cytoplasmic fraction is drawn from the oocyte when the distribution of mitochondria in the oocyte can be expected to be random, including for example at the germinal vesicle (GV) stage of the primary oocyte and /or at a stage from the metaphase II stage of meiosis of the secondary oocyte, to prior to syngamy.
  • GV germinal vesicle
  • the present inventors recognised that the method of isolating a cytoplasmic fraction is useful for studying the cytoplasm of other cells, for example embryonic cells, without impairing the developmental potential of these cells.
  • the invention provides a method of isolating a cytoplasmic fraction from an embryonic cell which does not impair the developmental potential of the cell, the method including the step of releasing a cytoplasmic fraction from the cell .
  • the volume of cytoplasmic fraction which is -released from the cell is about 5% of the volume of the cell. Preferably the volume is about 2% of the volume of the cell.
  • the method includes the steps of: a) inserting releasing means into the embryonic cell; b) drawing a cytoplasmic fraction which is about 5% of the volume of the embryonic cell into the releasing means; and c) withdrawing the releasing means from the cell so that the fraction is isolated in the releasing means .
  • the volume of the cytoplasmic fraction drawn into the releasing means is typically less than lOpL and preferably 8pL.
  • the releasing means includes at least an ICSI pipette and preferably the cytoplasmic fraction is drawn approximately lOO ⁇ m into the pipette.
  • the drawing of the cytoplasmic fraction from the embryonic cell into the releasing means typically forms an extrusion of cytoplasmic contents between the releasing means and the embryonic cell .
  • the cytoplasmic fraction is isolated from the embryonic cell by gently stretching or shearing the extrusion so as to separate the extrusion.
  • the cytoplasmic fraction is isolated by stretching the extrusion.
  • the cytoplasmic fraction contains cytoplasmic organelles, and includes or consists of a sample of the mitochondria and mitochondrial products of the embryonic cell.
  • a method of isolating a cytoplasmic fraction from an embryonic cell which does not impair the developmental potential of the cell is useful for detecting a nucleotide sequence, polymorphism or mutation in the mitochondrial genome, enabling the study of the nucleotide sequence, polymorphism or mutation in the context of the functional integrity of both the embryonic cell and the progeny descended from the embryonic cell.
  • the invention provides a method of detecting a nucleotide sequence, polymorphism or mutation in the mitochondrial genome of mitochondria located in an embryonic cell which does not impair the developmental potential of the cell, the method including the following steps: a) isolating a cytoplasmic fraction which includes mitochondria from the embryonic cell according to the method of the tenth aspect of the invention; and b) analysing the nucleotide sequence of the mitochondrial genome of the mitochondria in the cytoplasmic fraction for the presence of a nucleotide sequence, polymorphism or mutation in the mitochondrial genome.
  • the present inventors recognised that a method of isolating a cytoplasmic fraction without impairing the developmental potential of an embryonic cell is useful for predicting whether the progeny descended from the embryonic cell will or will not contain a nucleotide sequence, polymorphism or mutation of the mitochondrial genome which causes, or is suspected of causing, or is associated, with disease or dysfunction.
  • the invention provides a method for predicting whether the progeny descended from an embryonic cell will contain a nucleotide sequence, polymorphism or mutation in a mitochondrial genome which causes, or is suspected of causing, or is associated with, a disease or dysfunction, wherein the method does not impair the developmental potential of the cell, the method including the following steps: a) isolating a cytoplasmic fraction which includes mitochondria from the embryonic cell according to the method of the tenth aspect of the invention; and b) analysing the mitochondrial genome of the mitochondria in the fraction for the presence of the nucleotide sequence, polymorphism or mutation; wherein the presence of the nucleotide sequence, polymorphism or mutation indicates a likelihood that the progeny descended from the embryonic cell will contain the nucleotide sequence, polymorphism or mutation.
  • the invention provides a method of determining the level of heteroplasmy of mitochondrial genomes in an embryonic cell which does not impair the developmental potential of the cell, and which includes the following steps a) isolating a cytoplasmic fraction which includes mitochondria from the embryonic cell according to the method of the tenth aspect of the invention; and b) comparing the number of mitochondrial genomes in the fraction with a nucleotide sequence, polymorphism or mutation, with the number of genomes without the nucleotide sequence, polymorphism or mutation in the fraction.
  • the invention provides a method of determining whether the progeny descended from an embryonic cell are likely to suffer from a disease or dysfunction caused by, or suspected of being caused by or associated with, a nucleotide sequence, polymorphism or mutation in a mitochondrial genome, wherein the method does not impair the developmental potential of the cell, and includes the following steps : a) isolating a cytoplasmic fraction which includes mitochondria from the embryonic cell according to the method of the tenth aspect of the invention; and b) analysing the mitochondrial genome of the mitochondria in the fraction for the presence of the nucleotide sequence, polymorphism or mutation; determining that the progeny are likely to suffer from the disease or dysfunction where the analysis of the mitochondrial genome of mitochondria in the fraction demonstrates that the level of heteroplasmy of mitochondrial genomes with respect to the nucleotide sequence, polymorphism or mutation in the embryonic cell is at least the same as the level of heteroplasmy which is known to be associated with the manifestation of the disease
  • the method of isolating a cytoplasmic fraction from an embryonic cell which does not impair the developmental potential of the cell is particularly useful for selecting an embryo for embryo transfer which does not contain a nucleotide sequence, polymorphism or mutation which causes or is associated with disease or dysfunction in the progeny descended from the embryonic cell, prior to embryo transfer .
  • the invention provides a method of selecting an embryo for embryo transfer, which either: (i) does not contain a nucleotide sequence, polymorphism or mutation in the mitochondrial genome of mitochondria in an embryonic cell derived from the embryo, which causes, or is suspected of causing, or is associated with, a disease or dysfunction in the progeny descended from the embryonic cell or embryo; or (ii) has a level of heteroplasmy of mitochondrial genomes with respect to the nucleotide sequence, polymorphism or mutation which is less than the level of heteroplasmy which is known to be associated with the manifestation of the disease or dysfunction; wherein the method does not impair the developmental potential of the embryo, and includes the following steps : a) isolating a cytoplasmic fraction which includes mitochondria from the embryonic cell according to the method of the tenth aspect of the invention; and b) analysing the mitochondrial genome of the mitochondria in the fraction; and c) selecting the embryo for embryo transfer, provided that at least the degree of heteroplasm
  • the invention relates to a kit for use in the method of isolating a cytoplasmic fraction from an oocyte, or from an embryonic cell.
  • the kit includes at least one nucleotide probe specific for a nucleotide sequence, polymorphism or mutation in the mitochondrial genome of mitochondria in an oocyte or an embryonic cell, which causes, or is suspected of causing, or which is associated with a disease or dysfunction in the progeny descended from the fertilized oocyte or embryonic cell.
  • the at least one nucleotide probe is specific for any one of the nucleotide sequence mutations in the mitochondrial genome shown in Table 1 .
  • N any nucleotide
  • R either puRine (A,G)
  • the mitochondrial genome of mitochondria which are in the cytoplasmic fraction isolated according to the method of the tenth aspect of the invention can be analysed according to standard techniques. These techniques are exemplified further herein and include the polymerase chain reaction (PCR) , restriction fragment length polymorphism (RFLP) analysis, genomic hybridisation, nucleotide sequencing and gene function detection and/or measurement .
  • PCR polymerase chain reaction
  • RFLP restriction fragment length polymorphism
  • the invention provides nucleotides including the following sequences: ATP6F: TCACCACCCAACAATGAC
  • ATP6R TAAGGCGACAGCGATTTC .
  • oocyte means a female germ line cell including primary oocytes and secondary oocytes, and includes human oocytes.
  • Primary oocytes include germ cells at the GV (germinal vesicle) stage of meiosis.
  • Secondary oocytes include germ cells at the metaphase II stage of meiosis.
  • progeny descended from a fertilized oocyte means the individual which is generated from the fertilization of the female germ cell with the male germ cell.
  • “Individual” means the multicellular organism from the earliest stage of embryonic life (for example, the 2 cell stage) to adult life.
  • oes not impair the capacity of the oocyte to be fertilized means that the oocyte which has had a fraction of cytoplasm isolated, may be fertilized, or in other words, may undergo any one or more of the biochemical or cellular events which are associated with any one or more of the stages of fertilization, from the activation of the oocyte by entry of sperm, to the generation of a zygote and the formation of cleavage products of the zygote.
  • the oocyte may be fertilized in vitro by standard techniques, including for example ICSI and IVF .
  • embryonic cell includes a post-syngamous fusion product of the female and male germ cells, a zygote, and cleavage products of a zygote at any stage of development from the 2 cell stage to the stage of implantation.
  • progeny descended from an embryonic cell or “progeny descended from an embryo” means the individual which is generated from the post syngamous fusion product of the female and male germ cells.
  • “Individual” means the multi-cellular organism from the earliest stage of embryonic life (for example, the 2 cell stage) to adult life.
  • does not impair the developmental potential of the cell or “does not impair the developmental potential of the embryo” means that the embryonic cell which has had a fraction of cytoplasm isolated, and the embryo from which the embryonic cell may be derived, may undergo any one or more of the biochemical or cellular events which are associated, with cell differentiation and/or maturation.
  • Figure 1 shows a 400 by fragment amplified from the D-loop region of the mitochondrial genome derived from cytoplasmic biopsy samples of oocytes Al to A7 , using oligonucleotide primers L29 and H04.
  • Figure 2 shows amplification of a 271 bp fragment of mitochondrial genomes isolated from embryonic cells using primers corresponding to mtDNA positions 8201 and 8472.
  • oocytes were either germinal vesicle (GV) cells or were at the MI stage of meiosis, and were 6 hours postretrieval .
  • Human oocytes Bl to B8 were donated for research.
  • Human oocyte CI was designated at the patients request to be part of the study. This oocyte was at the Mil stage of meiosis and was 24 hours post-retrieval.
  • the pipette is a glass capillary drawn out to have an end diameter of approximately 7 ⁇ m with a bevelled tip.
  • the biopsy technique was performed as follows: ooplasm was drawn into the pipette to a distance of approximately lOO ⁇ m (approximately 8pl) ' . The pipette was then withdrawn from the oocyte, forming a thin ooplasmic bridge, which was then broken by stretching. Each ooplasmic biopsy was expelled directly into a PCR tube containing 20 ⁇ lof PCR buffer, Proteinase K and 20 mM DTT. Tubes were incubated either at 37°C overnight, or at 50°C for 30 minutes and then frozen. Both protocols were followed with heat inactivation of Proteinase K at 95°C for 10 minutes. 7Analysis of mitochondrial genome in oocyte cytoplasmic fractions
  • the mitochondrial genome in the oocyte cytoplasmic fractions was analysed by the polymerase chain reaction (PCR) .
  • reaction mixture contained 2.5 pmol of each primer, 200 ⁇ M of each dNTP, PCR buffer, milli-Q water and 0.5 units of Taq. All reactions were carried out in capped 0.2ml tube strips.
  • PCR cycling was performed in an FTS Thermal Sequencer (Corbett Research, Sydney, NSW) under the following conditions: initial denaturation at 93 °C for 5 minutes, followed by 24 to 40 cycles of 93 °C denaturation for 45 seconds, 60°C annealing for 1 minute and 72 °C extension for 1 minute; ending with a polishing step of 72 °C for 7 minutes, cooling to 15°C and holding at 4°C.
  • FTS Thermal Sequencer Cornbett Research, Sydney, NSW
  • initial denaturation at 93 °C for 5 minutes followed by 24 to 40 cycles of 93 °C denaturation for 45 seconds, 60°C annealing for 1 minute and 72 °C extension for 1 minute; ending with a polishing step of 72 °C for 7 minutes, cooling to 15°C and holding at 4°C.
  • Complete reaction mixtures lacking template DNA were included in all PCR reactions as negative controls.
  • the amplified DNA fragments from the D-loop region or the common deletion region were analysed by polyacrylamide gel electrophoresis on a 5% 37:1 acrylamide: bisacrylamide gel.
  • the Bl to B8 oocytes and the CI oocyte were fertilized by either ICSI or IVF according to standard protocols (1,2) .
  • the insemination was performed immediately after cytoplasmic biopsy of the oocyte.
  • Oocytes Al to A5 were not visibly affected by the cytoplasmic biopsy procedure and showed no signs of degeneration at 48 hours after biopsy.
  • Oocytes Bl to B3 and B5 to B8 showed no signs of degeneration at 48 hours after biopsy.
  • Oocyte CI showed no signs of degeneration at 48 hours after biopsy.
  • a single oocyte, - B4 degenerated at 17 hours after biopsy (Table 2) .
  • the cytoplasmic biopsy was obtained by using an ICSI pipette as described above. It was estimated that the volume of this cytoplasmic biopsy would be approximately 8pl . As the volume of an oocyte is approximately 500pl, the cytoplasmic biopsy removed is estimated to comprise approximately 2% of the cytoplasm. In a conservative estimate, there are approximately 100,000 mitochondrial genomes per oocyte (3) . In accordance with this estimate, the biopsy would remove approximately 1000 mitochondria. This amount of mitochondria is within the amplification capabilities of the polymerase chain reaction. PCR amplification from the D-loop region
  • a 400 bp D-loop fragment was amplified from the cytoplasmic biopsy of oocytes Al to A5 with 35 cycles of amplification (Figure 1) .
  • PN pronuclei observed All oocytes that were subject to the ICSI protocol, (except B4) were fertilized. Two pro-nuclei were observed in Bl, B3 and CI, and 3 pro-nuclei were observed in B3 at 17 hours after fertilization. The oocytes Bl, B3 and CI divided and progressed to the 2 cell stage at 48 hours post fertilization. No cell division was observed in B2 at 48 hours.
  • oocytes Al to A5 were matured in a medium formulated specifically for insemination, rather than oocyte maturation.
  • the cytoplasmic biopsy technique can be generally applied up to 24 hours post retrieval of the oocyte.
  • the biopsy technique is therefore able to be used together with known fertilization techniques, including for example, ICSI and IVF, which are generally used approximately 4 to 6 hours post retrieval of the oocyte.
  • fertilization techniques including for example, ICSI and IVF, which are generally used approximately 4 to 6 hours post retrieval of the oocyte.
  • insemination was performed immediately after biopsy, it is expected that the biopsy may be performed after insemination.
  • the amplification of fragments from the D loop region and the common deletion region of the mitochondrial genome from mitochondria in the cytoplasmic biopsy demonstrates that there is a sufficient source of template DNA in the biopsy sample for analysis by PCR, and that fragments of the order of from less than 0.5 kb to greater than 5 kb can be amplified from the sample.
  • the amplification of fragments from 2 independent loci of the mitochondrial genome suggests that mutations at other loci of the mitochondrial genome, in particular the mutations described in Table 1, can be amplified from the cytoplasmic biopsy sample using PCR and specific oligonucleotides .
  • Frozen research embryos (3 * two pronuclear embryos, 1 * 2 cell embryo and 1 * 4 cell embryo) were thawed and equilibrated in growth medium. Isolation of embryonic cell cytoplasmic fraction
  • the 3 single cell embryos and 1 cell from each of the multi cell embryos were biopsied using a standard ICSI pipette.
  • the pipette was introduced into the cytoplasmic region and an aliquot of cytoplasm corresponding to approximately 5% of the cell volume was withdrawn. This aliquot was delivered into a sucrose solution.
  • the embryos were returned to the incubator for continued growth. Analysis of mitochondrial genome in embryonic cell cytoplasmic fraction
  • cytoplasm The preparations of cytoplasm were made alkaline with Potassium Hydroxide and heated to 90°C for 5 minutes. After neutralizing with TrisHCI an aliquot was added to a reaction mix containing mitochondrial DNA specific primers designed to amplify a 271 base pair fragment corresponding to mtDNA positions 8201 and 8472. This region of the mitochondrial genome comprises part of the coding region for an oxidase and an ATPase. The mix was PCR amplified using a thermal cycler capable of real time monitoring of increasing fluorescence associated with the amplification of double stranded DNA. Reagent and medium blanks were negative for any fluorescence changes. Analysis of DNA copy number was by comparison to an external dilution of control human DNA.
  • Table 4 shows estimated mtDNA copies in biopsy samples

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Abstract

La présente invention concerne une méthode d'isolement d'une fraction cytoplasmique d'un ovocyte, n'altèrant pas la capacité de l'ovocyte à être fertilisé, et consistant à séparer une fraction cytoplasmique de l'ovocyte correspondant à environ 5 % du volume de l'ovocyte. L'invention concerne également une méthode d'isolement d'une fraction cytoplasmique d'une cellule embryonnaire, n'altérant pas le potentiel de développement de la cellule, ladite méthode consistant à séparer une fraction cytoplasmique de la cellule correspondant à environ 5 % du volume de la cellule.
EP00906062A 1999-02-23 2000-02-23 Isolement d'une fraction cytoplasmique sans alteration de la viabilite des cellules ovocytes et embryonnaires Withdrawn EP1155320A1 (fr)

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AUPP884199 1999-02-23
AUPP8841A AUPP884199A0 (en) 1999-02-23 1999-02-23 Female germ-line-cell mitochondrialgenome
PCT/AU2000/000125 WO2000050895A1 (fr) 1999-02-23 2000-02-23 Isolement d'une fraction cytoplasmique sans alteration de la viabilite des cellules ovocytes et embryonnaires

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