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WO2025191155A1 - Amélioration métabolique du trophectoderme humain - Google Patents

Amélioration métabolique du trophectoderme humain

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Publication number
WO2025191155A1
WO2025191155A1 PCT/EP2025/057066 EP2025057066W WO2025191155A1 WO 2025191155 A1 WO2025191155 A1 WO 2025191155A1 EP 2025057066 W EP2025057066 W EP 2025057066W WO 2025191155 A1 WO2025191155 A1 WO 2025191155A1
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WIPO (PCT)
Prior art keywords
embryo
ketoglutarate
alpha
trophectoderm
markers
Prior art date
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Pending
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PCT/EP2025/057066
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English (en)
Inventor
Jan Jakub ZYLICZ
Karlien Marie Francoise VAN NERUM
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Københavns Universitet
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Københavns Universitet
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Publication of WO2025191155A1 publication Critical patent/WO2025191155A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2517/00—Cells related to new breeds of animals
    • C12N2517/10—Conditioning of cells for in vitro fecondation or nuclear transfer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0604—Whole embryos; Culture medium therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]

Definitions

  • the present invention relates to assisted reproductive technology (ART), specifically in vitro fertilization (IVF) procedures.
  • ART assisted reproductive technology
  • IVF in vitro fertilization
  • the invention relates to the use of cell- membrane-permeable form of alpha-ketoglutarate (aKG) as a supplement to human IVF media, for improving trophectoderm (TE) quality, and methods thereof.
  • TE trophectoderm
  • nESC naive embryonic stem cells
  • TSC trophoblast stem cells
  • blastoids form a structure with epiblast cells surrounded by trophectodermal cells and are transcriptionally reminiscent of the bona fide human blastocyst ( Zhao et al., 2021).
  • 2D and 3D models of human pre-implantation development represent an excellent starting point for optimizing the quality of trophectodermal cells.
  • YAP allows the activation of key factors like CDX2 and EOMES, which, in turn, repress the pluripotency factors such as NANOG and OCT4 (Niwa et al., 2005).
  • This negative feedback loop allows cells to become specified to the trophectodermal lineage.
  • key markers of trophectoderm are not entirely conserved and include GATA3, CDX2 and NR2F2 while naive pluripotency is linked to high expression of NANOG, KLF17, and DPPA5 (Meistermann et al., 2021).
  • two other broad types of regulation allow for trophectoderm establishment. Firstly, epigenetic regulation is highly relevant.
  • mice histone H3 lysine 9 trimethylation is vital for the repression of trophectodermal markers within pluripotent cells (Dodge et al., 2004). In humans, this process is not conserved, and rather histone H3 lysine 27 trimethylation maintains stable silencing of the trophectodermal genes in nESC (Kumar et al., 2022; Zijlmans et al., 2022). Another pathway that modulates the trophectoderm induction in mice is metabolism. In mouse blastocysts, Hippo signaling is additionally modulated by metabolic regulation of YAP through glucose-dependent post-translational modifications (PTM) (Chi et al., 2020).
  • PTM glucose-dependent post-translational modifications
  • the inventors surprisingly found that human trophectoderm and epiblast differ drastically from a metabolic perspective.
  • the inventors Using 2D cell culture models and 3D blastoid models, the inventors have identified that the trophectoderm showed elevated levels of alpha-ketoglutarate (aKG), and, importantly, that addition of cell-permeable forms of alpha ketoglutarate in vitro promoted polarization (3D), aggregate patterning (3D), acquisition of trophectoderm fate (2D), and trophectoderm maturation (3D), while it downregulated cell pluripotency, through facilitation of histone deacetylation (2D).
  • the inventors further found that the robust trophectoderm establishment, after addition of cell-permeable forms of alpha ketoglutarate, did not negatively affect the development for the inner cell mass (ICM) in 3D blastoid models, but reduced the generation of primitive streak and extraembryonic mesoderm-like cells using 2D cell culture models.
  • ICM inner cell mass
  • 3D blastoid models displayed enhanced attachment and implantation potential following treatment with cell-permeable forms of alpha ketoglutarate.
  • the inventors provide novel uses of cell-permeable forms of alpha ketoglutarate, and methods making use thereof, in the field of IVF, in particular human IVF, resulting in improved embryo trophectoderm quality.
  • a first aspect of the present invention relates to the use of a cell membrane- permeable form of alpha-ketoglutarate (aKG) as a supplement to human in vitro fertilization (IVF) media.
  • aKG cell membrane- permeable form of alpha-ketoglutarate
  • IVF human in vitro fertilization
  • a second aspect of the present invention relates to the use of a cell membrane- permeable form of alpha-ketoglutarate (aKG) to improve human embryo trophectoderm quality.
  • a third aspect of the present invention relates to the use of a medium, supplemented with a cell membrane-permeable form of alpha-ketoglutarate to improve embryo trophectoderm quality.
  • a fourth aspect of the present invention relates to a method of culturing a preimplantation human embryo, wherein said method comprises culturing said embryo in a medium supplemented with a cell membrane-permeable form of alpha-ketoglutarate, and, optionally, measuring an improvement in trophectoderm quality of said embryo.
  • a fifth aspect of the present invention relates to a method of improving human embryo trophectoderm quality, comprising culturing a human embryo in a medium supplemented with a cell membrane-permeable form of alpha-ketoglutarate, and, optionally, measuring an improvement in trophectoderm quality of said embryo.
  • a sixth aspect of the present invention relates to a method of human in vitro fertilization which comprises cultivating culturing a pre-implantation human embryo in a medium supplemented with a cell membrane-permeable form of alpha-ketoglutarate, and, optionally, measuring an improvement in trophectoderm quality of said embryo.
  • a seventh aspect of the present invention relates to an in vitro method for obtaining an embryo with improved trophectoderm quality, said method comprising the steps of:
  • FIG. 1 96h dm-a-ketoglutarate treatment ameliorates 2D trophoblast stem cell (TSC) induction from human naive embryonic stem cells (nESC).
  • TSC trophoblast stem cell
  • A Schematic of human induced trophoblast stem cell (hiTSC) induction with 96h dm-aKG treatment.
  • B Immunofluorescence quantification of trophoblast (GATA3), pluripotency (NANOG) and primitive streak (TBXT) markers at day 3 of hiTSC induction, treated for 96h with 0, 2, or 4mM of dm-aKG.
  • C Expression levels of trophoblast (CDX2), pluripotency (KLF17) and extra- embryonic mesoderm (VIM) marker genes, determined by RT-qPCR.
  • FIG. 2 24h dm-a-ketoglutarate treatment ameliorates 2D trophoblast stem cell (TSC) induction from human naive embryonic stem cells (nESC).
  • TSC trophoblast stem cell
  • IF Immunofluorescence quantification of trophoblast (GATA3), pluripotency (NANOG) and primitive streak (TBXT) markers at day 3 of hiTSC induction, treated for 24h with 0, 2, or 4mM of dm-aKG.
  • C Expression levels of trophoblast (CDX2), pluripotency (KLF17) and extra- embryonic mesoderm (VIM) marker genes, determined by RT-qPCR.
  • Figure 3 Dm-a-ketoglutarate supplementation downregulates pluripotency in human naive embryonic stem cells.
  • Figure 4 Dm-a-ketoglutarate supplementation upregulates early epiblast markers in human naive embryonic stem cells.
  • FIG. 1 A. Combined beeswarm and box plots of log-normalized gene expression values of early epiblast markers (KLF3, PRAP1 and GDF15).
  • Figure 5 Dm-a-ketoglutarate supplementation enhances trophectoderm state of 2D trophoblast stem cell (TSC) induction from human naive embryonic stem cells (nESC).
  • TSC 2D trophoblast stem cell
  • Imputed annotation of in vitro samples from reference in vivo dataset CTB: cytotrophoblast, TE: trophectoderm, I NT: intermediate, PriS: primitive streak. Unassigned and ambiguous labels refer to cells with either none or with more than two imputed annotations respectively.
  • FIG. 6 Expression of selected lineage-specific marker genes across samples.
  • Figure 7 Dm-a-ketoglutarate supplementation aids in polarisation during human blastoid induction.
  • Figure 8 Dm-a-ketoglutarate supplementation aids in polarisation during human blastoid induction.
  • FIG. 10 Dm-a-ketoglutarate supplementation aids in robustness of blastoid growth without diminishing the epiblast-like population.
  • D Imputed developmental time of in vitro samples from reference in vivo dataset.
  • E embryonic day
  • CS Carnegie Stage. Unassigned and ambiguous labels refer to cells with either none or with more than two imputed stages respectively.
  • E Reference pseudotime trajectories of the in vivo epiblast (EPI: left) and trophectoderm (TE: right) related to the reference developmental time based on Zhao et al. 2024.
  • F-G Distribution of pseudotime values in nESC(f) and d3 hiTSC(g) cells which are related to the in vivo EPI and TE respectively. Pseudotime trajectories were identified from in vivo reference (E).
  • Figure 12 Increased aKG levels aid in polarisation and treatment of 40h aggregates with dm-aKG leads to apical polarisation
  • FIG. 13 Dm-aKG facilitates blastoid induction and TE maturation.
  • A. Quantification of blastoid induction efficiency according to cavitation. Data is based on brightfield images of d5 blastoids untreated or treated for 40h or 120h with 4mM dm-aKG. Right panel: Data is presented as the mean and data points of N 3 biological replicates.
  • Sample type is shown with single cells plotted as dots; boxes span the interquartile range (IQR), center line indicates the median, whiskers extend to values within 1.5 times the IQR.
  • Figure 14 40h dm-aKG treatment of blastoids leads to improved attachment and trophoblast lineage differentiation.
  • hiTSC human induced trophoblasts stem cells and includes human induced trophoblasts stem cells obtained from naive human embryonic stem cells (nESC).
  • alpha-ketoglutarate As used herein “alpha-ketoglutarate”, “aKG”, “aKG”, “2-oxoglutarate” are used interchangeably and refer to the carboxylate of a-ketoglutaric acid.
  • cell-membrane permeable compounds refer to compounds capable of being internalized by cells.
  • the term includes passive membrane diffusion and active internalization mechanisms, such as endocytosis.
  • ovum as used herein relates to an animal egg cell, and includes mammalian egg cells, such as human egg cells.
  • embryo comprises zygote or any post-zygotic derivative of a fertilized egg or ovum, for example the term embryo comprises blastocyst.
  • pre-implantation embryo refers to an embryo in any development stage prior to implantation in the uterus and includes embryos obtained by IVF. Typically following fertilization, human pre-implantation embryos will undergo a series of cell divisions, leading after approximately four days to the morula stage, before a cavity is formed in the pre-implantation embryo, at which point it is also referred to as a blastocyst. Cells of the blastocyst differentiate into the inner mass cells or the trophectoderm. The trophectoderm is the progenitor tissue of the placenta, which will give rise to epithelial trophoblast cells of the placenta, in turn mediating embryo (blastocyst) implantation in the uterus.
  • following refers to a timeframe starting at any timepoint after the initiation of said action step i.e. not necessarily after said action step has been stopped.
  • GATA3 refers to GATA protein binding 3
  • TXT T-Box Transcription Factor T
  • CDX2 Caudal Type Homeobox 2
  • KLF17 KLF Transcription Factor 17
  • VIM Vimentin
  • TBP TATA-Box Binding Protein
  • KLF4 KLF Transcription Factor 4
  • NANOG Nanog Homeobox
  • DPPA5 Developmental Pluripotency Associated 5
  • KLF3 refers to KLF Transcription Factor 3
  • PRAP1 refers to Proline Rich Acidic Protein 1
  • GDF15 refers to Growth Differentiation Factor 15
  • aPKC£ as used herein, refers to atypical protein kinase C zeta
  • YAP/TAZ as used herein refer to the Yes-Associated Protein also known as YAP1 , and the Transcriptional coactivator with
  • Gardner scale refers to the embryo/blastocyst scoring system commonly used in the field, in which morphological features such as embryo/blastocyst development and stage status, also referred to as expansion and hatching status, inner cell mass (ICM) quality, and trophectoderm (TE) quality, are evaluated and graded according to an alphanumeric scale. An example of which is in Gardner et a/. 1999.
  • the term also includes modifications and derivatives of the Gardner scoring system, for example, but not limited to, adaptations and derivatives of the system comprising a different number of grading levels for one or more of the features evaluated, or for example any adaptation or derivative of the system comprising at least a trophectoderm (TE) quality evaluation.
  • TE trophectoderm
  • normal karyotype refers to the typical chromosomal arrangement for a specific species.
  • normal human karyotype typically consists of 46 chromosomes arranged in 23 pairs, with two sex chromosomes (XX for females and XY for males) determining gender.
  • implantation rate or “implantation success rate” refers to the number of gestational sacs observed in the uterus of the recipient of the embryo(s) in an IVF procedure, divided by the number of embryos transferred to said uterus.
  • the gestational sacs are typically observed by non-invasive methods such as ultrasound.
  • polar trophectoderm is the area of the trophoblast that is located nearest to the inner cell mass, as opposed to the “mural trophectoderm”.
  • the present invention relates to the use of a cell-membrane-permeable form of alpha-ketoglutarate (aKG) as a supplement to human in vitro fertilization (IVF) media.
  • aKG cell-membrane-permeable form of alpha-ketoglutarate
  • IVF human in vitro fertilization
  • the cell membrane-permeable form of alpha-ketoglutarate may be as described in the Cell membrane-permeable form of alpha-ketoglutarate section herein.
  • the media may be as described in the IVF media section described herein.
  • Another aspect of the present invention relates to the use of a cell-membrane- permeable form of alpha-ketoglutarate (aKG) to improve human embryo trophectoderm quality.
  • aKG alpha-ketoglutarate
  • the human embryo provided may be as described in the Embryo types section herein.
  • the cell membrane-permeable form of alpha-ketoglutarate may be as described in the Cell membrane-permeable form of alpha-ketoglutarate section herein.
  • the measurement of the improvement of embryo trophectoderm quality may be as described in the Trophectoderm quality section herein.
  • a further aspect of the present invention relates to the use of a medium, supplemented with a cell membrane-permeable form of alpha-ketoglutarate to improve embryo trophectoderm quality.
  • the embryo provided may be as described in the Embryo types section herein.
  • the medium may be as described in the IVF media section described herein.
  • the cell membrane-permeable form of alpha-ketoglutarate may be as described in the Cell membrane-permeable form of alpha-ketoglutarate section herein.
  • the measurement of the improvement of embryo trophectoderm quality may be as described in the Trophectoderm quality section herein.
  • the cell-membrane- permeable form of alpha-ketoglutarate, or the medium comprising said cell-membrane- permeable form of alpha-ketoglutarate, such as the IVF medium comprising said cell- membrane-permeable form of alpha-ketoglutarate is used prior to the in vitro fertilization stage, for example on an ovum used for an IVF procedure.
  • the cell-membrane- permeable form of alpha-ketoglutarate, or the medium comprising said cell-membrane- permeable form of alpha-ketoglutarate, such as the IVF medium comprising said cell- membrane-permeable form of alpha-ketoglutarate is used simultaneously with, such as during, the fertilization step of an IVF procedure.
  • the cell-membrane- permeable form of alpha-ketoglutarate, or the medium comprising said cell-membrane- permeable form of alpha-ketoglutarate, such as the IVF medium comprising said cell- membrane-permeable form of alpha-ketoglutarate is used after the fertilization step of an IVF procedure, for example shortly after the fertilization step, such as on a preimplantation embryo in an IVF procedure.
  • the cell-membrane-permeable form of alpha-ketoglutarate, or the medium comprising said cell-membrane-permeable form of alpha-ketoglutarate, such as the IVF medium comprising said cell-membrane- permeable form of alpha-ketoglutarate is used prior to the in vitro fertilization stage, during the fertilization step, and during the in vitro post-fertilization steps of an IVF procedure.
  • said uses are prior to-, simultaneously with-, and/or after, embryo in vitro fertilization, preferably simultaneously with-, and/or after, embryo in vitro fertilization, even more preferably after embryo in vitro fertilization.
  • the cell- membrane-permeable form of alpha-ketoglutarate (aKG), or said medium comprising said alpha-ketoglutarate is beneficially only used shortly after fertilization, such as only contacted with an embryo at any early stage post-fertilization, such as only contacted with a pre-implantation embryo shortly after fertilization, such as only contacted with a pre-implantation human embryo shortly after the fertilization step of an IVF procedure.
  • said cell- membrane- permeable form of alpha-ketoglutarate (aKG), or said medium comprising thereof is only contacted with said embryo at the most 96h after embryo fertilization, such as at the most 72h, such as at the most 48h, such as at the most 24h, such as at the most 12h, such as at the most 10h, such as at the most 8h, such as at the most 6h, such as at the most 4h, such as at the most 2h, such as at the most 1 h, such as at the most 30min, such as at the most 10min, such as at the most 5min, such as at the most 1 min, after embryo fertilization.
  • the most 72h such as at the most 48h, such as at the most 24h, such as at the most 12h, such as at the most 12h, such as at the most 10h, such as at the most 8h, such as at the most 6h, such as at the most 4h, such as at the most 2h, such as at the most 1 h, such as at
  • the eggs are collected, fertilized with a sperm cell, for example using intracytoplasmic sperm injection (I CSI) or other suitable methods known in the art, and grown in IVF medium for a period of 0 to 6 days, typically 5 days, following fertilization, until the embryos reach the blastocyst stage.
  • I CSI intracytoplasmic sperm injection
  • the embryo may be transferred at earlier or later timepoints. The embryo with highest quality is then transferred into the uterus of a receiving individual.
  • the cell-membrane-permeable form of alpha-ketoglutarate (aKG), or said medium comprising thereof, of the present invention may be used, such as contacted with a pre-implantation embryo, preferably a human pre-implantation embryo in an IVF procedure, from the day of the egg fertilization leading to said embryo up until the day on which it is transferred to a recipient of an IVF procedure.
  • a pre-implantation embryo preferably a human pre-implantation embryo in an IVF procedure
  • said cell-membrane- permeable form of alpha-ketoglutarate (aKG), or said medium comprising thereof is contacted with said embryo for in the range of 0 to 6 days after embryo fertilization, such as for at least 1 h, such as for at least 2h, such as for at least 4h, such as for at least 6h, such as for at least 8h, such as for at least 10h, such as for at least 12h, such as for at least 24h, such as for at least 40h, such as for at least 48h, such as for at least 72h, such as for at least 4 days, such as for at least 5 days, such as for at least 6 days after embryo fertilization.
  • aKG alpha-ketoglutarate
  • said cell-membrane- permeable form of alpha-ketoglutarate (aKG), or said medium comprising aKG is only contacted with said embryo an embryo at the pronuclear stage, zygote stage, 2-cell stage, 4-cell stage, 8-cell stage, morula stage, early blastocyst stage and/or blastocyst stage.
  • Another aspect of the invention relates to a method of culturing a pre-implantation human embryo, wherein said method comprises culturing said embryo in a medium supplemented with a cell membrane-permeable form of alpha-ketoglutarate, and, optionally, measuring an improvement in trophectoderm quality of said embryo.
  • the human embryo provided may be as described in the Embryo types section herein.
  • the medium may be as described in the IVF media section described herein.
  • the cell membrane-permeable form of alpha-ketoglutarate may be as described in the Cell membrane-permeable form of alpha-ketoglutarate section herein.
  • the measurement of the improvement of embryo trophectoderm quality may be as described in the Trophectoderm quality section herein.
  • Another aspect of the present invention relates to a method of improving human embryo trophectoderm quality, comprising culturing a pre-implantation human embryo in a medium supplemented with a cell membrane-permeable form of alpha- ketoglutarate, and, optionally, measuring an improvement in trophectoderm quality of said embryo.
  • the embryo provided may be as described in the Embryo types section herein.
  • the medium may be as described in the IVF media section described herein.
  • the cell membrane-permeable form of alpha-ketoglutarate may be as described in the Cell membrane-permeable form of alpha-ketoglutarate section herein.
  • the measurement of the improvement of embryo trophectoderm quality may be as described in the Trophectoderm quality section herein.
  • Method of human in vitro fertilization Another aspect of the present invention relates to a method of human in vitro fertilization which comprises culturing a pre-implantation human embryo in a medium supplemented with a cell membrane-permeable form of alpha-ketoglutarate, and, optionally, measuring an improvement in trophectoderm quality of said embryo.
  • the embryo provided may be as described in the Embryo types section herein.
  • the medium may be as described in the IVF media section described herein.
  • the cell membrane-permeable form of alpha-ketoglutarate may be as described in the Cell membrane-permeable form of alpha-ketoglutarate section herein.
  • the measurement of the improvement of embryo trophectoderm quality may be as described in the Trophectoderm quality section herein.
  • said uses or methods are in vitro uses or methods.
  • said uses or methods are for obtaining an embryo with improved trophectoderm quality.
  • embryo trophectoderm quality is improved.
  • Another aspect of the present invention relates to an in vitro method for obtaining an embryo with improved trophectoderm quality, said method comprising the steps of:
  • the embryo provided may be as described in the Embryo types section herein.
  • the culture medium may be as described in the IVF media section described herein.
  • the cell membrane-permeable form of alpha-ketoglutarate may be as described in the Cell membrane-permeable form of alpha-ketoglutarate section herein.
  • the measurement of the improvement of embryo trophectoderm quality may be as described in the Trophectoderm quality section herein.
  • the cell-membrane-permeable form of alpha-ketoglutarate, or the medium comprising said cell-membrane-permeable form of alpha-ketoglutarate, such as the IVF medium comprising said cell-membrane-permeable form of alpha- ketoglutarate is further used for culturing prior to the in vitro fertilization stage, for example for culturing an ovum used for an IVF procedure.
  • the method further comprises, before the step of culturing said embryo in a medium supplemented with a cell membrane-permeable form of alpha-ketoglutarate, the steps of:
  • the inventors have found that the cell-membrane-permeable form of alpha-ketoglutarate exerts most of its function at the early aggregate stage, for example by aiding in initial polarization, aggregate patterning and TE maturation.
  • the step of culturing said embryo in a medium supplemented with the cell membrane- permeable form of alpha-ketoglutarate is initiated simultaneously with-, or afterfertilization.
  • the step of culturing said embryo in a medium supplemented with the cell membrane- permeable form of alpha-ketoglutarate is initiated at the most 96h after fertilization, such as at the most 72h, such as at the most 48h, such as at the most 24h, such as at the most 12h, such as at the most 10h, such as at the most 8h, such as at the most 6h, such as at the most 4h, such as at the most 2h, such as at the most 1h such as at the most 30min, such as at the most 10min, such as at the most 5min, such as at the most 1min, after fertilization.
  • the most 96h after fertilization such as at the most 72h, such as at the most 48h, such as at the most 24h, such as at the most 12h, such as at the most 10h, such as at the most 8h, such as at the most 6h, such as at the most 4h, such as at the most 2h, such as at the most 1h such as at the
  • the step of culturing said embryo in a medium supplemented with the cell membrane- permeable form of alpha-ketoglutarate is performed for in the range of 0 to 6 days, such as for at least 1 h, such as for at least 2h, such as for at least 4h, such as for at least 6h, such as for at least 8h, such as for at least 10h, such as for at least 12h, such as for at least 24h, such as for at least 40h, such as for at least 48h, such as for at least 72h, such as for at least 4 days, such as for at least 5 days, such as for at least 6 days after fertilization.
  • the step of culturing said embryo in a medium supplemented with cell membrane- permeable form of alpha-ketoglutarate is performed at the pronuclear stage, zygote stage, 2-cell stage, 4-cell stage, 8-cell stage, morula stage, early blastocyst stage and/or blastocyst stage.
  • the cell-membrane permeable form of alpha-ketoglutarate of the present invention exerts its functions when internalized by the recipient cells, for example an ovum or embryonic cells.
  • such mechanisms include, but not limited to simple diffusion, where molecules move across the cell membrane down their concentration gradient, and facilitated diffusion, which involves the assistance of specific carrier proteins.
  • such mechanisms typically include endocytosis (such as clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis), phagocytosis, and pinocytosis.
  • endocytosis such as clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis
  • phagocytosis phagocytosis
  • alpha ketoglutarate has a very low to no passive diffusion through the cell membrane, in part due to its hydrophilic nature and charged carboxy group.
  • the uses and methods of the present invention make use of esterified analogs of alpha ketoglutarate such as, but not limited to, dimethyl-alpha-ketoglutarate (dm-aKG), monomethyl alpha-ketoglutarate (mm-aKG), and octyl alpha ketoglutarate (octyl-aKG).
  • Other forms of alpha ketoglutarate which are cell-membrane permeable may be used.
  • the cell membrane-permeable form of alpha-ketoglutarate is selected from the group consisting of: dimethyl-alpha-ketoglutarate (dm-aKG), monomethyl alpha- ketoglutarate (mm-aKG), and octyl alpha ketoglutarate (octyl-aKG).
  • the cell membrane-permeable form of alpha-ketoglutarate is dimethyl-alpha-ketoglutarate (dm-aKG).
  • the cell membrane-permeable form of alpha-ketoglutarate does not negatively affect ovum and/or embryo cell viabilities.
  • the IVF medium comprises the cell membrane-permeable form of alpha- ketoglutarate at a concentration of at the most 10mM, such as at the most 5mM, such as at the most 4mM, such as at the most 3 mM, such as at the most 2mM, such as at the most 1mM, such as at the most 0.5mM, for instance in the range of 0.1-10mM, for instance in the range of 1-4mM, for instance in the range of 2-4mM, such as 3mM.
  • the most 10mM such as at the most 5mM, such as at the most 4mM, such as at the most 3 mM, such as at the most 2mM, such as at the most 1mM, such as at the most 0.5mM, for instance in the range of 0.1-10mM, for instance in the range of 1-4mM, for instance in the range of 2-4mM, such as 3mM.
  • the IVF medium comprises the cell membrane-permeable form of alpha-ketoglutarate at a concentration of at the most 4mM, preferably in the range of 2- 4mM.
  • Low or reduced pluripotency markers in the embryo such as embryo outer cells, or a fortiori in TE cells, is typically associated with a more advanced stage of differentiation or maturation.
  • the TE cells may exhibit characteristics such as enhanced adhesion, proliferation, or signaling capabilities, which are important for successful implantation and early embryonic development.
  • an upregulation of markers associated with TE lineage will be associated with improved TE quality.
  • the inventors have found in the uses and methods of the present invention that the cell membrane-permeable form of alpha-ketoglutarate enhanced differentiation into TE/trophoblast lineage by stabilizing the TE transcriptional programme.
  • the skilled person will know that the outer cells, or outer layer of cells of the embryo, will give rise to the trophectoderm cells.
  • the determination of the expression of the markers of the present invention associated with improved trophectoderm quality, the comparison of the expression, and/or the sorting on which said comparison is based is performed based on the measurement of the markers patterns as described herein, on the outer cells of the embryo.
  • the inventors have found that treatment with a cell membrane-permeable form of alpha-ketoglutarate specifically enhanced the competence of cells, such as nESCs, towards TE cells, such as TE/hiTSC-like GATA3+ve cells, but not towards off-target fates such as the primitive streak or extraembryonic mesoderm.
  • the present disclosure also demonstrates that dm-aKG pre-treatment in hiTSCs allowed for nearly homogenous induction of a transcriptional network reminiscent of in vivo early TE.
  • the present disclosure thus supports that dm-aKG has a targeted effect on TE lineage differentiation rather than affecting other lineages like primitive streak, or extraembryonic mesoderm.
  • the present disclosure also shows that the treatment of embryos with a cell membrane-permeable form of alpha-ketoglutarate led to differential expression of multiple markers related to TE maturity, such as upregulation of markers of mature polar TE (pTE: NR2F2, CCKBR, PTN), and downregulation of mural TE markers (mTE: ALPG, CITED4, TUBB4A).
  • pTE NR2F2, CCKBR, PTN
  • mTE ALPG, CITED4, TUBB4A
  • improved trophectoderm quality is measured as a downregulation of the expression of pluripotency markers, a downregulation of the expression of early epiblast markers, a downregulation of the expression of primitive streak markers, and/or a downregulation of the expression of extraembryonic mesoderm markers, and/or an upregulation of trophectoderm lineage markers, in the outer cells of the embryo, following culturing in a medium supplemented with the cell membrane- permeable form of alpha-ketoglutarate.
  • improved trophectoderm quality is measured as a downregulation of mural TE markers (mTE) and/or an upregulation of markers of mature polar TE (pTE).
  • mTE mural TE markers
  • pTE mature polar TE
  • the first TE specification step is apical polarization. Said polarization may be followed by increased atypical protein kinase C zeta (aPKC ) in outer cells of the embryo, in particular the apical domain of outer cells of the embryo, in turn initiating accumulation of nuclear Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ), initiating TE specification.
  • aPKC atypical protein kinase C zeta
  • YAP nuclear Yes-associated protein
  • TEZ PDZ-binding motif
  • improved trophectoderm quality is measured as increased apical polarization of the embryo outer cells, preferably wherein the apical polarisation is measured as increased levels of atypical protein kinase C zeta (aPKC£) in the embryo outer cells, even more preferably wherein the apical polarisation is measured as an increase in nuclear Yes-associated protein (YAP)/transcriptional coactivator with PDZ- binding motif (TAZ) expression, in embryo outer cells, following culturing in a medium supplemented with the cell membrane-permeable form of alpha-ketoglutarate
  • the step of measuring an improvement in trophectoderm quality comprises the steps of: determining the expression of pluripotency expression markers and/or trophectoderm lineage markers of said embryo; determining the expression of early epiblast markers, primitive streak markers, and/or extraembryonic mesoderm markers of said embryo; and/or determining the expression of apical polarization markers of the outer cells of said embryo, preferably wherein said apical polarization levels markers are atypical protein kinase C zeta (aPKC£) and/or Yes- associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ).
  • aPKC£ atypical protein kinase C zeta
  • YAP Yes- associated protein
  • TEZ transcriptional coactivator with PDZ-binding motif
  • the skilled person will know that in IVF procedures, several embryos are typically produced and embryos displaying the highest quality may be selected or sorted for implantation in the uterus of the recipient i.e. the receiving individual.
  • the step of sorting of the methods and in vitro methods of the present invention thus corresponds to a step of selecting said embryos displaying improved trophectoderm quality for implantation.
  • the step of measuring an improvement in trophectoderm quality comprises the steps of: determining the expression of pluripotency expression markers and/or trophectoderm lineage markers of said embryo, preferably of the outer cells of said embryo, prior to- and after initiation of the step of culturing in a medium supplemented with the cell membrane-permeable form of alpha-ketoglutarate; comparing the expression of said markers prior to- and after the initiation of the step of culturing in a medium supplemented with the cell membrane-permeable form of alpha-ketoglutarate, wherein a downregulation of the expression of pluripotency expression markers and/or an upregulation trophectoderm lineage markers following initiation of the step of culturing in a medium supplemented with the cell membrane-permeable form of alpha-ketoglutarate indicate improved embryo trophectoderm quality; and, optionally sorting embryos displaying downregulation of
  • the step of measuring an improvement in trophectoderm quality further comprises the steps of: determining the expression of early epiblast markers, primitive streak markers, and/or extraembryonic mesoderm markers of said embryo, preferably of the outer cells of said embryo, prior to- and after initiation of the step of culturing in a medium supplemented with the cell membrane-permeable form of alpha-ketoglutarate; comparing the expression of said markers prior to- and after initiation of the step of culturing in a medium supplemented with the cell membrane- permeable form of alpha-ketoglutarate, wherein an upregulation of the expression of early epiblast markers, downregulation of primitive streak markers, and/or downregulation of extraembryonic mesoderm markers indicate improved embryo trophectoderm quality; and, optionally sorting embryos displaying upregulation of the expression of early epiblast markers, downregulation of primitive streak markers, and/or downregulation of extraembryonic
  • the step of measuring an improvement in trophectoderm quality further comprises the steps of: determining the expression of mural TE markers (mTE) and/or mature polar TE (pTE) markers of said embryo, prior to- and after initiation of the step of culturing in a medium supplemented with the cell membrane- permeable form of alpha-ketoglutarate; comparing the expression of said markers prior to- and after initiation of the step of culturing in a medium supplemented with the cell membrane- permeable form of alpha-ketoglutarate, wherein an upregulation of the pTE markers and/or a downregulation of mTE markers, indicate improved embryo trophectoderm quality; and, optionally sorting embryos displaying upregulation of the expression of pTE markers and/or downregulation of mTE markers following initiation of the step of culturing in a medium supplemented with the cell membrane-permeable form of alpha-keto
  • mTE mural TE markers
  • pTE mature polar
  • the pluripotency expression markers are selected from the group consisting of: DPPA5, NANOG, OCT4 and KLF17, preferably the pluripotency expression markers are selected from the group consisting of: DPPA5, NANOG and OCT4, even more preferably the pluripotency expression markers are selected from the group consisting of NANOG and OCT4.
  • the trophectoderm lineage markers are selected from the group consisting of: GATA3, CDX2 and NR2F2, preferably the trophectoderm lineage markers are selected from the group consisting of: GATA3 and CDX2.
  • the early epiblast markers are selected from the group consisting of: KLF3, PRAP1, and GDF15.
  • the primitive streak marker is TBXT.
  • the extraembryonic mesoderm marker is VIM.
  • the mural TE markers are selected from the group consisting of: ALPG, CITED4, and TLIBB4A.
  • the mature polar TE (pTE) markers are selected from the group consisting of: NR2F2, CCKBR, and PTN.
  • the determination of the expression of the marker(s) is performed by quantitative reverse-transcription polymerase chain reaction (RT-qPCR), single-cell RNA sequencing (scRNA seq) or immunofluorescence (IF).
  • RT-qPCR quantitative reverse-transcription polymerase chain reaction
  • scRNA seq single-cell RNA sequencing
  • IF immunofluorescence
  • NANOG expression is maintained in the embryo inner cells, following supplementation with the cell membrane-permeable form of alphaketoglutarate.
  • FOS like 1, AP-1 transcription factor subunit (FOSL1) expression is upregulated, in the embryo, following supplementation with the cell membrane- permeable form of alpha-ketoglutarate.
  • the embryo size such as the polarized embryo size
  • said size is measured at blastocyst stage (day 5 post fertilization). Said measurement may also be done earlier, or later such as up to 6 days post fertilization.
  • a preferred physiological endpoint for evaluating embryo quality such as trophectoderm quality, and developmental potential, is the Gardner scale, as detailed hereafter.
  • aggregate patterning refers to the arrangement and distribution of cells within a cellular aggregate, such as a blastoid or an embryo, which is important for establishing functional structures and proper cellular differentiation during early embryo development.
  • improved trophectoderm quality is measured as an improvement of embryo patterning.
  • the process of embryo patterning includes, but is not limited to, apical polarization, the correct differentiation of embryonic cells into mature polar trophectoderm (pTE) and mural trophectoderm (mTE), and is often supported by an overall larger size, which reflects more effective cellular organization and patterning within the aggregate.
  • pTE polar trophectoderm
  • mTE mural trophectoderm
  • improved trophectoderm quality is measured as an improvement of trophectoderm maturation, such as an upregulation of markers of mature polar TE (pTE: NR2F2, CCKBR, PTN), and/or a downregulation of mural TE markers (mTE: ALPG, CITED4, TLIBB4A) following culturing in a medium supplemented with the cell membrane-permeable form of alpha-ketoglutarate.
  • pTE NR2F2, CCKBR, PTN
  • mTE ALPG, CITED4, TLIBB4A
  • the Gardner scoring or Gardner scale comprising morphological assessment of the embryo to provide a score made of an expansion grading, and inner cell mass grading and a trophectoderm quality grading, is typically used for the assessment of embryo quality, in IVF procedures.
  • Gardner scoring is typically done at blastocyst stage (day 5 post fertilization). Gardner scoring may also be done earlier, or up to 6 days post fertilization.
  • the embryo/blastocyst is given an expansion grade from 1 to 6, wherein grade 1 is the poorest and grade 6 is the highest quality.
  • ICM inner cell mass
  • the embryo/blastocyst is given a trophectoderm (TE) grade from C to A, wherein C is the poorest and A is the highest quality
  • TE trophectoderm
  • the resulting Gardner score of the embryo/blastocyst is typically expressed in the format “Expansion grade/ICM grade/TE grade”, i.e. 6CA for an embryo having the highest expansion grade, poorest ICM grade and highest TE grade.
  • improved trophectoderm quality is measured as an improved grade of the embryo according to Gardner scale, preferably an improved expansion grade, inner cell mass grade and/or trophectoderm quality grade, even more preferably an improved expansion grade and/or a trophectoderm grade, following culturing in a medium supplemented with the cell membrane-permeable form of alpha-ketoglutarate.
  • the step of measuring an improvement in trophectoderm quality comprises the step of determining the grade of the embryo according to Gardner scale, preferably wherein said grading comprises or consists of determining the expansion grade and/or a trophectoderm grade according to Gardner scale.
  • improved trophectoderm quality is measured as an increase in the range of 5-15%, such as 10% of the number of embryos that reach a Gardner score of 3BB, compared to age matched controls.
  • improved trophectoderm quality is measured as a grade of the embryo according to Gardner scale of at least 3BB , such as at least 3AB, such as at least 3BA, such as at least 3AA, such as at least 4BB, such as at least 4BA, such as at least 4AB, such as at least 4AA, such as at least 5BB, such as at least 5BA, such as at least 5AB, such as at least 5AA, such as at least 6BB, such as at least 6AB, such as at least 6BA, such as at least 6AA, and/or a trophectoderm quality grade of at least B, such as at least A, following culturing in a medium supplemented with the cell membrane-permeable form of alpha-ketoglutarate, preferably wherein the grade of the embryo is measured at in the range of day 5 to day 6 post-fertilization.
  • improved trophectoderm quality is measured as a grade of the embryo according to Gardner scale of at least 3BB at 5 days after fertilization. In other preferred embodiments, of the uses, the methods, or the in vitro methods of the present invention, improved trophectoderm quality is measured as a grade of the embryo according to Gardner scale of at least 4BB at 6 days after fertilization
  • the step of measuring an improvement in trophectoderm quality comprises the steps of: Performing imaging of said embryo after initiation of the step of culturing in a medium supplemented with the cell membrane-permeable form of alpha-ketoglutarate;
  • Determining the grade of the embryo according to Gardner scale preferably wherein said grading comprises or consists of determining the expansion grade and/or trophectoderm grade according to Gardner scale; and,
  • ⁇ a trophectoderm grade of at least C, such as at least B, such as at least A, following initiation of the step of culturing in a medium supplemented with the cell membrane- permeable form of alpha-ketoglutarate.
  • the step of measuring an improvement in trophectoderm quality comprises the steps of: Performing imaging of said embryo after initiation of the step of culturing in a medium supplemented with the cell membrane-permeable form of alpha-ketoglutarate;
  • Determining the grade of the embryo according to Gardner scale preferably wherein said grading comprises or consists of determining the expansion grade and/or trophectoderm grade according to Gardner scale; and,
  • At least 3BB such as at least 3AB, such as at least 3BA, such as at least 3AA, such as at least 4BB, such as at least 4BA, such as at least 4AB, such as at least 4AA, such as at least 5BB, such as at least
  • 5BA such as at least 5AB, such as at least 5AA, such as at least
  • 6BB such as at least 6AB, such as at least 6BA, such as at least
  • improved trophectoderm quality is measured as a grade of the embryo according to Gardner scale of at least 3BB at 5 days after fertilization
  • the step of sorting embryos comprises sorting embryos having a grade according to Gardner scale of at least 3BB 5 days after fertilization.
  • improved trophectoderm quality is measured as a grade of the embryo according to Gardner scale of at least 4BB at 6 days after fertilization
  • the step of sorting embryos comprises sorting embryos having a grade according to Gardner scale of at least 4BB 6 days after fertilization.
  • the imaging is live imaging. This may be for example the case for Gardner scoring of the embryo.
  • PGT Preimplantation Genetic Testing
  • PGT-A Preimplantation Genetic Testing for Aneuploidy
  • PGT-M monogenic disorders
  • PGT-SR Preimplantation Genetic Testing for Structural Rearrangements
  • PGT-SR Preimplantation Genetic Testing for Structural Rearrangements
  • PGT-A and PGT-SR are in particular associated with abnormal karyotypes.
  • Some genetic disorders identified by PGT-M may also be associated with abnormal karyotypes.
  • said methods may further comprise a step of PGT, on the embryo, prior to implantation, preferably at the blastocyst stage.
  • improved trophectoderm quality is measured among embryos presenting a normal karyotype.
  • IVF success rate and implantation rates are dependent on the age of individuals undergoing the IVF procedure, with success and implantation rates typically decreasing with age.
  • improved trophectoderm quality is compared to trophectoderm quality obtained in age-matched controls, such as controls age-matched with the ovum donor.
  • the attachment phase the first step of embryo implantation in the uterus, wherein the embryo makes contact with the uterine lining (endometrium) and starts to adhere is referred to as the attachment phase.
  • improved trophectoderm quality is measured as an improved embryo attachment rate following culturing in a medium supplemented with the cell membrane- permeable form of alpha-ketoglutarate.
  • the step of measuring an improvement in trophectoderm quality comprises the step of determining embryo attachment rate.
  • improved trophectoderm quality is measured as an embryo attachment rate of at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 100% following culturing in a medium supplemented with the cell membrane-permeable form of alpha-ketoglutarate.
  • the present disclosure supports an effect of dm-aKG treatment on the increase in the number of HLA-G+ extravillous trophoblast cells in the embryos purposed for implantation, for instance as shown in Example 7.
  • HLA-G+ is a marker of the trophoblast lineage that differentiates after attachment/implantation.
  • HLA-G+ expression may be measured to assess the ability of said embryos to implant, wherein HLA-G+ expression would be positively correlated with the ability of the embryo to implant. This may be possible for example, but not limited to, in experimental setups wherein the embryo is attached on an extracellular matrix or endometrial scaffold.
  • the present disclosure supports an effect of dm-aKG in improving the implantation success are of the embryo, such as the blastocyst stage embryo.
  • Implantation success rate can be measured clinically, non- invasively by methods known in the art, such as, but not limited to, ultrasound.
  • improved trophectoderm quality is measured as an improved embryo implantation success rate following culturing in a medium supplemented with the cell membrane-permeable form of alpha-ketoglutarate.
  • the step of measuring an improvement in trophectoderm quality comprises the step of determining embryo implantation success rate.
  • improved trophectoderm quality is measured as an embryo implantation success rate of at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 100% following culturing in a medium supplemented with the cell membrane-permeable form of alphaketoglutarate.
  • implantation success rate can be measured clinically, non-invasively by methods known in the art, such as ultrasound.
  • Implantation success rates of IVF procedures are very dependent on the species of the embryo used and the receiving organism. Implantation success rates will typically be very high in for instance mice (90% and above) whereas much lower in humans (30-50% in receiving individuals under 35 year old).
  • aged-matched controls such as aged- matched ovum donor and/or embryo recipient in IVF procedures, may be used for comparison purposes.
  • This may be performed at the zygote or pronuclear stage embryo, for example, but not limited to, to assess pronuclei morphology, or presence of a cytoplasmic halo.
  • the quality may also be assessed at cleavage stage (approximately 3 days after fertilization) to assess for example, but not limited to, cell (blastomere) number and/or regularity.
  • the assessment of quality may also be performed at blastocyst stage embryos (approximately 4 to 5 days after fertilization), where, typically, inner cell mass (ICM), trophoectoderm (TE) and/or the fluid cavity morphology is assessed.
  • ICM inner cell mass
  • TE trophoectoderm
  • control condition may be a condition in which an embryo or ovum is cultured in a medium without the additive to be tested, for example, a cell-membrane permeable form of alpha-ketoglutarate, for example by comparing the expression or grade before said additive is added to the ovum or embryo culture medium, and after said additive has been added to the ovum or embryo culture medium.
  • control condition and test condition may also be compared in parallel, for example by culturing an ovum or embryo in a medium comprising said additive to be tested, and another ovum or embryo in a medium without said additive to be tested. In any case, the media and conditions are otherwise kept similar.
  • the determination of the expression of markers, or the determination of a grade, associated with embryo quality, preferably associated with embryo trophectoderm quality is typically performed without control condition, at one or more timepoints after fertilization, such as 0 to 6 days after fertilization.
  • Embryo quality, preferably embryo trophectoderm quality may in such cases be assessed by standards, such as Gardner scoring, or in comparison to threshold values of marker expression associated with embryo trophectoderm quality.
  • the step of determining the expression and/or the grade is performed before, during, and/or after the step of culturing in a medium supplemented with the cell membrane-permeable form of alpha-ketoglutarate, preferably the step of determining the expression and/or the grade is performed before and/or in the range of 0 to 6 days, such as 1 day, such as 2 days, such as 3 days, such as 4 days, such as 5 days, after initiating the step of culturing in a medium supplemented with the cell membrane- permeable form of alpha-ketoglutarate.
  • the step of performing imaging and/or the step of determining the expression and/or the grade is performed in the range of 0 to 6 days after fertilization, such as 1 day after fertilization, such as 2 days after fertilization, such as 3 days after fertilization, such as 4 days after fertilization, such as 5 days after fertilization.
  • the embryos of the uses and methods of the present invention may be as described in this section.
  • the embryo is a non-human mammalian embryo.
  • the embryo is not a murine embryo, in particular not a mouse embryo and not a rat embryo.
  • the non-human mammal embryo is selected from the group consisting of: a bovine embryo, an equine embryo, a porcine embryo, a canine embryo, a feline embryo, an ovine embryo, a caprine embryo, a cervidae embryo.
  • the embryo is a human embryo.
  • the human embryo is a human embryo obtained by an in vitro fertilization procedure of an egg (ovum) by a sperm cell.
  • IVF/ART media suitable for culturing pre-implantation embryos, in particular for culturing human pre-implantation embryos, may be used as IVF media to which the cell membrane-permeable form of alphaketoglutarate is used as supplement or additive in the uses and methods of the present invention.
  • Said media may for instance be sequential culture media, used stepwise during the IVF procedure to mimic the changes of the uterine tract during embryo development, or single-step cell culture media, where the same medium is used from fertilization to embryo transfer.
  • sequential culture media it is preferred that at least the first medium used after fertilization is supplemented with the cell membrane-permeable form of alpha-ketoglutarate.
  • media used in IVF procedures may also be supplemented by the cell membrane-permeable form of alpha-ketoglutarate in the uses and methods of the present invention.
  • Such media include, but are not limited to media used for the fertilization step, such as ICSI media, cleavage media, and blastocyst media.
  • Other examples of media include media used for cryopreservation for storing embryos, eggs, or sperm, or sperm washing media used during IVF procedures.
  • the IVF medium further comprises one or more components selected from the group consisting of : salts, buffer, energy substrate, non-essential amino acids, essential amino acids, chelators, antibiotic and water.
  • the IVF medium further comprises one or more macromolecules, preferably said macromolecules are selected from the group consisting of: polymers, lipids, vitamins and proteins.
  • Such polymers may include, but are not limited to Polyvinylpyrrolidone (PVP), and/or polysaccharide polymers such as, but not limited to Hyaluronic acid (HA).
  • PVP Polyvinylpyrrolidone
  • HA Hyaluronic acid
  • IVF medium pH can be controlled using pH buffers, such as HEPES or MOPS, or inside cell culture incubators by controlling the percentage of CO2 in the incubator atmosphere to set the desired medium pH by equilibrium with bicarbonate (HCO3-) ions present in the cell culture medium.
  • pH buffers such as HEPES or MOPS
  • HCO3- bicarbonate
  • the IVF medium further comprises a pH indicator.
  • Example 1 Dm-a-ketoglutarate treatment ameliorates 2D trophoblast stem cell (TSC) induction from human naive embryonic stem cells (nESC).
  • TSC trophoblast stem cell
  • nESC human naive embryonic stem cells
  • nESC were depleted from the feeder layer by incubating the single cell suspension on 0.1% gelatin for 45-75 min.
  • Naive hESC were then seeded at a density of 1.8 x10 3 cells per cm 2 in a 6-well plate pre-coated with ECMatrixTM-511 Silk E8 Laminin Substrate (Merck Sigma Aldrich, CC161) in tt2iLG6 medium.
  • Naive hESC were cultured on gelatin-coated plates including a feeder layer of gammairradiated DR4 mouse embryonic fibroblasts (MEFs) (ATCC, SCRC-1045) in tt2iLG6 medium or PXGL medium.
  • tt2iLG6 medium consists of N2B27 basal medium supplemented with PD0325901 (1 pM, Axon Medchem, Axon 1408), CHIR99021 (0.3 pM, Axon Medchem, Axon1386), Go 6983 (2 pM, Axon Medchem, Axon 2466) and human leukemia inhibitory factor (hLIF, 10 ng/mL, Qkine, Qk036) 3 .
  • PXGL medium consists of N2B27 basal medium supplemented with PD0325901 (1 pM, Axon Medchem, Axon 1408), XAV939 (2 pM, Merck Sigma Aldrich, X3004), Go 6983 (2 pM, Axon Medchem, Axon 2466) and hLIF (10 ng/mL, Qkine, Qk036) 31 .
  • N2B27 basal medium contains DMEM/F12 with Glutamax (50%, Thermo Fisher Scientific, 31331028), neurobasal medium (50%, Fisher Scientific, 11570556), N2 supplement (1x, Merck Sigma Aldrich, SCM012), B27 supplement (0.5x, Thermo Fisher Scientific, 17504044), 2-mercaptoethanol (0.2 mM, Thermo Fisher Scientific, 31350010), and GlutaMAXTM (0.75 mM, Thermo Fisher Scientific, 25030024).
  • Cells were cultured in hypoxic conditions (5% CO2, 5% O2) and passaged as single cells every three to four days. Cells were cultured for 24 hours with Y27632 (10 pM, Axon Medchem, Axon 1683) after passaging. Medium was changed daily.
  • Ct comparative cycle threshold
  • cells were cultured in Ibidi microwell plates and fixed with 2% paraformaldehyde for 10 min at room temperature. Post fixation, paraformaldehyde solution was removed, and the samples were washed at least three times with 1X PBS. The samples were then permeabilised for 20 min using 0.2% Triton X-100 and afterwards blocked using blocking buffer containing 1% BSA, 0.1% Tween20 and 10% normal donkey serum in 1X PBS for at least 3 hours. The samples were then incubated overnight at 4 °C with primary antibodies diluted in blocking buffer. The next day, samples were washed with 1X PBS containing 0.1% Tween20 (PBST) at least three times for 10 min each.
  • PBST 0.1% Tween20
  • blastoid and aggregate IF the same protocol was followed with minor modifications. Namely, blastoid samples were fixed with 4% paraformaldehyde for 20 min at room temperature. Subsequently, the samples were washed for 3 times 10 minutes with PBST supplemented with BSA (0.3%). For image quantification of aggregates, sum projection of 5 consecutive confocal z-stacks was used. These stacks were 3 pm apart and at a central location of the aggregate. Nuclei were segmented in ilastik and curated in Imaged using the DAPI signal. aPKC£ signal was used to identify the whole ROI of each aggregate.
  • aPKC£ polarity quantification the area between the end of outer nuclei and the cell membrane was quantified and assigned as the apical signal. For each aggregate the average apical intensity was divided by the remaining non-nuclear signal. For YAP/TAZ quantifications signal was measured for each nucleus and they were categorised based on their location either within the most outer-layer or inner nuclei. Shown is average IF intensity normalised to the outer cell signal.
  • RNA data was normalized with default LogNormalize method, and HTO assay was normalized with centered log-ratio (CLR) transformation.
  • Seurat's HTODemux function was used to assign single cells back to their sample origins, resulting in 7001 cells classified as singlets.
  • cells with more than 15% mitochondrial counts or less than 7000 UMIs were removed, retaining the following number of cells per sample: 1158 nESC, 900 nESC+dm-aKG, 1577 hiTSC, 1711 hiTSC+dm-aKG.
  • the human embryo reference was established through the integration of previously published datasets, encompassing 5 human embryonic datasets spanning from early-stage in vitro cultured human blastocysts (Meistermann et al., 2021 ; Petropoulos et al., 2016; Yanagida et al., 2021) to 3D in vitro cultured human blastocysts (Xiang et al., 2020), and up to CS7 human gastrula (Tyser et al.,
  • final labels were assigned as follows: If >50% of labels for one cell agree, this is used as the final label; if two labels are exactly 50% each, both are used (e.g. “E6/E7”); all other cases are considered “ambiguous”; if a cell is not included in any neighbourhood, it is "unassigned”.
  • Dm-a-ketoglutarate treatment leads to an increase of GATA3+ TSC-like cells ( Figures 1-2, 6). Dm-a-ketoglutarate treatment leads to a decrease of remaining NANOG+ pluripotent cells during human TSC induction ( Figures 1-3, 6).
  • Dm-a-ketoglutarate treatment leads to a decrease in pluripotency markers in nESC.
  • Dm-a-ketoglutarate treatment leads to a reduction of off-target cell types such as TBXT+ primitive streak-like cells and VIM+ extra-embryonic mesoderm-like cells.
  • nESC cultured in PXGL were collected by incubation for 3 min with Accutase (Merck Sigma Aldrich, A6964) and were depleted from the feeder layer by incubation on 0.1% gelatin for at least 60 min.
  • Single cells were plated at a density of 80-85 cells per micro-well of a 24-well AggreWell TM 400 plate in N2B27 supplemented with Y27632 (10 pM, Axon Medchem, Axon1683) to aggregate for 16h.
  • half medium was changed with 2x PALLY consisting of N2B27 supplemented with PD0325901 (1 pM, Axon Medchem, Axon 1408), A83-01 (1 pM, Axon MedChem, Axon 1421), hLIF (10 ng/mL, Qkine, Qk036), oleoyl-L-a-lysophosphatidic acid sodium salt (LPA, 1 pM, Merck Sigma Aldrich, L7260) and Y27632 (10 pM, Axon Medchem, Axon1683). 24 hours later, half medium was changed with 1x PALLY.
  • blastoids were maintained for 56 hours in LY medium consisting of N2B27 supplemented with LPA (1 pM, Merck Sigma Aldrich, L7260) and Y27632 (10 pM, Axon Medchem, Axon1683).
  • LPA 1 pM, Merck Sigma Aldrich, L7260
  • Y27632 10 pM, Axon Medchem, Axon1683
  • Treatment of blastoid induction with dm-aKG was performed by directly diluting dimethyl 2-oxoglutarate (4mM, Merck Sigma Aldrich, 349631) in the culture medium. The medium was then equilibrated for 2-3 hours at 37°C in hypoxic conditions (5% CO2, 5% O2) before use.
  • Dm-a-ketoglutarate treatment leads to enhanced apical localization of polarity markers aPKC£ and YAP/TAZ ( Figures 7-8).
  • Example 2 The set-up for this planned experiment will be the same as in Example 2 (see Figure 3).
  • the experiment will provide the inventors with results on the transcriptional fidelity of blastoids treated with dm-a-ketoglutarate compared to the bona fide human blastocyst. Furthermore, the inventors will obtain information about the effect of enhanced polarization on the cell states of the 40h aggregates.
  • the inventors collaborate with an IVF clinic to uncover the effect of dm-a-ketoglutarate supplementation on human embryo development.
  • the human embryos will be subjected to live imaging, which is standard practice in the IVF clinic.
  • live imaging is standard practice in the IVF clinic.
  • the embryos will be collected for RNA sequencing to determine any potential changes in the expression of trophectodermal and epiblast marker genes.
  • the live cell imaging will also be used to assign any improvements in the Gardner scale of the embryos.
  • Example 5 dm-aKG metabolic treatment aids in establishment of TE cells
  • scRNAseq was performed as described in Example 1. To further verify if metabolic treatment aids in the establishment of TE-like cells, the inventors have mapped the scRNAseq results of nESc treated or untreated for 24h with 4mM dm-aKG and day 3 hiTSC induced from these 2 conditions. The scRNAseq results were mapped onto a stable reference LIMAP of human early development (Fig. 11 A)
  • nESCs both control and dm-aKG-treated cells were predominantly related to preimplantation early epiblast (82.6% and 87.6% respectively) (Fig. 11C).
  • dm-aKG treatment resulted in higher percentage of cells relating to E6 developmental stage or earlier (43.7% vs 22.6% in controls) (Fig. 11 D).
  • An earlier developmental phenotype in nESCs treated with dm-aKG was also confirmed by pseudotime trajectory analysis (Fig. 11 E,F,G). This result is in line with reduced expression of pluripotency genes, which in vivo still increase while the epiblast matures from E6 to E7-8.
  • scRNA-seq To assess transcriptional changes induced by dm-aKG, the inventors performed scRNA-seq on 40-hour aggregates. Cells originating from dm-aKG treatment established a unique transcriptional signature and were particularly enriched within clusters 2, 4 and 6 (Fig. 12A). Further analysis revealed a significant overlap between genes differentially expressed upon dm-aKG in aggregates and in the nESC context (Fig. 12B).
  • the inventors have performed scRNA-seq on d5 blastoids induced from the 40h 4mM dm-aKG treatment. This revealed two clearly separated populations of cells on LIMAP (Fig. 13B) where cells from control and dm- aKG treatment samples were largely intermingled (Data no shown). Based on marker gene expression the inventors identified the larger population as EPI-like cells while the smaller cluster was assigned as the TE-like cells (Fig. 13C).
  • dm-aKG-treated TE-like cells upregulated markers of mature polar TE (pTE: NR2F2, CCKBR, PTN), while downregulating mural TE markers (mTE: ALPG, CITED4, TUBB4A)(Fig. 13E).
  • pTE NR2F2, CCKBR, PTN
  • mTE ALPG, CITED4, TUBB4A
  • Example 7 Improved functionality of blastoids (nESC aggregates) treated with dm-aKG
  • blastoids treated with OmM or 4mM dm-aKG for 40h were selected with a P10 pipette and transferred either on 4-well IbiTreat p-plates (Ibidi, 80426) in IVC1 medium or on an extra-cellular matrix (ECM) mix in 8-well IbiTreat p-plates (Ibidi, 80826) in post-implantation medium.
  • IVC attachments were performed according to Shahbazi et al. with minor modifications (Shahbazi et al., 2016). In short, blastoids were cultured directly in IVC1 medium for 24h before assessing attachment.
  • IVC1 medium consists of advanced DMEM/F12 (Thermo Fisher Scientific, 11540446) supplemented with HI-FBS (20%, Thermo Fisher Scientific, 12389782), GlutaMAXTM (2 mM, Thermo Fisher Scientific, 25030024), ITS-X (1x, Gibco, 51500), - - estradiol (8 nM, Merck Sigma Aldrich, E2758), progesterone (200 ng/mL, Merck Sigma Aldrich, P0130) and N-acetyl-L-cysteine (25 pM, Merck Sigma Aldrich, A7250).
  • Attached structures were collected 4 days post attachment.
  • the attachments on the ECM mix were performed according to Karvas et al. with guided modifications (Karvas et al., 2023). Briefly, an 8-well IbiTreat p-plate (Ibidi, 80826) was coated with an ECM mix consisting of Cultrex UltimaMatrix (80%, R&D System, BME001-05) and hESC-qualified Matrigel (20%, CorningTM, 11573560) for at least 1h at 37°C. Blastoids were transferred on the solidified ECM mix in post-implantation medium.
  • Post attachment blastoids IF was performed following a similar protocol as blastoid and aggregate IF. Briefly, the samples were fixed with 4% paraformaldehyde for 3 hours at 4°C, while gradually removing ECM. Post fixation, paraformaldehyde solution was removed, and the samples were washed at least three times with 1X PBS. The samples were then permeabilised for 20 min using 0.3% Triton X-100 with 0.001% PVA (Merck Sigma Aldrich, 363170) and afterwards blocked using blocking buffer containing 0.001% PVA, 1% BSA, 0.1% Tween20 and 10% normal donkey serum in 1X PBS for at least 5 hours. The samples were then incubated for 48h at 4 °C with primary antibodies diluted in blocking buffer.
  • samples were washed with 1X PBS containing 0.1% Tween20 and 0.001% PVA (PBST) at least three times for 30 min each. After washing, the samples were incubated overnight with secondary antibodies diluted in blocking buffer in the dark at 4 °C. The samples were then washed with PBST three times for 30 min each and afterwards mounted using Vectashield mounting medium (VWR, VECTH-1000).
  • VWR Vectashield mounting medium
  • Fig. 14A ibiTreat microwells
  • Fig. 14B a significantly higher percentage of attached structures contained all three lineages (TE: GATA3; PrE: GATA6; EPI: NANOG) by day 4 of culture (Fig. 14C).
  • pTE mature polar TE
  • SCT syncytiotrophoblast
  • EVT extravillous trophoblast
  • 3D-cultured blastoids model human embryogenesis from pre-implantation to early gastrulation stages.
  • Polycomb repressive complex 2 shields naive human pluripotent cells from trophectoderm differentiation. Nature Cell Biology 2022 24:6, 24(6), 845-857. https://doi.Org/10.1038/S41556-022-00916-w Larsen, E. C., Christiansen, O. B., Kolte, A. M., & Mackion, N. (2013). New insights into mechanisms behind miscarriage. BMC Medicine, 11(1), 1-10. https://doi.Org/10.1186/1741-7015-11-154/FIGURES/2
  • Naive stem cell blastocyst model captures human embryo lineage segregation.
  • Reprogrammed iBIastoids contain amnion-like cells but not trophectoderm. BioRxiv, 2021.05.07.442980. https://doi.org/10.1101/2021.05.07.442980
  • said cell- membrane-permeable form of alpha-ketoglutarate (aKG), or said medium comprising thereof is first contacted with said embryo at the most 96h after embryo fertilization, such as at the most 72h, such as at the most 48h, such as at the most 24h, such as at the most 12h, such as at the most 10h, such as at the most 8h, such as at the most 6h, such as at the most 4h, such as at the most 2h, such as at the most 1 h, such as at the most 30min, such as at the most 10min, such as at the most 5min, such as at the most 1 min, after embryo fertilization.
  • aKG alpha-ketoglutarate
  • a method of culturing a pre-implantation human embryo comprising culturing said embryo in a medium supplemented with a cell membrane-permeable form of alpha-ketoglutarate, and, optionally, measuring an improvement in trophectoderm quality of said embryo.
  • a method of improving human embryo trophectoderm quality comprising culturing a pre-implantation human embryo in a medium supplemented with a cell membrane-permeable form of alpha-ketoglutarate, and, optionally, measuring an improvement in trophectoderm quality of said embryo.
  • a method of human in vitro fertilization which comprises culturing a preimplantation human embryo in a medium supplemented with a cell membrane- permeable form of alpha-ketoglutarate, and, optionally, measuring an improvement in trophectoderm quality of said embryo.
  • the use or the method according to any one of the preceding items, wherein said use or method is in vitro.
  • the use or the method according to any one of the preceding items, wherein said use or method is for obtaining an embryo with improved trophectoderm quality.
  • An in vitro method for obtaining an embryo with improved trophectoderm quality comprising the steps of: providing an embryo; culturing said embryo in a medium supplemented with a cell membrane- permeable form of alpha-ketoglutarate; and optionally, measuring an improvement in embryo trophectoderm quality, thereby obtaining an embryo with improved trophectoderm quality
  • the step of culturing said embryo in a medium supplemented with the cell membrane-permeable form of alpha-ketoglutarate is initiated at the most 96h after fertilization, such as at the most 72h, such as at the most 48h, such as at the most 24h, such as at the most 12h, such as at the most 10h, such as at the most 8h, such as at the most 6h, such as at the most 4h, such as at the most 2h, such as at the most 1h such as at the most 30min, such as at the most 10min, such as at the most 5min, such as at the most 1 min, after fertilization.
  • the most 96h after fertilization such as at the most 72h, such as at the most 48h, such as at the most 24h, such as at the most 12h, such as at the most 10h, such as at the most 8h, such as at the most 6h, such as at the most 4h, such as at the most 2h, such as at the most 1h such as at the
  • step of culturing said embryo in a medium supplemented with the cell membrane-permeable form of alpha-ketoglutarate is performed for in the range of 0 to 6 days, such as for at least 1 h, such as for at least 2h, such as for at least 4h, such as for at least 6h, such as for at least 8h, such as for at least
  • step of culturing said embryo in a medium supplemented with cell membrane-permeable form of alpha-ketoglutarate is performed at the pronuclear stage, zygote stage, 2-cell stage, 4-cell stage, 8-cell stage, morula stage, early blastocyst stage and/or blastocyst stage.
  • the cell membrane-permeable form of alpha- ketoglutarate is selected from the group consisting of: dimethyl-alpha- ketoglutarate (dm-aKG), monomethyl alpha-ketoglutarate (mm-aKG), and octyl alpha ketoglutarate (octyl-aKG). 21.
  • the cell membrane-permeable form of alphaketoglutarate is dimethyl-alpha-ketoglutarate (dm-aKG).
  • said medium comprises the cell membrane- permeable form of alpha-ketoglutarate at a concentration of at the most 10mM, such as at the most 5mM, such as at the most 4mM, such as at the most 3 mM, such as at the most 2mM, such as at the most 1 mM, such as at the most 0.5mM, for instance in the range of 0.1-10mM, for instance in the range of 1- 6mM, for instance in the range of 2-6mM, for instance in the range of 2-4mM, such as 3mM.
  • the step of measuring an improvement in trophectoderm quality comprises the steps of: determining the expression of pluripotency expression markers and/or trophectoderm lineage markers of said embryo, preferably in the outer cells of said embryo; determining the expression of early epiblast markers, primitive streak markers, and/or extraembryonic mesoderm markers of said embryo, preferably in the outer cells of said embryo; and/or determining the expression of apical polarization markers of the outer cells of said embryo, preferably wherein said apical polarization levels markers are atypical protein kinase C zeta (aPKC£) and/or Yes- associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ).
  • aPKC£ atypical protein kinase C zeta
  • YAP Yes- associated protein
  • TEZ transcriptional coactivator with PDZ-binding motif
  • the step of measuring an improvement in trophectoderm quality comprises the steps of: determining the expression of pluripotency expression markers and/or trophectoderm lineage markers of said embryo, preferably in the outer cells of said embryo, prior to- and after initiation of the step of culturing in a medium supplemented with the cell membrane-permeable form of alpha-ketoglutarate; comparing the expression of said markers prior to- and after the initiation of the step of culturing in a medium supplemented with the cell membrane-permeable form of alpha-ketoglutarate, wherein a downregulation of the expression of pluripotency expression markers and/or an upregulation trophectoderm lineage markers following initiation of the step of culturing in a medium supplemented with the cell membrane-permeable form of alpha-ketoglutarate indicate improved embryo trophectoderm quality; and, optionally sorting embryos displaying
  • the step of measuring an improvement in trophectoderm quality further comprises the steps of: determining the expression of early epiblast markers, primitive streak markers, and/or extraembryonic mesoderm markers of said embryo, preferably of the outer cells of said embryo, prior to- and after initiation of the step of culturing in a medium supplemented with the cell membrane-permeable form of alpha-ketoglutarate; comparing the expression of said markers prior to- and after initiation of the step of culturing in a medium supplemented with the cell membrane- permeable form of alpha-ketoglutarate, wherein a downregulation of the expression of early epiblast markers, a downregulation of primitive streak markers, and/or downregulation of extraembryonic mesoderm markers, indicate improved embryo trophectoderm quality; and, optionally sorting embryos displaying downregulation of the expression of early epiblast markers, a downregulation of primitive streak markers, and/or down
  • mTE mural TE markers
  • pTE mature polar TE
  • the pluripotency expression markers are selected from the group consisting of: DPPA5, NANOG, OCT4 and KLF17, preferably wherein the pluripotency expression markers are selected from the group consisting of: DPPA5, NANOG and OCT4, even more preferably wherein the pluripotency expression markers are selected from the group consisting of NANOG and OCT4.
  • mural TE markers are selected from the group consisting of: ALPG, CITED4, and TLIBB4A.
  • pTE mature polar TE
  • trophectoderm quality is measured as an improvement of trophectoderm maturation, such as an upregulation of markers of mature polar TE (pTE: NR2F2, CCKBR, PTN), and/or a downregulation of mural TE markers (mTE: ALPG, CITED4, TLIBB4A) following culturing in a medium supplemented with the cell membrane-permeable form of alpha-ketoglutarate.
  • pTE NR2F2, CCKBR, PTN
  • mTE ALPG, CITED4, TLIBB4A
  • improved trophectoderm quality is measured as an improved grade of the embryo according to Gardner scale, preferably an improved expansion grade, inner cell mass grade and/or trophectoderm quality grade, even more preferably an improved expansion grade and/or a trophectoderm grade, following culturing in a medium supplemented with the cell membrane-permeable form of alpha-ketoglutarate.
  • the method or the in vitro method according to any one of items 8 to 42, wherein the step of measuring an improvement in trophectoderm quality comprises the step of determining the grade of the embryo according to Gardner scale, preferably wherein said grading comprises or consists of determining the expansion grade and/or a trophectoderm grade according to Gardner scale.
  • a grade of the embryo according to Gardner scale of at least 3BB such as at least 3AB, such as at least 3BA, such as at least 3AA, such as at least 4BB, such as at least 4BA, such as at least 4AB, such as at least 4AA, such as at least 5BB, such as at least 5BA, such as at least 5AB, such as at least 5AA, such as at least 6BB, such as at least 6AB, such as at least 6BA, such as at least 6AA, and/or a trophectoderm quality grade of at least B, such as at least A, following culturing in a medium supplemented with the cell membrane-permeable form of alphaketoglutarate, preferably wherein the grade of the embryo is measured at in the range of day 5 to day 6 post-fertilization.
  • the method or the in vitro method according to any one of items 8 to 44, wherein the step of measuring an improvement in trophectoderm quality comprises the steps of:
  • grading comprises or consists of determining the expansion grade and/or trophectoderm grade according to Gardner scale; and, optionally sorting embryos having:
  • 3BB such as at least 3AB, such as at least 3BA, such as at least 3AA, such as at least 4BB, such as at least 4BA, such as at least
  • 4AB such as at least 4AA, such as at least 5BB, such as at least
  • 5BA such as at least 5AB, such as at least 5AA, such as at least
  • 6BB such as at least 6AB, such as at least 6BA, such as at least
  • the imaging is live imaging.
  • step of determining the expression and/or the grade is performed before, during, and/or after the step of culturing in a medium supplemented with the cell membrane-permeable form of alpha-ketoglutarate, preferably wherein the step of determining the expression and/or the grade is performed before and/or in the range of 0 to 6 days after initiating the step of culturing in a medium supplemented with the cell membrane-permeable form of alphaketoglutarate.
  • non-human mammal embryo is selected from the group consisting of: a bovine embryo, an equine embryo, a porcine embryo, a canine embryo, a feline embryo, an ovine embryo, a caprine embryo, a cervidae embryo.
  • the IVF medium further comprises one or more components selected from the group consisting of : salts, buffer, energy substrate, non-essential amino acids, essential amino acids, chelators, antibiotic and water.
  • the IVF medium further comprises one or more macromolecules, preferably wherein said macromolecules are selected from the group consisting of: polymers, lipids, vitamins and proteins.
  • the IVF medium further comprises a pH indicator.

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Abstract

La présente invention concerne le domaine de la médecine reproductive et de la fécondation in vitro. Plus particulièrement, la présente invention concerne l'utilisation d'une forme d'alpha-cétoglutarate perméable à la membrane comme complément à des milieux de fécondation in vitro (FIV) humaine. La présente invention concerne en outre l'utilisation d'une forme d'alpha-cétoglutarate perméable à la membrane pour améliorer la qualité du trophectoderme embryonnaire humain, et l'utilisation d'un milieu complémenté par une forme d'alpha-cétoglutarate perméable à la membrane cellulaire pour améliorer la qualité du trophectoderme embryonnaire, de préférence la qualité du trophectoderme embryonnaire humain. La présente invention concerne également des procédés d'obtention d'embryons présentant une qualité de trophectoderme améliorée, consistant à cultiver lesdits embryons dans un milieu complémenté par une forme d'alpha-cétoglutarate perméable à la membrane.
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