WO2013113658A1 - Milieux - Google Patents

Milieux Download PDF

Info

Publication number
WO2013113658A1
WO2013113658A1 PCT/EP2013/051581 EP2013051581W WO2013113658A1 WO 2013113658 A1 WO2013113658 A1 WO 2013113658A1 EP 2013051581 W EP2013051581 W EP 2013051581W WO 2013113658 A1 WO2013113658 A1 WO 2013113658A1
Authority
WO
WIPO (PCT)
Prior art keywords
csf
culture medium
embryo culture
human embryo
concentration
Prior art date
Application number
PCT/EP2013/051581
Other languages
English (en)
Inventor
George Anthony THOUAS
David Kenneth GARDNER
Original Assignee
Vitrolife Sweden Ab
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from GBGB1201628.3A external-priority patent/GB201201628D0/en
Priority claimed from GBGB1217833.1A external-priority patent/GB201217833D0/en
Application filed by Vitrolife Sweden Ab filed Critical Vitrolife Sweden Ab
Publication of WO2013113658A1 publication Critical patent/WO2013113658A1/fr

Links

Classifications

    • 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
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
    • 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
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
    • 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
    • C12N2501/90—Polysaccharides
    • C12N2501/905—Hyaluronic acid

Definitions

  • the present invention relates generally to mammalian in vitro fertilization (IVF) and to culture media and processes useful for carrying out fertilization and embryo development.
  • IVF mammalian in vitro fertilization
  • the invention provides a culture medium comprising G-CSF that supports the growth and development of embryos to the blastocyst stage and significantly increases the likelihood of successful pregnancy in IVF.
  • In vitro fertilization is a technique used to overcome various forms of male and female infertility.
  • the process involves fertilizing an oocyte with a sperm in vitro and subsequently transferring the developing embryo into the female body.
  • IVF Steptoe, P.C. and Edwards, R.G., Lancet 2(8085): 366 (1978)
  • the process faces continuing challenges of low implantation and pregnancy rates.
  • the reported success rate for IVF was only 8 to 10%. This low success rate was due, at least in part, to the poor quality of the embryo culture media.
  • Simple media are those, such as Earle's and human tubal fluid (HTF), which are balanced salt solutions with added carbohydrate energy sources such as pyruvate, lactate and glucose.
  • Complex media such as Ham's F-10, further include non-essential and essential amino acids as well as other additives, such as vitamins, antibiotics and proteins.
  • IVF culture media are intended to support embryo development up to the 8-cell stage or beyond in a single medium
  • the trend has been to optimize separate culture media to support the developing embryo at different stages of development.
  • This has led to the widespread use of sequential culture medium in IVF.
  • a sequential culture media system may use one culture medium for the growth of an embryo from a one- cell zygote to an eight-cell embryo during the first 48 hours of development and another culture medium to grow the eight-cell embryo to the blastocyst stage.
  • the sequential culture media are designed to more closely mimic the female reproductive tract during in vivo embryo growth.
  • the media compositions typically differ with respect to components such as amino acids and sugars to improve optimization of the media to support the embryonic growth and development.
  • the benefits of sequential culture media also show how specialized embryo culture media should be to improve IVF outcomes.
  • G-CSF granulocyte colony-stimulating factor
  • CSF3 colony-stimulating factor 3
  • G-CSF granulocyte colony-stimulating factor
  • CSF3 colony-stimulating factor 3
  • G-CSF differs from other CSF in terms of size, structure and activity.
  • the cytokines have separate receptors and are coded by different genes. Although cytokine systems appear to provide for redundancy in some aspects, it is not possible to predict the effect of one cytokine or growth factor based on results with another cytokine or growth factor. For a growth factor to be active on a cell, the cell must present a receptor molecule. G-CSF activity on neutrophils is not species specific.
  • G-CSF G-CSF
  • Salmassi et al 2005, Human Reproduction, 20:2434-2440. They found that the G-CSF level in serum varied during the menstrual cycle and the levels differed depending on if pregnancy occurred or not. The increase in G-CSF level pre-oocyte retrieval was dependent on how the female responded to the hormonal stimulation, with the highest levels in those females who responded the strongest, i.e. required the lowest levels of FSH and with more than eleven oocytes. Serum concentration of G-CSF in high responders was about 70 pg/ml.
  • the G-CSF concentration was higher in the follicular fluid (FF) and reached a concentration of more than 100 pg/ml.
  • Sarapik et al 2011, Clinical and Developmental Immunology, 2012: Article ID 606459
  • this study did not find a connection between G-CSF levels and any specific cause of infertility or predictability of G- CSF for IVF outcome.
  • the individual concentration of G-CSF in FF differed substantially between 0 up to almost 5 ng/ml.
  • Dominguez et al (2008, Human Reproduction, 23: 1993-2000), used protein arrays to measure changes in medium concentration of various proteins in medium conditioned with implanted versus non implanted blastocysts.
  • G-CSF was among the analyzed proteins and the medium content did not differ in relation to implanted or non-implanted embryos. This was in contrast to GM-CSF (granulocyte-macrophage colony- stimulating factor), which was decreased in the medium for the implanted blastocysts.
  • GM-CSF granulocyte-macrophage colony- stimulating factor
  • G-CSF is used in culture media for certain cell types, mainly in proliferation and differentiation of haematopoeitic cells.
  • the present invention provides improved culture media for human embryos.
  • the invention further provides methods for culturing mammalian embryos in the culture media.
  • the culture media are characterized by a well-defined G-CSF concentration range, of O.lng/ml to lOOng/ml. This corresponds to, or is near to, the range previously measured in human follicular fluid, i.e. is physiological or near physiological conditions.
  • range is important to reduce the risk of inducing subsequent effects of growth factors that could create problems at any time later in life.
  • protein has a synergistic effect with G-CSF making the defined concentrations acceptable to achieve the desired effect on embryo development.
  • the human culture medium includes water, inorganic salts, at least one energy source, one or more macro molecules and G-CSF at a concentration of 0.1 ng/ml to 100 ng/ml.
  • the invention can also be seen as the use of G-CSF in physiological or near physiological conditions in an embryo culture that includes water, inorganic salts, at least one energy source and one or more macromolecules.
  • the G-CSF concentration can be from 1 ng/ml to 100 ng/ml and preferably the G-CSF concentration is 1 ng/ml to 10 ng/ml.
  • the medium further includes one or more amino acids.
  • the effect of G-CSF is synergistically improved with addition of a protein.
  • the protein can preferentially be human serum albumin or its recombinant counterparts.
  • the culture media may optionally include other ingredients, such as, but not limited to, growth factors, other cytokines, hormones, vitamins, antioxidants and antibiotics.
  • the culture media may be tailored to support the growth of human embryos from a first developmental stage to a second developmental stage.
  • a culture medium may be designed to support a mammalian embryo up to the 4-cell stage, up to the 8-cell stage or up to the blastocyst stage.
  • the various culture media encompassed by the present invention may include different components at varying concentrations.
  • the present invention additionally provides a method of culturing human embryos, the method comprising culturing embryos in the presence of a medium comprising G-CSF.
  • the method comprises culturing embryos in the presence of a medium comprising G-CSF at a concentration of 0.1 to 100 ng/ml or 1 to 100 ng/ml or preferably 1 to 10 ng/ml.
  • the medium may further comprise a macromolecule which acts synergistically with G-CSF.
  • the macromolecule can be a protein, preferably serum albumin, more preferably human serum albumin.
  • FIG. 1 presents data showing blastocyst development of mouse embryos in vitro at different concentrations of G-CSF with and without protein (recombinant Human Serum Albumin (rHSA)).
  • rHSA Human Serum Albumin
  • FIG. 2 presents data showing cell numbers in the inner cell mass (ICM), trophectoderm (TE) and combined ICM and TE of blastocysts cultured with 100 ng/ml of G- CSF versus controls.
  • ICM inner cell mass
  • TE trophectoderm
  • FIG. 3 Shows differentially stained embryos for ICM and TE of embryos cultured with and without addition of G-CSF.
  • FIG. 4 Development of individually cultured Swiss outbred embryos to Day 4 at a narrower dose range of G-CSF in GlTM/G2TM media. With Recombumin® in the media (5mg/ml), blastocyst rate is approximately double with as little as lng/ml of the factor.
  • FIG. 6 shows percentages of mouse blastocysts that were resorbed and that reached fetus stage at day 15.5 of gestation following day 4 transfer to day 4 recipient females.
  • FIG. 7a shows mean fetal and placenta weights of mouse fetuses at day 15.5 of gestation and FIG. 7b shows mean fetal crown-rump length of mouse fetuses at day 15.5 of gestation.
  • the present invention provides IVF culture media compositions comprising G-CSF in concentrations that support the development of gametes, zygotes and/or embryos in vitro prior to implantation. More specifically, the present IVF culture media comprise G-CSF at a concentration of 0.1 to 100 ng/ml. Preferably, the G-CSF is present at a concentration of 1 to 100 ng/ml; more preferably 1 to 10 ng/ml.
  • the culture media and uses thereof described herein have significant noncommercial and non-industrial uses (e.g., research) in addition to (or as an alternative to) commercial and industrial uses.
  • This invention is not intended to support commercial uses of human embryos which are prohibited by law.
  • culture medium is used throughout the present specification to refer to aqueous media containing salts and carbohydrates prepared to a defined osmolarity and pH which is used for in vitro culture of human embryos such as, gametes, zygotes, cleavage stage embryos, morulas and blastocysts.
  • Embryo culture media can be prepared in the laboratory by any practitioner skilled in the art and are also commercially available.
  • the composition of embryo transfer media is based on embryo culture media although transfer media generally comprises higher concentrations of hyaluronic acid i.e. 0.3 mg/ml to 5 mg/ml and preferably only recombinant proteins.
  • a transfer medium preferably comprises recombinant human albumin as the protein source.
  • the human culture medium must be suitable for culturing human embryos, so must not contain any components which may harm human embryos. In particular, the medium does not generally contain serum.
  • the term "embryo” is used to refer to cells in all stages of development from a fertilized egg up to the first 5 or 6 days or to the expanded or hatched blastocysts stages.
  • the invention is based, at least in part, on the inventors' discovery that culture media comprising G-CSF substantially improves the success rate of IVF procedures compared to culture media that omit G-CSF.
  • culture media comprising G-CSF substantially improves the success rate of IVF procedures compared to culture media that omit G-CSF.
  • protein is also included in the medium there is a synergistic effect between G-CSF and the protein. This synergy means that a much lower concentration of G-CSF provides adequate response.
  • the improvement provided by the present inventions is, when shown as an increase in blastocyst development from B6CBA(F1) mouse zygotes, such that a 100 ng/ml concentration of G-CSF results in 100% blastocyst development in a protein free medium.
  • a 100 ng/ml concentration of G-CSF results in 100% blastocyst development in a protein free medium.
  • 1 ng/ml of G-CSF is required.
  • Even 0.1 ng/ml provides close to 100% blastocyst development in the protein comprising medium.
  • the blastocyst development increased from about 25% in medium with or without protein and without G- CSF to 45% in medium containing protein and 1 ng/ml G-CSF. Without protein in the medium 100 ng/ml G-CSF was required to reach 45% blastocyst development.
  • Improvements provided by the present media may be measured by one or more of several parameters. These include increased survival rates or developmental rates for blastocysts up to the 4- to 6-day stage, blastocysts with a greater number of cells, blastocysts having larger inner cell masses and blastocyst having inner cell masses that make up a greater percent of the total and/or percentage of hatched blastocysts.
  • the improvement in embryo development resulting from the use of the present culture media may be quantified by comparing embryos cultured in an improved medium containing G-CSF at physiological or near physiological levels with embryos cultured in a control medium which differs from the improved medium only in that G-CSF is absent from the control.
  • the present culture media may provide at least a 5%, at least a 10%, or even at least a 15% increase in one or more of the following: (1) percent compacting on day 3; (2) percent total blastocyst development on day 4; (3) percent total blastocyst development on day 5; and (4) percent blastocyst hatching on day 5.
  • the present media may provide blastocysts with a total inner cell number and/or an increase in the percent of the total mass due to the inner cell masses which are at least 10% higher, or even at least 15% higher, than those of blastocysts cultured in the control medium.
  • the culture media of the present invention may be optimized to support embryo development at various stages.
  • the media may be optimized to culture an embryo up to the 4-cell stage, up to the 8-cell stage, from the 4-cell stage to the 8-cell stage, up to the blastocyst stage, or from the 4- or 8- cell stage to the blastocyst stage.
  • the nature and concentration of the essential and non-essential ingredients of the culture media may vary, however, in each culture medium that includes G-CSF, the concentration of G-CSF should be physiological or near physiological and the medium should preferably comprise protein.
  • the G-CSF may be present in one of the sequential media and absent in others.
  • the culture media of the present invention preferably comprises one or more proteins having a synergistic effect with G-CSF.
  • Suitable proteins include globulins, glycoproteins, and serum albumin, particularly human serum albumin. Recombinant serum albumin can be used.
  • the proteins can be added to the media at a concentration of 0.1 mg/ml to 20 mg/ml, preferably 0.5 mg/ml to 15 mg/ml and more preferably lmg/ml to 10 mg/ml.
  • the media may comprise one or more
  • a macromolecule having a synergistic effect with G-CSF.
  • a macromolecule is a term used to contrast a micromolecule, which is smaller in size and molecular weight.
  • Macromolecules usually refer to large biological polymers, such as nucleic acids and proteins, which are made up of small monomers linked together.
  • the macromolecules may be present in combination with one or more proteins having a synergistic effect with G-CSF or may be used as an alternative to the proteins.
  • Suitable macromolecules include, but are not limited to, extracellular matrix components such as heparin sulfate, fibronectin, laminin, collagen, elastin, hyaluronic acid and vitronectin or carbohydrate polymers such as dextran, or cellulose or starch derivatives such as hydroxy ethyl starch, or synthetic polymers such as polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG).
  • the macromolecules can be added to the media at a concentration of 0.01 mg/ml to 20 mg/ml, preferably 0.1 mg/ml to 10 mg/ml.
  • a typical culture medium for culturing embryos can include water, one or more inorganic salts, one or more energy sources, and one or more amino acids (including nonessential and, optionally, essential amino acids), as core ingredients.
  • Typical energy sources in an embryo culture medium include carbohydrate energy sources, such as pyruvate, lactate and glucose.
  • Suitable inorganic salts include, CaCl 2 ⁇ 2H 2 0, CuS0 4 ⁇ 5H 2 0, FeS0 4 ⁇ 7H 2 0, KC1, MgS0 4 , NaCl, NaHC0 3 , Na 2 HP0 4 , ZnS0 4 ⁇ 7H 2 0, and KH 2 P0 4 .
  • Non-essential amino acids include alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine, and tryosine.
  • Essential amino acids include histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine.
  • the culture media can include other additives.
  • vitamins such as biotin, pantothenate, folic acid, niacinamide, pyridoxine, riboflavin, and thiamine
  • growth factors such as penicillin and streptomycin.
  • antibiotics such as penicillin and streptomycin.
  • the antibiotic would be penicillin G or streptomycin sulfate or a combination thereof.
  • Albumin e.g., human serum albumin or recombinant human albumin
  • polyvinylpyrolidone e.g., polyvinylpyrolidone, hyaluronan, chelators, such as EDTA
  • buffers such as HEPES buffer or MOPS buffer
  • miscellaneous ingredients that also may be included in the culture media include, but are not limited to, one or more of choline chloride, hypoxanthine, inositol, thymidine, cyanocobalamine, cysteamine, phenol red, lipoic acid and glutathione.
  • the concentration of the core ingredients and other additives in a culture medium may vary depending upon the stage of embryo development for which the medium is optimized.
  • Typical concentrations for the inorganic salts in the culture media may be about 100 mM to about 150 mM or about 110 mM to about 140 mM.
  • Typical concentrations for the energy sources in the media may be about 5 mM to about 40 mM, about 5 mM to about 30 mM, or about 5 mM to about 15 mM.
  • Typical concentrations for the total amount of amino acids in the culture media may be about 0.1 mM to about 15 mM or about 0.5 mM to about 12 mM, or about 0.5 mM to about 6 mM.
  • concentrations thereof may vary depending on the developmental stage of the cells in the culture media.
  • the vitamins, growth factors, hormones and other miscellaneous ingredients in the culture medium tend to be added at fairly low concentrations, e.g., 1 mM or lower, 0.5 mM or lower, or even 0.1 mM or lower.
  • the present culture media may be formulated by adding G-CSF to existing embryo culture media to provide a G-CSF concentration of 0.1 ng/ml to 100 ng/ml, preferably 1 ng/ml to 10 ng/ ml.
  • the G-CSF is preferably human recombinant G-CSF, however other G- CSF preparations could be used.
  • Human recombinant G-CSF or Filgrastim (a G-CSF analog) preparations are today in clinical use for inducing neutrophil proliferation in cancer patients and to treat severe chronic neutropenia. Filgrastim is today sold under the tradename Neupogen ® .
  • G-CSF can be added to any embryo culture medium a concentration of 0.1- lOOng/ml as long as the culture medium contains protein.
  • G-CSF can be added to commercially-available simple culture media, such as human tubal fluid (HTF),
  • G-CSF may also be added to more complex embryo culture media designed to support early embryo growth.
  • GlTM culture medium available from Vitro life. This medium is designed to support the development of cleavage stage embryos to around the 8-cell stage. The medium contains carbohydrates, amino acids, and chelators to support the early embryo. The complete formulation for the GlTM medium is provided in Table 1.
  • G-CSF may be added to commercially available IVF culture medium capable of supporting embryos to beyond day 3 (8-cell stage). These culture media have been designed to carry embryos to the blastocyst stage prior to implantation.
  • G-CSF may be added to these media to enhance the blastocyst development.
  • One example includes a-modified essential medium ( MEM), described in Desai et ah, Human Reprod 12: 328-335 (1997).
  • a second example includes HECM-6 medium plus pantothenate. McKiernan SH and Bavister BD, Human Reprod 15: 157-164 (2000).
  • Other examples include the G2TM media available from Vitrolife.
  • G2TM is a medium that is designed to support the development of the embryo from around the 8-cell stage (day 3) to the blastocyst stage.
  • the medium (Table 2) contains carbohydrates, amino acids, and vitamins to support the later stage embryo.
  • 8-cell stage embryos are transferred from an embryo culture medium optimized to support early stage growth (i.e., up to the 8-cell stage) supplemented with G-CSF to an embryo culture medium optimized to support later stage growth (i.e., up to the blastocyst stage) G-CSF may or may not be added to the latter media.
  • the present culture media also may be formulated by adding G-CSF to existing gamete (oocyte or sperm) culture or freezing media. Tables 3-5 below provide gamete culture media in which G-CSF may be included to provide for improved cellular
  • Table A Effect of G-CSF on blastocyst formation (%b) and hatching (%hb) in the presence and absence of human serum albumin (HSA).
  • Example 2 ICM and TE cell proliferation with and without G-CSF
  • Flushed B6CBA(F1) mouse zygotes were grown to D3 in groups in GlTM with 5mg/ml regular HSA. Zygotes were then grown in groups from D3-5 with 5mg/ml
  • Recombumin® with 100 ng/ml of human recombinant granulocyte colony- stimulating factor (hrGCSF) compared to controls.
  • the cells in the inner cell mass (ICM) and trophectoderm (TE) were calculated and results showed that when grouped in G2TM containing 100 ng/ml GCSF, compared to GCSF-free controls, the blastocysts showed significantly more inner cell mass cells (29 vs. 22, P ⁇ 0.05) and hence more cells overall (113 vs. 102, PO.001). See Fig 2 and Fig 3 and Table B below.
  • Table B Effect of G-CSF on ICM, TE and Total cell number.
  • G-CSF-containing culture media the effect of G-CSF-containing culture media on human embryo development was examined.
  • Patients expected to have a good prognosis for IVF have half of their embryos cultured in GlTM culture media free of G-CSF (i.e., the control), while the other half are cultured in GlTM medium containing 1 ng/ml G- CSF (i.e., the G-CSF medium of the invention).
  • Example 7 Effect of G-CSF on fetal and placental weight and fetal crown-rump length in mice.
  • Fetuses developed to day 15.5 of gestation in example 6 were examined postmortem for fetal and placental weight (Fig. 7a) and crown-rump length (Fig. 7b). No statistical differences in fetal development between in vivo control and G-CSF treated blastocysts were observed. The fetal weight was significantly higher (P ⁇ 0,05) in the G-CSF group compared to the in vitro control group.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Gynecology & Obstetrics (AREA)
  • Reproductive Health (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne une composition et un procédé pour la fécondation in vitro qui utilise des milieux de culture comprenant G-CSF. Des milieux de culture d'embryons qui comprennent G-CSF sont aptes à permettre un développement amélioré de blastocystes et agissent en synergie avec les protéines dans les milieux. L'effet synergique offre l'opportunité d'utiliser des concentrations physiologiques ou pratiquement physiologiques de G-CSF, tout en permettant une réponse de développement de blastocyste complète.
PCT/EP2013/051581 2012-01-31 2013-01-28 Milieux WO2013113658A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB1201628.3 2012-01-31
GBGB1201628.3A GB201201628D0 (en) 2012-01-31 2012-01-31 Media
GB1217833.1 2012-10-05
GBGB1217833.1A GB201217833D0 (en) 2012-10-05 2012-10-05 Media

Publications (1)

Publication Number Publication Date
WO2013113658A1 true WO2013113658A1 (fr) 2013-08-08

Family

ID=47628154

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2013/051581 WO2013113658A1 (fr) 2012-01-31 2013-01-28 Milieux

Country Status (1)

Country Link
WO (1) WO2013113658A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2898062A1 (fr) * 2012-09-19 2015-07-29 Scarpellini, Fabio Utilisation de g-csf dans une culture d'embryons in vitro
US11639494B2 (en) 2015-08-24 2023-05-02 Vitrolife Sweden Ab Culture medium

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999067364A1 (fr) * 1998-06-19 1999-12-29 Luminis Pty. Ltd. Procede et milieu pour la culture in vitro d'embryons humains
US20040082062A1 (en) * 1998-06-19 2004-04-29 Sarah Robertson Method and medium for in vitro culture of human embryos
WO2005039505A2 (fr) * 2003-10-24 2005-05-06 Nora, Llc Compositions et methodes pour une grossesse saine
US20090226397A1 (en) * 2003-10-24 2009-09-10 Nora Therapeutics, Inc. Compositions and methods for reducing the likelihood of implantation failure or miscarriage in recipients of artificial insemination

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999067364A1 (fr) * 1998-06-19 1999-12-29 Luminis Pty. Ltd. Procede et milieu pour la culture in vitro d'embryons humains
US20040082062A1 (en) * 1998-06-19 2004-04-29 Sarah Robertson Method and medium for in vitro culture of human embryos
WO2005039505A2 (fr) * 2003-10-24 2005-05-06 Nora, Llc Compositions et methodes pour une grossesse saine
US7470662B2 (en) 2003-10-24 2008-12-30 Nora Therapeutics, Inc. Method of reducing the likelihood of implantation failure during an assisted reproduction in a female subject in a need thereof
US20090226397A1 (en) * 2003-10-24 2009-09-10 Nora Therapeutics, Inc. Compositions and methods for reducing the likelihood of implantation failure or miscarriage in recipients of artificial insemination

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
A. SALMASSI: "Is granulocyte colony-stimulating factor level predictive for human IVF outcome?", HUMAN REPRODUCTION, vol. 20, no. 9, 1 January 2005 (2005-01-01), pages 2434 - 2440, XP002453576, ISSN: 0268-1161, DOI: 10.1093/humrep/dei071 *
AL NAIB ET AL., JOURNAL OF REPRODUCTIVE IMMUNOLOGY, vol. 91, 2011, pages 31 - 40
CLARK ET AL., SCIENCE, vol. 236, 1987, pages 1229 - 1237
DESAI ET AL., HUMAN REPROD, vol. 12, 1997, pages 328 - 335
DOMINGUEZ, HUMAN REPRODUCTION, vol. 23, 2008, pages 1993 - 2000
LEDEE N ET AL: "Levels of follicular G-CSF and interleukin-15 appear as noninvasive biomarkers of subsequent successful birth in modified natural in vitro fertilization/intracytoplasmic sperm injection cycles", FERTILITY AND STERILITY, ELSEVIER SCIENCE INC, NEW YORK, NY, USA, vol. 95, no. 1, 1 January 2011 (2011-01-01), pages 94 - 98, XP027563204, ISSN: 0015-0282, [retrieved on 20101216] *
MCKIERNAN SH; BAVISTER BD, HUMAN REPROD, vol. 15, 2000, pages 157 - 164
SALMASSI, HUMAN REPRODUCTION, vol. 20, 2005, pages 2434 - 2440
STEPTOE, P.C.; EDWARDS, R.G., LANCET, vol. 2, no. 8085, 1978, pages 366
W. WURFEL ET AL: "High pregnancy rates with administration of granulocyte colony-stimulating factor in ART-patients with repetitive implantation failure and lacking killer-cell immunglobulin-like receptors", HUMAN REPRODUCTION, vol. 25, no. 8, 1 August 2010 (2010-08-01), pages 2151 - 2152, XP055059222, ISSN: 0268-1161, DOI: 10.1093/humrep/deq106 *
WURFEL, HUMAN REPRODUCTION, vol. 25, 2010, pages 2151 - 2153

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2898062A1 (fr) * 2012-09-19 2015-07-29 Scarpellini, Fabio Utilisation de g-csf dans une culture d'embryons in vitro
US20150225694A1 (en) * 2012-09-19 2015-08-13 Fabio Scarpellini Use of G-CSF In In Vitro Embryo Culture
US9890356B2 (en) * 2012-09-19 2018-02-13 Fabio Scarpellini Use of G-CSF in in vitro embryo culture
US11639494B2 (en) 2015-08-24 2023-05-02 Vitrolife Sweden Ab Culture medium

Similar Documents

Publication Publication Date Title
Trounson et al. Current status of IVM/IVF and embryo culture in humans and farm animals
US11639494B2 (en) Culture medium
JP4532493B2 (ja) 細胞培養培地
Chian et al. Maturational and developmental competence of cumulus-free immature human oocytes derived from stimulated and intracytoplasmic sperm injection cycles
Furnus et al. Effect of hyaluronic acid on development of in vitro produced bovine embryos
AU2017371402B2 (en) Compositions and methods for maturation of oocytes in vitro
CN111254109B (zh) 牛卵母细胞的体外受精和胚胎培养的方法和运输培养液
Lim et al. Roles of growth factors in the development of bovine embryos fertilized in vitro and cultured singly in a defined medium
EP2228439A1 (fr) Supplément de milieu de culture d'embryons et de gamètes de mammifères, et méthode d'utilisation correspondant
Hasler et al. In vitro fertilization
Kanda et al. Development of in vitro fertilized feline embryos in a modified Earle's balanced salt solution: influence of protein supplements and culture dishes on fertilization success and blastocyst formation
WO2013113658A1 (fr) Milieux
CA2477743C (fr) Maturation in vitro d'ovocytes humains immatures
JP5859543B2 (ja) 胚培養用組成物
JP2019213554A (ja) 胚の培養方法および培地
Penitente Filho Improved in vitro production systems for bovine embryos.
Zahmatkesh et al. A comparative study of EGF effects on in vitro bovine embryo development in monoculture and sequential media
US20040082062A1 (en) Method and medium for in vitro culture of human embryos
WO2013149957A1 (fr) Milieux de culture améliorés pour cellules embryonnaires
Sunde et al. Culture Media and Embryo Culture
Sjoblom et al. Use Oocyte of Medium and Embryo Supplements Culture for
Zander-Fox et al. Amino acids and ammonium
Fakhrildin Effect of Cumulus cell co-culture and Protein Supplement on Success of in vitro Fertilization and Development of Pre-implanted Embryos in mice
Kim et al. In Vitro Development of Mouse IVF/IVC Embryo Treated with Epidermal Growth Factor (EGF).

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13701771

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13701771

Country of ref document: EP

Kind code of ref document: A1