AU588339B2 - Culture media for in vitro fertilization and embryo transfer - Google Patents

Culture media for in vitro fertilization and embryo transfer

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Publication number
AU588339B2
AU588339B2 AU59696/86A AU5969686A AU588339B2 AU 588339 B2 AU588339 B2 AU 588339B2 AU 59696/86 A AU59696/86 A AU 59696/86A AU 5969686 A AU5969686 A AU 5969686A AU 588339 B2 AU588339 B2 AU 588339B2
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AU
Australia
Prior art keywords
sodium
potassium
culture medium
chloride
ions
Prior art date
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Ceased
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AU59696/86A
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AU5969686A (en
Inventor
Patrick James Quinn
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Luminis Pty Ltd
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Luminis Pty Ltd
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Priority to AU59696/86A priority Critical patent/AU588339B2/en
Publication of AU5969686A publication Critical patent/AU5969686A/en
Application granted granted Critical
Publication of AU588339B2 publication Critical patent/AU588339B2/en
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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
    • C12N5/0608—Germ cells
    • C12N5/0609—Oocytes, oogonia
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars

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

Description

CULTURE MEDIA FOR IN VITRO FERTILIZATION AND EMBRYO TRANSFER
The invention relates to culture media and more particularly to culture media for in vitro fertilization and embryo transfer.
The process ofi n vitro fertilization of human oocytes, cleavage of embryos and embryo transfer require that a culture medium be used to support the embryo for a period of up to three or four days during the various processes necessary for fertilization and early incubation before embryo transfer and reimplantatlon.
In the natural process of fertilization for a human oocyte the oocyte is supported within the mother within a fluid known as human tubal fluid andi t i s the object of the present invention to provide a culture medium as a synthetic human tubal fluid.
Approximation of the culture conditions as close as possible to those found in the natural environment of the gametes may be most likely to yield the best results. Using this rationale early workers have formulated a culture media similar in biochemical composition to human tubal fluid with varying rates of success. Examples of these include Tyrodes Medium T6, WM1 (Hoppe and Pitts), Modified Earles, and Hams F 10.
One important characteristic of synthetic human tubal fluids appears to the ratio of sodium ions to potassium ions. For natural human tubal fluid this valve is approximately 18. Earlier attempts such as Tyrodes Medium T6 have a value of over 100. We have found that values in between these are most advantageous.
In the present invention we have devised a synthetic culture medium which is believed to approximate human tubal fluid but with desirable additional components and variations in the actual composition, including ratios of concentrations of sodium ions to potassium ions. In one form therefore the present invention may be said to reside in a culture medium for in vitro fertilization and embryo transfer comprising the following compounds in the following ranges of concentration;
Sodium chloride (NaCI) 96.5 - 106.7 mM Potassium chloride (KCl) 4.46 - 4.92mM
Magnesium sulphate (MgSO47H2O) 0.18 - 0.22mM
Potassium phosphate monobasic (KH2PO4) 0.35 - 0.39mM
Calcium chloride 2 hydrate (CaCl22H2O) 1.94 - 2.14mM
Sodium bicarbonate (NaHCO3) 23.7 - 26.3mM
Glucose 2.64 - 2.92mM
Sodium Pyruvate 0.31 - 0.35mM
Sodium Lactate 20.3 - 22.5mM Penicillin 95 - 105 units/m l
Phenol red 5 - 15 micrograms/m l
In a preferred embodiment of the invention, the ratio of sodium ion concentration to potassium ion concentration is in the range from 28 to 32.
In a further preferred embodiment the ratio of concentrations of sodium ions to potassium ions is 29.3.
In one preferred embodiment of the i nvention the synthetic human tubal fluid may have the following composition;
Sodium Chloride (NaCI) 101.6 mM
Potassium chloride (KCl) 4.69 mM Magnesium sulphate (MgSO47H2O) 0.20mM
Potassium phosphate monobasic (KH2PO4) 0.37mM
Calcium chloride 2 hydrate
(CaCl22H2O) 2.04mM Sodium bicarbonate (NaHCO3) 25.0mM
Glucose 2.78mM Sodium pyruvate 0.33mM
Sodium lactate 21.4mM
Penicillin 100 units/ml
Phenol red 10 micrograms/ml
In another form the invention may be said to reside in a method of assisting with the in vitro fertilization of human oocytes including the step of handling the human oocytes in a culture medium, the culture medium being comprised of the compounds listed below in the range of compositions listed as follows;
Sodium chloride (NaCI ) 96.5 - 106.7mM
Potassium chloride (KCl ) 4.46 - 4.92mM
Magnesium sulphate (MgSO4 7H2O) 0.18 - 0.22mM
Potassium phosphate monobasic
(KH2PO4) 0.35 - 0.39mM
Calcium chloride 2 hydrate
(CaCl 2 2H2 O) 1.94 - 2.14mM
Sodium bicarbonate (NaHCO3) 23.7 - 26.3mM
Glucose 2.64 - 2.92mM
Sodium pyruvate 0.31 - 0.35mM
Sodium lactate 20.3 - 22.5mM
Penicillin 95 - 105 units/ml
Phenol red 5 - 15 micrograms/ml
In a preferred embodiment of this method of the invention, the ratio of sodium ion concentration to potassium ion concentration is in the range of from 28 to 32.
In a further preferred form of the invention, the ratio concentration of sodium ions to potassium ions is 29.3.
In a further preferred form of the invention, the method includes the step of handling the oocytes in a culture medium comprising;
Sodium Chloride (NaCI) 101.6 mM Potassium chloride (Kcl) 4.69 mM
Magnesium sulphate (MgSO47H2O) 0.20mM
Potassium phosphate monobasic (KH2PO4) 0.37mM
Calcium chloride 2 hydrate
(CaCl22H2O) 2.04mM
Sodium bicarbonate (NaHCO3) 25.0mM
Glucose 2.78mM
Sodium pyruvate 0.33mM
Sodium lactate 21.4mM
Penicillin 100 units/ml
Phenol red 10 micrograms/ml
In a further form thei nvention may be said to reside in a culture medium for thei n vitro fertilization of human oocytesi ncluding sodium potassiumi ons wherein the ratio of sodium ions to potassium ions is i n the range of from 28 to 32.
In a preferred embodiment of this form of the invention, the ratio of sodium ions to potassium ions is 29.3.
This then generally describes the nature of the present invention and it will be seen that by this invention there is provided a culture medium which is not exactly the same as natural human tubal fluid, but which is capable of supporting in vitro fertilization.
To more clearly assist with the understanding of this invention reference will now be made to a preferred embodiment and tests to determine the efficacy of the preferred embodiment.
In one preferred embodiment synthetic human tubal fluid culture medium is as given in Table 1 below (marked synthetic HTF).
The medium may be prepared by using rainwater which has been distilled in glass six times. The bicarbonate-buffered medium is gassed for a minimum of five minutes with humidified 5% oxygen 5% carbon dioxide 90% nitrogen mixture and sterilized by passage through a 0.45-0.2 micrometre filter membrane (Millipore, Sydney, Australia or Amicon Sterilet Adelaide, Australia) and then stored at 4°C for up to two weeks before use. A minimum of six hours or preferably the day before being used the bicarbonate buffered medium is gassed again for two to three minutes with the same gas mixture as above and the protein component is added.
In a similar way a known culture medium Tyrodes Medium T6 having a composition as given in Table 1 below was also prepared.
T6 FT
l O O l . O O
100 100
Tests have been carried out using both mouse embryo development in vitro and with initiation of human pregnancy in an endeavour to discover which components of the T6 medium and the synthetic human tubal fluid according to this invention might be responsible for observed differences in mouse embryo development in vitro and initiation of human pregnancies. The results show that for human pregnancy initiation almost three times as many pregnancies occurred when fertilization and culture were carried out using the synthetic human tubal fluid of the present invention over the T6 medium.
In comparison of the two compositions a greatest difference in composition of the two media are their Na+/K+ ratios. We refer to these as sodium/potassium for the rest of the specification.
The ratios of concentrations of sodium ions to potassium ions for these are as follows:
Synthetic Human Tubal Fluid according to the present invention - 29.3
T6 - 105
When media are tested with sodium/potassium leveis varying from 150.5/ 1.42 millimoles to 148.2/5.06 millimoles results showed that there was significant linear and quadratic responses in the number of embryos developing to expanded blastocysts with increasing levels of K+.
In medium containing the potassium levels of T6 medium 75% of the zygotes developed which was significantly fewer than the 95-100% embryos developing when the potassium level was 2.3 to 5.1 millimoles which is the range for the synthetic human tubal fluid of the present invention.
The greatest number of mouse zygotes developing to expanded blastocysts when cultured In synthetic human tubal fluid medium of the present invention compared to T6 medium was paralleled by the three fold increase of the number of pregnancies initiated in those patients whose gametes had been fertilized and cultured in the medium in the present invention rather than T6 medium.
The present invention therefore provides a synthetic human tubal fluid culture medium which is more than just a direct replication of naturally occurring human tubal fluid but has enhanced viability. Potassium chloride (Kcl) 4.69 mM
Magnesium sulphate (MgSO47H2O) 0.20mM
Potassium phosphate monobasic (KH2PO4) 0.37mM
Calcium chloride 2 hydrate
(CaCl22H2O) 2.04mM
Sodium bicarbonate (NaHCO3) 25.0mM
Glucose 2.78mM
Sodium pyruvate 0.33mM
Sodium lactate 21.4mM
Penicillin 100 units/ml
Phenol red 10 micrograms/ml
In a further form the invention may be said to reside i n a culture medium for the in vitro fertilization of human oocytes i ncluding sodium potassium i ons wherein the ratio of sodium i ons to potassium ions i s in the range of from 28 to 32.
In a preferred embodiment of this form of the invention, the ratio of sodium ions to potassiumi ons is 29.3.
This then generally describes the nature of the present invention and it will be seen that by this invention there is provided a culture medium which i s not exactly the same as natural human tubal fluid, but which is capable of supporting in vitro fertilization.
To more clearly assist with the understanding of this invention reference will now be made to a preferred embodiment and tests to determine the efficacy of the preferred embodiment.
In one preferred embodiment synthetic human tubal fluid culture medium i s as given in Table 1 below (marked synthetic HTF).
The medium may be prepared by using rainwater which has been distilled in glass six times. The bicarbonate-buffered medium is gassed for a minimum of five minutes with humidified 5% oxygen 5% carbon dioxide 90% nitrogen mixture and sterilized by passage through a 0.45-0.2 micrometre filter membrane (Millipore, Sydney, Australia or Amlcon Sterilet Adelaide, Australia) and then stored at 4°C for up to two weeks before use. A minimum of six hours or preferably the day before being used the bicarbonate buffered medium is gassed again for two to three minutes with the same gas mixture as above and the protein component is added.
In a similar way a known culture medium Tyrodes Medium T6 having a composition as given in Table 1 below was also prepared.
T6
HFT
l O O O . O O
100 100
Tests have been carried out using both mouse embryo development in vitro and with initiation of human pregnancy in an endeavour to discover which components of the T6 medium and the synthetic human tubal fluid according to this invention might be responsible for observed differences in mouse embryo development in vitro and initiation of human pregnancies. The results show that for human pregnancy initiation almost three times as many pregnancies occurred when fertilization and culture were carried out

Claims (10)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:
1. A culture medium for in vitro fertilization of human oocytes comprising:
Sodium chloride (NaCI) 96.5 - 106.7 mM Potassium chloride (KCl) 4.46 - 4.92mM Magnesium sulphate (MgSO47H2O) 0.18 - 0.22mM
Potassium phosphate monobasic (KH2PO4) 0.35 - 0.39mM
Calcium chloride 2 hydrate (CaCl22H2O) 1.94 - 2.14mM
Sodium bicarbonate (NaHCO3) 23.7 - 26.3mM
Glucose 2.64 - 2.92mM
Sodium Pyruvate 0.31 - 0.35mM
Sodium Lactate 20.3 - 22.5mM
Penicillin 95 - 105 units/m l
Phenol red 5 - 15 micrograms/m l
2. A culture medium in Claim 1 wherein the ratio of sodium ion concentration to potassium ion concentration is in the range of from 28 to 32.
3. A culture medium as in Claim 2 wherein the ratio of concentrations of sodium ions to potassium ions is approximately 29.3.
4. A culture medium as in Claim 1 comprising approximately;
Sodium Chloride (NaCI) 101.6 mM Potassium chloride (KCl) 469 mM Magnesium sulphate (MgSO47H2O) 0.20mM
Potassium phosphate monobasic (KH2PO4) 0.37mM
Calcium chloride 2 hydrate
(CaCl22H2O) 2.04mM Sodium bicarbonate (NaHCO3) 25.0mM Glucose 2.78 mM Sodium pyruvate 0.33mM Sodium lactate 21.4mM
Penicillin 100 units/ml
Phenol red 10 micrograms/ml
5. A method of assisting with the in vitro fertilization of human oocytes including the steps of handling the oocytes in a culture medium, the culture medium being of the compounds being listed below in the range of compositions as follows;
Sodium chloride (NaCI) 96.5 - 106.7 mM
Potassium chloride (KCl) 4.46 - 4.92mM
Magnesium sulphate (MgSO47H2O) 0.18 - 0.22mM
Potassium phosphate monobasic (KH2PO4) 0.35 - 0.39mM Calcium chloride 2 hydrate
(CaCl22H2O) 1.94 - 2.14mM
Sodium bicarbonate (NaHCO3) 23.7 - 26.3mM
Glucose 2.64 - 2.92mM
Sodium Pyruvate 0.31 - 0.35mM Sodium Lactate 20.3 - 22.5mM
Penicillin 95 - 105 units/m l
Phenol red 5 - 15 micrograms/m l
6. A method as in Claim 5 wherein the ratio of sodium ion concentration to potassium ion concentration is in the range of from 28 to 32.
7. A method as in Claim 6 wherein the ratio of concentration of sodium ions to potassium ions is approximately 29.3.
8. A method as in Claim 5 wherein the culture medium has a concentration as follows;
Sodium Chloride (NaCI) 101.6 mM
Potassium chloride (Kcl) 4.69 mM Magnesium sulphate (MgSO47H2O) 0.20mM
Potassium phosphate monobasic (KH2PO4) 0.37mM Calcium chloride 2 hydrate
(CaCl22H2O) 2.04mM Sodium bicarbonate (NaHCO3) 25.0mM Glucose 2.78mM
Sodium pyruvate 0.33mM Sodium lactate 21.4mM Penicillin 100 units/ml Phenol red 10 micrograms/ml
9. A culture medium for the in vitro fertilization of human oocytes including sodium and potassium ions wherein the ratio of sodium ions to potassium ions is in the range of from 28 to 32.
10. A culture medium as in Claim 9 wherein the ratio of sodium Ions to potassium ions is approximately 29.3.
AU59696/86A 1985-06-12 1986-06-12 Culture media for in vitro fertilization and embryo transfer Ceased AU588339B2 (en)

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Application Number Priority Date Filing Date Title
AU59696/86A AU588339B2 (en) 1985-06-12 1986-06-12 Culture media for in vitro fertilization and embryo transfer

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Application Number Priority Date Filing Date Title
AUPH100985 1985-06-12
AUPH1009 1985-06-12
AU59696/86A AU588339B2 (en) 1985-06-12 1986-06-12 Culture media for in vitro fertilization and embryo transfer

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AU5969686A AU5969686A (en) 1987-01-07
AU588339B2 true AU588339B2 (en) 1989-09-14

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU628796B2 (en) * 1987-12-30 1992-09-24 W.R. Grace & Co.-Conn. In vitro culture of bovine embryos

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3128228A (en) * 1960-03-04 1964-04-07 Ustav Ser A Ockovacich Latek Tissue culture medium
AU8338082A (en) * 1981-02-27 1982-09-14 Amf Inc. Tissue culture medium
AU8853282A (en) * 1981-10-05 1983-04-14 Alcon Laboratories, Inc. Opthalmic irrigating solution

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3128228A (en) * 1960-03-04 1964-04-07 Ustav Ser A Ockovacich Latek Tissue culture medium
AU8338082A (en) * 1981-02-27 1982-09-14 Amf Inc. Tissue culture medium
AU8853282A (en) * 1981-10-05 1983-04-14 Alcon Laboratories, Inc. Opthalmic irrigating solution

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU628796B2 (en) * 1987-12-30 1992-09-24 W.R. Grace & Co.-Conn. In vitro culture of bovine embryos

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