CULTURE MEDIA FOR IN VITRO FERTILIZATION AND EMBRYO TRANSFER

Abstract: A culture medium for in vitro fertilization of human oocytes which comprises: NaCl (96.5 - 106.7 mM) KCl(4.46 - 4.92 mM) MgSO4.7H2O (0.18 - 0.22 mM) KH2PO4 (0.35 - 0.39 mM) CaCl2.2H2O (1.94 - 2.14 mM) NaHCO3 (23.7 - 26.3 mM) Glucose (2.64 - 2.92 mM) Sodium Pyruvate (0.31 - 0.35 mM) Sodium Lactate (20.3 - 22.5 mM) Penicillin (95 - 105 units/ml) Phenol red (5 - 15 micrograms/ml). Culture media wherein the ratio of sodium ions to potassium ions is in the range of from 28 to 32 are preferred.

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 of In 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 relmplantatlon. 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 and It Is 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 luids 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 (KC1) 4.46 - 4.92mM Magnesium sulphate ( gSθ 7H2θ) 0.18 - 0.22mM Potassium phosphate monobasic (KH2PO4) 0.35 - 0.39mM Calcium chloride 2 hydrate (CaCl22H2θ) 1.94 - 2.14mM Sodium bicarbonate (NaHC03) 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 Invention the synthetic human tubal fluid may have the following composition; Sodium Chloride (NaCI) 101.6 mM Potassium chloride (KC1) 4.69 mM Magnesium sulphate (MgSθ 7H2θ) 0.20mM Potassium phosphate monobasic (KH2PO4) 0.37mM Calcium chloride 2 hydrate (CaCl 2 2H 2 0) 2.04mM Sodium bicarbonate (NaHCθ3) 25.0mM Glucose 2.78mM Sodium pyruvate 0.33mM Sodium lactate 2l .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 - 10δ.7mM Potassium chloride (KC1 ) 4.46 - 4.92mM Magnesium sulphate (MgS0 7H2O) 0.18 - 0.22mM Potassium phosphate monobasic (KH 2 P0 4 ) 0.35 - 0.39mM Calcium chloride 2 hydrate (CaC1 2 2H 2 0) 1.94 - 2.14mM Sodium bicarbonate (NaHC03) 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 (MgSθ47H2θ) 0.20mM Potassium phosphate monobasic (KH2PO4) 0.37mM Calcium chloride 2 hydrate (CaCl 2 2H 2 0) 2.04mM Sodium bicarbonate (NaHC03) 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 in a culture medium for the In vitro fertilization of human oocytes Including sodium potassium Ions wherein the ratio of sodium ions to potassium ions is In 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 1s 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 (Mlllipore, 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. TAPLE 1 COMPONENT SYNTHETIC Ifi HEI NaCI 101.6 99.4 KC1 4.69 1.42 MgS0 4 .7H 2 0 0.20 0.71 KH2PO4 0.37 - CaCl 2 «2H 2 0 2.04 1.78 NaHC0 3 25 - 25 • Glucose 2.78 5.56 Na pyruvate 0.33 0.47 Na lactate 21.4 24.9 Penicillin l OO U/ml lOO U/ml Streptomycin SO4 - 50 ug/ml Phenol red 0.001% (w/v) 0.001 % (w/v) 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 levels 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 Tδ 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 (MgS047H20) 0.20mM Potassium phosphate monobasic (KH2PO4) 0.37mM Calcium chloride 2 hydrate (CaCl 2 2H 2 0) 2.04mM Sodium bicarbonate (NaHCθ3) 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 In a culture medium for the in vitro fertilization of human oocytes Including sodium potassium Ions wherein the ratio of sodium Ions to potassium ions Is in 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 (Milllpore, Sydney, Australia or Amlcon Sterl let 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. TABLE 1 COMPONENT SYNTHETIC IS t£I NaCI 101.6 99.4 KC1 4.69 1.42 KH 2 P0 0.37 - CaCl 2 «2H 2 0 2.04 1.78 NaHC0 3 25 25 Glucose 2.78 5.56 Na pyruvate 0.33 0.47 Na lactate 21.4 24.9 Penicillin lOO U/ml l OO U/ml Streptomycin SO4 - 50 ug/ml Phenol red 0.001 (w/v) 0.001 % (w/v) 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: 1. A culture medium for in vitro fertilization of human oocytes comprising: Sodium chloride (NaCI) 96.5 - 106.7 mM Potassium chloride (KC1) 4.46 - 4.92mM Magnesium sulphate (MgS047H 2 0) 0.18 - 0.22mM Potassium phosphate monobasic (KH2PO4) 0.35 - 0.39mM Calcium chloride 2 hydrate (CaCl 2 2H2θ) 1.94 - 2.14mM Sodium bicarbonate ( aHCθ3) 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 (KC1) 469 mM Magnesium sulphate (MgS0 7H20) 0.20mM Potassium phosphate monobasic (KH2PO4) 0.37mM Calcium chloride 2 hydrate (CaCl 2 2H 2 0) 2.04mM Sodium bicarbonate (NaHC03) 25.0mM Glucose 2.78rnM 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 (KC1) 4.46 - 4.92mM Magnesium sulphate (MgS047H 2 0) 0.18 - 0.22mM Potassium phosphate monobasic (KH2PO4) 0.35 - 0.39mM Calcium chloride 2 hydrate (CaCl22H2θ) 1.94 - 2.14mM Sodium bicarbonate (NaHC03) 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 (MgSθ47H2θ) 0.20mM Potassium phosphate monobasic (KH2PO4) 0.37mM Calcium chloride 2 hydrate (CaCl 2 2H 2 0) 2.04mM Sodium bicarbonate (NaHC03) 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.

Inventors: QUINN PATRICK JAMES, QUINN, PATRICK, JAMES
Applicants: LUMINIS PTY LTD, QUINN PATRICK JAMES, LUMINIS PTY. LTD., QUINN, PATRICK, JAMES
Application year: 1986
Main classification: C12N5
IPC codes: C12N5