US20050208651A1 - Vitro maturation of immature human oocytes - Google Patents
Vitro maturation of immature human oocytes Download PDFInfo
- Publication number
- US20050208651A1 US20050208651A1 US10/506,713 US50671305A US2005208651A1 US 20050208651 A1 US20050208651 A1 US 20050208651A1 US 50671305 A US50671305 A US 50671305A US 2005208651 A1 US2005208651 A1 US 2005208651A1
- Authority
- US
- United States
- Prior art keywords
- culture medium
- oocyte
- growth factor
- oocytes
- human
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 210000000287 oocyte Anatomy 0.000 title claims abstract description 225
- 230000035800 maturation Effects 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 claims abstract description 89
- 238000000338 in vitro Methods 0.000 claims abstract description 31
- 108010073521 Luteinizing Hormone Proteins 0.000 claims abstract description 26
- 102000009151 Luteinizing Hormone Human genes 0.000 claims abstract description 26
- 229940040129 luteinizing hormone Drugs 0.000 claims abstract description 26
- 230000000638 stimulation Effects 0.000 claims abstract description 25
- 230000002611 ovarian Effects 0.000 claims abstract description 18
- 238000012258 culturing Methods 0.000 claims abstract description 13
- 230000001939 inductive effect Effects 0.000 claims abstract description 7
- 239000001963 growth medium Substances 0.000 claims description 75
- 229940024606 amino acid Drugs 0.000 claims description 44
- 235000001014 amino acid Nutrition 0.000 claims description 44
- 150000001413 amino acids Chemical class 0.000 claims description 44
- 229940088594 vitamin Drugs 0.000 claims description 41
- 229930003231 vitamin Natural products 0.000 claims description 41
- 235000013343 vitamin Nutrition 0.000 claims description 41
- 239000011782 vitamin Substances 0.000 claims description 41
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims description 34
- 210000001771 cumulus cell Anatomy 0.000 claims description 30
- 235000002639 sodium chloride Nutrition 0.000 claims description 27
- 150000003839 salts Chemical class 0.000 claims description 25
- 102000018233 Fibroblast Growth Factor Human genes 0.000 claims description 21
- 108050007372 Fibroblast Growth Factor Proteins 0.000 claims description 21
- 229940126864 fibroblast growth factor Drugs 0.000 claims description 21
- 239000003102 growth factor Substances 0.000 claims description 20
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 claims description 20
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 19
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 claims description 19
- 108010062540 Chorionic Gonadotropin Proteins 0.000 claims description 18
- 102000011022 Chorionic Gonadotropin Human genes 0.000 claims description 18
- 229940084986 human chorionic gonadotropin Drugs 0.000 claims description 18
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 claims description 18
- 102000009024 Epidermal Growth Factor Human genes 0.000 claims description 17
- 101800003838 Epidermal growth factor Proteins 0.000 claims description 17
- 102000004877 Insulin Human genes 0.000 claims description 17
- 108090001061 Insulin Proteins 0.000 claims description 17
- 229940116977 epidermal growth factor Drugs 0.000 claims description 17
- 229940125396 insulin Drugs 0.000 claims description 17
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 claims description 16
- 229940028334 follicle stimulating hormone Drugs 0.000 claims description 16
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 claims description 15
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 claims description 14
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 claims description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 13
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 13
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 12
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 12
- 101000766306 Homo sapiens Serotransferrin Proteins 0.000 claims description 12
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 12
- XNSAINXGIQZQOO-SRVKXCTJSA-N protirelin Chemical compound NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H]1NC(=O)CC1)CC1=CN=CN1 XNSAINXGIQZQOO-SRVKXCTJSA-N 0.000 claims description 12
- 229940054269 sodium pyruvate Drugs 0.000 claims description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 9
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 9
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 9
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 9
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims description 9
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 9
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 9
- 235000020776 essential amino acid Nutrition 0.000 claims description 9
- 239000003797 essential amino acid Substances 0.000 claims description 9
- 229960000890 hydrocortisone Drugs 0.000 claims description 9
- 229940082569 selenite Drugs 0.000 claims description 9
- MCAHWIHFGHIESP-UHFFFAOYSA-L selenite(2-) Chemical compound [O-][Se]([O-])=O MCAHWIHFGHIESP-UHFFFAOYSA-L 0.000 claims description 9
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 8
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 8
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 8
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 8
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 8
- 229940088597 hormone Drugs 0.000 claims description 8
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 claims description 7
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims description 7
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 7
- 239000004472 Lysine Substances 0.000 claims description 7
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 claims description 7
- 239000004473 Threonine Substances 0.000 claims description 7
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 7
- 229960000304 folic acid Drugs 0.000 claims description 7
- 235000019152 folic acid Nutrition 0.000 claims description 7
- 239000011724 folic acid Substances 0.000 claims description 7
- 239000005556 hormone Substances 0.000 claims description 7
- 229960000310 isoleucine Drugs 0.000 claims description 7
- 229960003136 leucine Drugs 0.000 claims description 7
- 229960005190 phenylalanine Drugs 0.000 claims description 7
- 229960002477 riboflavin Drugs 0.000 claims description 7
- 235000019192 riboflavin Nutrition 0.000 claims description 7
- 239000002151 riboflavin Substances 0.000 claims description 7
- 229960003495 thiamine Drugs 0.000 claims description 7
- 229960002898 threonine Drugs 0.000 claims description 7
- 229960004295 valine Drugs 0.000 claims description 7
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 claims description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
- 235000019743 Choline chloride Nutrition 0.000 claims description 6
- 239000004471 Glycine Substances 0.000 claims description 6
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 6
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 6
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 6
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 claims description 6
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 6
- 229960003767 alanine Drugs 0.000 claims description 6
- 229960001230 asparagine Drugs 0.000 claims description 6
- 229960005261 aspartic acid Drugs 0.000 claims description 6
- 229960002685 biotin Drugs 0.000 claims description 6
- 235000020958 biotin Nutrition 0.000 claims description 6
- 239000011616 biotin Substances 0.000 claims description 6
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical compound [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 claims description 6
- 229960003178 choline chloride Drugs 0.000 claims description 6
- 229960003067 cystine Drugs 0.000 claims description 6
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 239000001103 potassium chloride Substances 0.000 claims description 6
- 229960002429 proline Drugs 0.000 claims description 6
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 6
- 229960001153 serine Drugs 0.000 claims description 6
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 229960004441 tyrosine Drugs 0.000 claims description 6
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims description 5
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 5
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 5
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 5
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 5
- 229960002885 histidine Drugs 0.000 claims description 5
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims description 5
- 235000018977 lysine Nutrition 0.000 claims description 5
- 229930182817 methionine Natural products 0.000 claims description 5
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 5
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 5
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 5
- 239000004474 valine Substances 0.000 claims description 5
- 239000004475 Arginine Substances 0.000 claims description 4
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 4
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 4
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 4
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims description 4
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 4
- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 claims description 4
- 235000004279 alanine Nutrition 0.000 claims description 4
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 4
- 235000009697 arginine Nutrition 0.000 claims description 4
- 229960003121 arginine Drugs 0.000 claims description 4
- 235000009582 asparagine Nutrition 0.000 claims description 4
- 235000003704 aspartic acid Nutrition 0.000 claims description 4
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 4
- 235000013922 glutamic acid Nutrition 0.000 claims description 4
- 239000004220 glutamic acid Substances 0.000 claims description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 4
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 claims description 4
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims description 4
- 235000019190 thiamine hydrochloride Nutrition 0.000 claims description 4
- 239000011747 thiamine hydrochloride Substances 0.000 claims description 4
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 3
- 239000000556 agonist Substances 0.000 claims description 3
- CBUWTGCATVNMJE-UHFFFAOYSA-N 6,6-dimethylheptanal Chemical compound CC(C)(C)CCCCC=O CBUWTGCATVNMJE-UHFFFAOYSA-N 0.000 claims description 2
- 108700012941 GNRH1 Proteins 0.000 claims description 2
- 229940090343 human menopausal gonadotrophin Drugs 0.000 claims description 2
- 229960004407 chorionic gonadotrophin Drugs 0.000 claims 1
- 239000002609 medium Substances 0.000 description 69
- 230000004720 fertilization Effects 0.000 description 26
- 210000002257 embryonic structure Anatomy 0.000 description 15
- 201000010065 polycystic ovary syndrome Diseases 0.000 description 13
- 206010036049 Polycystic ovaries Diseases 0.000 description 12
- 210000002459 blastocyst Anatomy 0.000 description 11
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 10
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 10
- 108010057021 Menotropins Proteins 0.000 description 8
- 230000036512 infertility Effects 0.000 description 8
- 231100000535 infertility Toxicity 0.000 description 8
- 208000000509 infertility Diseases 0.000 description 8
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 7
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 7
- 229960005309 estradiol Drugs 0.000 description 7
- 229930182833 estradiol Natural products 0.000 description 7
- 210000001672 ovary Anatomy 0.000 description 7
- 210000004508 polar body Anatomy 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 102000004338 Transferrin Human genes 0.000 description 6
- 108090000901 Transferrin Proteins 0.000 description 6
- 210000001161 mammalian embryo Anatomy 0.000 description 6
- 239000012581 transferrin Substances 0.000 description 6
- 238000002604 ultrasonography Methods 0.000 description 6
- 238000003776 cleavage reaction Methods 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 229960004452 methionine Drugs 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 229940056360 penicillin g Drugs 0.000 description 5
- 230000037452 priming Effects 0.000 description 5
- 230000001850 reproductive effect Effects 0.000 description 5
- 230000007017 scission Effects 0.000 description 5
- 229960005322 streptomycin Drugs 0.000 description 5
- 229960004799 tryptophan Drugs 0.000 description 5
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 4
- 206010033266 Ovarian Hyperstimulation Syndrome Diseases 0.000 description 4
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 4
- RADKZDMFGJYCBB-UHFFFAOYSA-N Pyridoxal Chemical compound CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 4
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 210000000805 cytoplasm Anatomy 0.000 description 4
- 230000013020 embryo development Effects 0.000 description 4
- 229960002989 glutamic acid Drugs 0.000 description 4
- 238000003306 harvesting Methods 0.000 description 4
- 239000002480 mineral oil Substances 0.000 description 4
- 235000010446 mineral oil Nutrition 0.000 description 4
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 4
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 3
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 3
- 230000000740 bleeding effect Effects 0.000 description 3
- 230000002357 endometrial effect Effects 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 229960003646 lysine Drugs 0.000 description 3
- 230000002175 menstrual effect Effects 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 230000027758 ovulation cycle Effects 0.000 description 3
- 230000001817 pituitary effect Effects 0.000 description 3
- 230000035935 pregnancy Effects 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 235000019157 thiamine Nutrition 0.000 description 3
- 239000011721 thiamine Substances 0.000 description 3
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- 201000005670 Anovulation Diseases 0.000 description 2
- 206010002659 Anovulatory cycle Diseases 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 2
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 2
- 201000009273 Endometriosis Diseases 0.000 description 2
- 102000003745 Hepatocyte Growth Factor Human genes 0.000 description 2
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 description 2
- 102000008100 Human Serum Albumin Human genes 0.000 description 2
- 108091006905 Human Serum Albumin Proteins 0.000 description 2
- 108010003272 Hyaluronate lyase Proteins 0.000 description 2
- 102000001974 Hyaluronidases Human genes 0.000 description 2
- 238000003744 In vitro fertilisation Methods 0.000 description 2
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 2
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 2
- 235000019766 L-Lysine Nutrition 0.000 description 2
- FFEARJCKVFRZRR-UHFFFAOYSA-N L-Methionine Natural products CSCCC(N)C(O)=O FFEARJCKVFRZRR-UHFFFAOYSA-N 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 2
- 229930064664 L-arginine Natural products 0.000 description 2
- 235000014852 L-arginine Nutrition 0.000 description 2
- 239000004158 L-cystine Substances 0.000 description 2
- 235000019393 L-cystine Nutrition 0.000 description 2
- 229930182816 L-glutamine Natural products 0.000 description 2
- 229930182844 L-isoleucine Natural products 0.000 description 2
- 239000004395 L-leucine Substances 0.000 description 2
- 235000019454 L-leucine Nutrition 0.000 description 2
- 229930195722 L-methionine Natural products 0.000 description 2
- 229930182821 L-proline Natural products 0.000 description 2
- 208000035752 Live birth Diseases 0.000 description 2
- MJVAVZPDRWSRRC-UHFFFAOYSA-N Menadione Chemical compound C1=CC=C2C(=O)C(C)=CC(=O)C2=C1 MJVAVZPDRWSRRC-UHFFFAOYSA-N 0.000 description 2
- 206010027339 Menstruation irregular Diseases 0.000 description 2
- 108010025020 Nerve Growth Factor Proteins 0.000 description 2
- 102000015336 Nerve Growth Factor Human genes 0.000 description 2
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 2
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 2
- 102000013275 Somatomedins Human genes 0.000 description 2
- 108010009583 Transforming Growth Factors Proteins 0.000 description 2
- 102000009618 Transforming Growth Factors Human genes 0.000 description 2
- 231100000552 anovulation Toxicity 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- RMRCNWBMXRMIRW-BYFNXCQMSA-M cyanocobalamin Chemical compound N#C[Co+]N([C@]1([H])[C@H](CC(N)=O)[C@]\2(CCC(=O)NC[C@H](C)OP(O)(=O)OC3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)C)C/2=C(C)\C([C@H](C/2(C)C)CCC(N)=O)=N\C\2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O RMRCNWBMXRMIRW-BYFNXCQMSA-M 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 229960002449 glycine Drugs 0.000 description 2
- 229960002773 hyaluronidase Drugs 0.000 description 2
- 230000001788 irregular Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229940053128 nerve growth factor Drugs 0.000 description 2
- 229960003966 nicotinamide Drugs 0.000 description 2
- 235000005152 nicotinamide Nutrition 0.000 description 2
- 239000011570 nicotinamide Substances 0.000 description 2
- 239000000186 progesterone Substances 0.000 description 2
- 229960003387 progesterone Drugs 0.000 description 2
- 229940087667 prometrium Drugs 0.000 description 2
- 229960003581 pyridoxal Drugs 0.000 description 2
- 235000008164 pyridoxal Nutrition 0.000 description 2
- 239000011674 pyridoxal Substances 0.000 description 2
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 2
- 229960003471 retinol Drugs 0.000 description 2
- 235000020944 retinol Nutrition 0.000 description 2
- 239000011607 retinol Substances 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 210000001082 somatic cell Anatomy 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 239000003104 tissue culture media Substances 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- QZNNVYOVQUKYSC-JEDNCBNOSA-N (2s)-2-amino-3-(1h-imidazol-5-yl)propanoic acid;hydron;chloride Chemical compound Cl.OC(=O)[C@@H](N)CC1=CN=CN1 QZNNVYOVQUKYSC-JEDNCBNOSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 206010014733 Endometrial cancer Diseases 0.000 description 1
- 206010014759 Endometrial neoplasm Diseases 0.000 description 1
- 229910000608 Fe(NO3)3.9H2O Inorganic materials 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 239000000579 Gonadotropin-Releasing Hormone Substances 0.000 description 1
- 102000006771 Gonadotropins Human genes 0.000 description 1
- 108010086677 Gonadotropins Proteins 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 108010082302 Human Follicle Stimulating Hormone Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- ABSPRNADVQNDOU-UHFFFAOYSA-N Menaquinone 1 Natural products C1=CC=C2C(=O)C(CC=C(C)C)=C(C)C(=O)C2=C1 ABSPRNADVQNDOU-UHFFFAOYSA-N 0.000 description 1
- 125000003047 N-acetyl group Chemical group 0.000 description 1
- 102000005348 Neuraminidase Human genes 0.000 description 1
- 108010006232 Neuraminidase Proteins 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108010020661 Profasi Proteins 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- 101000857870 Squalus acanthias Gonadoliberin Proteins 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- 238000003639 Student–Newman–Keuls (SNK) method Methods 0.000 description 1
- 102000002070 Transferrins Human genes 0.000 description 1
- 108010015865 Transferrins Proteins 0.000 description 1
- MECHNRXZTMCUDQ-UHFFFAOYSA-N Vitamin D2 Natural products C1CCC2(C)C(C(C)C=CC(C)C(C)C)CCC2C1=CC=C1CC(O)CCC1=C MECHNRXZTMCUDQ-UHFFFAOYSA-N 0.000 description 1
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 description 1
- 206010047998 Withdrawal bleed Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000002730 additional effect Effects 0.000 description 1
- 208000030961 allergic reaction Diseases 0.000 description 1
- 150000003862 amino acid derivatives Chemical class 0.000 description 1
- 238000001949 anaesthesia Methods 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 229960002104 cyanocobalamin Drugs 0.000 description 1
- 235000000639 cyanocobalamin Nutrition 0.000 description 1
- 239000011666 cyanocobalamin Substances 0.000 description 1
- GVJHHUAWPYXKBD-UHFFFAOYSA-N d-alpha-tocopherol Natural products OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 210000004696 endometrium Anatomy 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229960002061 ergocalciferol Drugs 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000003325 follicular Effects 0.000 description 1
- 210000001733 follicular fluid Anatomy 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229960001031 glucose Drugs 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 229960002743 glutamine Drugs 0.000 description 1
- 150000004676 glycans Chemical group 0.000 description 1
- XLXSAKCOAKORKW-AQJXLSMYSA-N gonadorelin Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)NCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 XLXSAKCOAKORKW-AQJXLSMYSA-N 0.000 description 1
- 239000002622 gonadotropin Substances 0.000 description 1
- 229940035638 gonadotropin-releasing hormone Drugs 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 201000010066 hyperandrogenism Diseases 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000009027 insemination Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000000938 luteal effect Effects 0.000 description 1
- 230000001785 maturational effect Effects 0.000 description 1
- 230000021121 meiosis Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 208000025661 ovarian cyst Diseases 0.000 description 1
- 230000011599 ovarian follicle development Effects 0.000 description 1
- 210000003101 oviduct Anatomy 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000007793 ph indicator Substances 0.000 description 1
- SHUZOJHMOBOZST-UHFFFAOYSA-N phylloquinone Natural products CC(C)CCCCC(C)CCC(C)CCCC(=CCC1=C(C)C(=O)c2ccccc2C1=O)C SHUZOJHMOBOZST-UHFFFAOYSA-N 0.000 description 1
- 235000019175 phylloquinone Nutrition 0.000 description 1
- 239000011772 phylloquinone Substances 0.000 description 1
- MBWXNTAXLNYFJB-NKFFZRIASA-N phylloquinone Chemical compound C1=CC=C2C(=O)C(C/C=C(C)/CCC[C@H](C)CCC[C@H](C)CCCC(C)C)=C(C)C(=O)C2=C1 MBWXNTAXLNYFJB-NKFFZRIASA-N 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229960001898 phytomenadione Drugs 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 235000008160 pyridoxine Nutrition 0.000 description 1
- 239000011677 pyridoxine Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 239000011669 selenium Substances 0.000 description 1
- 229940091258 selenium supplement Drugs 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000012090 serum-supplement Substances 0.000 description 1
- 239000001540 sodium lactate Substances 0.000 description 1
- 229940005581 sodium lactate Drugs 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000010959 steel Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229960001295 tocopherol Drugs 0.000 description 1
- 235000010384 tocopherol Nutrition 0.000 description 1
- 229930003799 tocopherol Natural products 0.000 description 1
- 239000011732 tocopherol Substances 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 235000001892 vitamin D2 Nutrition 0.000 description 1
- 239000011653 vitamin D2 Substances 0.000 description 1
- MECHNRXZTMCUDQ-RKHKHRCZSA-N vitamin D2 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)/C=C/[C@H](C)C(C)C)=C\C=C1\C[C@@H](O)CCC1=C MECHNRXZTMCUDQ-RKHKHRCZSA-N 0.000 description 1
- 235000005282 vitamin D3 Nutrition 0.000 description 1
- 239000011647 vitamin D3 Substances 0.000 description 1
- QYSXJUFSXHHAJI-YRZJJWOYSA-N vitamin D3 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-YRZJJWOYSA-N 0.000 description 1
- 235000012711 vitamin K3 Nutrition 0.000 description 1
- 239000011652 vitamin K3 Substances 0.000 description 1
- 229940011671 vitamin b6 Drugs 0.000 description 1
- 229940021056 vitamin d3 Drugs 0.000 description 1
- 229940041603 vitamin k 3 Drugs 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 229910001868 water Inorganic materials 0.000 description 1
- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 1
Images
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
- C12N5/0608—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
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
- C12N2500/24—Iron; Fe chelators; Transferrin
- C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
-
- 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
- C12N2500/32—Amino acids
-
- 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
- C12N2500/38—Vitamins
-
- 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
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- 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
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- 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
- C12N2501/30—Hormones
- C12N2501/31—Pituitary sex hormones, e.g. follicle-stimulating hormone [FSH], luteinising hormone [LH]; Chorionic gonadotropins
-
- 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
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
-
- 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
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
- C12N2501/392—Sexual steroids
-
- 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
Definitions
- the present invention relates to the field of assisted reproductive technology (ART), particularly to methods for culturing immature human oocytes so that they mature to metaphase-II stage capable of being fertilized and forming a viable embryo.
- ART assisted reproductive technology
- HCG human chorionic gonadotropin
- the harvested oocyte is then fertilized with the sperm.
- fertilization is effected by intracytoplasmic sperm injection (ICSI).
- ICSI intracytoplasmic sperm injection
- M-II metaphase-II
- a M-II oocyte is ready to accept the sperm and be fertilized.
- Metaphase-II is characterized by exclusion of one polar body from the cytoplasm, and is typically detected by visual identification under a microscope.
- the harvested oocyte remains surrounded by cumulus cells that support and promote the final maturation and development of the oocyte.
- the cumulus cells interfere with ICSI and obscure microscopic observation of the oocyte. Therefore, prior to ICSI, it is necessary to denude the oocyte of cumulus cells to permit determination of the level of oocyte maturity. This is generally accomplished by enzymatic and mechanical stripping of the cumulus cells.
- PCOS polycystic ovarian syndrome
- IVM media and methods have involved the use of human follicular fluid, in an effort to mimic the conditions within the follicle. In view of the risk of transmission of disease, allergic reaction, etc., use of biological fluids in IVM techniques is prohibited in most countries. Instead, the use of a “chemically-defined medium”, wherein the chemical composition of all of the ingredients is known, is preferred.
- TCM-199 tissue culture medium 199
- TCM-199 is a complex medium that contains many components, and was designed for in vitro culture of somatic cells, not germ cells. Notably, TCM-199 does not contain any growth factors.
- TCM-199 was first used for sheep oocyte IVM (Moor & Trounson, 1977), and has since been used routinely for bovine oocyte IVM (Brackett & Zuelke, 1993).
- the present invention provides in vitro maturation methods that are useful for culturing immature human oocytes to maturity.
- the invention provides a method for in vitro maturation of immature human oocytes, comprising the steps of:
- step (a) the subject has not been treated with a gonadotrophin releasing hormone agonist, human menopausal gonadotrophin, HCG, or follicle stimulating hormone (FSH).
- a gonadotrophin releasing hormone agonist human menopausal gonadotrophin, HCG, or follicle stimulating hormone (FSH).
- FSH follicle stimulating hormone
- step (a) comprises administering to the subject HCG or luteinizing hormone (LH), or both.
- HCG is administered in an amount of about 5000 to about 20,000 IU, more preferably about 10,000 IU.
- the immature human oocyte is preferably an M-I stage or GV stage ooctye.
- the oocyte preferably is cultured until it reaches M-II.
- the immature human oocyte is essentially free of cumulus cells and is cultured in a culture medium comprising:
- At least one growth factor is at least one growth factor.
- the growth factor is fibroblast growth factor or epidermal growth factor, or both.
- the culture medium further comprises at least one hormone, e.g. insulin.
- the culture medium comprises from 0.5 mg/L to 50 mg/L insulin.
- the culture medium further comprises human transferrin, preferably from 5 mg/L to 500 mg/L human transferrin.
- the culture medium comprises from 0.0001 mg/L to 0.001 mg/L fibroblast growth factor.
- the culture medium comprises from 0.0001 to 0.01 mg/L epidermal growth factor.
- the culture medium comprises one or more vitamins, preferably biotin, D-Ca pantothenate, choline chloride, folic acid, i-inositol, nicotinamide, pyroxidal-HCl, riboflavin, and thiamine-HCl.
- vitamins preferably biotin, D-Ca pantothenate, choline chloride, folic acid, i-inositol, nicotinamide, pyroxidal-HCl, riboflavin, and thiamine-HCl.
- the culture medium may further comprise hydrocortisone or selenite, or both.
- Preferred inorganic salts include CaCl 2 , KCl, MgSO 4 , NaCl, NaHCO 3 and NaH 2 PO4-H 2 O.
- Preferred energy sources include D-glucose and sodium pyruvate, or both D-glucose and sodium pyruvate.
- the amino acids comprise alanine, arginine, asparagine, aspartic acid, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
- the culture medium comprises the inorganic salts, amino acids, vitamins and other components as set forth in Table 1, and wherein each inorganic salt, amino acid, vitamin and other component is present in an amount of ⁇ 50% (weight/volume), more preferably ⁇ 10% (weight/volume), of the amount specified in Table 1.
- the oocyte has a cumulus that is intact or at least partially intact, and/or the oocyte is cultured in the presence of cumulus cells.
- the oocyte may be cultured in a culture medium that is essentially free of one or more of epidermal growth factor, fibroblast growth factor, human transferrin, insulin, selenite, and hydrocortisone.
- the oocyte is cultured in a culture medium comprising the inorganic salts, amino acids, vitamins and other components as set forth in Table 2, and wherein each inorganic salt, amino acid, vitamin and other component is present in an amount of ⁇ 50% (weight/volume), more preferably ⁇ 10% (weight/volume) of the amount specified in Table 2.
- the culture medium consists essentially of the inorganic salts, amino acids, vitamins and other components identified in Table 2.
- the culture medium consists of the inorganic salts, amino acids, vitamins and other components identified in Table 2.
- the invention provides a method for in vitro maturation of immature human oocytes, comprising culturing an immature human oocyte in a culture medium comprising:
- At least one growth factor is at least one growth factor.
- This embodiment is particularly useful when the immature human oocyte is essentially free of cumulus cells.
- the invention provides a method for in vitro maturation of immature human oocytes, comprising culturing an immature human oocyte in a culture medium comprising the inorganic salts, amino acids, vitamins and other components as set forth in Table 2.
- This embodiment is particularly useful when the immature human oocyte is cultured in the presence of cumulus cells.
- the culture medium consists essentially of the inorganic salts, amino acids, vitamins and other components as set forth in Table 2.
- the culture medium consists of the inorganic salts, amino acids, vitamins and other components as set forth in Table 2.
- each inorganic salt, amino acid, vitamin and other component is present in an amount of ⁇ 50%, more preferably ⁇ 10% (weight/volume), of the amount specified in Table 2.
- certain of the components listed in the “other components” sections of Table 1 and 2 may be absent from the medium.
- Components that may be absent include, without limitation, phenol red, penicillin G, streptomycin, and human serum albumin. This may apply wherever reference is made herein to Table 1 or 2.
- FIG. 1 shows fertilization rates of M-I stage oocytes derived from stimulation and ICSI cycles following 6 h, 24 h or 48 h of maturation, respectively.
- FIG. 2 shows cleavage rates of M-I stage oocytes derived from stimulation and ICSI cycles following 6 h, 24 h or 48 h of maturation, respectively.
- FIGS. 3A and 3B show embryos developing to the 8-cell stage (A) and the blastocyst stage (B) following 6 h, 24 h or 48 h of maturation of M-I stage oocytes derived from stimulation and ICSI cycles.
- FIG. 4 shows fertilization rates of GV stage oocytes derived from stimulation and ICSI cycles following 24 h or 48 h of maturation.
- FIG. 5 shows cleavage rates of GV stage oocytes derived from stimulation and ICSI cycles following 24 h or 48 h of maturation.
- FIGS. 6A and 6B show embryos developing to the 8-cell stage (A) and the blastocyst stage (B) following 24 h or 48 h of maturation of GV stage oocytes derived from stimulation and ICSI cycles.
- FIG. 7 shows a blastocyst derived from GV stage oocytes following 24 h of maturation in vitro and illustrates that there is no difference in morphology from oocytes matured in vivo.
- the scale bar indicates 5 ⁇ m length.
- any female human subject who possesses viable oocytes is a candidate for IVM therapy.
- the subject will suffer from some form of infertility.
- the subject may experience normal oocyte production but have an impediment to fertilization, as in, e.g. PCOS or PCOS-like ovaries.
- IVM is also useful in women infertility with endometriosis, blockage of either or both fallopian tubes, etc.
- numerous follicles mature simultaneously in the ovaries, without the appearance of a dominant follicle.
- the subject's menstrual cycle may be regular, irregular or non-existent.
- IVM may be especially useful in women who are susceptible to or suffer from OHSS and are therefore not suitable candidates for traditional in vitro fertilization techniques involving an ovarian stimulation protocol.
- the subject may be an individual who does not suffer from infertility or have any reproductive impediment whatsoever, but for whom an in vitro fertilization method nevertheless remains desirable, as in, e.g. cases of male factor infertility.
- IVM in accordance with the invention does not necessarily involve an ovarian stimulation protocol.
- One of two “ovarian stimulation protocols” is usually used in conventional IVF, a “long protocol” or a “short protocol.”
- the long protocol has two steps. The first step involves pre-treating the subject for two or three weeks with GnRHa to down-regulate pituitary activity. Once pituitary suppression has been achieved, in the second step, HMG or FSH is administered to induce the development of multiple follicles.
- the short protocol involves only the ovarian stimulation step, but not the down-regulation step.
- Ovarian stimulation is required in conventional IVF techniques to permit the oocytes to mature to the M-II stage prior to harvest.
- ovarian stimulation need not be used. That is, the subject need not be pre-treated with GnRHa's, HMG and/or FSH.
- LH Luteinizing Hormone
- an increase in endogenous levels of LH is induced in the subject (also described herein as “priming”). This may be accomplished by e.g. administering to the subject an effective amount of human chorionic gonadotropin (HCG) (Profasi Serono, Oakville, Ontario, Canada) or LH, either of which stimulate endogenous LH production.
- HCG human chorionic gonadotropin
- Baseline concentration of LH in the plasma of premenopausal women is usually about 2-4 mIU/ml, and at midcycle peak may be up to 25-35 mIU/ml.
- the expression “inducing in a female human subject an increase in endogenous luteinizing hormone levels” means increasing the plasma LH concentration above the baseline concentration.
- plasma LH concentration is increased at least 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or 2000% above baseline concentration.
- HCG or LH may vary depending on the patient and the circumstances and may be determined by those of skill in the art.
- Subcutaneous injection is a preferred mode of administration although other modes of administration may be used.
- a single injection of about 5000 to about 20,000 IU of HCG is generally sufficient, with a single dose of about 10,000 IU of HCG being preferred.
- commencement of menstrual bleeding In the case of a subject having a regular menstrual cycle, the time for inducement may be determined relative to the commencement of menstrual bleeding, which is considered “day 0”.
- commencement of menstrual bleeding i.e. the initiation of “day 0”
- progesterone available from e.g. Prometrium; Schering, Pointe-Claire, Quebec, Canada
- intravaginally in an amount of approximately 300 mg/day for 10 days.
- the ovaries are examined by ultrasound to assess the size and the number of follicles.
- the dominant follicle reaches a diameter of at least about 16 mm by day 10-14.
- a dominant follicle is present (e.g. as in normal ovarian function, as in the case of a healthy subject or a subject suffering from e.g. endometriosis or tubal blockage)
- the increase in endogenous LH activity is induced (e.g. by administration of HCG) when the dominant follicle reaches a diameter of about 15-17 mm, preferably at least about 16 mm.
- inducement of an increase in endogenous LH levels preferably occurs when the follicles are less than about 12 mm in diameter.
- Oocytes are generally retrieved from about 32 to about 40 hours after priming, more preferably about 34 to 38 hours after priming and even more preferably about 36 hours after priming.
- Means for retrieving oocytes are known in the art.
- transvaginal ultrasonographically-guided oocyte collection is done using a 17-20 gauge, preferably 19 gauge single-lumen aspiration needle at an aspiration pressure of from about 5 to about 10 kPa, preferably about 7.5 kPa.
- Immature human oocyte means a human oocyte that has not yet reached metaphase-II (M-II). As discussed previously, metaphase-II is characterized by exclusion of one polar body from the cytoplasm. Immature human oocytes used in the invention are typically at the germinal vesicle (GV) or metaphase-I (M-I) stage.
- GV germinal vesicle
- M-I metaphase-I
- Oocytes may be cultured with their cumulus intact, in a form known as a “cumulus-oocyte-complex” (COC), or oocytes may be partially or entirely denuded from cumulus cells.
- COC can be stripped with 85 IU/ml hyaluronidase in HEPES buffered medium and mechanically pipetted until oocytes are denuded.
- An oocyte that is “essentially free of cumulus cells” is an oocyte that is associated with sufficiently few cumulus cells that the cumulus cells have no detectable physiological effect on the oocyte.
- Culture conditions for human oocytes are known in the art and are not critical to the invention. Suitable culture conditions include e.g. culturing the oocytes at 37° C. in an atmosphere of 95% air and 5% CO 2 at high humidity, e.g. 100% humidity. A “triple gas” atmosphere of 5% O 2 , 5% CO 2 and 90% N 2 may also be used. Mineral oil may be overlaid on the medium to control evaporation and/or temperature. Oocytes are typically cultured in a well containing 1 ml of culture medium or more, or may be cultured in 10 ⁇ l of culture medium or less in a droplet in a culture dish.
- Immature oocytes may be cultured for e.g. about 24 to about 48 hours. Because about 60% of oocytes typically reach maturity (M-II) after 24 hours of culture, when oocytes are cultured with an intact or partially intact cumulus, they may be denuded at about 24 hours and observed under a microscope to assess oocyte maturity. The physiological influence of the cumulus cells on the oocyte persists for only about the first 12 hours in culture, so denuding the oocyte after about 24 hours in culture has little or no negative effect.
- oocytes Due to the influence of cumulus cells on the maturation of oocytes, somewhat different culture media may be used when the oocytes are cultured for the first 12-24 hours in the presence of cumulus cells. This can occur e.g. when the oocytes are cultured as COCs or partially denuded or when denuded oocytes are co-cultured in the presence of cumulus cells.
- the inorganic salts, essential and non-essential amino acids, and energy source in the IVM medium are not critical to the invention. Suitable salts, amino acids and energy sources are known in the art.
- Inorganic salts are provided to buffer the pH of the medium within a range preferably of about 7.2-7.4 and to maintain correct osmolarity of the medium with the oocytes.
- Suitable inorganic salts and concentrations thereof as used in culture media are known in the art.
- Typical inorganic salts include CaCl 2 , KCl, MgSO 4 , NaCl, NaHCO 3 , NaH 2 PO4.H 2 O, FE(NO 3 ) 3 .9H 2 O, KH 2 PO 4 , Na.acetate, Na 2 H 2 PO 4 , etc.
- the IVM medium contains at least one amino acid or source thereof.
- at least the essential amino acids, or sources thereof are included in IVM medium.
- “Essential” amino acids are those amino acids not synthesized in the oocyte and that are essential for protein synthesis.
- the essential amino acids are generally considered to be isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine.
- a commercial amino acid mixture, containing the eight essential amino acids as well as some or all of the remaining non-essential amino acids may conveniently be used.
- Non-naturally occuring amino acids or amino acid derivatives as are known in the art may also be included in the IVM medium.
- the IVM medium of the invention comprises alanine, arginine, asparagine, aspartic acid, cystine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
- the IVM medium preferably contains vitamins as are known in the art for inclusion in culture media.
- Vitamins that may be included in the media include, without limitation, vitamins A1 (retinol), A2 (an alternative form of retinol), B1 (thiamine), B2 (riboflavin), B6 (pyridoxine), B9 (folic acid), B12 (cyanocobalamin), B17, C (ascorbic acid), D, D2 (calciferol), D3 (cholecalciferol), E (tocopherol), H (biotin), K, K1 (phylloquinone), K2, K3 (menadione), P, etc.
- a particularly preferred combination of vitamins comprises biotin, D-Ca pantothenate, choline chloride, folic acid, i-inositol, nicotinamide, pyroxidal HCl, riboflavin, and thiamine-HCl.
- the IVM medium contains at least one growth factor (GF).
- growth factors are proteins that bind to receptors on the cell surface, with the primary result of activating cellular proliferation and/or differentiation. Many growth factors are quite versatile, stimulating cellular division in numerous different cell types, while others are specific to a particular cell-type. Growth factors may act on membranes of oocytes to positively stimulate oocyte maturation (Maruo et al., 1993; Goud et al., 1998).
- Useful growth factors in the context of the present invention include those selected from the following growth factor superfamilies: epidermal growth factor (EGF) family; platelet derived growth factor (PDGF) family; insulin-like growth factor (IGF) family; nerve growth factor (NGF) family; transforming growth factor (TGF) family; fibroblast growth factor (FGF) family; hepatocyte growth factor (HGF) family; hemapoietic growth factors; and cytokines.
- EGF epidermal growth factor
- PDGF platelet derived growth factor
- IGF insulin-like growth factor
- NGF nerve growth factor
- TGF transforming growth factor
- FGF fibroblast growth factor
- HGF hepatocyte growth factor
- hemapoietic growth factors and cytokines.
- the growth factors in IVM medium comprise fibroblast growth factor (FGF) and epidermal growth factor (EGF).
- FGF and EGF are readily available from commercial sources, and may be purchased together in a cell growth supplement.
- the IVM medium preferably contains from 0.0001 mg/L to 0.001 mg/L FGF, more preferably from 0.0002 mg/L to 0.001 mg/L FGF, and even more preferably about 0.0005 mg/L FGF.
- the IVM medium preferably contains from 0.0001 to 0.01 mg/L EGF, more preferably from 0.0005 mg/L to 0.002 mg/L EGF, and even more preferably about 0.001 mg/L EGF.
- the IVM medium preferably also contains at least one hormone.
- Preferred hormones include insulin, estradiol, follicle-stimulating hormone (FSH) and luteinizing hormone (LH).
- FSH follicle-stimulating hormone
- LH luteinizing hormone
- Human menopausal gonadotropin (HMG) can generally be substituted for FSH
- human chorionic gonadotropin (HCG) can generally be substituted for LH.
- the IVM medium comprises insulin. Normally, insulin is involved in energy metabolism and amino acid transportation into cells.
- the IVM medium preferably contains from 0.5 mg/L to 50 mg/L insulin, more preferably 0.25 mg/L to 10 mg/L insulin, and even more preferably about 5 mg/L insulin.
- the IVM medium further comprises estradiol, LH and FSH.
- the amount of estradiol is preferably about 1 ⁇ g/ml ⁇ 10%, ⁇ 20%, ⁇ 30%, ⁇ 40% or ⁇ 50%.
- the amount of each of FSH and LH is preferably about 0.08 IU/ml ⁇ 10%, ⁇ 20%, ⁇ 30%, ⁇ 40% or ⁇ 50%.
- the IVM medium also contains human transferrin (TF).
- TF is a 75 kDa glycoprotein containing 679 amino acids and two glycan chains. For each, the last residue is a sialic (N-acetyl neuraminic) acid, which can be hydrolyzed by neuraminidase. TF not only transports iron in all extracellular fluid but also exerts many additional properties, including cell growth stimulation (Gross-Weege et al., 1986).
- the IVM medium preferably contains from from 5 mg/L to 500 mg/L TF, more preferably from 25 mg/L to 100 mg/L TF, and even more preferably about 50 mg/L TF.
- growth factors, hormones and transferrins discussed above may be naturally occurring, synthetic or recombinant, and encompass biologically active fragments, variants, derivatives and homologs of these substances that retain at least some of the biological activity of the naturally-occurring, synthetic or recombinantly-produced substances.
- a protein may include one or more amino acid insertions, deletions, substitutions or modifications without substantially reducing its biological activity.
- energy source is not critical to the invention, and may be e.g. glucose, sodium pyruvate, lactate, or a mixture of some or all of these energy sources.
- the IVM medium may additional contain additional components such as selenite or selenium and hydrocortisone.
- Buffers for controlling the pH of the medium may be added, such as Hepes, as may be pH indicators such as phenol red.
- Antibiotics such as penicillin G and streptomycin may be added to the IVM medium to prevent contamination.
- Synthetic Serum Supplement may be included in the IVM medium as a source of protein for the oocyte and to prevent cells from adhering to glassware during in vitro culture.
- SSS has the advantage of having received regulatory approval for human IVF, and is commercially available.
- the IVM medium includes the components set forth in Table 1.
- the absolute and relative quantities of each component can vary substantially, for example by up to ⁇ 10%, ⁇ 20%, ⁇ 30%, ⁇ 40%, ⁇ 50% weight by volume.
- Culture media as described above are also useful for culturing immature oocytes that have a cumulus that is intact or at least partially intact, and/or that are co-cultured with cumulus cells. But the presence of the cumulus cells in the first 12-24 hours of culture eliminates the need for growth factors, so the culture medium need not contain growth factors such as fibroblast growth factor, epidermal growth factor, etc.
- the culture medium described in the preceding section preferably contains human transferrin, insulin, selenite and hydrocortisone, these are not of substantial benefit when culturing oocytes in the presence of cumulus cells, and are therefore generally absent from the culture medium.
- complex culture media such as tissue culture media (e.g. TCM-199)
- tissue culture media e.g. TCM-199
- complex culture media such as TCM-199
- oocytes that are essentially free of cumulus cells are cultured in media that consists of, or consists essentially of the components listed in Table 1
- oocytes that are cultured in the presence of cumulus cells are cultured in media that consists of or consists essentially of the components listed in Table 2.
- fertilization is accomplished by intracytoplasmic sperm injection (ICSI).
- ICSI intracytoplasmic sperm injection
- spermatozoa can be prepared by gradient separation by centrifugation or by “swim-up” followed by washing with a suitable medium such as HTF supplemented with 10% SSS.
- a single spermatozoon may be injected into an M-II oocyte.
- the oocyte may be transferred to e.g. fertilization medium supplemented with 10% SSS in a tissue culture dish. Fertilization can be detected by the appearance of two distinct pronuclei and two polar bodies approximately 16-18 hours after ICSI.
- fertilized oocytes may be cultured in fertilization media until about 72 hours after ICSI and then transferred to e.g. embryo developmental medium (Vitrolife, Göteborg, Sweden) in a tissue culture dish under mineral oil for an additional 48 hours.
- embryo developmental medium e.g. embryo developmental medium (Vitrolife, Göteborg, Sweden) in a tissue culture dish under mineral oil for an additional 48 hours.
- a total of 521 immature oocytes (213 oocytes at GV stage and 308 oocytes at M-I stage) were collected from 183 women (age range between 28 to 40 years) who underwent controlled ovarian stimulation and ICSI cycles under informed consent.
- Ovarian stimulation was performed with GnRHa, HMG or FSH and HCG using standard protocols.
- oocyte retrieval was carried out using an ultrasound-guided trans-vaginal probe.
- the collected COC were cultured in human tubal fluid medium (HTF; Irvine Scientific) supplemented with 10% synthetic serum substitute (SSS; Irvine Scientific) and placed in a 5% CO 2 incubator (37° C. with high humidity) for at least 1 h before they were stripped from their cumulus cells.
- HIF human tubal fluid medium
- SSS synthetic serum substitute
- COC were stripped with 85 IU/ml hyaluronidase in HTF medium and mechanical pipetting. All oocytes were completely denuded from their cumulus cells. Denuded oocytes were observed under an inverted microscope to assess for oocyte maturity. The mature oocytes were determined by the presence of a first polar body extrusion (1PB). The oocytes with a visual polar body (M-II) were provided for clinical application to perform the ICSI procedure immediately. Immature oocytes were defined by the absence of 1PB and then identified as GV or M-I stages depending upon whether the oocytes contained a visible GV in the cytoplasm under microscope. Immature oocytes without a GV in the cytoplasm were considered as M-I stage. These immature oocytes were used for in vitro maturation.
- PB first polar body extrusion
- M-II visual polar body
- the immature oocytes were cultured in HTF medium supplemented with 10% SSS (Irvine Scientific) placed in a 37° C. and 5% CO 2 incubator before transferred to the maturation medium.
- the time of the immature oocytes were transferred to maturation medium is defined as the starting point for maturation in culture. Normally, it takes approximately 3-4 h from oocyte retrieval to the start of oocyte maturation.
- the immature oocytes were washed at least twice in maturation medium.
- the immature oocytes were cultured in an organ tissue culture dish (60 ⁇ 15 mm; Falcon) containing 1 ml of maturation medium (TCM-199 or IVM-medium) at 37° C. in an atmosphere of 5% CO 2 and 95% air with high humidity.
- TCM-199 or IVM-medium 1 ml of maturation medium
- a maximum of 5 oocytes (1 to 5) were placed in each organ tissue culture dish.
- the maturity of the oocytes was determined under the microscope at 6 h for M-I stage oocytes and then at 24 h and 48 h for both M-I and GV stage oocytes.
- ICSI Intracytoplasmic Sperm Injection
- sperm for ICSI were prepared by PureSperm (Nidacon, Sweden) gradient separation (45%, 70% and 90%) at 560 g for 20 min. Following gradient separation, the sperm pellet was washed twice (200 g) with 2 ml of HTF medium (Irvine Scientific) supplemented with 10% SSS.
- each oocyte was transferred into a 20 ⁇ l droplet of HTF medium supplemented with 10% SSS in a tissue culture dish (35 ⁇ 10 mm; Falcon) under mineral oil (Sigma).
- Fertilization was assessed 16-18 h after ICSI for the appearance of two distinct pronuclei and two polar bodies. Since the oocytes were not matured at the same time following culture, the sperm samples were kept in 5 ml Falcon tubes (tight tube cap) containing 0.5-1.0 ml HTF medium supplemented with 10% SSS at room temperature for further insemination by ICSI at 6 h, 24 h or 48 h, respectively.
- Fertilized oocytes (with two pronuclei) were cultured in HTF medium supplemented with 10% SSS until 72 h after ICSI, and then each embryo was transferred into a 20 ⁇ l droplet of G2.2 medium (VitroLife, Sweden) in a tissue culture dish (35 ⁇ 10 mm; Falcon) under mineral oil (Sigma) further culture for 48 h. Embryo development was recorded at 24 h intervals following culture until day 5 (120 h). All embryos were destroyed on day 5 following consent instruction from the patients.
- FIG. 1 shows the fertilization rates of the oocytes matured in the IVM-medium or TCM-199 at 6 h (93.8% vs. 90.1%), 24 h (89.2% vs. 84.9%) and 48 h (50.0% vs. 0.0%), respectively.
- the treatment cycles was initiated by the administration of intra-vaginal progesterone (Prometrium; Schering, Pointe-Clair, Quebec, Canada) in a dose of 300 mg daily for 10 days. Withdrawal bleeding occurred within 3 days after the last dose.
- intra-vaginal progesterone Prometrium; Schering, Pointe-Clair, Quebec, Canada
- Oocyte retrieval was performed on day 10 to 14 of the cycle. Before oocyte collection 36 hours, the patients were given s.c. 10,000 IU of human chorionic gonadotropin (HCG) for priming.
- HCG human chorionic gonadotropin
- Transvaginal ultrasound-guided oocyte collection was performed using a specially designed 19 G single-lumen aspiration needle (Cook, Australia) with an aspiration pressure of 7.5 kPa. Aspiration of all small follicles was performed under local anaesthesia. Oocytes were collected in 10 ml culture tubes (Falcon, USA) containing 2.0 ml warm 0.9% saline contained with 2 IU/ml heparin (Baxter, Toronto, Ontario, Canada).
- COCs cumulus-oocyte complexes
- the mature oocytes (metaphase-II stage) were inseminated by ICSI. Fertilization was assessed 18 h after ICSI for the appearance of two distinct pronuclei and two polar bodies. Embryo transfer was performed on day 2 or 3 after ICSI. Since the oocytes were inseminated either 24 or 48 hours following culture, the developmental stages of embryos were variable.
- estradiol Estace; Roberts Pharmaceutical, Mississauga, Canada
- estradiol Estace; Roberts Pharmaceutical, Mississauga, Canada
- the endometrial thickness on the day of oocyte retrieval was ⁇ 4 mm, a 10 mg dose was administered; if it was >6 mm, a 6 mg dose was given.
- Luteal support was provided by 400 mg intravaginal progesterone (Prometrium) twice daily for 16 days starting from the day after oocyte collection.
- Inorganic Salt CaCl 2 200.0000 KCl 400.0000 MgSO 4 98.0000 NaCl 6800.0000 NaHCO 3 1250.0000 NaH 2 PO 4 .H 2 O 125.0000 Amino Acids: L-Alanine 8.9000 L-Arginine 126.4000 L-Asparagine 13.2000 L-Aspartic Acid 13.3000 L-Cystine 24.0000 L-Glutamic Acid 14.7000 L-Glutamine 292.0000 Glycine 7.5000 L-Histidine.HCI.H 2 O 42.0000 L-Isoleucine 52.4000 L-Leucine 52.4000 L-Lysine.HCI 72.5000 L-Methionine 15.1000 L-Phenylalanine 33.0000 L-Proline 11.5000 L-Serine 10.5000 L-Threonine 47.6000 L-Tryptophan 10.2000 L-Tyrosine 36.0000 L-Valine 46.8000 Vitamins: Biotin 0.0100 D-Ca Pant
- Pincus G, Enzmann E V The comparative behaviour of mammalian eggs in vivo and in vitro. 1. The activation of ovarian eggs. J Exp Med 1935; 62:665-675.
Abstract
The invention provides methods for in vitro maturation of immature human oocytes. In one aspect, the invention provides a method for in vitro maturation of immature human oocytes, comprising the steps of: (a) inducing in a female human subject an increase in endogenous luteinizing hormone levels, said subject having not undergone an ovarian stimulation protocol prior to said inducing step; (b) obtaining from said subject an immature oocyte; and (c) culturing said oocyte until maturity.
Description
- This application claims the benefit of U.S. provisional patent application No. 60/362,395, filed Mar. 8, 2002, which is incorporated herein by reference.
- The present invention relates to the field of assisted reproductive technology (ART), particularly to methods for culturing immature human oocytes so that they mature to metaphase-II stage capable of being fertilized and forming a viable embryo.
- Since the first successful human pregnancy by way of in vitro fertilization (IVF) was achieved in 1978, ART has helped thousands of thousands of women to overcome infertility problem
- Normally, during the follicular phase of a woman's reproductive cycle, only a single follicle grows to the preovulatory stage and releases its oocyte for potential fertilization. However, current IVF treatment requires that multiple oocytes be harvested. Therefore, women are normally pre-treated for approximately two or three weeks with gonadotropin releasing hormone agonists (GnRHa). After pituitary suppression has been achieved, human menopausal gonadotropin (HMG) or purified follicle-stimulating hormone (FSH) is administered to induce development of multiple follicles. Once two leading follicles have reached a diameter of at least about 18-20 mm, the patient is typically given 5,000 to 10,000 IU of human chorionic gonadotropin (HCG) to trigger final oocyte maturation. Approximately 36 hours later, a large number of oocytes (10 on average) are collected.
- The harvested oocyte is then fertilized with the sperm. In many instances of human infertility, fertilization is effected by intracytoplasmic sperm injection (ICSI). Successful IVF generally requires that the oocyte have reached the metaphase-II (M-II) stage before fertilization is attempted. A M-II oocyte is ready to accept the sperm and be fertilized. Metaphase-II is characterized by exclusion of one polar body from the cytoplasm, and is typically detected by visual identification under a microscope.
- The harvested oocyte remains surrounded by cumulus cells that support and promote the final maturation and development of the oocyte. The cumulus cells interfere with ICSI and obscure microscopic observation of the oocyte. Therefore, prior to ICSI, it is necessary to denude the oocyte of cumulus cells to permit determination of the level of oocyte maturity. This is generally accomplished by enzymatic and mechanical stripping of the cumulus cells.
- Unfortunately, not all of the oocytes have matured to the M-II stage when they are harvested in ovarian stimulation. As a result of follicular asynchrony, in general, at least 10-15% of oocytes are still immature, and are at the germinal vesicle (GV) or metaphase-I (M-I) stage, at the time of harvest. These immature oocytes, denuded of cumulus cells, are discarded at most IVF clinics.
- It would be desirable if these immature GV and M-I stage oocytes, presently wasted, could be brought to maturity, in vitro, and then successfully fertilized. Moreover, it would be desirable to be able to harvest immature oocytes from unstimulated ovaries and mature them to the M-II stage by way of in vitro maturation (IVM), thereby avoiding the entire regimen of treatment of women with GnRHa and stimulation of ovaries with gonadotropin. These procedures of ovarian stimulation require frequent blood sampling and ultrasound monitoring, at considerable cost. Moreover, current IVF treatment causes substantial discomfort and, in some cases, results in ovarian hyperstimulation syndrome (OHSS) and/or even death. There is also anxiety that the long-term effects of repeated ovarian stimulation may increase the risk of ovarian, endometrial and breast cancer.
- Thus, there is substantial interest in developing IVF techniques that rely upon in vitro maturation of oocytes, rather than harvesting mature oocytes by ovarian stimulation. But only a few live births have been obtained from cumulus-denuded oocytes matured in vitro (Nagy et al., 996; Jaroudi et al., 1997; Edirisinghe et al., 1997). Clearly then, there is an opportunity for improving the maturational and developmental competence of cumulus-denuded immature oocytes matured in vitro.
- Recovery of immature oocytes followed by IVM of these immature oocytes is a potentially useful treatment for women with polycystic ovarian syndrome (PCOS) related infertility. PCOS is one of the most common reproductive disorders in women of childbearing age. It has a heterogeneous presentation, which is clinically characterized by anovulation and hyperandrogenism, and on pelvic ultrasound examination shows numerous antral follicles within the ovaries (Adams et al., 1986). There is a significantly higher risk of OHSS in these group of women compared with normal ovaries (MacDougall et al., 1993). Although the first pregnancy and live birth from immature oocytes retrieved from women with PCOS followed by IVM has been reported, the success rates are still low because oocyte maturation rates are relatively low.
- Some attempts at developing IVM media and methods have involved the use of human follicular fluid, in an effort to mimic the conditions within the follicle. In view of the risk of transmission of disease, allergic reaction, etc., use of biological fluids in IVM techniques is prohibited in most countries. Instead, the use of a “chemically-defined medium”, wherein the chemical composition of all of the ingredients is known, is preferred.
- Mammalian immature oocytes removed from antral follicles will resume meiosis spontaneously without hormonal stimulation when cultured in a simple medium (Pincus & Enamann, 1935). However, oocyte maturation in vitro is profoundly affected by culture conditions.
- Most attempts at human IVM have employed tissue culture medium 199 (TCM-199) as the maturation medium for immature oocytes (Trounson et al., 1994; Barnes et al., 1995; Chian et al., 1999a, 1999b, 2000, 2001). TCM-199 is a complex medium that contains many components, and was designed for in vitro culture of somatic cells, not germ cells. Notably, TCM-199 does not contain any growth factors. TCM-199 was first used for sheep oocyte IVM (Moor & Trounson, 1977), and has since been used routinely for bovine oocyte IVM (Brackett & Zuelke, 1993).
- Although different culture media have been used to mature human oocytes (Shea et al., 1975; Cha et al., 1991; Trounson et al., 1994; Cha & Chian, 1998; Chian et al., 1999a, 1999b, 2000), it has been indicated recently that there is no apparent benefit for oocyte maturation, fertilization and embryonic development from use of these culture media (Trounson et al., 1998; 2001).
- Thus, there remains a need for methods for maturing human oocytes in vitro.
- The present invention provides in vitro maturation methods that are useful for culturing immature human oocytes to maturity.
- In a broad aspect, the invention provides a method for in vitro maturation of immature human oocytes, comprising the steps of:
- (a) inducing in a female human subject an increase in endogenous luteinizing hormone levels, the subject having not undergone an ovarian stimulation protocol prior to the inducing step;
- (b) obtaining from the subject an immature oocyte; and
- (c) culturing the oocyte until maturity.
- Preferably, prior to step (a), the subject has not been treated with a gonadotrophin releasing hormone agonist, human menopausal gonadotrophin, HCG, or follicle stimulating hormone (FSH).
- In one embodiment, step (a) comprises administering to the subject HCG or luteinizing hormone (LH), or both. In a preferred embodiment, HCG is administered in an amount of about 5000 to about 20,000 IU, more preferably about 10,000 IU.
- The immature human oocyte is preferably an M-I stage or GV stage ooctye. The oocyte preferably is cultured until it reaches M-II.
- In one embodiment, the immature human oocyte is essentially free of cumulus cells and is cultured in a culture medium comprising:
- at least one inorganic salt;
- essential amino acids or a source thereof;
- an energy source; and
- at least one growth factor.
- In one embodiment, the growth factor is fibroblast growth factor or epidermal growth factor, or both.
- In one embodiment, the culture medium further comprises at least one hormone, e.g. insulin. In a preferred embodiment, the culture medium comprises from 0.5 mg/L to 50 mg/L insulin.
- In one embodiment, the culture medium further comprises human transferrin, preferably from 5 mg/L to 500 mg/L human transferrin.
- In one embodiment, the culture medium comprises from 0.0001 mg/L to 0.001 mg/L fibroblast growth factor.
- In one embodiment, the culture medium comprises from 0.0001 to 0.01 mg/L epidermal growth factor.
- In one embodiment, the culture medium comprises one or more vitamins, preferably biotin, D-Ca pantothenate, choline chloride, folic acid, i-inositol, nicotinamide, pyroxidal-HCl, riboflavin, and thiamine-HCl.
- The culture medium may further comprise hydrocortisone or selenite, or both.
- Preferred inorganic salts include CaCl2, KCl, MgSO4, NaCl, NaHCO3 and NaH2PO4-H2O.
- Preferred energy sources include D-glucose and sodium pyruvate, or both D-glucose and sodium pyruvate.
- In one embodiment, the amino acids comprise alanine, arginine, asparagine, aspartic acid, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
- In one embodiment, the culture medium comprises the inorganic salts, amino acids, vitamins and other components as set forth in Table 1, and wherein each inorganic salt, amino acid, vitamin and other component is present in an amount of ±50% (weight/volume), more preferably ±10% (weight/volume), of the amount specified in Table 1.
- In another embodiment, the oocyte has a cumulus that is intact or at least partially intact, and/or the oocyte is cultured in the presence of cumulus cells.
- In this embodiment, the oocyte may be cultured in a culture medium that is essentially free of one or more of epidermal growth factor, fibroblast growth factor, human transferrin, insulin, selenite, and hydrocortisone.
- In one embodiment the oocyte is cultured in a culture medium comprising the inorganic salts, amino acids, vitamins and other components as set forth in Table 2, and wherein each inorganic salt, amino acid, vitamin and other component is present in an amount of ±50% (weight/volume), more preferably ±10% (weight/volume) of the amount specified in Table 2.
- In one embodiment, the culture medium consists essentially of the inorganic salts, amino acids, vitamins and other components identified in Table 2.
- In another embodiment, the culture medium consists of the inorganic salts, amino acids, vitamins and other components identified in Table 2.
- In another embodiment, the invention provides a method for in vitro maturation of immature human oocytes, comprising culturing an immature human oocyte in a culture medium comprising:
- at least one inorganic salt;
- essential amino acids or a source thereof;
- an energy source; and
- at least one growth factor.
- This embodiment is particularly useful when the immature human oocyte is essentially free of cumulus cells.
- In another embodiment, the invention provides a method for in vitro maturation of immature human oocytes, comprising culturing an immature human oocyte in a culture medium comprising the inorganic salts, amino acids, vitamins and other components as set forth in Table 2. This embodiment is particularly useful when the immature human oocyte is cultured in the presence of cumulus cells.
- In a preferred embodiment, the culture medium consists essentially of the inorganic salts, amino acids, vitamins and other components as set forth in Table 2.
- In another preferred embodiment, the culture medium consists of the inorganic salts, amino acids, vitamins and other components as set forth in Table 2.
- In particularly preferred embodiments, each inorganic salt, amino acid, vitamin and other component is present in an amount of ±50%, more preferably ±10% (weight/volume), of the amount specified in Table 2.
- In another embodiment, certain of the components listed in the “other components” sections of Table 1 and 2 may be absent from the medium. Components that may be absent include, without limitation, phenol red, penicillin G, streptomycin, and human serum albumin. This may apply wherever reference is made herein to Table 1 or 2.
- By the methods of the invention, increases may be obtained in one or more of:
- (a) total number of oocytes reaching M-II stage;
- (b) total number of oocytes fertilized;
- (c) total number of embryos developing to the 8-cell stage; and
- (d) total number of blastocysts formed; as measured at 6 hours, 12 hours, 24 hours or 48 hours after commencement of maturation in IVM media in accordance with the invention. Increases in one or more of (a) to (d) of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or 500% may be advantageously obtained.
-
FIG. 1 shows fertilization rates of M-I stage oocytes derived from stimulation and ICSI cycles following 6 h, 24 h or 48 h of maturation, respectively. -
FIG. 2 shows cleavage rates of M-I stage oocytes derived from stimulation and ICSI cycles following 6 h, 24 h or 48 h of maturation, respectively. -
FIGS. 3A and 3B show embryos developing to the 8-cell stage (A) and the blastocyst stage (B) following 6 h, 24 h or 48 h of maturation of M-I stage oocytes derived from stimulation and ICSI cycles. -
FIG. 4 shows fertilization rates of GV stage oocytes derived from stimulation and ICSI cycles following 24 h or 48 h of maturation. -
FIG. 5 shows cleavage rates of GV stage oocytes derived from stimulation and ICSI cycles following 24 h or 48 h of maturation. -
FIGS. 6A and 6B show embryos developing to the 8-cell stage (A) and the blastocyst stage (B) following 24 h or 48 h of maturation of GV stage oocytes derived from stimulation and ICSI cycles. -
FIG. 7 shows a blastocyst derived from GV stage oocytes following 24 h of maturation in vitro and illustrates that there is no difference in morphology from oocytes matured in vivo. The scale bar indicates 5 μm length. - Candidates for IVM
- Any female human subject who possesses viable oocytes is a candidate for IVM therapy. Typically, the subject will suffer from some form of infertility. For instance, the subject may experience normal oocyte production but have an impediment to fertilization, as in, e.g. PCOS or PCOS-like ovaries. IVM is also useful in women infertility with endometriosis, blockage of either or both fallopian tubes, etc. In typical PCOS, numerous follicles mature simultaneously in the ovaries, without the appearance of a dominant follicle. The subject's menstrual cycle may be regular, irregular or non-existent. IVM may be especially useful in women who are susceptible to or suffer from OHSS and are therefore not suitable candidates for traditional in vitro fertilization techniques involving an ovarian stimulation protocol.
- Alternatively, the subject may be an individual who does not suffer from infertility or have any reproductive impediment whatsoever, but for whom an in vitro fertilization method nevertheless remains desirable, as in, e.g. cases of male factor infertility.
- Absence of Ovarian Stimulation Protocol
- Unlike in conventional in vitro fertilization methods, IVM in accordance with the invention does not necessarily involve an ovarian stimulation protocol. One of two “ovarian stimulation protocols” is usually used in conventional IVF, a “long protocol” or a “short protocol.” The long protocol has two steps. The first step involves pre-treating the subject for two or three weeks with GnRHa to down-regulate pituitary activity. Once pituitary suppression has been achieved, in the second step, HMG or FSH is administered to induce the development of multiple follicles. The short protocol involves only the ovarian stimulation step, but not the down-regulation step.
- Ovarian stimulation is required in conventional IVF techniques to permit the oocytes to mature to the M-II stage prior to harvest. In the present invention, wherein oocytes are harvested at an immature stage, i.e. prior to reaching the M-II stage, ovarian stimulation need not be used. That is, the subject need not be pre-treated with GnRHa's, HMG and/or FSH.
- Increasing Endogenous Levels of Luteinizing Hormone (LH)
- Prior to retrieving immature oocytes, an increase in endogenous levels of LH is induced in the subject (also described herein as “priming”). This may be accomplished by e.g. administering to the subject an effective amount of human chorionic gonadotropin (HCG) (Profasi Serono, Oakville, Ontario, Canada) or LH, either of which stimulate endogenous LH production. Baseline concentration of LH in the plasma of premenopausal women is usually about 2-4 mIU/ml, and at midcycle peak may be up to 25-35 mIU/ml. The expression “inducing in a female human subject an increase in endogenous luteinizing hormone levels” means increasing the plasma LH concentration above the baseline concentration. Preferably plasma LH concentration is increased at least 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or 2000% above baseline concentration.
- The dosage and mode of administration of HCG or LH may vary depending on the patient and the circumstances and may be determined by those of skill in the art. Subcutaneous injection is a preferred mode of administration although other modes of administration may be used. A single injection of about 5000 to about 20,000 IU of HCG is generally sufficient, with a single dose of about 10,000 IU of HCG being preferred.
- In the case of a subject having a regular menstrual cycle, the time for inducement may be determined relative to the commencement of menstrual bleeding, which is considered “
day 0”. In a subject having an irregular or absent menstrual cycle, commencement of menstrual bleeding (i.e. the initiation of “day 0”) can be achieved by procedures known in the art, e.g. by the administration of progesterone (available from e.g. Prometrium; Schering, Pointe-Claire, Quebec, Canada), e.g. intravaginally, in an amount of approximately 300 mg/day for 10 days. - Typically, on day three and day eight, the ovaries are examined by ultrasound to assess the size and the number of follicles. Usually, the dominant follicle reaches a diameter of at least about 16 mm by day 10-14.
- In instances where a dominant follicle is present (e.g. as in normal ovarian function, as in the case of a healthy subject or a subject suffering from e.g. endometriosis or tubal blockage) the increase in endogenous LH activity is induced (e.g. by administration of HCG) when the dominant follicle reaches a diameter of about 15-17 mm, preferably at least about 16 mm.
- If a dominant follicle is not present, as in cases of e.g. PCOS, inducement of an increase in endogenous LH levels preferably occurs when the follicles are less than about 12 mm in diameter.
- Retrieval of Immature Oocytes
- Oocytes are generally retrieved from about 32 to about 40 hours after priming, more preferably about 34 to 38 hours after priming and even more preferably about 36 hours after priming. Means for retrieving oocytes are known in the art. In one embodiment, transvaginal ultrasonographically-guided oocyte collection is done using a 17-20 gauge, preferably 19 gauge single-lumen aspiration needle at an aspiration pressure of from about 5 to about 10 kPa, preferably about 7.5 kPa.
- Culture of Immature Oocytes
- As used herein, the term “immature human oocyte” means a human oocyte that has not yet reached metaphase-II (M-II). As discussed previously, metaphase-II is characterized by exclusion of one polar body from the cytoplasm. Immature human oocytes used in the invention are typically at the germinal vesicle (GV) or metaphase-I (M-I) stage.
- Oocytes may be cultured with their cumulus intact, in a form known as a “cumulus-oocyte-complex” (COC), or oocytes may be partially or entirely denuded from cumulus cells. COC can be stripped with 85 IU/ml hyaluronidase in HEPES buffered medium and mechanically pipetted until oocytes are denuded. An oocyte that is “essentially free of cumulus cells” is an oocyte that is associated with sufficiently few cumulus cells that the cumulus cells have no detectable physiological effect on the oocyte.
- Culture conditions for human oocytes are known in the art and are not critical to the invention. Suitable culture conditions include e.g. culturing the oocytes at 37° C. in an atmosphere of 95% air and 5% CO2 at high humidity, e.g. 100% humidity. A “triple gas” atmosphere of 5% O2, 5% CO2 and 90% N2 may also be used. Mineral oil may be overlaid on the medium to control evaporation and/or temperature. Oocytes are typically cultured in a well containing 1 ml of culture medium or more, or may be cultured in 10 μl of culture medium or less in a droplet in a culture dish.
- Immature oocytes may be cultured for e.g. about 24 to about 48 hours. Because about 60% of oocytes typically reach maturity (M-II) after 24 hours of culture, when oocytes are cultured with an intact or partially intact cumulus, they may be denuded at about 24 hours and observed under a microscope to assess oocyte maturity. The physiological influence of the cumulus cells on the oocyte persists for only about the first 12 hours in culture, so denuding the oocyte after about 24 hours in culture has little or no negative effect.
- Culture Medium for Oocytes Essentially Free of Cumulus Cells
- Due to the influence of cumulus cells on the maturation of oocytes, somewhat different culture media may be used when the oocytes are cultured for the first 12-24 hours in the presence of cumulus cells. This can occur e.g. when the oocytes are cultured as COCs or partially denuded or when denuded oocytes are co-cultured in the presence of cumulus cells.
- The inorganic salts, essential and non-essential amino acids, and energy source in the IVM medium are not critical to the invention. Suitable salts, amino acids and energy sources are known in the art.
- Inorganic salts are provided to buffer the pH of the medium within a range preferably of about 7.2-7.4 and to maintain correct osmolarity of the medium with the oocytes. Suitable inorganic salts and concentrations thereof as used in culture media are known in the art. Typical inorganic salts include CaCl2, KCl, MgSO4, NaCl, NaHCO3, NaH2PO4.H2O, FE(NO3)3.9H2O, KH2PO4, Na.acetate, Na2H2PO4, etc.
- The IVM medium contains at least one amino acid or source thereof. Preferably, at least the essential amino acids, or sources thereof, are included in IVM medium. “Essential” amino acids are those amino acids not synthesized in the oocyte and that are essential for protein synthesis. The essential amino acids are generally considered to be isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. A commercial amino acid mixture, containing the eight essential amino acids as well as some or all of the remaining non-essential amino acids may conveniently be used. Non-naturally occuring amino acids or amino acid derivatives as are known in the art may also be included in the IVM medium. In a particularly preferred embodiment, the IVM medium of the invention comprises alanine, arginine, asparagine, aspartic acid, cystine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
- The IVM medium preferably contains vitamins as are known in the art for inclusion in culture media. Vitamins that may be included in the media include, without limitation, vitamins A1 (retinol), A2 (an alternative form of retinol), B1 (thiamine), B2 (riboflavin), B6 (pyridoxine), B9 (folic acid), B12 (cyanocobalamin), B17, C (ascorbic acid), D, D2 (calciferol), D3 (cholecalciferol), E (tocopherol), H (biotin), K, K1 (phylloquinone), K2, K3 (menadione), P, etc. A particularly preferred combination of vitamins comprises biotin, D-Ca pantothenate, choline chloride, folic acid, i-inositol, nicotinamide, pyroxidal HCl, riboflavin, and thiamine-HCl.
- The IVM medium contains at least one growth factor (GF). Broadly speaking, growth factors are proteins that bind to receptors on the cell surface, with the primary result of activating cellular proliferation and/or differentiation. Many growth factors are quite versatile, stimulating cellular division in numerous different cell types, while others are specific to a particular cell-type. Growth factors may act on membranes of oocytes to positively stimulate oocyte maturation (Maruo et al., 1993; Goud et al., 1998). Useful growth factors in the context of the present invention include those selected from the following growth factor superfamilies: epidermal growth factor (EGF) family; platelet derived growth factor (PDGF) family; insulin-like growth factor (IGF) family; nerve growth factor (NGF) family; transforming growth factor (TGF) family; fibroblast growth factor (FGF) family; hepatocyte growth factor (HGF) family; hemapoietic growth factors; and cytokines.
- In a preferred embodiment, the growth factors in IVM medium comprise fibroblast growth factor (FGF) and epidermal growth factor (EGF). FGF and EGF are readily available from commercial sources, and may be purchased together in a cell growth supplement. The IVM medium preferably contains from 0.0001 mg/L to 0.001 mg/L FGF, more preferably from 0.0002 mg/L to 0.001 mg/L FGF, and even more preferably about 0.0005 mg/L FGF. The IVM medium preferably contains from 0.0001 to 0.01 mg/L EGF, more preferably from 0.0005 mg/L to 0.002 mg/L EGF, and even more preferably about 0.001 mg/L EGF.
- The IVM medium preferably also contains at least one hormone. Preferred hormones include insulin, estradiol, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Human menopausal gonadotropin (HMG) can generally be substituted for FSH, and human chorionic gonadotropin (HCG) can generally be substituted for LH. In a preferred embodiment, the IVM medium comprises insulin. Normally, insulin is involved in energy metabolism and amino acid transportation into cells. The IVM medium preferably contains from 0.5 mg/L to 50 mg/L insulin, more preferably 0.25 mg/L to 10 mg/L insulin, and even more preferably about 5 mg/L insulin. In a particularly preferred embodiment, the IVM medium further comprises estradiol, LH and FSH. The amount of estradiol is preferably about 1 μg/ml ±10%, ±20%, ±30%, ±40% or ±50%. The amount of each of FSH and LH is preferably about 0.08 IU/ml ±10%, ±20%, ±30%, ±40% or ±50%.
- In a preferred embodiment, the IVM medium also contains human transferrin (TF). TF is a 75 kDa glycoprotein containing 679 amino acids and two glycan chains. For each, the last residue is a sialic (N-acetyl neuraminic) acid, which can be hydrolyzed by neuraminidase. TF not only transports iron in all extracellular fluid but also exerts many additional properties, including cell growth stimulation (Gross-Weege et al., 1986). The IVM medium preferably contains from from 5 mg/L to 500 mg/L TF, more preferably from 25 mg/L to 100 mg/L TF, and even more preferably about 50 mg/L TF.
- The growth factors, hormones and transferrins discussed above may be naturally occurring, synthetic or recombinant, and encompass biologically active fragments, variants, derivatives and homologs of these substances that retain at least some of the biological activity of the naturally-occurring, synthetic or recombinantly-produced substances. By way of a non-limiting example, a protein may include one or more amino acid insertions, deletions, substitutions or modifications without substantially reducing its biological activity.
- The choice of energy source is not critical to the invention, and may be e.g. glucose, sodium pyruvate, lactate, or a mixture of some or all of these energy sources.
- The IVM medium may additional contain additional components such as selenite or selenium and hydrocortisone.
- Buffers for controlling the pH of the medium may be added, such as Hepes, as may be pH indicators such as phenol red.
- Antibiotics such as penicillin G and streptomycin may be added to the IVM medium to prevent contamination.
- Synthetic Serum Supplement (SSS) may be included in the IVM medium as a source of protein for the oocyte and to prevent cells from adhering to glassware during in vitro culture. SSS has the advantage of having received regulatory approval for human IVF, and is commercially available.
- In a particularly preferred embodiment, the IVM medium includes the components set forth in Table 1. The absolute and relative quantities of each component can vary substantially, for example by up to ±10%, ±20%, ±30%, ±40%, ±50% weight by volume.
- Culture Medium for Oocytes in the Presence of Cumulus Cells
- Culture media as described above are also useful for culturing immature oocytes that have a cumulus that is intact or at least partially intact, and/or that are co-cultured with cumulus cells. But the presence of the cumulus cells in the first 12-24 hours of culture eliminates the need for growth factors, so the culture medium need not contain growth factors such as fibroblast growth factor, epidermal growth factor, etc.
- Also, whereas the culture medium described in the preceding section preferably contains human transferrin, insulin, selenite and hydrocortisone, these are not of substantial benefit when culturing oocytes in the presence of cumulus cells, and are therefore generally absent from the culture medium.
- More generally, complex culture media, such as tissue culture media (e.g. TCM-199), contain many components that are designed specifically for somatic cell culture in vitro rather than for oocyte culture. The use of complex culture media such as TCM-199 provides poorer results than if less complex media as described herein are used. Thus, in particularly preferred embodiments, oocytes that are essentially free of cumulus cells are cultured in media that consists of, or consists essentially of the components listed in Table 1; and oocytes that are cultured in the presence of cumulus cells are cultured in media that consists of or consists essentially of the components listed in Table 2.
- The term “comprising” is intended to be inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The term “consisting of” is intended to exclude any element, step, or ingredient that is not specified, but for impurities ordinarily associated with specified ingredients or materials. The term “consisting essentially of” is intended to occupy a middle ground between “comprising” and “consisting of” and to exclude other than the specified materials or steps and those that do not materially affect the basic and novel characteristics of the invention herein.
- Fertilization of Oocytes and Culture of Embryos
- Techniques for fertilizing oocytes and culturing embryos are known in the art.
- In one embodiment, fertilization is accomplished by intracytoplasmic sperm injection (ICSI). Generally, spermatozoa can be prepared by gradient separation by centrifugation or by “swim-up” followed by washing with a suitable medium such as HTF supplemented with 10% SSS. A single spermatozoon may be injected into an M-II oocyte. Following ICSI, the oocyte may be transferred to e.g. fertilization medium supplemented with 10% SSS in a tissue culture dish. Fertilization can be detected by the appearance of two distinct pronuclei and two polar bodies approximately 16-18 hours after ICSI.
- In one embodiment, fertilized oocytes may be cultured in fertilization media until about 72 hours after ICSI and then transferred to e.g. embryo developmental medium (Vitrolife, Göteborg, Sweden) in a tissue culture dish under mineral oil for an additional 48 hours.
- As used herein, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.
- The invention is further illustrated by reference to the following non-limiting examples.
- Source of Immature Oocytes
- A total of 521 immature oocytes (213 oocytes at GV stage and 308 oocytes at M-I stage) were collected from 183 women (age range between 28 to 40 years) who underwent controlled ovarian stimulation and ICSI cycles under informed consent. Ovarian stimulation was performed with GnRHa, HMG or FSH and HCG using standard protocols.
- 36 hours after HCG injection, oocyte retrieval was carried out using an ultrasound-guided trans-vaginal probe. The collected COC were cultured in human tubal fluid medium (HTF; Irvine Scientific) supplemented with 10% synthetic serum substitute (SSS; Irvine Scientific) and placed in a 5% CO2 incubator (37° C. with high humidity) for at least 1 h before they were stripped from their cumulus cells.
- COC were stripped with 85 IU/ml hyaluronidase in HTF medium and mechanical pipetting. All oocytes were completely denuded from their cumulus cells. Denuded oocytes were observed under an inverted microscope to assess for oocyte maturity. The mature oocytes were determined by the presence of a first polar body extrusion (1PB). The oocytes with a visual polar body (M-II) were provided for clinical application to perform the ICSI procedure immediately. Immature oocytes were defined by the absence of 1PB and then identified as GV or M-I stages depending upon whether the oocytes contained a visible GV in the cytoplasm under microscope. Immature oocytes without a GV in the cytoplasm were considered as M-I stage. These immature oocytes were used for in vitro maturation.
- The immature oocytes were cultured in HTF medium supplemented with 10% SSS (Irvine Scientific) placed in a 37° C. and 5% CO2 incubator before transferred to the maturation medium. The time of the immature oocytes were transferred to maturation medium is defined as the starting point for maturation in culture. Normally, it takes approximately 3-4 h from oocyte retrieval to the start of oocyte maturation.
- In vitro Maturation of the Immature Oocytes
- Immature oocytes were divided into two groups. One was the GV stage oocytes (n=213), another was the M-I stage oocytes (n=308). Each group of oocytes was randomly cultured in one of two maturation media respectively: (1) TCM-199 (GIBCO, Catalogue No. 11153) or, (2) IVM-medium (Table 1). Both TCM-199 and IVM-medium were supplemented with 10% SSS (Irvine Scientific), 0.075 IU/ml FSH +LH (Humegon, Ontario, Canada) and, 1.0 μg/ml estradiol (Sigma). Additional supplements to TCM-199 included 0.25 mM sodium pyruvate (Sigma), 0.05 unit/ml penicillin G and streptomycin (GIBCO).
- Before culture, the immature oocytes were washed at least twice in maturation medium. The immature oocytes were cultured in an organ tissue culture dish (60×15 mm; Falcon) containing 1 ml of maturation medium (TCM-199 or IVM-medium) at 37° C. in an atmosphere of 5% CO2 and 95% air with high humidity. A maximum of 5 oocytes (1 to 5) were placed in each organ tissue culture dish.
- Following culture, the maturity of the oocytes was determined under the microscope at 6 h for M-I stage oocytes and then at 24 h and 48 h for both M-I and GV stage oocytes.
- Intracytoplasmic Sperm Injection (ICSI)
- In vitro matured oocytes were inseminated by ICSI with sperm of the patient's husband. All semen samples were obtained from the ejaculate for clinical utilization. The remaining samples were donated for this experiment under informed consent.
- Sperm for ICSI were prepared by PureSperm (Nidacon, Sweden) gradient separation (45%, 70% and 90%) at 560 g for 20 min. Following gradient separation, the sperm pellet was washed twice (200 g) with 2 ml of HTF medium (Irvine Scientific) supplemented with 10% SSS.
- A single sperm was injected into each M-II oocyte. Following ICSI, each oocyte was transferred into a 20 μl droplet of HTF medium supplemented with 10% SSS in a tissue culture dish (35×10 mm; Falcon) under mineral oil (Sigma).
- Fertilization was assessed 16-18 h after ICSI for the appearance of two distinct pronuclei and two polar bodies. Since the oocytes were not matured at the same time following culture, the sperm samples were kept in 5 ml Falcon tubes (tight tube cap) containing 0.5-1.0 ml HTF medium supplemented with 10% SSS at room temperature for further insemination by ICSI at 6 h, 24 h or 48 h, respectively.
- Culture of Embryos
- Fertilized oocytes (with two pronuclei) were cultured in HTF medium supplemented with 10% SSS until 72 h after ICSI, and then each embryo was transferred into a 20 μl droplet of G2.2 medium (VitroLife, Sweden) in a tissue culture dish (35×10 mm; Falcon) under mineral oil (Sigma) further culture for 48 h. Embryo development was recorded at 24 h intervals following culture until day 5 (120 h). All embryos were destroyed on day 5 following consent instruction from the patients.
- Statistical Analysis
- Statistical significant differences in the percentages of oocyte maturation, fertilization and development between the groups were determined and compared by Student-Newman-Keuls' test (Steel & Torrie, 1980). A P-value of <0.05 was considered statistically significant.
- Results
- As shown in Table 3, 52.0% and 46.1% of M-I stage oocytes were mature 6 h after culture in the IVM medium and TCM-199, respectively. Following further culture for 24 h and 48 h, there were no differences in oocyte maturation rates between IVM-medium (76.0% and 78.6%) and TCM-199 (67.5% and 70.8%).
- As shown in Table 4, the final fertilization rates also were not different between these two media (90.0% vs. 84.4%).
-
FIG. 1 shows the fertilization rates of the oocytes matured in the IVM-medium or TCM-199 at 6 h (93.8% vs. 90.1%), 24 h (89.2% vs. 84.9%) and 48 h (50.0% vs. 0.0%), respectively. - The final cleavage rates of the fertilized oocytes were not different between the oocytes matured in these two media (Table 4; 97.3% vs. 98.9%), and there were also no differences in these two media when the oocytes were matured 6 h (100.0% vs. 98.4%) and 24 h (93.9% vs. 100.0%) respectively (
FIG. 2 ). - There were no significant differences in the embryos that developed to the 8-cell stage between the oocytes matured in IVM-medium (63.7%) and TCM-199 (57.1%), and the percentages of embryos that developed to the 8-cell stage were also not different when the oocytes matured 6 h (66.7% vs. 60.3%), 24 h (54.6% vs. 50.0%) and 48 h (0.0% vs. 0.0%) in these two media, respectively (
FIG. 3 a). - However, total blastocyst formation rates were significantly different (P<0.05) between the IVM-medium and TC-199 respectively (Table 4; 19.6% vs. 7.7%). Furthermore, the percentage of blastocyst formation was different (P<0.05) for these two media when the oocytes matured 6 h (24.0% vs. 7.9%) after maturation in culture (
FIG. 3 b). - However, there were no differences between these two media when the oocytes matured 24 h (9.7% vs. 7.1%) and 48 h (0.0% vs. 0.0%), respectively.
- As shown in Table 5, when GV stage oocytes were matured in vitro, there were significant differences (P<0.01) in the maturation rates between the IVM-medium and TCM-199 at 24 h (50.1% vs. 34.0%) and 48 h (75.7% vs. 55.7%) respectively.
- However, as shown in Table 6, the final fertilization rates were not different between the IVM-medium (86.4%) and TCM-199 (78.0%).
- There were also no differences in the fertilization rates between these two media when the oocytes were matured 24 h (88.9% vs. 83.3%) and 48 h (81.5% vs. 69.6%) (
FIG. 4 ). - Final cleavage rates were not different between these two media (Table 6, 100.0% vs. 95.6%). The cleavage rates of the fertilized oocytes were also not different between these two media when the oocytes matured 24 h (100.0% vs. 96.7%) and 48 h (100.0% vs. 93.8%) (
FIG. 5 ). - However, there were significant differences (P<0.05) in embryos that developed to the 8-cell stage and blastocyst stage between the IVM-medium and TCM-199 (Table 6, 58.6% vs. 40.9% and 12.9% vs. 0.0%, respectively).
- When oocytes were matured for 24 h, percentages of embryos that developed to the 8-cell stage (
FIG. 6 a; 75.0% vs. 51.7%) and the blastocyst stage (FIG. 6 b; 18.8% vs. 0.0%) were significantly different (P<0.05) between the IVM-medium and TCM-199, respectively. - However, there were no differences in the embryos that developed to the 8-cell stage (
FIG. 6 a; 22.7% vs. 20.0%) and blastocyst (FIG. 6 b; 0.0% vs. 0.0%) between these two media when the oocytes were matured for 48 h. - A total of 40 women with PCOS underwent 40 completed IVM treatment cycles. All patients were under 40 years of age (mean 32.3±3.9) and some patients presented irregular menstrual cycles or anovulation. All patients had a minimum 2-year history of infertility.
- If the patients had irregular menstrual cycles, the treatment cycles was initiated by the administration of intra-vaginal progesterone (Prometrium; Schering, Pointe-Clair, Quebec, Canada) in a dose of 300 mg daily for 10 days. Withdrawal bleeding occurred within 3 days after the last dose.
- On
day 2 or 3 following the onset of menstrual bleeding, the patients underwent a baseline ultrasound scan to ensure that no ovarian cysts were present. Transvaginal ultrasound scans were repeated onday 8 to exclude the development of a dominant follicle. The size of all follicles on ultrasound scan had to be <10 mm in diameter onday 8 of the cycle. - Oocyte retrieval was performed on day 10 to 14 of the cycle. Before oocyte collection 36 hours, the patients were given s.c. 10,000 IU of human chorionic gonadotropin (HCG) for priming.
- Transvaginal ultrasound-guided oocyte collection was performed using a specially designed 19 G single-lumen aspiration needle (Cook, Australia) with an aspiration pressure of 7.5 kPa. Aspiration of all small follicles was performed under local anaesthesia. Oocytes were collected in 10 ml culture tubes (Falcon, USA) containing 2.0 ml warm 0.9% saline contained with 2 IU/ml heparin (Baxter, Toronto, Ontario, Canada).
- Following oocyte collection, cumulus-oocyte complexes (COCs) were cultured in an organ tissue culture dish (60×15 mm; Falcon) containing 1 ml of modified IVM-medium (Table 2) at 37° C. in an atmosphere of 5% CO2 and 95% air with high humidity.
- Following culture of 24 and 48 hours, the mature oocytes (metaphase-II stage) were inseminated by ICSI. Fertilization was assessed 18 h after ICSI for the appearance of two distinct pronuclei and two polar bodies. Embryo transfer was performed on
day 2 or 3 after ICSI. Since the oocytes were inseminated either 24 or 48 hours following culture, the developmental stages of embryos were variable. - For the preparation of the endometrium, the patients were given estradiol (Estace; Roberts Pharmaceutical, Mississauga, Canada) depending on the endometrial thickness on the day of oocyte retrieval in divided doses, starting on the day of oocyte retrieval. If the endometrial thickness on the day of oocyte retrieval was <4 mm, a 10 mg dose was administered; if it was >6 mm, a 6 mg dose was given. Luteal support was provided by 400 mg intravaginal progesterone (Prometrium) twice daily for 16 days starting from the day after oocyte collection.
- A total of 653 immature oocytes were collected from 40 women with PCOS who underwent IVM treatment. Following culture of 48 hours, 81.5% (532/653) of oocytes became mature. Following embryo transfer, 13 patients became pregnant (32.5%=13/40). Results are tabulated in Table 7.
TABLE 1 Composition of oocyte in vitro maturation (IVM) medium. mg/L Inorganic Salt: CaCl2 200.0000 KCl 400.0000 MgSO4 98.0000 NaCl 6800.0000 NaHCO3 1250.0000 NaH2PO4.H2O 125.0000 Amino Acids: L-Alanine 8.9000 L-Arginine 126.4000 L-Asparagine 13.2000 L-Aspartic Acid 13.3000 L-Cystine 24.0000 L-Glutamic Acid 14.7000 L-Glutamine 292.0000 Glycine 7.5000 L-Histidine.HCI.H2O 42.0000 L-Isoleucine 52.4000 L-Leucine 52.4000 L-Lysine.HCI 72.5000 L-Methionine 15.1000 L-Phenylalanine 33.0000 L-Proline 11.5000 L-Serine 10.5000 L-Threonine 47.6000 L-Tryptophan 10.2000 L-Tyrosine 36.0000 L-Valine 46.8000 Vitamins: Biotin 0.0100 D-Ca Pantothenate 1.0000 Choline Chloride 1.0000 Folic Acid 1.0000 i-Inositol 2.0000 Nicotinamide 1.0000 Pyridoxal.HCI 1.0000 Riboflavin 0.1000 Thiamine.HCI 1.0000 Other Components: D-Glucose 1000.0000 Sodium Pyruvate 110.0000 Insulin 5.0000 Human Transferrin 50.0000 Selenite 0.0052 Hydrocortisone 0.0036 FGF 0.0005 EGF 0.0010 Phenol Red 5.0000 Penicillin G 50.0000 units Streptomycin 50.0000 units FSH 0.075 IU/ml LH 0.075 IU/ml estradiol 1.0 μg/ml -
TABLE 2 Modified Composition of oocyte in vitro maturation (IVM) medium. mg/L Inorganic Salt: CaCl2 200.0000 KCl 400.0000 MgSO4 98.0000 NaCl 6800.0000 NaHCO3 1250.0000 NaH2PO4.H2O 125.0000 Amino Acids: L-Alanine 8.9000 L-Arginine 126.4000 L-Asparagine 13.2000 L-Aspartic Acid 13.3000 L-Cystine 24.0000 L-Glutamic Acid 14.7000 L-Glutamine 292.0000 Glycine 7.5000 L-Histidine.HCl.H2O 42.0000 L-Isoleucine 52.4000 L-Leucine 52.4000 L-Lysine.HCl 72.5000 L-Methionine 15.1000 L-Phenylalanine 33.0000 L-Proline 11.5000 L-Serine 10.5000 L-Threonine 47.6000 L-Tryptophan 10.2000 L-Tyrosine 36.0000 L-Valine 46.8000 Vitamins: D-Ca Pantothenate 1.0000 Choline Chloride 1.0000 Folic Acid 1.0000 i-Inositol 2.0000 Nicotinamide 1.0000 Pyridoxal.HCl 1.0000 Riboflavin 0.1000 Thiamine.HCl 1.0000 Other Components: D-Glucose 1000.0000 Sodium Pyruvate 110.0000 Estradiol 1.0000 Phenol Red 5.0000 FSH 75.0000 IU LH 75.0000 IU Human serum albumin 1000.0000 Penicillin G 50.0000 units Streptomycin 50.0000 μg -
TABLE 3 Maturation of immature human oocytes (metaphase-I stage) cultured 6 h, 24 h or 48 h in the IVM-medium or TCM-199. Culture No. of oocytes No. of oocytes matured at (%): media examined 6 h 24 h 48 h IVM medium 154 80 (52.0) 117 (76.0) 121 (78.6) TCM-199 154 71 (46.1) 104 (67.5) 109 (70.8) -
TABLE 4 Fertilization and embryonic development of immature human oocytes (metaphase-I stage) matured in the IVM-medium or TCM-199. No. of No. of No. of embryos oocytes oocytes No. of oocytes developed to (%): Culture media inseminated fertilized (%) cleaved (%) 8-cell stage Blastocyst IVM medium 121 110 (90.0) 107 (97.3) 68 (63.6) 21 (19.6)a TCM-199 109 92 (84.4) 91 (98.9) 52 (57.1) 7 (7.7)b
abDifferent letters indicate significant differences within columns (P < 0.05).
-
TABLE 5 Maturation of immature human oocytes (germinal vesicle stage) cultured 24 h or 48 h in the IVM-medium or TCM-199. No. of oocytes No. of oocytes Culture No. of oocytes matured at 24 h matured at 48 h media examined (%) (%) IVM medium 107 54 (50.1)a 81 (75.7)a TCM-199 106 36 (34.0)b 59 (55.7)b
abDifferent letters indicate significant differences within columns (P < 0.05).
-
TABLE 6 Fertilization and embryonic development of immature human oocytes (germinal vesicle stage) matured in the IVM-medium or TCM-199. No. of No. of No. of embryos Culture oocytes oocytes No. of oocytes developed to (%): media inseminated fertilized (%) cleaved (%) 8-cell stage Blastocyst IVM medium 81 70 (86.4) 70 (100.0) 41 (58.6)a 9 (12.9)a TCM-199 59 46 (78.0) 44 (95.6) 18 (40.9)b 0 (0.0)b
abDifferent letters indicate significant differences within columns (p < 0.05).
-
TABLE 7 Results of in vitro maturation and fertilization of oocytes using modified IVM-medium followed by embryo transfer in women with polycystic ovaries (PCO) or polycystic overy syndrome (PCOS) (from January 2002 to September 2002 at McGill Reproductive Center).* Cycles 40 Age - yrs 32.3 ± 3.9 No. of oocytes collected Total 653 Mean 15.9 ± 5.9 No. of oocytes matured (%) 532 (81.5) No. of oocytes fertilized (%) 336 (63.2) No. of oocytes cleaved (%) 329 (97.9) No. of embryos transferred Total 154 Mean 3.9 ± 0.6 No. of clinical pregnancies (%) 13 (32.5) No. of implantation (%) 17 (11.0)
*Plus-minus values are mean ± SD.
- Adams J, Polson D, Frank S. Prevalence of polycystic ovaries in women with anovulation or idiopathic hirsutism. Br Med J 1986; 293:355-359.
- Barnes F L, Crombie A, Gardner D K, Kausche A, Lacham-Kaplan O, Suikkari A A, Tiglias J, Wood C, Trounson A O. Blastocyst development and birth after in vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching. Hum Reprod 1995; 10:3243-3247.
- Brackett B G, Zuelke K A. Analysis of factors involved in the in vitro production of bovine embryos. Theriogenology 1993; 39:43-64.
- Cha K Y, Koo J J, Ko J J, Choi D H, Han S Y, Yoon T K. Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulated cycles, their culture in vitro and their transfer in a donor oocyte program. Fertil Steril 1991; 55:109-113.
- Cha K Y, Chian R C. Maturation in vitro of immature human oocytes for clinical use. Hum Reprod Update; 4:103-120.
- Chian R C, Niwa K, Sirard M A. Effects of cumulus cells on male pronuclear formation and subsequent early development of bovine oocytes in vitro. Theriogenology 1994; 41:1499-1508.
- Chian R C, Sirard M A. Effects of cumulus cells and follicle-stimulating hormone during in vitro maturation on parthenogenetic activation of bovine oocytes. Mol Reprod Dev 1995; 42:425-431.
- Chian R C, Park S E, Park E H, Son W Y, Chung H M, Lim J G, Ko J J, Cha K Y. Molecular and structural characteristics between immature human oocytes retrieved from stimulated and unstimulated ovaries. Gormel V, Leung P C K (eds), In Vitro Fertilization and Assisted Reproduction; Monduzzi, Bologna, pp 315-319.
- Chian R C, Buckett W M, Too L L, Tan S L. Pregnancies resulting from in vitro matured oocytes retrieved from patients with polycystic ovary syndrome after priming with human chorionic gonadotropin.
Fertil Steril 1999; 72:639-642. - Chian R C, Gulekli B, Buckett W M, Tan S L. Priming with human chorionic gonadotropin before retrieval of immature oocytes in women with infertility due to the polycystic ovary syndrome. New
Eng J Med 1999; 341:1624-1626. - Chian R C, Ao A, Clarke H J, Tulandi T, Tan S L. Production of steroids from human cumulus cells treated with different concentrations of gonadotropins during culture in vitro.
Fertil Steril 1999; 71:61-66. - Chian R C, Buckett W M, Tulandi T, Tan S L. Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome. Hum Reprod 2000; 15:165-170.
- Chian R C, Gulekli B, Buckett W M, Tan S L. Pregnancy and delivery after cryopreservation of zygotes produced by in-vitro matured oocytes retrieved from a woman with polycystic ovarian syndrome. Hum Reprod 2001; 16:1700-1702.
- Edirisinghe W R, Junk S M, Matson P L, Yovich J L. Birth from cryopreserved embryos following in vitro maturation of oocytes and intracytoplasmic sperm injection. Hum Reprod 1997; 12:1056-1058.
- Goud P T, Goud A P, Qian C, Laverge H, Van der Elst J, De Sutter P, Dhont M. In-vitro maturation of human germinal vesicle stage oocytes: role of cumulus cells and epidermal growth factor in the culture medium. Hum Reprod 1998; 13:1638-1644.
- Gross-Weege W, Theobald K, Konig W. Inhibition of histamine release from rat peritoneal mast cells by a factor from human serum: identification as transferring. Agents Actions 1986; 19:10-17.
- Jaroudi K A, Hollanders J M G, Elnour A M, Roca G L, Atared A M, Coskun S. Embryo development and pregnancies from in vitro matured and fertilized human oocytes.
Hum Reprod 1999; 14:1749-1751. - MacDougal M J, Tan S L, Balen A, et al. A controlled study comparing patients with and without polycystic ovaries undergoing in vitro fertilization. Hum Reprod 1993; 8:233-237.
- Maruo T, Ladines-Llave C A, Samoto T, Matsuo H, Manalo A S, Ito H, Mochizuki M. Expression of epidermal growth factor and its receptor in the human ovary during follicular growth and regression. Endocrinology 1993; 132:924-931.
- Moor R M, Trounson A O. Hormonal and follicular factors affecting maturation of sheep oocytes in vitro and their subsequent developmental capacity. J Reprod Fertil 1977; 49:101-109.
- Nagy Z P, Cecile J, Liu J, Loccuifer A, Devroey P, Van Steirteghem A. Pregnancy and birth after intracytoplasmic sperm injection of in vitro matured germinal vesicle stage oocytes: case report. Fertil Steril 1996; 65:1047-1050.
- Pincus G, Enzmann E V. The comparative behaviour of mammalian eggs in vivo and in vitro. 1. The activation of ovarian eggs. J Exp Med 1935; 62:665-675.
- Shea B F, Baker R D, Latour J P. Human follicular oocytes and their maturation in vitro. Fertil Steril 1975; 26:1075-1082.
- Steel R G D, Torrie J H. Principles and procedures of statistics: a biometrical approach. New York, McGraw-Hill, 1980; ppl72-194.
- Trounson A, Wood C, Kausche A. In vitro maturation and fertilization and developmental competence of oocytes recovered from untreated polycystic ovarian patients. Fertil Steril 1994; 62:353-362.
- Trounson A O, Anderiesz C, Jones G M, Kausche A, Lolatgis N, Wood C. Oocyte maturation. Hum Reprod 1998; 13:52-62.
- Trounson A, Anderiesz C, Jones G. Maturation of human oocytes in vitro and their developmental competence. Reproduction 2001; 121:51-75.
- All references cited in this specification are herein incorporated by reference as if each individual reference were specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such reference by virtue of prior invention.
- Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
Claims (68)
1. A method for in vitro maturation of immature human oocytes, comprising the steps of:
(a) inducing in a female human subject an increase in endogenous luteinizing hormone levels, said subject having not undergone an ovarian stimulation protocol prior to said inducing step;
(b) obtaining from said subject an immature oocyte; and
(c) culturing said oocyte until maturity.
2. The method according to claim 1 , wherein step (a) comprises administering to said subject human chorionic gonadotrophin or luteinizing hormone, or both.
3. The method according to claim 1 , wherein step (a) comprises administering to said subject human chorionic gonadotropin in an amount of about 5000 to about 20,000 IU.
4. The method according to claim 1 , wherein said immature human oocyte comprises an M-I stage ooctye.
5. The method according to claim 1 , wherein said immature human oocyte comprises a GV stage oocyte.
6. The method according to claim 1 , wherein said immature human oocyte is cultured until it reaches M-II.
7. The method according to claim 1 , wherein, in step (c), said immature human oocyte is essentially free of cumulus cells and is cultured in a culture medium comprising:
at least one inorganic salt;
essential amino acids or a source thereof;
an energy source; and
at least one growth factor.
8. The method according to claim 7 , wherein said at least one growth factor is selected from the group consisting of fibroblast growth factor and epidermal growth factor.
9. The method according to claim 7 , wherein said culture medium comprises both fibroblast growth factor and epidermal growth factor.
10. The method according to claim 7 , wherein said culture medium further comprises at least one hormone.
11. The method according to claim 10 , wherein said hormone comprises insulin.
12. The method according to claim 11 , wherein said culture medium comprises from 0.5 mg/L to 50 mg/L insulin.
13. The method according to claim 7 , wherein said culture medium further comprises human transferrin.
14. The method according to claim 13 , wherein said culture medium comprises from 5 mg/L to 500 mg/L human transferrin.
15. The method according to claim 7 , wherein said culture medium comprises from 0.0001 mg/L to 0.001 mg/L fibroblast growth factor.
16. The method according to claim 7 , wherein said culture medium comprises from 0.0001 to 0.01 mg/L epidermal growth factor.
17. The method according to claim 7 , wherein said culture medium comprises one or more vitamins.
18. The method according to claim 17 , wherein said one or more vitamins comprise biotin, D-Ca pantothenate, choline chloride, folic acid, i-inositol, nicotinamide, pyroxidal.HCl, riboflavin, and thiamine-HCl.
19. The method according to claim 7 , wherein said culture medium comprises hydrocortisone.
20. The method according to claim 7 , wherein said culture medium comprises selenite.
21. The method according to claim 7 , wherein said inorganic salts comprise CaCl2, KCl, MgSO4, NaCl, NaHCO3, NaH2PO4.H20.
22. The method according to claim 7 , wherein said energy source comprises D-glucose, or sodium pyruvate, or both D-glucose and sodium pyruvate.
23. The method according to claim 7 , wherein said amino acids comprise alanine, arginine, asparagine, aspartic acid, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
24. The method according to claim 7 , wherein said culture medium comprises the inorganic salts, amino acids, vitamins and other components as set forth in Table 1, and wherein each inorganic salt, amino acid, vitamin and other component is present in an amount of ±50% (weight/volume) of the amount specified in Table 1.
25. The method according to claim 24 , wherein each inorganic salt, amino acid, vitamin and other component is present in said culture medium in an amount of ±10% (weight/volume) of the amount specified in Table 1.
26. The method according to claim 1 , wherein, in step (c), said oocyte has a cumulus that is intact or at least partially intact, and/or said oocyte is cultured in the presence of cumulus cells.
27. The method according to claim 26 , wherein said oocyte is cultured in a culture medium that is essentially free of epidermal growth factor.
28. The method according to claim 26 , wherein said oocyte is cultured in a culture medium that is essentially free of fibroblast growth factor.
29. The method according to claim 26 , wherein said oocyte is cultured in a culture medium that is essentially free of human transferrin.
30. The method according to claim 26 , wherein said oocyte is cultured in a culture medium that is essentially free of insulin.
31. The method according to claim 26 , wherein said oocyte is cultured in a culture medium that is essentially free of selenite.
32. The method according to claim 26 , wherein said oocyte is cultured in a culture medium that is essentially free of hydrocortisone.
33. The method according to claim 26 , wherein said oocyte is cultured in a culture medium that is essentially free of epidermal growth factor, fibroblast growth factor, human transferrin, insulin, selenite, and hydrocortisone.
34. The method according to claim 26 , wherein said oocyte is cultured in a culture medium comprising the inorganic salts, amino acids, vitamins and other components as set forth in Table 2, and wherein each inorganic salt, amino acid, vitamin and other component is present in an amount of ±50% (weight/volume) of the amount specified in Table 2.
35. The method according to claim 34 , wherein each inorganic salt, amino acid, vitamin and other component is present in said culture medium in an amount of ±10% (weight/volume) of the amount specified in Table 2.
36. The method according to claim 26 , wherein said oocyte is cultured in a culture medium consisting essentially of the inorganic salts, amino acids, vitamins and other components as set forth in Table 2.
37. The method according to claim 26 , wherein said oocyte is cultured in a culture medium consisting of the inorganic salts, amino acids, vitamins and other components as set forth in Table 2.
38. The method according to claim 1 , wherein, prior to step (a), said subject has not been treated with a gonadotrophin releasing hormone agonist, human menopausal gonadotrophin or follicle stimulating hormone.
39. A method for in vitro maturation of immature human oocytes, comprising culturing an immature human oocyte in a culture medium comprising:
at least one inorganic salt;
essential amino acids or a source thereof;
an energy source; and
at least one growth factor.
40. The method according to claim 39 , wherein said at least one growth factor is selected from the group consisting of fibroblast growth factor and epidermal growth factor.
41. The method according to claim 39 , wherein said culture medium comprises both fibroblast growth factor and epidermal growth factor.
42. The method according to claim 39 , wherein said culture medium further comprises at least one hormone.
43. The method according to claim 42 , wherein said hormone comprises insulin.
44. The method according to claim 43 , wherein said culture medium comprises from 0.5 mg/L to 50 mg/L insulin.
45. The method according to claim 39 , wherein said culture medium further comprises human transferrin.
46. The method according to claim 45 , wherein said culture medium comprises from 5 mg/L to 500 mg/L human transferrin.
47. The method according to claim 39 , wherein said culture medium comprises from 0.0001 mg/L to 0.001 mg/L fibroblast growth factor.
48. The method according to claim 39 , wherein said culture medium comprises from 0.0001 to 0.01 mg/L epidermal growth factor.
49. The method according to claim 39 , wherein said culture medium comprises one or more vitamins.
50. The method according to claim 49 , wherein said one or more vitamins comprise biotin, D-Ca pantothenate, choline chloride, folic acid, i-inositol, nicotinamide, pyroxidal HCl, riboflavin, and thiamine-HCl.
51. The method according to claim 39 , wherein said culture medium comprises hydrocortisone.
52. The method according to claim 39 , wherein said culture medium comprises selenite.
53. The method according to claim 39 , wherein said inorganic salts comprise CaCl2, KCl, MgSO4, NaCl, NaHCO3, NaH2PO4 .H2O.
54. The method according to claim 39 , wherein said energy source comprises D-glucose, or sodium pyruvate, or both D-glucose and sodium pyruvate.
55. The method according to claim 39 , wherein said amino acids comprise alanine, arginine, asparagine, aspartic acid, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
56. The method according to claim 39 , wherein said culture medium comprises the inorganic salts, amino acids, vitamins and other components as set forth in Table 1, and wherein each inorganic salt, amino acid, vitamin and other component is present in an amount of ±50% (weight/volume) of the amount specified in Table 1.
57. The method according to claim 55 , wherein each inorganic salt, amino acid, vitamin and other component is present in said culture medium in an amount of ±10% (weight/volume) of the amount specified in Table 1.
58. The method according to claim 39 , wherein said culture medium consists essentially of the inorganic salts, amino acids, vitamins and other components as set forth in Table 1.
59. The method according to claim 39 , wherein said culture medium consists of the inorganic salts, amino acids, vitamins and other components as set forth in Table 1.
60. The method according to claim 57 or 58 , wherein each inorganic salt, amino acid, vitamin and other component is present in an amount of ±50% (weight/volume) of the amount specified in Table 1.
61. The method according to claim 57 or 58 , wherein each inorganic salt, amino acid, vitamin and other component is present in an amount of ±10% (weight/volume) of the amount specified in Table 1.
62. The method according to claim 39 , wherein said immature human oocyte is essentially free of cumulus cells.
63. A method for in vitro maturation of immature human oocytes, comprising culturing an immature human oocyte in a culture medium comprising the inorganic salts, amino acids, vitamins and other components as set forth in Table 2.
64. The method according to claim 62 , wherein said culture medium consists essentially of the inorganic salts, amino acids, vitamins and other components as set forth in Table 2.
65. The method according to claim 62 , wherein said culture medium consists of the inorganic salts, amino acids, vitamins and other components as set forth in Table 2.
66. The method according to claim any one of claims 62-64, wherein each inorganic salt, amino acid, vitamin and other component is present in an amount of ±50% (weight/volume) of the amount specified in Table 2.
67. The method according to any one of claims 62-64, wherein each inorganic salt, amino acid, vitamin and other component is present in an amount of ±10% (weight/volume) of the amount specified in Table 2.
68. The method according to claim 62 , wherein said oocyte is cultured in the presence of cumulus cells
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/506,713 US20050208651A1 (en) | 2002-03-08 | 2003-03-07 | Vitro maturation of immature human oocytes |
US11/969,522 US7790459B2 (en) | 2002-03-08 | 2008-01-04 | In vitro maturation of immature human oocytes |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US36239502P | 2002-03-08 | 2002-03-08 | |
US10/506,713 US20050208651A1 (en) | 2002-03-08 | 2003-03-07 | Vitro maturation of immature human oocytes |
PCT/CA2003/000323 WO2003076600A2 (en) | 2002-03-08 | 2003-03-07 | In vitro maturation of immature human oocytes |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/969,522 Division US7790459B2 (en) | 2002-03-08 | 2008-01-04 | In vitro maturation of immature human oocytes |
Publications (1)
Publication Number | Publication Date |
---|---|
US20050208651A1 true US20050208651A1 (en) | 2005-09-22 |
Family
ID=27805171
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/506,713 Abandoned US20050208651A1 (en) | 2002-03-08 | 2003-03-07 | Vitro maturation of immature human oocytes |
US11/969,522 Expired - Lifetime US7790459B2 (en) | 2002-03-08 | 2008-01-04 | In vitro maturation of immature human oocytes |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/969,522 Expired - Lifetime US7790459B2 (en) | 2002-03-08 | 2008-01-04 | In vitro maturation of immature human oocytes |
Country Status (6)
Country | Link |
---|---|
US (2) | US20050208651A1 (en) |
EP (1) | EP1483368A2 (en) |
JP (1) | JP2005518823A (en) |
AU (1) | AU2003208232B2 (en) |
CA (1) | CA2477743C (en) |
WO (1) | WO2003076600A2 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110709505A (en) * | 2017-04-12 | 2020-01-17 | 派乔易股份有限公司 | Cytokine-free adjuvants, in particular for in vitro fertilization or for cell culture media for follicles, male germ cells or embryos |
CN114149962A (en) * | 2021-11-25 | 2022-03-08 | 艾尔斯(浙江)医学科技有限公司 | Human oocyte in vitro fertilization pre-culture method, series culture solution and application |
Families Citing this family (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DK1697504T3 (en) * | 2003-11-26 | 2011-11-14 | Merck Serono Sa | Use of IL-6 type cytokines to mature oocytes |
AU2005292362B2 (en) * | 2004-09-30 | 2012-05-31 | Merck Serono Sa | Use of IL-17- for maturation of oocytes |
JP2008514239A (en) * | 2004-09-30 | 2008-05-08 | アプライド リサーチ システムズ エーアールエス ホールディング ナームロゼ フェンノートシャップ | Use of pregnancy-specific glycoprotein for oocyte maturation |
US9566256B2 (en) | 2008-09-22 | 2017-02-14 | Biochemics, Inc. | Transdermal drug delivery using an osmolyte and vasoactive agent |
US9278233B2 (en) | 2008-12-04 | 2016-03-08 | Biochemics, Inc. | Methods and compositions for tattoo removal |
AT510810B1 (en) * | 2010-12-09 | 2017-09-15 | Gonadosan Gmbh | COMBINATION PROCEDURE FOR IMPROVING FEMALE FERTILITY |
BR112018068474B1 (en) * | 2016-02-24 | 2024-01-30 | Universitá Degli Studi Di Milano, Unimi | FOLLICULAR SYSTEM FOR IN VITRO OOCYTE MATURATION AND KIT |
FR3061208B1 (en) * | 2016-12-23 | 2024-04-05 | Patrick Choay Sas | ADJUVANTS FOR CELL CULTURE MEDIA, PARTICULARLY FOR IN VITRO FERTILIZATION, OR FOR THE CULTURE OF FOLLICLES, MALE GERM CELLS OR EMBRYOS |
CN110643569B (en) * | 2019-11-29 | 2020-05-26 | 广州达瑞生殖技术有限公司 | Granular cell stripping liquid and preparation method thereof |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4987080A (en) * | 1987-11-03 | 1991-01-22 | Grob Howard S | Method for in vitro maturation of oocytes |
US5563059A (en) * | 1993-02-23 | 1996-10-08 | Genentech, Inc. | Use of human inhibin and human activin to increase the number of mature primate oocytes |
US5882928A (en) * | 1997-03-11 | 1999-03-16 | Oocytechs Research Corporation | In vitro maturation and fertilization of mammalian oocytes |
US6211429B1 (en) * | 1997-06-18 | 2001-04-03 | The Curators Of The University Of Missouri | Complete oocyte activation using an oocyte-modifying agent and a reducing agent |
US6281013B1 (en) * | 1999-02-24 | 2001-08-28 | Novo Nordisk A/S | Treatment of Infertility |
US20010028878A1 (en) * | 1998-06-22 | 2001-10-11 | Svend Lindenberg | In vitro maturation of human oocytes in one day |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IL94184A0 (en) | 1989-05-01 | 1991-01-31 | Granada Biosciences Inc | In vitro maturation of bovine oocytes |
PT723453E (en) | 1993-08-03 | 2000-07-31 | Celtrix Pharma | METHOD OF TREATMENT OF REPRODUCTIVE DISORDERS |
TR200101538T2 (en) | 1998-11-30 | 2001-12-21 | Ivf Sciences Colorado, Inc. | In vitro fertilization system and sequential culture medium. |
BR0008468A (en) | 1999-02-24 | 2002-02-05 | Novo Nordisk As | Method for human in vitro fertilization, use of a hypothalamic hormone and / or pituitary hormone or an agonist or antagonist of the same or an active derivative thereof, pharmaceutical kit in single dosage form, and any new aspect or combination of aspects |
JP2001017160A (en) | 1999-07-07 | 2001-01-23 | Fuso Pharmaceutical Industries Ltd | Medium composition for in vitro fertilization |
AU1692901A (en) | 1999-11-25 | 2001-06-04 | Novo Nordisk A/S | Treatment of human infertility |
AU2001248282A1 (en) | 2000-04-06 | 2001-10-23 | Novo-Nordisk A/S | Synchronization of the cytoplasmatic and the nuclear maturation of oocytes in vitro |
-
2003
- 2003-03-07 EP EP03706185A patent/EP1483368A2/en not_active Withdrawn
- 2003-03-07 JP JP2003574807A patent/JP2005518823A/en active Pending
- 2003-03-07 CA CA2477743A patent/CA2477743C/en not_active Expired - Fee Related
- 2003-03-07 WO PCT/CA2003/000323 patent/WO2003076600A2/en active Application Filing
- 2003-03-07 AU AU2003208232A patent/AU2003208232B2/en not_active Ceased
- 2003-03-07 US US10/506,713 patent/US20050208651A1/en not_active Abandoned
-
2008
- 2008-01-04 US US11/969,522 patent/US7790459B2/en not_active Expired - Lifetime
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4987080A (en) * | 1987-11-03 | 1991-01-22 | Grob Howard S | Method for in vitro maturation of oocytes |
US5563059A (en) * | 1993-02-23 | 1996-10-08 | Genentech, Inc. | Use of human inhibin and human activin to increase the number of mature primate oocytes |
US5882928A (en) * | 1997-03-11 | 1999-03-16 | Oocytechs Research Corporation | In vitro maturation and fertilization of mammalian oocytes |
US6211429B1 (en) * | 1997-06-18 | 2001-04-03 | The Curators Of The University Of Missouri | Complete oocyte activation using an oocyte-modifying agent and a reducing agent |
US20010028878A1 (en) * | 1998-06-22 | 2001-10-11 | Svend Lindenberg | In vitro maturation of human oocytes in one day |
US20020115211A1 (en) * | 1998-06-22 | 2002-08-22 | Medi-Cult A/S | A method for in vitro maturation of human gametes |
US6281013B1 (en) * | 1999-02-24 | 2001-08-28 | Novo Nordisk A/S | Treatment of Infertility |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110709505A (en) * | 2017-04-12 | 2020-01-17 | 派乔易股份有限公司 | Cytokine-free adjuvants, in particular for in vitro fertilization or for cell culture media for follicles, male germ cells or embryos |
CN114149962A (en) * | 2021-11-25 | 2022-03-08 | 艾尔斯(浙江)医学科技有限公司 | Human oocyte in vitro fertilization pre-culture method, series culture solution and application |
Also Published As
Publication number | Publication date |
---|---|
AU2003208232B2 (en) | 2008-05-22 |
WO2003076600A2 (en) | 2003-09-18 |
EP1483368A2 (en) | 2004-12-08 |
US7790459B2 (en) | 2010-09-07 |
JP2005518823A (en) | 2005-06-30 |
US20080176324A1 (en) | 2008-07-24 |
AU2003208232A1 (en) | 2003-09-22 |
WO2003076600A3 (en) | 2004-02-05 |
CA2477743A1 (en) | 2003-09-18 |
CA2477743C (en) | 2011-11-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7790459B2 (en) | In vitro maturation of immature human oocytes | |
Canipari | Oocyte–granulosa cell interactions | |
Chian et al. | Maturational and developmental competence of cumulus-free immature human oocytes derived from stimulated and intracytoplasmic sperm injection cycles | |
Bevers et al. | Regulation and modulation of oocyte maturation in the bovine | |
Wynn et al. | Pretreatment with follicle stimulating hormone promotes the numbers of human oocytes reaching metaphase II by in-vitro maturation. | |
Mermillod et al. | Aspects of follicular and oocyte maturation that affect the developmental potential of embryos | |
Kim et al. | In vitro maturation, fertilization, and development of human germinal vesicle oocytes collected from stimulated cycles | |
Sirisathien et al. | Influences of epidermal growth factor and insulin-like growth factor-I on bovine blastocyst development in vitro | |
Luvoni et al. | Factors involved in vivo and in vitro maturation of canine oocytes | |
Rodrigues et al. | Embryonic development of in vitro matured and in vitro fertilized dog oocytes | |
Jaroudi et al. | Pregnancy after transfer of embryos which were generated from in-vitro matured oocytes. | |
Smitz et al. | Oocyte in-vitro maturation and follicle culture: current clinical achievements and future directions | |
AU2017371402B2 (en) | Compositions and methods for maturation of oocytes in vitro | |
Tibary et al. | Update on reproductive biotechnologies in small ruminants and camelids | |
Salha et al. | Dynamics of human follicular growth and in-vitro oocyte maturation | |
JP2011507530A (en) | Protein-free gamete and embryo handling media products and culture media products | |
Chian et al. | In vitro maturation of immature human oocytes for clinical application | |
Trounson | The production of ruminant embryos in vitro | |
Mikkelsen et al. | Maternal serum supplementation in culture medium benefits maturation of immature human oocytes | |
KR20190052542A (en) | A follicular fluid replacement medium for in vitro Maturation of oocytes and The Use thereof | |
Huang et al. | Biochemical compositions of follicular fluid and the effects of culture conditions on the in vitro development of pig oocytes | |
US20010028878A1 (en) | In vitro maturation of human oocytes in one day | |
Jee et al. | Influence of well defined protein source on in vitro maturation of human oocyte: human follicular fluid versus human serum albumin | |
Heng et al. | Effects of granulosa coculture on in-vitro oocyte meiotic maturation within a putatively less competent murine model | |
Heng et al. | Investigations of oocyte in vitro maturation within a mouse model |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: MCGILL UNIVERSITY, CANADA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:CHIAN, RI-CHANG;REEL/FRAME:015269/0822 Effective date: 20030407 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |