Transcript Slide 1
A Unified Approach to Human Embryo Culture Don Rieger, Ph.D. Vice President, Research & Development LifeGlobal LLC [email protected] Overview I. A Single Medium vs. Sequential Media II. The global® Family of Media III. A Unified Approach to Culture A Single Medium vs. Sequential Media from Zygote to Blastocyst A Single Medium vs. Sequential Media for Embryo Culture “… in order to optimize mammalian embryo development in culture, sequential media are required, each designed to meet the changing requirements of the developing embryo.” (Gardner and Lane, In Principles of Cloning, 187-213, 2002) “The popularity of this conviction is due to its intuitive appeal, but critical examination shows that it is not supported by direct experimental evidence” (Biggers et al., Hum Reprod 20, 3376-84, 2005) A single medium can be used for culture from the zygote to the blastocyst stage Changing the medium is a stress on the embryo The Development of global® Medium Year Investigators SOM 1991 Lawitts & Biggers KSOM 1994 Erbach et al. KSOMaa 2000 Biggers et al. global® (Ala-Gln) 2002 Wiemer et al. global® (Gly-Gln) 2006 (Biggers et al., 2004) global® Formulation Salts NaCl KCl CaCl2 KH2PO4 MgSO4 Buffer Energy Substrates Non-Essential AA's Glutamine Dipeptide Essential AA's Chelator Indicator Antibiotic NaHCO3 Glucose NaLactate NaPyruvate 10 NEAA's Gly-Gln 9 EAA's EDTA Phenol Red Gentamicin Greenblatt et al. Toronto, ON, Canada % of Zygotes or Transfers (Human Reprod. 20, Suppl. 1, i21, Abstr. O-058, 2005) 75 P = 0.0001 Global G1/G2 N.S. 50 N.S. 25 0 Blastocysts Preg. Rate Impl. Rate Zech et al. Bregenz, Austria % of Embryos or Transfers (Human Reprod. 21, Suppl. 1, i162, 2006) 60 P < 0.05 P < 0.01 Global G1/G2 40 P < 0.05 20 0 Blastocysts Preg. Rate Impl. Rate Angus et al. Houston, TX, USA (Fertil. Steril 86, Suppl. 2: S229, 2006) % of Embryos or Transfers Global P < 0.05 60 N.S. G1/G2 N.S. 40 20 0 Blastocysts +ve hCG Ongoing Matsubara et al. Hiroshima, Japan % of Zygotes or Transfers (Proc. 24th Ann. Meet. Japan Soc. Fertil. Implant., 206, 2006 ) Global 75 P < 0.001 BAS1/BAS2 N.S. 50 N.S. 25 0 > 6 cells Day 3 Blastocysts Day 5 Pregnancy Rate Sepulveda et al. Lima, Peru (Proc. Red Latinamer. Reprod. Asist., 49-50, 2007) % of Zygotes or Transfers Global ECM/Multiblast P = 0.036 75 P = 0.003 P = 0.019 50 25 0 Blastocysts Day 5 Ongoing Pregnancy Rate Implantation Rate Vansteenbrugge et al. Namur, Belgium (Fertil. Steril 88, Suppl 1: S319, 2007) % of Embryos or Transfers Global 60 P < 0.001 N.S. Blastocysts Day 5 Ongoing Pregnancy Rate G1/G2 N.S. 40 20 0 Implantation Rate Conclusion A single medium (Global) is as good as, or better than, sequential media for development of the human embryo to the blastocyst stage The ® global Family of Media The global® Family of Media Objective To maintain, as closely as possible, the same medium throughout all stages of gamete and embryo handling and culture In order to Minimize the stress of changing from one medium to another 1. ® global for Fertilization Energy Metabolism of the COC and Sperm global® for Fertilization Formulation Components Salts Buffer Energy Substrates Non-Essential AA's Glutamine Dipeptide Essential AA's Chelator Indicator Antibiotic NaCl KCl CaCl2 global® for Fertilization NaCl KCl CaCl2 KH2PO4 KH2PO4 MgSO4 MgSO4 NaHCO3 Glucose NaLactate NaPyruvate 10 NEAA's Gly-Gln 9 EAA's EDTA Phenol Red Gentamicin NaHCO3 Glucose (High) NaLactate NaPyruvate 10 NEAA's Gly-Gln 9 EAA's EDTA Phenol Red Gentamicin global® 2. global® w/HEPES Acid-Base Buffer Systems global® w/HEPES Formulation Components Salts Buffer Energy Substrates Non-Essential AA's Glutamine Dipeptide Essential AA's Chelator Indicator Antibiotic NaCl KCl CaCl2 global® w/HEPES NaCl KCl CaCl2 KH2PO4 KH2PO4 MgSO4 MgSO4 NaHCO3 Glucose NaLactate NaPyruvate 10 NEAA's Gly-Gln 9 EAA's EDTA Phenol Red Gentamicin HEPES Glucose NaLactate NaPyruvate 10 NEAA's Gly-Gln 9 EAA's EDTA Phenol Red Gentamicin global® 3. PGD Biopsy Medium The PGD Biopsy Procedure The Effect of Biopsy Medium on Pregnancy Rate Preg. Rate (% of retrievals) P < 0.05 50 40 30 20 10 0 Simple No Biopsy Biop.Medium Time Period 1 No Biopsy Supplemented Biop. Medium Time Period 2 (Hill & Li, The Clinical Embryologist 7, 11-12, 2004) PGD Biopsy Medium Formulation Components global® PGD Biopsy NaCl KCl CaCl2 KH2PO4 MgSO4 NaCl KCl Non-Essential Amino Acids NaHCO3 Glucose NaLactate NaPyruvate 10 NEAA's HEPES Glucose NaLactate NaPyruvate 10 NEAA's Glutamine Dipeptide Glycyl-glutamine Glycyl-glutamine Essential Amino Acids 9 EAA's 9 EAA's Chelator EDTA EDTA Indicator Phenol Red Phenol Red Antibiotic Gentamicin Gentamicin Salts Buffer Energy Substrates Osmolyte Protein KH2PO4 Sucrose HSA The Unified Approach to Culture ART Procedures – Day 0 Classical IVF ICSI global® for Fertilization global® w/HEPES Culture global® Day 1 Day 1 ART Procedures – Day 1 Classical IVF Post-ICSI Culture global® for Fertilization global® Culture fresh droplets of global® Day 3 ART Procedures – Day 3 Transfer global® Culture global® PGD Biopsy PGD Biopsy Medium Cryopreservation Assisted Hatching Culture fresh droplets of global® Day 5 global® w/HEPES ART Procedures – Day 5 and 6 Culture Transfer global® Cryopreservation global® Culture fresh droplets of global® Transfer global® Day 6 Cryopreservation Thank You Biggers JD (2001) Research in the canine block. Int J Dev Biol 45, 469-76.