a unified approach to human embryo culture don rieger phd and klaus wiemer ph.d. life global,...
TRANSCRIPT
A Unified Approach to Human Embryo Culture
Don Rieger PhD and Klaus Wiemer Ph.D.
Life Global, Northwest Center for Reproductive
Sciences
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
Medium Year InvestigatorsSOM 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)
The Development of global®
global® Formulation
NaClKClCaCl2KH2PO4
MgSO4
Buffer NaHCO3
GlucoseNaLactateNaPyruvate
Non-Essential AA's 10 NEAA'sGlutamine Dipeptide Gly-GlnEssential AA's 9 EAA'sChelator EDTAIndicator Phenol RedAntibiotic Gentamicin
Salts
Energy Substrates
The global® Family of Media
The global® Family of Media
ObjectiveTo 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 global®global® forFertilization
NaCl NaClKCl KClCaCl2 CaCl2KH2PO4 KH2PO4
MgSO4 MgSO4
Buffer NaHCO3 NaHCO3
Glucose Glucose (High)NaLactate NaLactateNaPyruvate NaPyruvate
Non-Essential AA's 10 NEAA's 10 NEAA'sGlutamine Dipeptide Gly-Gln Gly-GlnEssential AA's 9 EAA's 9 EAA'sChelator EDTA EDTAIndicator Phenol Red Phenol RedAntibiotic Gentamicin Gentamicin
Salts
Energy Substrates
2. global® w/HEPES
Acid-Base Buffer Systems
global® w/HEPES Formulation
Components global®global®
w/HEPESNaCl NaClKCl KClCaCl2 CaCl2KH2PO4 KH2PO4
MgSO4 MgSO4
Buffer NaHCO3 HEPES
Glucose GlucoseNaLactate NaLactateNaPyruvate NaPyruvate
Non-Essential AA's 10 NEAA's 10 NEAA'sGlutamine Dipeptide Gly-Gln Gly-GlnEssential AA's 9 EAA's 9 EAA'sChelator EDTA EDTAIndicator Phenol Red Phenol RedAntibiotic Gentamicin Gentamicin
Salts
Energy Substrates
3. PGD Biopsy Medium
The PGD Biopsy Procedure
The Effect of Biopsy Medium on Pregnancy Rate
0
10
20
30
40
50
Pre
g. R
ate
(%
of
retr
ieva
ls)
Time Period 1 Time Period 2
P < 0.05
No Biopsy Simple
Biop.Medium
Supplemented
Biop. Medium
No Biopsy
(Hill & Li, The Clinical Embryologist 7, 11-12, 2004)
PGD Biopsy Medium Formulation
Components global® PGD BiopsyNaCl NaClKCl KClCaCl2KH2PO4 KH2PO4
MgSO4
Buffer NaHCO3 HEPES
Glucose GlucoseNaLactate NaLactateNaPyruvate NaPyruvate
Non-Essential Amino Acids 10 NEAA's 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
Osmolyte Sucrose
Protein HSA
Salts
Energy Substrates
The Unified Approachto Culture
Day 1
ART Procedures – Day 0
Classical IVFglobal® for Fertilization
ICSIglobal® w/HEPES
Cultureglobal®
Day 1
ART Procedures – Day 1
Classical IVFglobal® for Fertilization
Post-ICSI Cultureglobal®
Culturefresh droplets of
global®
Day 3
ART Procedures – Day 3
Cultureglobal®
Day 5
Culturefresh droplets of
global®
Assisted Hatchingglobal® w/HEPES
PGD BiopsyPGD Biopsy Medium
CryopreservationTransferglobal®
Transferglobal®
Cryopreservation
ART Procedures – Day 5 and 6
Cultureglobal® CryopreservationTransfer
global®
Culturefresh droplets of
global®
Day 6
Thank You