A Unified Approach to Human Embryo Culture

Don Rieger, Ph.D.Vice President, Research & Development

LifeGlobal LLCDonRieger@LIfeGlobal.com

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

Medium Year InvestigatorsSOM 1991 Lawitts & BiggersKSOM 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® FormulationNaClKClCaCl2KH2PO4

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

Greenblatt et al.Toronto, ON, Canada

(Human Reprod. 20, Suppl. 1, i21, Abstr. O-058, 2005)

Blastocysts Preg. Rate Impl. Rate0

25

50

75

% o

f Zyg

otes

or T

rans

fers Global G1/G2

P = 0.0001

N.S.

N.S.

Zech et al.Bregenz, Austria

(Human Reprod. 21, Suppl. 1, i162, 2006)

Blastocysts Preg. Rate Impl. Rate0

20

40

60

% o

f Em

bryo

s or

Tra

nsfe

rs Global

G1/G2P < 0.01P < 0.05

P < 0.05

Angus et al.Houston, TX, USA

(Fertil. Steril 86, Suppl. 2: S229, 2006)

Blastocysts +ve hCG Ongoing0

20

40

60

% o

f Em

bryo

s or

Tra

nsfe

rs

Global

G1/G2P < 0.05

N.S.

N.S.

Matsubara et al.Hiroshima, Japan

(Proc. 24th Ann. Meet. Japan Soc. Fertil. Implant., 206, 2006 )

PregnancyRateDay 3 Day 5

0

25

50

75

% o

f Zyg

otes

or T

rans

fers

Global

BAS1/BAS2

N.S.

P < 0.001

N.S.

> 6 cells Blastocysts

Sepulveda et al.Lima, Peru

(Proc. Red Latinamer. Reprod. Asist., 49-50, 2007)

0

25

50

75

% o

f Zyg

otes

or T

rans

fers

Global ECM/Multiblast

P = 0.019

P = 0.036

P = 0.003

OngoingPregnancy

Rate

ImplantationRate

BlastocystsDay 5

Vansteenbrugge et al.Namur, Belgium

(Fertil. Steril 88, Suppl 1: S319, 2007)

0

20

40

60

% o

f Em

bryo

s or

Tra

nsfe

rs

Global G1/G2

P < 0.001 N.S. N.S.

OngoingPregnancy

Rate

ImplantationRate

BlastocystsDay 5

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

ObjectiveTo maintain, as closely as possible, the same medium throughout all stages of

gamete and embryo handling and culture

In order toMinimize 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/HEPES

NaCl NaClKCl KClCaCl2 CaCl2KH2PO4 KH2PO4

MgSO4 MgSO4

Buffer NaHCO3 HEPESGlucose 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

Preg

. Rat

e (%

of r

etrie

vals

)

Time Period 1 Time Period 2

P < 0.05

No Biopsy SimpleBiop.Medium

SupplementedBiop. 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 HEPESGlucose 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 SucroseProtein 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

Biggers JD (2001) Research in the canine block. Int J Dev Biol 45, 469-76.