a unified approach to human embryo culture don rieger phd and klaus wiemer ph.d. life global,...

A Unified Approach to Human Embryo Culture

Don Rieger PhD and Klaus Wiemer Ph.D.

Life Global, Northwest Center for Reproductive

Sciences

kwiemer@nwreprosci.com

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