Sequential Culture

Patrick Quinn PhD, HCLDpatrick.quinn@coopersurgical.com

Sage In-vitro Fertilization, Inc

Redmond, Oregon, USA

Preimplantation DevelopmentPreimplantation Development

Pre-compactionPre-compaction D1-D3, zygote D1-D3, zygote 12/16-cell12/16-cell In oviductIn oviduct maternal mRNAmaternal mRNA UndifferentiatedUndifferentiated Single cellsSingle cells Energy metabolism PEnergy metabolism P G G Amino acids NEAAAmino acids NEAA Vitamins NoVitamins No No growthNo growth Growth factors?Growth factors?

Post- compactionPost- compaction Morula Morula Blastocyst Blastocyst In uterusIn uterus Embryonic mRNAEmbryonic mRNA ICM, trophectodermICM, trophectoderm Transport epitheliumTransport epithelium PP G G NEAA + EAANEAA + EAA YesYes Growth, ie expansionGrowth, ie expansion YesYes

Sequential Culture MediaSequential Culture Media

Imitative Principle Imitative Principle

In vitro = In vivoIn vitro = In vivo

Location of embryo changesLocation of embryo changes

Content of tract secretion changesContent of tract secretion changes

Mimicing In vivo ConditionsMimicing In vivo Conditions

1.1. Fertilization.Fertilization.

Spermatozoa require glucose; need at least 2.8 mM. Spermatozoa require glucose; need at least 2.8 mM. Quinn 1995 JARG 12:97-105Quinn 1995 JARG 12:97-105

2.2. If using ICSI, transfer injected oocytes directly to If using ICSI, transfer injected oocytes directly to Cleavage Medium that only has 0.1 mM glucoseCleavage Medium that only has 0.1 mM glucose

Fertilization has to be separated from Fertilization has to be separated from embryo culture in terms of culture embryo culture in terms of culture conditions.conditions.

Quinn’s Advantage Sequential Media

QA Cleavage

Medium

QA

Fer

tili

zati

on

Me

diu

m

QA Blastocyst Medium

Ionic CompositionEnergy SourcesAmino Acids pHOsmolalityVitaminsGrowth Factors

Components of ART Culture Media

Components of ART Culture Media

Sage IVF1. Inorganic Salts: NaCl, KCl, MgSO4, KH2PO4, NaHCO3,

EDTA Variation in Ca/Mg during fertilization and embryo

development.2. Energy Sources: Sodium pyruvate, calcium-L+-lactate,

glucose, sodium citrate. Only the bioactive L+ isomer of lactate present3. Amino Acids: non-essential and essential, plus taurine,

alanyl glutamine4. pH: Specified under set CO2 level – 7.3 for Fertilization

and Blastocyst medium, 7.2 for Cleavage medium5. Osmolarity: 265 mosmoles/Kg6. Vitamins: in Blastocyts medium7. Other: Phenol red8. Antibiotic: Gentamicin

Ionic Composition – Ca/Mg ratios

Energy Sources – Ca lactate

Amino Acids – Al-Gln, no alanine

pH – defined with a specific % CO2

Osmolality – range between 265-280

Vitamins – in blastocyst medium; role ill-defined

Components of ART Culture Media

Other Highlights• Sodium citrate in Fertilization & Cleavage

Medium

• Lactate present as calcium lactate

• EDTA present in precompaction but not

post compaction media

• Pantothenate, choline, inositol and other

vitamins present in blastocyst medium

• Osmolality 265 mOsm/Kg

Concentration of Pyruvate, Lactate & Concentration of Pyruvate, Lactate & Glucose in Human Reproductive Tract Glucose in Human Reproductive Tract

FluidsFluids

Site Cycle phase Pyruvate L (+) Lactate

Glucose

G1 Oviduct Mid-cycle 0.32 10.5 0.5

G2 Uterus All phases 0.10 5.9 3.2

Gardner et al 1996

PyruvatePyruvate Included in nearly all ART mediaIncluded in nearly all ART media

Required for early development?Required for early development?

Amino acids can replace (Bavister)Amino acids can replace (Bavister)

This illustrates the plasticity in embryo This illustrates the plasticity in embryo metabolismmetabolism

PyruvatePyruvate QA Fertilization & Cleavage QA Fertilization & Cleavage

MediumMedium

0.33 mM0.33 mM

QA Blastocyst MediumQA Blastocyst Medium

0.1 mM0.1 mM

LactateLactate L + isomer biologically active, ie metabolizedL + isomer biologically active, ie metabolized

lactatelactate Calcium-L-lactate = Ca Calcium-L-lactate = Ca

lactatelactate 2.04 mM = 4.08 mM L (+) lactate2.04 mM = 4.08 mM L (+) lactate

= 8.16 mM DL-lactate (HTF = 21.4 mM)= 8.16 mM DL-lactate (HTF = 21.4 mM)

Extra D (-) lactate Extra D (-) lactate effects pHi effects pHi

GlucoseGlucose Required for spermRequired for sperm PrecompactionPrecompaction glycolysis is best, glycolysis is best, low Glu, + EDTAlow Glu, + EDTA some required for pentose phosphate some required for pentose phosphate

pathwaypathway Postcompaction - need Postcompaction - need glycolysis for glycolysis for

growth & differentiationgrowth & differentiation

GlucoseGlucose QA Fertilization MediumQA Fertilization Medium

2.78 mM - sperm function, cumulus/corona cells2.78 mM - sperm function, cumulus/corona cells

QA Cleavage MediumQA Cleavage Medium

0.1 mM - pentose phosphate pathway and other 0.1 mM - pentose phosphate pathway and other metabolismmetabolism

QA Blastocyst MediumQA Blastocyst Medium

2.78 mM - increased glycolysis2.78 mM - increased glycolysis

Calcium/Magnesium Interactions in Early Embryonic Development

• Early hamster embryo development is disrupted by increased free Ca-i

• Increased Ca-i can be inhibited by > Mg2+ levels in the medium, the presence of the Ca-channel blocker, nifedipine and the intracellular Ca chelator, BAPTA

• All of these treatments increased hamster embryo development

• BUT, high Mg inhibits sperm capacitation

Lane & Bavister, Biol Reprod 59:1000-1007, 1998

Rogers & Yanagimachi, Biol Reprod 15:614-619, 1976

Why vary the Mg2+ concentration?

High Mg2+ decreases uptake of exogenous Ca2+.

Therefore use with embryos to prevent damage to mitochondria and subsequent abnormal energy metabolism.

But keep Mg2+ low in Fertilization medium as sperm require a Ca2+ spike to undergo capacitation and acrosome reaction

Magnesium Concentrations in Media

QA Fertilization Medium

0.2 mM

QA Cleavage & Blastocyst Medium

2 mM

AMINO ACIDS IN ART MEDIA

• Non-essential: 7 + Gln - Used before and after compaction - cleavage rate precompaction - blastocoel, trophectoderm,

hatching postcompaction

• Essentials: 12 - Used after compaction - ICM

Amino Acids Non-Essential Essential

Alanine omitted Arginine 0.1 mM

Asparagine 0.1 mM Histidine 0.1 mM

Aspartate 0.1 mM Leucine 0.2 mM

Glutamate omitted Lysine 0.2 mM

Glycine 0.1 mM Threonine 0.2 mM

Proline 0.1 mM Valine 0.4 mM

Serine 0.1 mM

Taurine 0.1 mM

Alanyl-glutamine 1.0 mM

Amino AcidsCannot get maximum growth rates of 1-cell mouse zygotes to fully expanded blastocysts when medium contains essential amino acids, eg Blastocyst Medium.

Mouse embryos have to be cultured in Cleavage Medium (non-essential amino acids) and then placed in Blastocyst Medium to get good rates of blastocyst formation.

Highly likely that human embryos would react the same.

Role of Amino Acids in Preimplantation Development

Non Essentials

Role - Increase mitotic rate

Mechanisms

(i) Regulators of energy metabolism

(ii) Osmolytes; maintain intracellular physiology in high osmotic pressure of oviduct fluid

(iii) Buffer pHi

Essential

Role - Stimulate differentiation of ICM

Mechanism - unknown

Tricarboxylic Acid (TCA) Cycle Krebs Cycle

AMMONIUM PRODUCTION IN ART MEDIA

• Dependent on levels of aa’s

• Stabilized dipeptides, eg. Ala-Gln

• Pyruvate acts as sink Alanine

Ammonium production from different culture media

Lane & Gardner, BOR 69:1109-17, 2003

0

0.1

0.2

0.3

0.4

0.5

0.6

0 24 48 72 96 120

Culture period (h)

Am

mo

niu

m C

on

cen

trat

ion

(m

M)

G1.2/G2.2

QA

P1/Blast

KSOMaa-wellKSOMaa-drop

No significant difference between QA and G.2 series

Appearance of Alanine During Incubation of

Bovine Embryos

0

0.51

1.52

2.5

33.5

4

Z 4 C-16 M B

Stage of Development

Ala

nine

App

eara

nce

(pm

ol/e

mbr

yo/h

)

Partridge & Leese, Reprod Fertil Devel., 8:945-50, 1996

Pyruvate Acting as a Sink for Ammonium Production During

In Vitro Culture

transaminaseAlanine Pyruvate + NH3

Vitamins

• Vitamins in Eagle’s MEM (+ AAs) maintain normal metabolic activity in mouse and rat blastocysts, and normal implantation rates and fetal weight

• In hamsters, only pantothenate stimulates 1-cell blastocyst. Several water soluble vitamins stimulate expansion and hatching

• Included in postcompaction media, eg G2, Blastocyst Medium, QA Blastocyst Medium

• Exact requirements and specificity for human embryos is unknown

8-cell embryos; D3QA Fertilization & Cleavage Media

Grade A( = 4); 0-5% fragmentation

Late Day 4 Blastocysts in medium containing Growth Factor

Day 5 Blastocysts in medium containing Growth Factor

Microdrop culture to enhance effects of autocrine/paracrine growth factors

produced by embryo itself

Microdrops in 60 mm diameter dishes, Falcon #353802. Overlay with 9 mL of oil

30 uL drops used for washing 2PN embryos

10 uL drops used for culture of individual embryos

4-well Nunc Multi dish

50 uL to 1.0

mL

Oil ART-4008Cleavage Medium ART-1026 or Protein Plus Cleavage Medium ART-1526

Place NO MORE NO MORE than 6 than 6

oocytes /embryos in a dish!!

Group Culture for same effect

IF YOU WANT TO DO GROUP CULTURE OF EMBRYOS RATHER THAN SINGLE EMBRYO CULTURE, PREPARE THE FOLLOWING TYPES OF DISHES FOR D1-3 AND D3-5/6 WITH THE APPROPRIATE MEDIUM.

HOWEVER, it is important to stress that for best pH and temperature control, Place NO MORE than 6 NO MORE than 6 embryos in a dish!!

Place the following microdrops in 60 mm diameter dishes, Falcon #353802. Overlay with 9 mL of oil. Prepare no more than two dishes at a time!

30 uL drops used for washing embryos

30 uL drop used for culture of grouped embryos

1 2 3

A

B

CWash the embryos through the two 30 uL drops 1 and 2 in each row and then place in the third drop 3 for culture. Up to 6 embryos can be cultured in one 30 uL drop.

Life Global Comparisons

Life Global Comparisons

Original version 2006 Version 3, 2006

Be careful about what some ART media companies claim and/or tell you

How different is the protocol of Single Step Culture from Sequential

Culture?

One still has to change embryos on Day 3 to fresh medium.

It is the composition of the media that is different.

How different is the protocol of Single Step Culture from Sequential

Culture?

Commercially, Sage can provide both options of culture.

Several labs have used the Sage Cleavage Medium for culture from ICSI to D3, then change to fresh Cleavage Medium for D3 to D5/6, and have obtained high quality blastocysts.

Lynette Scott, BostonSerdar Coskun, Saudi Arabia

The unanswered question of this debate is that there has been no real trial of the two culture systems. And, there may be subtle differences between different programs, eg patients, stimulation protocols, laboratory procedures, so each laboratory should do its own trial to compare the different culture systems.

Comparison of Different

Media Systems

Media Comparisons

Two Phase Trials Phase I: Within patient, sibling oocyte split between two media sources in vitro data parameters, eg fertilization, cleavage rate, morphology.

Phase II:Between patient, randomized allocation to media in vivo data, eg PR & IR.

Media Comparisons

Two Phase Trials In both Phase I and II, try to make the

comparisons as balanced as possible, eg:

1. Same protein supplement.2. Same pH, O2 and, if possible, same

incubator.3. Prospective randomization of

everything.4. Same embryologist, physician etc

working on a within patient comparison.

Design of Study

1. Have a Balanced Comparison in time and patients. For example, one week (or month etc) with Sage media, one week with current medium. Make patients similar in age, diagnosis, etc.

2. Compare for 3-4 time periods with a minimum of 20 patients in each group.

3. Repeat comparison if problems arise, eg fertilization, development and pregnancy rates decline (<10%) for no apparent reason.

Laboratory and Clinical End Points

Lab End Points• Fertilization Rate• Development Rate• Degree of

Fragmentation• Lab specific

endpoints– Multinucleate– PN score– blastocyst

development

Clinical End Points

• Implantation Rate

Comparison of media with protein supplement as SPS or HSA

Suggested design:

• Patients ≤ 38 years old

• 1st or 2nd cycle

• No severe male or female pathology

• Minimum of 10 embryos to do a within patient 50:50 split

• Attempt to do ETs with embryos from a single medium group.

The unanswered question of this debate is that there has been no real trial of the two culture systems. And, there may be subtle differences between different programs, eg patients, stimulation protocols, laboratory procedures, so each laboratory should do its own trial to compare the different culture systems.

Only in this way can a lab get appropriate evidence as to whether Single Step Culture versus Sequential Culture system is best for them.