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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.
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