Developments in embryo culture media. Presentation from the RHG Congress 2018.

1. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
Developments in embryo
culture media
Dr Christine Leary
Consultant Embryologist
The Hull IVF Unit & CCMR Hull York Medical School

2. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
AimsAims
Role of embryo culture medium
The individual components
Different culture systems
Future developments

3. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
Role of culture mediaRole of culture media
• Facilitate “normal” fertilization and support
“normal” embryo development in vitro
• Avoid factors that can have a negative
influence on embryo development – short
term stress & longer term epigenetic
effects.

4. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
Background/ HistoryBackground/ History
•Originally based on simple media developed for organ explant and somatic cell
culture.
•Analysis of tubal and uterine fluid -characterized by high potassium and
chloride concentrations. Carbohydrate levels vary between the oviduct and the
uterus and within the cycle. High concentrations of non-essential amino acids
and glutamine. Change in pH along tract.
•Animal studies – mouse, hamster

5. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
Metabolic and nutritional requirementsMetabolic and nutritional requirements
• Stage specific
• Species specific
• Before 1997-
single media
formulations
• Animal research –different nutritional and
ionic requirements – for different stages;
• Gamete maturation, activation, fertilisation,
genomic activation, cleavage, compaction,
blastocyst formation

6. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
Preimplantation development
Blastocoel
cavity
ICM
TE
5-9 days, depending on species
• Genetic
material
combined
• Increase
cell number
• Genome
activated
• Cell-cell
junctions
begin to
form
• Differentiation
• Protein synthesis
• Na+
/K+
ATPase
• True growth
• Prep for implantation
Cell number
Mitochondrial number fixed

7. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
Glucose - summary
Glucose consumption
Lactate production
Relative contribution of PPP

8. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
Metabolic studiesMetabolic studies
• Glucose- Gardner &
Leese 1987, Lane &
Gardner 1996, Butcher et
al 1998, Gardner et al
2001, 2011
• Pyruvate- Conaghan
et al 1993, Turner et al
1994
• Amino acids-
Houghton et al 2002,
Brison et al 2004
Glucose uptake on Day 4 (A) & 5 (B) of embryonic development
and pregnancy outcome (positive fetal heart beat).
Gardner D K et al. Hum. Reprod. 2011;humrep.der143

9. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
Common protocolsCommon protocols
• One step -non-renewal
• Single formulation – renewed d2/3
• Two step – sequential
‘Meet the changing needs of the embryo’ – change levels of glucose, complement of
amino acids and chelators or ‘Let the embryo decide’ For review- see Biggers and Summers
2008 –Fertil. Steril. 90(3), 473-483

10. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
Physical Parameters &Physical Parameters &
CompositionComposition
• Osmolarity (275-
305mOsm/L)
• pH (7.2-7.5)
• Temperature
• CO2 concentration
• Oxygen-tension
• Light
• Humidity
• Endotoxin free
• Storage conditions
• Volume of culture medium &
consumables
• Oil overlay
• Small ions
• Energy substrates
• Macromolecules;
carbohydrates, lipids, fatty
acids, protein, amino acids,
• Antibiotics,
• Cofactors, vitamins,
hormones, growth factors,
hyaluronic acid
– What concentrations… ?

11. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
Composition - IonsComposition - Ions
• Balanced Salt solution;
– Na, K, Cl, Ca, Pi, Mg,HCO3
– Concentrations/ balance differ
•Water = 99% of the content, purity =pyrogen-free

12. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
Composition - EnergyComposition - Energy
Carbohydrate – energy
source
• Pyruvate, lactate,
glucose
Amino acids
• Essential and non-essential
• Stability of Glutamine
• Stage specific –
– High glucose, crabtree effect (premature stimulation
of glycolysis)
– Interactions (inhibition alleviated by the addition of
amino acids and EDTA)
– Non essential until 8 cell stage, essental amino acid
stimulate cleavage post 8 cell stage

13. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
Protein supplementProtein supplement
• Required for;
– sperm capacitation
– Handling
– Chelators
– Regulators of redox
potential
– Lipid and peptide
carriers
– Source of fixed
nitrogen
– Bind lipids
– Stabilize growth
factors
• Risks
– BSA –CJD
– Whole serum- large
fetuses
– Recombinant –viral
transmutations
• HSA ; 4.5% albumin
• SSS; 84% HSA, 16% α,β
globulins

14. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
Growth factorsGrowth factors
• Insulin
• LIF
• EGF/TGFα
• TGFβ
• PDGG
• HB-EGF
• Benefits of group
culture- autocrine,
paracrine factors
• For review see Richter 2008
Current Opin Obs Gyn 20 (3)
292-304

15. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
Consequences of inappropriate conditionsConsequences of inappropriate conditions
• Cellular stress, cell death
• Retarded cleavage
• Cleavage arrest
• Cytoplasmic blebbing
• Impaired energy production
• ‘Quite embryo hypothesis’- Leese 2012 Reproduction 143,
417-427
• Inadequate genome activation and transcription
• Altered expression of key genes- influence birth
weight.

16. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF UnitBirthweight distributions of live born singletons resulting from embryo culture in either Vitrolife
or Cook sequential media.The graph depicts the percentage of newborns per birthweight
category.
Dumoulin J C et al. Hum. Reprod. 2010;25:605-612

17. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
Relevance of differencesRelevance of differences
in compositionin composition

18. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
How to choose?How to choose?
Criteria
1)Performance? – laboratory, clinical results,
long-term safety, cost benefit analysis.
2)Documentation? - Manufacturer research &
design philosophy, product information,
composition, quality control and assurance, CE
marking, FDA approval, reliability, supply,
distributor, transport, shelf life.

19. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
PerformancePerformance
Is your aim to obtain:
•A high fertilization rate ?
•The maximum number of top grade embryos?
•A high rate of blastocyst formation?
•A high pregnancy rate per fresh embryo
transferred?
•A low miscarriage rate?
•A high cumulative delivery rate (fresh + frozen)?
•A low rate of multiple pregnancy rate?

20. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
Documentation -Quality controlDocumentation -Quality control
• GMP
• Licensing
• LAL testing
• Sperm survival
• Mouse embryo toxicity
• Certificates of analysis
• CE marking

21. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
Future Studies & DevelopmentsFuture Studies & Developments
• What components are needed, beneficial,
detrimental?
• Are animal models of any value?
• Ability to generate data from large prospective
studies and ability to follow up of off-spring, but ?
relevance for the human situation.
• Use of donated supernumerary human
embryos?

22. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
Culture
medium
•Glucose
•Pyruvate
•Lactate
•Oxygen
•Amino acids
•Tryglycerides
BiomarkersBiomarkers
Uptake Production
Pyruvate
Lactate
Glucose
Amino acids
Lipid
Oxygen
H20
CO2
Lactate
NH4
+
Enzymes
Hormones
Other factors
Cytokines
Amino acids
Proteins

23. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
Substrate utilisation in embryos from normal weightSubstrate utilisation in embryos from normal weight
and overweight and obese women (owob)and overweight and obese women (owob)

24. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
Individualised culture mediumIndividualised culture medium

25. East Riding Fertility Services, The Hull IVF UnitEast Riding Fertility Services, The Hull IVF Unit
ConclusionConclusion
• Significant developments, progress ongoing
• Need appropriate models and surrogate end
points – Possibly;
• Fertilization rate (2PN rate)
• Cleavage rate (correct kinetics), morphology (empirical)
• Chromosome pattern (karyotype), Expression patterns
(transcriptome), Metabolism (metabolome)
Likely a combination of all.
• Your success criteria (performance/
documentation) will define your selection
parameters and influence on your current and
future culture strategy.