The Influence of Culture Medium on Embryonic Viability Klaus E. Wiemer PhD Director of Embryology and Reproductive Sciences
Developmental Difference In Vivo vs. In Vitro
Developmental Environment
Attempts to Mimic In Vivo Environment Culture Well EGF Oil PDFα ILGF
Micro droplet Oil Overlay 30mm culture dish Oil Culture medium 4 cell embryo
Micro droplet Oil Overlay VOC s Filtration Oil Issues Plastic Ware Media
Factors affecting Culture Conditions! Culture Environment! Outside variables! Oil! Clinical Influences
Effect of Styrene and Toluene on Embryonic Development Type of Contamination % Contaminant Level Blastocyst Styrene.2, 1.7, 17, 174 ppm 94 " 100% Toluene.5, 4.5, 45, 445 ppm 94 " 100% w/o BSA " 94 " 100% w/o oil overlay >17 & >45ppm " 0% Embryonic block 4 " 8 cell stage
Total Organic Carbon and Embryonic Development!Impact of TOC levels on human embryonic development is unclear!possible to have ion free H 2 O with!toc levels!toc effects """" environmental!h 2 O/Media TOC levels < 5 ppb
QC Issues for Oil! Maintenance of droplet "integrity changes with Lot #! Source of endotoxins or other contaminants! Mouse embryo tested! Hybridoma tested (Lite Oil, Life Global)
The Interaction of Clinical Variables on Outcomes! Assessing the impact of follicular stimulation! Thorough evaluation of embryos # Normal # Something wrong?! Cryopreservation! Assessment of transfer
Assessment of Follicular Stimulation! Length of stimulation A. E2 level B. No. of mature oocytes C. Dosage of gonadotrophin! Oocyte response during ICSI A. 3-6 hrs post-retrieval 1. Immediate breakage 2. Incidence of dismorphic qualities
Thoughts on Embryo Culture Medium
Classifications of Culture Medium!Simple:!Complex: Balanced salt solutions containing glucose and pyruvate in varying quantities Balanced salt solutions containing many additional components including amino acids
Complex Culture Formulations Ham s F-10 Ham s F-12 MEM! Based on tissue culture needs! Contain potential embryo toxic products! Support early embryonic development! Sub-optimal post-genomic activation development (D3 )
Alternative Solutions for Early Embryonic Development! Complex culture formulation specifically designed to support early (Day 0 " 3) embryonic development! Contains glucose and all amino acids! Suitable for Day 2 or 3 transfers
Emerging Thoughts on Blastocyst Culture
Blastocyst Culture Methods! Sequential Culture Media! Phase I simple salt solutions supplemented with pyruvate, lactate, glucose, some amino acids # 1st 2 days of culture after fertilization # Up to genomic activation! Phase II complex solution contains, amino acids, glucose, vitamins # Supports embryonic differentiation
Blastocyst Culture Alternative Thoughts?! The use of one culture medium to support entire embryonic development from Day 1 to Day 5! This culture medium contains all necessary amino acids, glucose and vitamins to support embryonic differentiation throughout development! Embryo uses what it needs!
Beneficial Effects of Amino Acids on Embryonic Development
The Importance of Amino Acids In Vivo! Found in varying quantities in the reproductive tract! Unfertilized ovum contain a significant free amino acid pool-alanine, glycine, etc Schultz et al., 1981
Proposed Functions of Intracellular AA! Precursors for biosynthesis! Energy sources! Regulators of energy metabolism! Osmolytes! Buffers of intracellular ph! Chelators of heavy metals Summers and Biggers 2003
Essential vs. Nonessential AA! Term coined following nutritional studies! Adapted from cell culture studies Rose 1938 Eagle 1959! Traditional division of amino acids in embryo development is over simplistic and might mask important subtle effects! Embryos in culture should be exposed to all amino acids and allowed to choose which to consume or release Houghton et al., 2002
Effects of AA Supplementation on Mouse Embryonic Development! Enhanced development to the blastocyst stage! Enhanced blastocyst hatching! Proliferation of cells within the trophectoderm and ICM! Increased allocation of cells to the ICM! Better organization of the extracellular matrix Biggers el al., 2000
Impact of AA on Morphological Aspects of Human Embryos! Greater number of blastomeres present at Day 3 Freeman and Rieger 2004, Wiemer et al., unpublished data! Better overall morphological scoring on Day 3 Neal et al., 2004! Higher incidence of compaction Biggers and Racowsky 2004, Neal et al., 2004, Wiemer et al., unpublished data! Required for efficient rates of high quality blastocyst formation
Further Observations Regarding AA (present throughout culture)! More efficient blastocyst development from 8-10 cell embryos! Accelerated rates of blastocoel cavity formation on Day 4 PM! Higher incidence of expanded/hatching blastocysts on Day 5 Wiemer et al., unpublished data
Embryo grown in AA free Medium
Embryo grown in AA supplemented medium
Glutamine- what is all the fuss about?! Possible toxic effect on embryos due to ammonium production! No evidence of toxicity associated with L-Glutamine on embryonic or fetal development! Alanyl L-Glutamine is more stable; used in commercial media despite lack of evidence Lane and Gardner 2004 Biggers 2004 a,b! Glycyl L-Glutamine shown to improve blastocyst quality
Amino Acid Supplementation! Amino Acids provide many beneficial roles for in vitro embryonic development! Current classification of amino acids for embryo culture is inadequate! All amino acids should be present during preimplantation embryo culture to maximize development
Extended Embryo Culture! Addition of growth factors: # Platelet derived growth factor # Insulin-like growth factors # LIF # Various other cytokines
In Summary! Many outside variables influence In Vitro development! Proper QC protocols must be in place to maximize embryonic potential! Culture media can be classified based upon its components and use! Amino Acids are beneficial for optimum development! Additional supplementation with cytokines might improve development