The Influence of Culture Medium on Embryonic Viability. Klaus E. Wiemer PhD Director of Embryology and Reproductive Sciences

Transcription

1 The Influence of Culture Medium on Embryonic Viability Klaus E. Wiemer PhD Director of Embryology and Reproductive Sciences

2 Developmental Difference In Vivo vs. In Vitro

3 Developmental Environment

4 Attempts to Mimic In Vivo Environment Culture Well EGF Oil PDFα ILGF

5 Micro droplet Oil Overlay 30mm culture dish Oil Culture medium 4 cell embryo

6 Micro droplet Oil Overlay VOC s Filtration Oil Issues Plastic Ware Media

7 Factors affecting Culture Conditions! Culture Environment! Outside variables! Oil! Clinical Influences

8 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

9 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

10 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)

11 The Interaction of Clinical Variables on Outcomes! Assessing the impact of follicular stimulation! Thorough evaluation of embryos # Normal # Something wrong?! Cryopreservation! Assessment of transfer

12 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

13 Thoughts on Embryo Culture Medium

14 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

15 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 )

16 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

17 Emerging Thoughts on Blastocyst Culture

18 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

19 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!

20 Beneficial Effects of Amino Acids on Embryonic Development

21 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

22 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

23 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

24 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

25 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

26 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

27 Embryo grown in AA free Medium

28 Embryo grown in AA supplemented medium

29 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

30 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

31 Extended Embryo Culture! Addition of growth factors: # Platelet derived growth factor # Insulin-like growth factors # LIF # Various other cytokines

32 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