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Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. One-step versus Two-step culture of embryos: Is there a difference? Liow Swee Lian O & G Partners Fertility Centre Gleneagles Hospital SINGAPORE Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. White, PR (1954) The cultivation of animal and plant cells Witkowski JA (1979) Med History 23: 279-296 Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Historical background Ross Granville Harrison (1907): • Short note on “Observations on the living developing nerve fibre” that described his research on the growth and development of the nervous system. • Fragments of tadpole spinal cord were incubated in a clot of lymph in a hollow glass-slide • Nerve fibres grew out from the explants by active movements of the nerve fibre tips • First tissue culture technique Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. The potential of the tissue culture technique devised by Harrison was immediately recognized. Abercrombie has described this work as an "astonishing stride forward in the history of biology". M. Abercrombie, 'Ross Granville Harrison, 1870-1959', Biogr. Mem. Fellows R. Soc. Lond., 1961, 7: 111-126. J. M. Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. W H and M R Lewis (1911) commented: ``It is to be hoped that an artificial medium will be found as satisfactory as the plasma, for the advantages are obvious if one can work with a known medium in the investigation of the many new problems, which suggest themselves.'' Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Culture Media Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Conditions for Chemically defined culture media • Reproducible at different times and different laboratories. • Can be varied in a controlled manner. • Free of unknown biological activities, such as, enzymes and growth factors that may affect the response being studied. Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. • Chemically-defined media were designed for plant cells (White, 1946) and animal cells (Fischer, 1947). • Early studies of culture media for mammalian embryos were done in the rabbit and the mouse. • Whitten (1956): 8-cell mouse embryos developed to blastocysts in a simple chemically defined medium. Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Compositions of culture media Based largely on 3 physiological salines 1. Earle's balanced salt solution (Earle,1943), 2. Krebs-Ringer bicarbonate (Krebs and Henseleit, 1932), and 3. Tyrode's solution (Tyrode, 1910). Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Culture Medium Systems Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Culture of human preimplantation embryos • Early IVF days, embryos were cultured from zygote to day 2 (4-cell) or day 3 (8-cell) embryo stage Zygote (2PN-stage) 4-cell stage (Day 2) 8-cell stage (Day 3) Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Culture of human preimplantation embryos • Late 1990s, extended culture of zygote to blastocyst was introduced – To select more robust embryos for ET – Higher implantation rate – Reduce multiple gestation – Culture of PGD-embryos to blastocysts – Generation of embryonic stem cells 8-cell stage (Day 3) Morula (Day 4) Blastocyst (Day 5) Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Extended culture of zygote to blastocyst Three protocols 1. non renewal single medium protocol uninterrupted culture using one medium throughout the 5 days of culture 2. renewal single medium protocol interrupted culture where one medium is used throughout but is renewed on the third day of culture 3. sequential media protocol Interrupted culture where two media of different composition are used sequentially Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Justifications for sequential media 1. The changing energy requirements of the preimplantation embryo and the inhibitory effect of glucose on early cleavage stage embryos 2. The inhibitory effect of EDTA on blastocyst development and the inner cell mass (ICM) 3. The chemical breakdown of L-glutamine(Gln) in aqueous solution and the accumulation of ammonia and its effects on embryo development 4. The role of amino acids on preimplantation embryo development Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Utilization of energy substrates by preimplantation embryos during in vitro development PYRUVATE GLUCOSE MOUSE oocyte +++ + MOUSE zygote to 8-cell stage +++ + + +++ HUMAN oocyte +++ + HUMAN zygote to 8-cell stage +++ + HUMAN 8-cell to blastocyst ++ +++ MOUSE 8-cell to blastocyst Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Glucose utilization during different stages of preimplantation embryo in vitro development Martin KL et al (1993) J Reprod Fertil 99:259-266 Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. The role of EDTA in culture medium ethylenediaminetetraacetic acid (EDTA) Positive •used as an anti-oxidant against free oxygen radicals • to overcome 2-cell block in mouse embryos Negative • decreased cell count in mouse blastocysts • smaller fetuses ØHence, EDTA is absent in second step in sequential media. ØHowever, reducing EDTA concentration from 0.1 mmol/L to 0.01 mmol/L can avoid deleterious effects observed at higher EDTA concentrations (Biggers et al, 2008) Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Glutamine (Gln) •added in the culture media at 1 mmol/L • overcomes 2-cell block in mouse (Chatot et al, 1989) • reduce intracellular H2O2 and prevents DNA damage (Suzuki et al, 2007) • breakdown of Gln in aqueous solution • build-up of ammonium over time • ammonium has harmful effecs on embryos Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Levels of ammonium build-up in culture over time L-alanyl-L-glutamine (AlaGln) or glycyl-L-glutamine (GlyGln) can be used to replace Gln Hashimoto et al (2008) J Reprod Dev 54 (5): 370-374 Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. One-step versus Two-step (sequential) culture media Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Macklon et al (2002) Hum Reprod 17: 2700-2705 Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Macklon et al (2002) Hum Reprod 17: 2700-2705 Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Macklon et al (2002) Hum Reprod 17: 2700-2705 Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Macklon et al (2002) Hum Reprod 17: 2700-2705 Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Wirleitner et al (2010) RBM Online 21:776-782 Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Wirleitner et al (2010) RBM Online 21:776-782 Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Wirleitner et al (2010) RBM Online 21:776-782 Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Summary • Recent studies indicate that embryos grown in single medium were comparable to sequential medium • Less stress imposed on embryos in – Single medium – similar composition and osmolarity – Sequential medium – different compositions and possibly different osmolarity Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Summary Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Summary Designing culture medium qThe `back-to-nature' approach Ø the concentration of a substance incorporated into a medium should approximate the concentration to which the embryo is naturally exposed. qThe ‘let-the-embryo-choose’ prinicple Øto vary the concentrations of each compound separately, keeping the concentrations of the other components constant. ü The ‘back-to-nature ‘ principle would be more preferable to ‘let- the-embryo choose’ principle in designing chemically-defined culture medium