Download One-step versus Two-step culture of embryos: Is there a difference?

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