Historical Development of Embryo Culture Media

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Historical Development of Embryo Culture Media

• John D Biggers D.Sc., Ph.D.
• Harvard Medical School

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Steps in Achieving IVFET

• In vitro fertilization
• Culture of the preimplantation stages
• Embryo transfer into surrogate mothers

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PIONEER OF EMBRYO TRANSFER
Heape W (1891) Preliminary note on the
transplantation and growth of mammalian ova
within a uterine foster-mother. Proc Roy Soc Lond
B Biol Sci 48, 257-458.
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The questions Albert Brachet tried to answer by
culturing preimplantation mammalian embryos

• Is an egg or a young blastocyst capable of
• development outside the maternal organism?
• 2. If so, can it adapt itself to an artificial
  medium by creating special organs, or will it
  form its normal fixation and nutritional
  structures?

Brachet A (1912) Développement in vitro de
blastodermes et de jeunes embryons de mammifères.
C R Hebd Seances Acad Sci 155, 1191-1193.
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• Professor Albert Brachet
• First attempt to culture a mammalian embryo

Brachet A (1913) Recherches sur le déterminisme
héréditaire de lœf des mammifères. Développement
in vitro des jeunes vésicules blastodermiques de
Lapin. Arch Biol (Lièges) 32, 205-248.
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Gregory Pincus
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Media used by Hammond
Compound Flushing medium (mg) Culture medium (mg)
NaCl 880 880
KCl 30 30
CaCl2 25 25
MgCl2 5 5
NaH2PO4 10 0
Glucose 108 108
Thin egg white ? ?
Egg yolk 0 ?
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John Hammond Jr.

• Recovery and culture of tubal mouse ova. Nature
  163, 28-29 (1949)

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Hammonds Results

• The occurrence of development depends on the
  embryonic stage placed in culture
• Two cell preimplantation mouse embryos do not
  develop
• Eight cell preimplantation mouse embryos
  developed into blastocysts.

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WESLEY K. WHITTEN Culture of Tubal Mouse Ova
Nature 177, 96 (1956) John Curtin School of
Medical Research, Australian National University,
Canberra
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Comparison of Hammonds and Whittens media
Component Hammond (mmol/l) Whitten (mmol/l)
NaCl 150.5 94.59
KCl 4.02 4.78
NaH2PO4 0.83 -
KH2PO4 - 1.19
MgCl2 0.52 -
MgSO4 - 1.19
CaCl2 2.25 1.71
NaHCO3 - 25.07
Glucose 6.00 5.56
BSA 0 1 mg/ml
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Whittens Results

• Confirmed Hammonds findings using the flushing
  medium containing egg white
• Observed that Krebs-Ringer bicarbonate provided
  better control of pH and that egg white can be
  replaced with crystalline bovine albumin (98 in
  0.4 BSA 87 in 0.1BSA)
• Demonstrated that mouse eight-cell embryos, but
  not two cell embryos, can develop in a chemically
  defined environment.

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Reasons for Using a Chemically Defined
Medium(Modified from Lewis and Lewis ,1911)

• Can be easily reproduced at different times and
  in different laboratories.
• Can be varied in a controlled manner by selecting
  compounds and their concentrations.
• Are free of unknown enzyme activities, and
  hormones and growth factors, which may interfere
  with the responses being studied.

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ANNE McLAREN and J. D. BIGGERS Nature 182, 877 -
878 (1958) Successful Development and Birth of
Mice cultivated in vitro as Early Embryos Royal
Veterinary College, London, N.W.1. Aug. 21.
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McLarens and Biggers Results

• Confirmed Whittens observations that eight-cell
  mouse embryos would develop in supplemented
  Krebs Ringer solution (87 blastocysts)
• Blastocysts that developed in vitro could give
  rise to fetuses and newborn mice after transfer
  into the uterus of surrogate mothers.

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Min Chueh Chang
PIONEERS OF IN VITRO FERTILIZATION
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Capacitation
The need for ejaculated spermatozoa to mature
in the female genital tract before they are able
to fertilize an ovum

• Austin CR (1951) Observations on the penetration
  of the sperm into the egg. Australian J. Scient.
  Res. B 4, 581
• Chang, MC (1951) Fertilizing capacity of
  spermatozoa deposited into the Fallopian tubes.
  Nature 168, 697.

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Chang MC (1959) Fertilization of Rabbit Ova in
vitro. Nature 184, 466 467 No. ova
266 No. that
cleaved normally 55 (21) No.
transplanted into surrogate mothers
36 No. live-born
15 (42)
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Impacts

• Physiology of embryos
• Genetic control of the development of embryos
• Genetic modification of embryos
• Stem cells and therapeutic cloning
• Reproductive cloning
• Freezing and storage of embryos
• Human IVF/ET
• Preimplantation genetic diagnosis
• Public policy issues

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ANDRZEJ K. TARKOWSKI Mouse Chimæras Developed
from Fused Eggs Nature 190, 857 - 860 (1961)
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Biggers JD, Whittingham DG, Donahue RP (1967) The
pattern of energy metabolism in the mouse oocyte
and zygote. PNAS 58, 560-567 (Also includes data
of Ralph Brinster, Joan Thomson)
Pyruvate oocyte, 1-, 2-, 8-cell Lactate 2-,
8-cell Glucose 8-cell
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Towards IVFET

• Whitten WK, Biggers JD (1968) Complete
  development in vitro of the pre-implantation
  stages of the mouse in a simple chemically
  defined medium. J Reprod Fertil 17, 399-401.
• Whitingham DG (1968) Fertilization of mouse eggs
  in vitro. Nature 220, 592-593.
• Steptoe PC, Edwards RG (1978) Birth after the
  reimplantation of a human embryo. Lancet 2, 366.
  

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First media to be tested in human

• Simple media are used, including Earles
  solution with pyruvate, a medium designed to
  support mouse homologous human serum (7.5
  8.6). We prefer Earles solution containing
  pyruvate , with serum from each patient.
• Selection was based on comercially available
  media.
• Edwards RG (1981) Test tube babies. Nature 293,
  253-256.

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First specifically designed chemically defined
culture media for human embryos

• Complex medium (B2/B3)
• Menezo Y, Testart J, Perrone D (1984) Serum
  is not necessary in human in vitro fertilization,
  early embryo culture, and transfer. Fertil Steril
  42, 750-755.
• Simple medium (HTF)
• Quinn P, Kerin JF, Warnes GM (1985) Improved
  pregnancy rate in human in vitro fertilization
  with the use of a medium based on the composition
  of human tubal fluid. Fertil Steril 44, 493-498.

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Back to Nature

• Quinn P, Kerin JF, Warnes GM (1985) Improved
  pregnancy rate in human in vitro fertilization
  with the use of a medium based on the composition
  of human tubal fluid. Fertil Steril 44, 493-498.
• Leese HJ (1998) Human embryo culture back to
  nature. J Assist Reprod Genet 15, 466-468.
• Leese HJ (2002) Quiet please, do not disturb
  a hypothesis of embryo metabolism and viability.
  BioEssays 24, 845-849.

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There is no guarantee that back-to-nature
works

• K concentration
• Osmolarity
• Hydrogen ion concentration (pH)

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Let the Embryos Choose(Assayed by abolishing the
2-cell block)

• Lawitts JA, Biggers JD (1991) Optimization of
  mouse embryo culture media using simplex methods.
  J Reprod Fertil 91, 543-556.
• (Medium SOM)
• Lawitts JA, Biggers JD (1993) Culture of
  preimplantation embryos. Methods Enzymol 225,
  153-164. (Medium KSOM)

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Two media initially developed for the culture of
mouse preimplantation embryos

• G1/G2. (Based on the toxicological studies and
  theBack to nature principle)
• Gardner DK, Lane M (1993) Amino acids and
  ammonium production regulate mouse embryo
  development in culture. Biol Reprod 48, 377-385
• KSOMAA. (Based on the Let the embryos choose
  principle.)
• Biggers JD, McGinnis LK, Raffin M (2000)
  Amino acids and preimplantation development of
  the mouse in protein free potassium simplex
  optimized medium. Biol Reprod 63, 281-293.

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Culture Human Preimplantation Embryos to the
Blastocyst

• G1/G2
• Requires a two-step protocol using two media
  of different composition sequentially.
• Gardner DK, Lane M (1997) Culture and
  selection of viable human blastocysts a feasible
  proposition for human IVF. Hum Reprod Uptake 3,
  367-382.
• KSOMAA
• Requires a one-step using a single medium.
• Biggers JD, Racowsky C (2002) The
  development of fertilized human ova to the
  blastocyst stage in KSOMAA is a two-step
  protocol necessary? Reprod Biomed Online 5,
  133-140.

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Development of Human Zygotes to the Blastocyst
after five days in Culture
Two-step culture
P-1/CCM
81/140 (57.9)
One-step culture KSOMAA
51/82 (62.2)
Biggers JD, Racowsky C (2002) The development of
fertilized human ova to the blastocyst stage in
medium KSOMAA Is a two-step protocol necessary?
Reprod Biomed Online 5, 133-140.
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Some Comments

• The introduction of G1/G2 led to at least six
  other commercially available two step protocols
  for the culture of human preimplantation embryos.
• Several newer versions of G1/G2 have been
  developed.
• KSOMAA is the basis of Global which can be
  used as a one step protocol.

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Summary of Clinical Tests comparing Global with
some sequential protocols

• The rate of blastocyst development was greater
  when a single-step protocol was used compared to
  two-step protocol in7 studies.
• Four comparisons were statistically significant
  three comparisons were not significantly
  different.
• The pregnancy rate using either a one-step
  protocol or a two-step protocol were not
  significantly different.
• A single step protocol is at least as effective
  as two-step protocols.

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Biological characteristics of a non-renewal
single medium, a renewal single medium, and a
sequential medium
Characteristic Single medium (Non renewed) Single medium (Renewed) Sequential media
Leaves embryos undisturbed Yes No No
Accumulated endogenous growth factors Left in place Lost Lost
Replacement of essential nutrients No Yes Yes
Accumulated toxins Left in place Removed Removed
Relative environmental stress to embryos Low Moderate High
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Logistical characteristics of a non-renewal
single medium, a renewal single medium, and a
sequential medium
Characteristic Single mediumNon (renewed) Single medium (Renewed) Sequential media
Required quality control One medium One medium Two media
Relative labor intensity Low Moderate High
Relative cost Low Low High
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Conclusions

• G1/G2 and KSOMAA are based entirely on basic
  research using mice the notion that the mouse
  is a poor model for the human cannot be
  sustained.
• It is unlikely that chemically defined media will
  ever completely match the natural conditions in
  which preimplantation embryos develop.
  Preimplantation embryos have defense mechanisms
  which allow them to adapt to artificial
  environments.
• The development of media is an ongoing process
  which will focus more on the study of the
  longterm epigenetic effects produced by
  artificial chemically defined media.
• Basic biological research done from 1891-1968 set
  the stage for many advances in reproductive and
  developmental biology leading to IVFET in humans.