Historical Development of
Embryo Culture Media
John D Biggers D.Sc., Ph.D.
Harvard Medical School
Steps in Achieving IVFET
• In vitro fertilization
• Culture of the preimplantation stages
• Embryo transfer into surrogate mothers
PIONEER OF
EMBRYO TRANSFER
Heape W (1891)
Preliminary note on
the transplantation
and growth of
mammalian ova within
a uterine fostermother.
Proc Roy Soc Lond B
Biol Sci 48, 257-458.
The questions Albert Brachet tried to answer
by culturing preimplantation mammalian
embryos
1. 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.
Professor Albert Brachet
First attempt to culture a mammalian embryo
Brachet A (1913) Recherches sur le déterminisme héréditaire de l’œf des
mammifères. Développement in vitro des jeunes vésicules
blastodermiques de Lapin. Arch Biol (Lièges) 32, 205-248.
Gregory Pincus
Media used by Hammond
Compound
NaCl
KCl
Flushing medium
(mg%)
880
30
Culture medium
(mg%)
880
30
CaCl2
MgCl2
NaH2PO4
Glucose
25
5
10
108
25
5
0
108
Thin egg white
Egg yolk
?
0
?
?
John Hammond Jr.
• Recovery and culture of tubal mouse ova.
Nature 163, 28-29 (1949)
Hammond’s 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.
WESLEY K.
WHITTEN
Culture of Tubal Mouse
Ova
Nature 177, 96 (1956)
John Curtin School of Medical
Research, Australian National
University, Canberra
Comparison of Hammond’s and Whitten’s 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
Whitten’s Results
 Confirmed Hammond’s findings using the
flushing medium containing egg white
 Observed that Kreb’s-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 0.1%BSA)
 Demonstrated that mouse eight-cell embryos,
but not two cell embryos, can develop in a
chemically defined environment.
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.
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.
McLaren’s and Biggers’ Results
• Confirmed Whitten’s observations that
eight-cell mouse embryos would
develop in supplemented Kreb’s 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.
Min Chueh Chang
PIONEERS OF IN VITRO FERTILIZATION
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.
Chang MC (1959) Fertilization of Rabbit
Ova in vitro. Nature 184, 466 – 467
No. ova
No. that cleaved normally
266
55 (21%)
No. transplanted into surrogate
mothers
36
No. live-born
15 (42%)
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
ANDRZEJ K. TARKOWSKI
Mouse Chimæras Developed
from Fused Eggs
Nature 190, 857 - 860 (1961);
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
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.
First media to be tested in human
 “Simple media are used, including Earle’s solution
with pyruvate, a medium designed to support
mouse homologous human serum (7.5 – 8.6%).
We prefer Earle’s 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.
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, 750755.
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.
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.
There is no guarantee that
“back-to-nature” works
• K+ concentration
• Osmolarity
• Hydrogen ion concentration (pH)
Two-dimensional Concentration-Response Surface
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)
Two media initially developed for the culture
of mouse preimplantation embryos
 G1/G2. (Based on the toxicological studies and
the“Back 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, 281293.
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.
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.
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.
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 twostep protocols.
BIOLOGICAL CHARACTERISTICS OF A NON-RENEWAL SINGLE
MEDIUM, A RENEWAL SINGLE MEDIUM, AND A SEQUENTIAL
MEDIUM
Characteristic
Single
Single
Sequential
medium
medium
media
(Non
(Renewed)
renewed)
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
LOGISTICAL CHARACTERISTICS OF A NON-RENEWAL
SINGLE MEDIUM, A RENEWAL SINGLE MEDIUM, AND A
SEQUENTIAL MEDIUM
Characteristic
Single
Single
Sequential
mediumN medium
media
on
(Renewed)
(renewed)
Required quality control
One medium
One medium
Two media
Relative labor intensity
Low
Moderate
High
Relative cost
Low
Low
High
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.