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

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.

PIONEER OF EMBRYO TRANSFER

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 BrachetFirst 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 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 ?

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 ResultsConfirmed 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

• 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.

The need for ejaculated spermatozoa to ‘mature’ in the female genital tract before they are able to fertilize an ovum

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%)

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, 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.

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, 281-293.

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.

One-step cultureKSOMAA

Two-step culture

DEVELOPMENT OF HUMAN ZYGOTES TO THE BLASTOCYST AFTER FIVE DAYS IN CULTURE

P-1/CCM

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.

51/82 (62.2%)

81/140 (57.9%)

Some CommentsThe 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 two-step protocols.

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

BIOLOGICAL CHARACTERISTICS OF A NON-RENEWAL SINGLE MEDIUM, A RENEWAL SINGLE MEDIUM, AND A SEQUENTIAL

MEDIUM

CharacteristicSingle

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

LOGISTICAL CHARACTERISTICS OF A NON-RENEWAL SINGLE MEDIUM, A RENEWAL SINGLE MEDIUM, AND A

SEQUENTIAL MEDIUM

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.