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Historical Development of Embryo Culture Media John D Biggers D.Sc., Ph.D. Harvard Medical School.

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Presentation on theme: "Historical Development of Embryo Culture Media John D Biggers D.Sc., Ph.D. Harvard Medical School."— Presentation transcript:

1 Historical Development of Embryo Culture Media John D Biggers D.Sc., Ph.D. Harvard Medical School

2 Steps in Achieving IVFET In vitro fertilization Culture of the preimplantation stages Embryo transfer into surrogate mothers

3 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, PIONEER OF EMBRYO TRANSFER

4 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,

5 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,

6 Gregory Pincus

7 Media used by Hammond CompoundFlushing medium (mg%) Culture medium (mg%) NaCl880 KCl30 CaCl 2 25 MgCl 2 55 NaH 2 PO Glucose108 Thin egg white?? Egg yolk0?

8 John Hammond Jr. Recovery and culture of tubal mouse ova. Nature 163, (1949)

9 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.

10 WESLEY K. WHITTEN Culture of Tubal Mouse Ova Nature 177, 96 (1956) John Curtin School of Medical Research, Australian National University, Canberra

11 Comparison of Hammond’s and Whitten’s media ComponentHammond (mmol/l) Whitten (mmol/l) NaCl KCl NaH 2 PO KH 2 PO MgCl MgSO CaCl NaHCO Glucose BSA01 mg/ml

12 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.

13 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.

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

15 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.

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17 Min Chueh Chang PIONEERS OF IN VITRO FERTILIZATION

18 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

19 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%)

20 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

21 ANDRZEJ K. TARKOWSKI Mouse Chimæras Developed from Fused Eggs Nature 190, (1961);

22 Biggers JD, Whittingham DG, Donahue RP (1967) The pattern of energy metabolism in the mouse oocyte and zygote. PNAS 58, (Also includes data of Ralph Brinster, Joan Thomson) Pyruvate – oocyte, 1-, 2-, 8-cell Lactate – 2-, 8-cell Glucose – 8-cell

23 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, Whitingham DG (1968) Fertilization of mouse eggs in vitro. Nature 220, Steptoe PC, Edwards RG (1978) Birth after the reimplantation of a human embryo. Lancet 2, 366.

24 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,

25 First specifically designed chemically defined culture media for human embry os 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, 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,

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28 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, Leese HJ (1998) Human embryo culture: back to nature. J Assist Reprod Genet 15, Leese HJ (2002) Quiet please, do not disturb: a hypothesis of embryo metabolism and viability. BioEssays 24,

29 There is no guarantee that “back-to-nature” works K+ concentration Osmolarity Hydrogen ion concentration (pH)

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32 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, (Medium SOM) Lawitts JA, Biggers JD (1993) Culture of preimplantation embryos. Methods Enzymol 225, (Medium KSOM)

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34 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, KSOM AA. (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,

35 Culture Human Preimplantation Embryos to the Blastoc yst 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, KSOM AA Requires a one-step using a single medium. Biggers JD, Racowsky C (2002) The development of fertilized human ova to the blastocyst stage in KSOM AA : is a two-step protocol necessary? Reprod Biomed Online 5,

36 One-step culture KSOM AA 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 KSOM AA : Is a two-step protocol necessary? Reprod Biomed Online 5, /82 (62.2%) 81/140 (57.9%)

37 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. KSOM AA is the basis of Global® which can be used as a one step protocol.

38 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.

39 Characteristic Single medium (Non renewed) Single medium (Renewed) Sequential media Leaves embryos undisturbed YesNo Accumulated endogenous growth factors Left in placeLost Replacement of essential nutrients NoYes Accumulated toxins Left in placeRemoved Relative environmental stress to embryos LowModerateHigh BIOLOGICAL CHARACTERISTICS OF A NON-RENEWAL SINGLE MEDIUM, A RENEWAL SINGLE MEDIUM, AND A SEQUENTIAL MEDIUM

40 Characteristic Single mediumN on (renewed ) Single medium (Renewed) Sequential media Required quality control One medium Two media Relative labor intensityLowModerateHigh Relative costLow High LOGISTICAL CHARACTERISTICS OF A NON-RENEWAL SINGLE MEDIUM, A RENEWAL SINGLE MEDIUM, AND A SEQUENTIAL MEDIUM

41 Conclusions G1/G2 and KSOM AA 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 set the stage for many advances in reproductive and developmental biology leading to IVFET in humans.


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