Semin Reprod Med 2000; 18(2): 205-218
DOI: 10.1055/s-2000-12559
Copyright © 2000 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel.: +1(212) 584-4662

Embryo Nutrition and Energy Metabolism and Its Relationship to Embryo Growth, Differentiation, and Viability

David K. Gardner1 , Thomas B. Pool2 , Michelle Lane1
  • 1Colorado Center for Reproductive Medicine, Englewood, Colorado
  • 2Fertility Center of San Antonio, San Antonio, Texas
Further Information

Publication History

Publication Date:
31 December 2000 (online)

ABSTRACT

Over the past decade there has been a resurgence of interest in the culture media used in clinical in vitro fertilization. Unfortunately, during this time more confusion than consensus appears to have developed regarding the composition of these media. In order to facilitate a clearer understanding of this field, it is important to understand the role of specific medium components and how their use is regulated by the embryo. The roles of the key nutrients glucose, pyruvate, lactate, and amino acids during the preimplantation period have therefore been presented. Analysis of how the embryo regulates the utilization of such nutrients has led to a clearer understanding of the embryo's requirements during the dynamic period of preimplantation development. From such information, sequential culture media have been developed along with novel noninvasive tests of embryonic viability. It is proposed that continued studies on the human embryo will lead to further improvements in embryo culture conditions and the optimization of viability assays, culminating in the ability to transfer single embryos for the majority of, if not all patients.

REFERENCES

  • 1 Leese H J. Metabolism of the preimplantation mammalian embryo. In: Millligan SR, ed. Oxford Reviews of Reproductive Biology Vol. 13. London: Oxford University Press 1991: 35-72
  • 2 Leese H J. Metabolic control during preimplantation mammalian development.  Hum Reprod Update . 1995;  1 63-72
  • 3 Gardner D K, Lane M. Embryo culture systems. In: Trounson A, Gardner DK, eds. Handbook of In Vitro Fertilization Boca Raton, FL: CRC Press 1993: 85-114
  • 4 Gardner D K, Lane M. Culture and selection of viable blastocyst: a feasible proposition for human IVF?.  Hum Reprod Update . 1997;  3 367-382
  • 5 Gardner D K, Lane M. Embryo culture systems. In: Trounson A, Gardner DK, eds. Handbook of In Vitro Fertilization 2nd ed. Boca Raton, FL: CRC Press 1999: 205-264
  • 6 Rieger D. Relationship between energy metabolism and development of the early embryo.  Theriogenology . 1992;  37 75-93
  • 7 Gardner D K. Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture.  Theriogenology . 1998;  49 83-102
  • 8 Leese H J, Barton A M. Pyruvate and glucose uptake by mouse ova and preimplantation embryos.  J Reprod Fertil . 1984;  72 9-13
  • 9 Gardner D K, Leese H J. Non-invasive measurement of nutrient uptake by single cultured preimplantation mouse embryos.  Hum Reprod . 1986;  1 25-27
  • 10 Hardy K, Hooper M AK, Handyside A H, Rutherford A J, Winston R ML, Leese H J. Non-invasive measurement of glucose and pyruvate uptake by individual human oocytes and preimplantation embryos.  Hum Reprod . 1989;  4 188-191
  • 11 Gott A L, Hardy K, Winston R M, Leese H J. Non-invasive measurement of pyruvate and glucose uptake and lactate production by single human preimplantation embryos.  Hum Reprod . 1990;  5 104-108
  • 12 Gardner D K, Selwood L, Lane M. Nutrient uptake and culture of Sminthopsis macroura (Stripe-faced Dunnart) embryos.  Reprod Fertil Dev . 1996;  8 685-690
  • 13 Lane M, Gardner D K. Lactate regulates pyruvate uptake and metabolism in the preimplantation mouse embryo.  Biol Reprod . 2000;  62 16-22
  • 14 Gardner D K, Lane M, Batt P A. Uptake and metabolism of pyruvate and glucose by individual pre-attachment sheep embryos developed in vivo.  Mol Reprod Dev . 1993;  36 313-319
  • 15 Gardner D K, Leese H J. The role of glucose and pyruvate transport in regulating nutrient utilization by preimplantation mouse embryos.  Development . 1988;  104 423-429
  • 16 Pool T B, Atiee S H, Martin J E. Oocyte and embryo culture: basic concepts and recent advances.  Infertil Reprod Med Clin North Am . 1998;  9 181-203
  • 17 Menke T M, McLaren A. Mouse blastocysts grown in vivo and in vitro: carbon dioxide production and trophoblast outgrowth.  J Reprod Fertil . 1970;  23 117-127
  • 18 Gardner D K, Leese H J. Concentrations of nutrients in mouse oviduct fluid and their effects on embryo development and metabolism in vitro.  J Reprod Fertil . 1990;  88 361-368
  • 19 Lane M, Gardner D K. Amino acids and vitamins prevent culture induced metabolic perturbations and associated loss of viability of mouse blastocysts.  Hum Reprod . 1998;  13 991-997
  • 20 Lane M, Gardner D K. Embryo homeostasis. In: Gardner DK, Lane M, eds. ART and the Human Blastocyst New York: Springer Verlag 2001
  • 21 Lane M. Mechanisms for managing cellular and homeostatic stress in vitro.  Theriogenology. In press
  • 22 Gardner D K, Lane M. Culture of viable human blastocysts in defined sequential serum-free media.  Hum Reprod . 1998;  13(suppl 3) 148-159
  • 23 Gardner D K, Lane M, Calderon I, Leeton J. Environment of the preimplantation human embryo in vivo: metabolite analysis of oviduct and uterine fluids and metabolism of cumulus cells.  Fertil Steril . 1996;  65 349-353
  • 24 Carlson D, Blasck D L, Howe G R. Oviduct secretion in the cow.  J Reprod Fertil . 1970;  22 549-552
  • 25 Nichol R, Hunter R HF, Gardner D K, Leese H J, Cooke G M. Concentrations of energy substrates in oviductal fluid and blood plasma of pigs during the peri-ovulatory period.  J Reprod Fertil . 1992;  96 699-707
  • 26 Vella P, Lane M, Gardner D K. Induction of glycolysis in the day-3 mouse embryo by glucose.  Biol Reprod . 1997;  57(Suppl) 26
  • 27 Hogan A, Heyner S, Charron M J. Glucose transporter gene expression in early mouse embryos.  Development . 1991;  113 363-372
  • 28 Aghayan M, Rao L V, Smith R M. Developmental expression and cellular localization of glucose transporter molecules during mouse preimplantation development.  Development . 1992;  115 305-312
  • 29 Dan-Goor M, Sasson S, Davarashvili A, Almagor M. Expression of glucose transporter and glucose uptake in human oocytes and preimplantation embryos.  Hum Reprod . 1997;  12 2508-2510
  • 30 Brinster R L. Hexokinase activity in the preimplantation mouse embryo.  Enzymologia . 1968;  34 304-308
  • 31 Brinster R L. Phosphofructokinase activity in the preimplantation mouse embryo.  Wilhelm Roux Arch Entwicklungsmech Org . 1971;  166 300-302
  • 32 Chi MM-Y, Manchester J Y, Yang V C, Curato A D, Strickler R C, Lowry O H. Contrast in levels of metabolic enzymes in human and mouse ova.  Biol Reprod . 1988;  39 295-307
  • 33 Martin K L, Hardy K, Winston R ML, Leese H J. Activity of enzymes of energy metabolism in single human preimplantation embryos.  J Reprod Fertil . 1993;  99 259-266
  • 34 Biggers J D, Gardner D K, Leese H J. Control of carbohydrate metabolism in preimplantation mammalian embryos. In: Rosenblum IY, Heyner S, eds. Regulation of Growth in Development Boca Raton, FL: CRC Press 1989: 19-32
  • 35 Barbehenn E K, Wales R G, Lowry O H. The explanation for the blockade of glycolysis in early mouse embryos.  Proc Natl Acad Sci U S A . 1974;  71 1056-1060
  • 36 Barbehenn E K, Wales R G, Lowry O H. Measurement of metabolites in single preimplantation embryos; a new means to study metabolic control in early embryos.  J Embryol Exp Morphol . 1978;  43 29-46
  • 37 Gardner D K, Leese H J. Assessment of embryo metabolism and viability. In: Trounson A, Gardner DK, eds. Handbook of In Vitro Fertilization 2nd ed. Boca Raton, FL: CRC Press 1999: 347-371
  • 38 Seshagiri P B, Bavister B D. Glucose and phosphate inhibit respiration and oxidative metabolism in cultured hamster eight-cell embryos: evidence for the ``Crabtree effect''.  Mol Reprod Dev . 1991;  30 105-111
  • 39 Crabtree H G. Observations on the carbohydrate metabolism of tumours.  Biochem J . 1929;  23 536-545
  • 40 Koobs D H. Phosphate mediation of the Crabtree and Pasteur effects.  Science . 1972;  178 127-133
  • 41 Auerbach S, Brinster R L. Lactate dehydrogenase isozymes in the early mouse embryo.  Exp Cell Res . 1967;  46 89-92
  • 42 Auerbach S, Brinster R L. Lactate dehydrogenase isozymes in mouse blastocyst cultures.  Exp Cell Res . 1968;  53 313-315
  • 43 Edwards L E, Gardner D K. Characterisation of hexokinase kinetics in the preimplantation mouse embryo.  (abstract)(Melbourne, Vic)  Proc Fertil Soc Aust . 1995;  14 28
  • 44 Wilson J E. Hexokinases.  Rev Physiol Biochem Pharmacol . 1995;  126 65-198
  • 45 Leese H J, Biggers J D, Mroz E A, Lechene C. Nucleotides in a single mammalian ovum or preimplantation embryo.  Anal Biochem . 1984;  140 443-448
  • 46 Rozell M D, Williams J E, Butler J E. Changes in concentration of adenosine triphosphate and adenosine diphosphate in individual preimplantation sheep embryos.  Biol Reprod . 1992;  47 866-870
  • 47 Gardner D K, Lane M. Alleviation of the 2-cell block in CF1 mouse embryos is associated with an increase in the ATP:ADP ratio and subsequent inhibition of PFK.  Biol Reprod . 1997;  57(suppl 1) 216(abstract)
  • 48 Gardner D K, Lane M. Alleviation of the ``2-cell block'' and development to the blastocyst of CF1 mouse embryos: role of amino acids, EDTA and physical parameters.  Hum Reprod . 1996;  11 2703-2712
  • 49 Gardner D K, Lane M. The 2-cell block in CF1 mouse embryos is associated with an increase in glycolysis and a decrease in tricarboxylic acid (TCA) cycle activity: alleviation of the 2-cell block is associated with the restoration of in vivo metabolic pathway activities.  Biol Reprod . 1993;  49(suppl 1) 152(abstract)
  • 50 Rieger D, Guay P. Measurement of the metabolism of energy substrates in individual bovine blastocysts.  J Reprod Fertil . 1988;  83 585-591
  • 51 Hume D A, Weidemann M J. Role and regulation of glucose metabolism in proliferating cells.  J Natl Cancer Inst . 1979;  62 3-8
  • 52 Reitzer L J, Wice B M, Kennel D. The pentose cycle: control and essential function in HeLa cell nucleic acid synthesis.  J Biol Chem . 1980;  255 5616-5626
  • 53 Morgan M J, Faik P. Carbohydrate metabolism in cultured animal cells.  Biosci Rep . 1981;  1 669-686
  • 54 Steeves T E, Gardner D K. Metabolism of glucose, pyruvate, and glutamine during the maturation of oocytes derived from pre-pubertal and adult cows.  Mol Reprod Dev . 1999;  54 92-101
  • 55 Mastroianni Jr L, Jones R. Oxygen tension within rabbit fallopian tube.  J Reprod Fertil . 1965;  9 99-102
  • 56 Ross R N, Graves C N. O2 levels in the female rabbit reproductive tract.  J Anim Sci . 1974;  39 994
  • 57 Maas D HA, Storey B T, Mastroianni Jr L. Oxygen tension in the oviduct of the rhesus monkey (Macaca mulatta).  Fertil Steril . 1976;  27 1312-1317
  • 58 Fischer B, Bavister B D. Oxygen tension in the oviduct and uterus of rhesus monkeys, hamsters and rabbits.  J Reprod Fertil . 1993;  99 673-679
  • 59 Mills R M, Brinster R L. Oxygen consumption of pre-implantation mouse embryos.  Exp Cell Res . 1967;  47 337-344
  • 60 Houghton F D, Thompson J G, Kennedy C J, Leese H J. Oxygen consumption and energy metabolism of the early mouse embryo.  Mol Reprod Dev . 1996;  44 476-485
  • 61 Thompson J G, Partridge R J, Houghton F D, Cox C I, Leese H J. Oxygen uptake and carbohydrate metabolism by in vitro derived bovine embryos.  J Reprod Fertil . 1996;  106 299-306
  • 62 Rogers P AW, Murphy C R, Gannon B J. Absence of capillaries in the endometrium surrounding the implanting rat blastocyst.  Micron . 1982;  13 373-374
  • 63 Rogers P AW, Murphy C R, Gannon B J. Changes in the spatial organization of the uterine vasculature during implantation in the rat.  J Reprod Fertil . 1982;  65 211-214
  • 64 Ludwig T E, Lane M, Bavister B D. Effect of hexoses on hamster embryo development in culture.  Biol Reprod . 1999;  60(suppl 1) 314(abstract)
  • 65 Schini S A, Bavister B D. Two-cell block to development of cultured hamster embryos is caused by phosphate and glucose.  Biol Reprod . 1988;  39 1183-1192
  • 66 Seshagiri P B, Bavister B D. Phosphate is required for inhibition by glucose of development of hamster 8-cell embryos in vitro.  Biol Reprod . 1989;  40 607-614
  • 67 Thompson J G, Simpson A C, Pugh P A, Tervit H R. Requirement for glucose during in vitro culture of sheep preimplantation embryos.  Mol Reprod Dev . 1992;  31 253-257
  • 68 Kim J H, Niwa K, Lim J M, Okuda K. Effects of phosphate, energy substrates, and amino acids on development of in vitro-matured, in vitro-fertilized bovine oocytes in a chemically defined, protein-free culture medium.  Biol Reprod . 1993;  48 1320-1325
  • 69 Conaghan J, Handyside A H, Winston R ML, Leese H J. Effects of pyruvate and glucose on the development of human preimplantation embryos in vitro.  J Reprod Fertil . 1993;  99 87-95
  • 70 Coates A, Rutherford A J, Hunter H, Leese H J. Glucose-free medium in human in vitro fertilization and embryo transfer: a large-scale, prospective, randomized clinical trial.  Fertil Steril . 1999;  72 229-232
  • 71 Bavister B D. Glucose and culture of human embryos.  Fertil Steril . 1999;  72 233-234
  • 72 Carrillo A J, Lane B, Pridman D D. Improved clinical outcomes for in vitro fertilization with delay of embryo transfer from 48 to 72 hours after oocyte retrieval: use of glucose- and phosphate-free media.  Fertil Steril . 1998;  69 329-334
  • 73 Leese H J, Barton A M. Production of pyruvate by isolated mouse cumulus cells.  J Exp Zool . 1985;  234 231-236
  • 74 Butcher L, Coates A, Martin K L, Rutherford A J, Leese H J. Metabolism of pyruvate by the early human embryo.  Biol Reprod . 1998;  58 1054-1056
  • 75 Biggers J D, Whittingham D G, Donahue R P. The pattern of energy metabolism in the mouse oocyte and zygote.  Proc Natl Acad Sci U S A . 1967;  58 560-567
  • 76 Lane M, Gardner D K. Regulation of substrate utilization in mouse embryos by the malate-aspartate shuttle.  Biol Reprod . 2000;  62(suppl 1) 371
  • 77 Gardner D K, Sakkas D. Mouse embryo cleavage, metabolism and viability: role of medium composition.  Hum Reprod . 1993;  8 288-295
  • 78 Kouridakis K, Gardner D K. Pyruvate in embryo culture media acts as antioxidant.  (Melbourne, Vic)  Proc Fertil Soc Aust . 1995;  14 29
  • 79 O'Fallon J V, Wright Jr W R. Pyruvate revisited: a non-metabolic role for pyruvate in preimplantation embryo development.  Theriogenology . 1995;  43 288
  • 80 Gibb C A, Poronnik P, Day M L, Cook D I. Control of cytosolic pH in two-cell mouse embryos: roles of H+-lactate cotransport and Na+/H+ exchange.  Am J Physiol . 1997;  273 C404-C419
  • 81 Edwards L E, Williams D A, Gardner D K. Intracellular pH of the preimplantation mouse embryo: effects of extracellular pH and weak acids.  Mol Reprod Dev . 1998;  50 434-442
  • 82 Whitten W K, Biggers J D. Complete development in vitro of the preimplantation stages of the mouse embryo in a simple chemically defined medium.  J Reprod Fertil . 1968;  17 399-401
  • 83 Miller J GO, Schultz G A. Amino acid content of preimplantation rabbit embryos and fluids of the reproductive tract.  Biol Reprod . 1987;  36 125-129
  • 84 Casslen B G. Free amino acids in human uterine fluid: possible role of high taurine concentration.  J Reprod Med . 1987;  32 181-184
  • 85 Moses D F, Matkovic M, Cabrera Fisher E, Martinez A G. Amino acid contents of sheep oviductal and uterine fluids.  Theriogenology . 1997;  47 336
  • 86 Schultz G A, Kaye P L, McKay D J, Johnson M H. Endogenous amino acids pool sizes in mouse eggs and preimplantation embryos.  J Reprod Fertil . 1981;  61 387-393
  • 87 Van Winkle J L. Amino acid transport in developing animal oocytes and early conceptuses.  Biochim Biophys Acta . 1988;  947 173-208
  • 88 Gwatkin R BL. Amino acid requirements for attachment and outgrowth of the mouse blastocyst in vitro.  J Cell Comp Physiol . 1966;  68 335-344
  • 89 Spindle A I, Pedersen R A. Hatching, attachment and outgrowth of mouse blastocysts in vitro: fixed nitrogen requirements.  J Exp Zool . 1973;  186 305-318
  • 90 Gwatkin R BL, Haidri A A. Requirements for the maturation of hamster oocytes in vitro.  Exp Cell Res . 1973;  76 1-7
  • 91 Juetten J, Bavister B D. The effects of amino acids, cumulus cells, and bovine serum albumin on in vitro fertilization and first cleavage of hamster eggs.  J Exp Zool . 1983;  227 487-490
  • 92 Zhang X, Armstrong D T. Presence of amino acids and insulin in a chemically defined medium improves development of 8-cell rat embryos in vitro and subsequent implantation in vivo.  Biol Reprod . 1990;  42 662-668
  • 93 Mehta T S, Kiessling A A. Development potential of mouse embryos conceived in vitro and cultured in ethylenediaminetetraacetic acid with or without amino acids or serum.  Biol Reprod . 1990;  43 600-606
  • 94 Gardner D K, Lane M. Amino acids and ammonium regulate mouse embryo development in culture.  Biol Reprod . 1993;  48 377-385
  • 95 Lane M, Gardner D K. Increase in postimplantation development of cultured mouse embryos by amino acids and induction of fetal retardation and exencephaly by ammonium ions.  J Reprod Fertil . 1994;  102 305-312
  • 96 Gardner D K, Lane M, Spitzer A, Batt P A. Enhanced rates of cleavage and development for sheep zygotes cultured to the blastocyst stage in vitro in the absence of serum and somatic cells: amino acids, vitamins, and culturing embryos in groups stimulate development.  Biol Reprod . 1994;  50 390-400
  • 97 Lane M, Gardner D K. Differential regulation of mouse embryo development and viability by amino acids.  J Reprod Fertil . 1997;  109 153-164
  • 98 Steeves T E, Gardner D K. Temporal and differential effects of amino acids on bovine embryo development in culture.  Biol Reprod . 1999;  61 731-740
  • 99 Eagle H. Amino acids metabolism in mammalian cell cultures.  Science . 1959;  130 423-437
  • 100 Epstein C J, Smith S A. Amino acid uptake and protein synthesis in preimplantation mouse embryos.  Dev Biol . 1973;  33 171-184
  • 101 Crosby I M, Gandolfi F, Moor R M. Control of protein synthesis during early cleavage of sheep embryos.  J Reprod Fertil . 1988;  82 769-775
  • 102 Rieger D, Loskutoff N M, Betteridge K J. Developmentally related changes in the metabolism of glucose and glutamine by cattle embryos produced and co-cultured in vitro.  J Reprod Fertil . 1992;  95 585-595
  • 103 Van Winkle J L, Haghighat N, Campione A L. Glycine protects preimplantation mouse conceptuses from a detrimental effect on development of the inorganic ions in oviductal fluid.  J Exp Zool . 1990;  253 215-219
  • 104 Edwards L E, Williams D A, Gardner D K. Intracellular pH of the mouse preimplantation embryo: amino acids act as buffers of intracellular pH.  Hum Reprod . 1998;  13 3441-3448
  • 105 Liu Z, Foote R H. Development of bovine embryos in KSOM with added superoxide dismutase and taurine and with five and twenty percent O2 .  Biol Reprod . 1995;  53 786-790
  • 106 Lindenbaum A. A survey of naturally occurring chelating ligands.  Adv Exp Med Biol . 1973;  40 67-77
  • 107 Ho Y, Wigglesworth K, Eppig J J, Schultz R M. Preimplantation development of mouse embryos in KSOM: augmentation by amino acids and analysis of gene expression.  Mol Reprod Dev . 1995;  41 232-238
  • 108 Doherty A S, Mann M R, Tremblay K D, Bartolomei M S, Schultz R M. Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo.  Biol Reprod . 2000;  62 1526-1535
  • 109 Gardner D K, Georgiou E. Glycine and proline reduce the time of the first three cleavage divisions in cultured mouse embryos.  Theriogenology . 1998;  49 200
  • 110 Barnes F L, Crombie A, Gardner D K. Blastocyst development and birth after in-vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching.  Hum Reprod . 1995;  10 3243-3247
  • 111 Bavister B D, McKiernan S H. Regulation of hamster embryo development in vitro by amino acids. In: Bavister BD, ed. Preimplantation Embryo Development New York: Springer-Verlag 1993: 57-72
  • 112 Dumoulin J CM, Evers J LH, Bras M, Pieters M E C H, Geraedts J PM. Positive effect of taurine on preimplantation development of mouse embryos in vitro.  J Reprod Fertil . 1992;  94 373-380
  • 113 Dumoulin J CM, Van Wissen C P L, Menheere P C A P, Michiels A J C H, Geraedts J PM, Evers J LH. Taurine acts as an osmolyte in human and mouse oocytes and embryos.  Biol Reprod . 1997;  56 739-744
  • 114 Devreker F, Van den Bergh M, Biramane J, Winston R L, Englert Y, Hardy K. Effects of taurine on human embryo development in vitro.  Hum Reprod . 1999;  14 2350-2356
  • 115 Lane M, Gardner D K. Non-essential amino acids and glutamine decrease the time of the first three cleavage divisions and increase compaction of mouse zygotes in vitro.  J Assist Reprod Genet . 1997;  14 398-403
  • 116 Renard J P, Philippon A, Menezo Y. In vitro glucose uptake of glucose by bovine blastocysts.  J Reprod Fertil . 1980;  58 161-164
  • 117 Rieger D. The measurement of metabolic activity as an approach to evaluating viability and diagnosing sex in early embryos.  Theriogenology . 1984;  21 138-149
  • 118 Gardner D K, Leese H J. Assessment of embryo viability prior to transfer by the non-invasive measurement of glucose uptake.  J Exp Zool . 1987;  242 103-105
  • 119 Gardner D K, Pawelczynski M, Trounson A O. Nutrient uptake and utilization can be used to select viable day-7 bovine blastocysts after cryopreservation.  Mol Reprod Dev . 1996;  44 472-475
  • 120 Lane M, Gardner D K. Selection of viable mouse blastocysts prior to transfer using a metabolic criterion.  Hum Reprod . 1996;  11 1975-1978
  • 121 Conaghan J, Hardy K, Handyside A H, Winston R ML, Leese H J. Selection criteria for human embryo transfer: a comparison of pyruvate uptake and morphology.  J Assist Reprod Genet . 1993;  10 21-30
  • 122 Gardner D K, Lane M, Stevens J, Schlenker T, Schoolcraft W B. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer.  Fertil Steril . 2000;  73 1155-1158
  • 123 Gardner D K, Lane M, Stevens J, Schoolcraft W B. Non-invasive assessment of human embryo nutrient consumption as a measure of developmental potential.  Fertil Steril . 2000;  74(suppl 1) S32
  • 124 Gardner D K. Mammalian embryo culture in the absence of serum or somatic cell support.  Cell Biol Int . 1994;  18 1163-1179
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