Non-invasive amino acid turnover predicts human embryo ...

Human Reproduction Vol.17, No.4 pp. 999–1005, 2002

Non-invasive amino acid turnover predicts human embryo developmental capacity Franchesca D.Houghton1,3, Judith A.Hawkhead1, Peter G.Humpherson1, Jan E.Hogg2, Adam H.Balen2, Anthony J.Rutherford2 and Henry J.Leese1 1Department

of Biology, University of York, PO Box 373, York YO10 5YW and 2Assisted Conception Unit, Clarendon Wing, Leeds General Infirmary, Leeds LS1 9NS, UK

3To

whom correspondence should be addressed. E-mail: [email protected]

BACKGROUND: IVF is limited by low success rates and a confounding high multiple birth rate contributing to prematurity, increased neonatal mortality and child handicap. These problems could be overcome if single embryos of known developmental competence could be selected for transfer on day 2/3 of development, but current methods, which rely on morphological appearance, are poor predictors of viability. METHODS: We have measured noninvasively the depletion/appearance (i.e. turnover) of a physiological mixture of 18 amino acids by single human embryos during in-vitro culture using high performance liquid chromatography. RESULTS: From the time of transfer (day 2/3), embryos with future competence to develop to the blastocyst stage (day 5/6) exhibit amino acid flux patterns distinct from those of embryos with similar morphological appearance which arrest. Significantly, the profiles of Ala, Arg, Gln, Met and Asn flux predict blastocyst potentiality at >95%. The amino acid most consistently depleted throughout development by those embryos which form blastocysts was leucine. Of the amino acids which were produced, the most striking was alanine, which appeared in increasing amounts throughout development. CONCLUSIONS: Non-invasive amino acid profiling has the potential to select developmentally competent single embryos for transfer, thereby increasing the success rate and eliminating multiple births in IVF. Key words: amino acids/developmental potential/human preimplantation embryo culture/IVF

Introduction Human zygotes produced by IVF can develop to the blastocyst stage in simple salt solutions containing carbon sources such as pyruvate and glucose, a macromolecule such as albumin (Conaghan et al., 1998), and in some cases, maternal serum (Bolton et al., 1989). These systems result in blastocyst yields of 17–23% (Bolton et al., 1989; Conaghan et al., 1998). In order to improve rates of blastocyst formation, mixtures of non-essential and essential amino acids are widely added to human embryo culture media (Devreker et al., 2001). The regime most favoured is supplementation with non-essential amino acids during early cleavage, up to the 8-cell stage, followed by non-essential as well as essential amino acids for the 8-cell to morula and morula to blastocyst transitions (Gardner and Lane, 1997); a sequential system which increases blastocyst yields to ~50% (Schoolcraft et al., 1999). The mechanisms by which amino acids promote human preimplantation development are largely unknown. In early embryos of other species, notably the mouse, amino acids have been shown to serve a variety of functions: in the synthesis of proteins and nucleotides (Epstein and Smith, 1973; Alexiou and Leese, 1992), as sources of energy (Lane and Gardner, 1997, 1998), as osmolytes (Van Winkle and Campione, 1996; Dumoulin et al., 1997; Dawson et al., 1998), as antioxidants © European Society of Human Reproduction and Embryology

(Nasr-Esfahani et al., 1992), pH regulators (Bavister and McKiernan, 1993; Edwards et al., 1998), chelators (Van Winkle et al., 1990) and as precursors of signalling molecules such as nitric oxide (Wu et al., 2000). Amino acid transport mechanisms in early mouse embryos have been studied extensively, with up to 15 different systems identified (Van Winkle, 2001). Many of the uptake processes involve co-transport with Na⫹, a dependency which increases throughout the preimplantation period (Tasca, 2001). There are relatively few reports on the role of specific amino acids during early human embryo development: glutamine addition increases the proportion of embryos that reach the blastocyst stage (Devreker et al., 1998); taurine acts as an osmolyte (Dumoulin et al., 1997), but provides no additional benefit over glutamine in development to the blastocyst stage (Devreker et al., 1999); glycine has also been shown to be involved in osmoregulation (Hammer et al., 2000). In these studies, amino acids have been administered singly, whereas in the female reproductive tract, the preimplantation embryo will be exposed to a complete mixture (Leese et al., 1979; Miller and Schultz, 1987). To take account of this, Lamb and Leese measured non-invasively the depletion or appearance of 18 out of a mixture of 20 amino acids by small numbers of mouse blastocysts (Lamb and Leese, 1994). The same 999

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approach was applied to bovine embryos from the zygote to blastocyst stage (Partridge and Leese, 1996; Jung et al., 1998). The data showed that amino acid depletion/appearance differed between the two species and, in the case of the bovine, with stage of development. A notable feature of bovine amino acid metabolism was the appearance of alanine in increasing amounts throughout development (Partridge and Leese, 1996). Subsequently, data from Donnay et al. suggested that alanine was derived from ammonia via transamination with pyruvate (Donnay et al., 1999). We have now examined the depletion or appearance of amino acids brought about by single, spare, human embryos conceived by IVF as a first approach to understanding the mechanisms by which mixtures of amino acids promote human preimplantation development. It should be emphasised that this approach is not intended to be a strict, kinetic analysis of amino acid uptake; rather, it represents the way in which embryos modify the composition of a mixture of amino acids present at concentrations believed to be close to physiological. Amino acid profiles have been obtained for human embryos from day 2–3 post-insemination and during the compacting 8cell to morula and morula to blastocyst transitions. Our data indicate that amino acid depletion/appearance differ with embryo stage and with embryo quality assessed in terms of developmental capacity. Specifically, we have identified groups of amino acids whose depletion or appearance, measured on day 2 post-fertilization, predict the ability of human embryos to develop to the blastocyst.

Materials and methods Spare human embryos donated to research were obtained with informed consent from patients undergoing IVF at the Assisted Conception Unit, Leeds General Infirmary. Ethical approval for the work was granted by the Human Fertilisation and Embryology Authority and the Ethics Committees of the collaborating institutions. Ovarian stimulation and oocyte collection were performed as previously described (Balen, 2001). In general, a long pituitary desensitization protocol was used, with intranasal nafarelin followed by gonadotrophin stimulation with either HMG (Menogon; Ferring Pharmaceuticals Ltd, Langley, Berks, UK) or recombinant FSH (Puregon; Organon Laboratories Ltd, Cambridge, UK). Briefly, oocytes were collected by follicular aspiration 36 h after HCG administration and cultured at 37°C in 5% CO2 in Medi-Cult IVF medium under oil. The oocyte–cumulus complexes were inseminated with a final concentration of 70 000 motile sperm per ml at ~40h post-HCG and incubated overnight until fertilization was confirmed by the presence of two pronuclei (day 1 post-insemination). Prior to being used for research, zygotes were cultured in 70 µl drops of Medi-Cult IVF medium under oil, as above. A maximum of three embryos was transferred on day 2 post-insemination and any remaining surplus embryos, from day 2 to day 5 post-fertilization, were transported to York in the same medium at 37°C. Developmental grade (Table I) and stage were recorded and the embryos cultured individually in 4 µl drops of EBSS at 37°C in 5% CO2 in air for 24 h. The embryos were then transferred every 24 h to fresh 4 µl drops until the blastocyst stage. The EBSS was supplemented with 0.5% HSA, 1 mmol/l glucose, 5 mmol/l lactate, 0.47 mmol/l pyruvate and a physiological mixture of amino acids (Tay et al., 1997). The spent medium was stored at –80°C. Following

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Table I. Human embryo scoring system Embryo grade

Classification

1 2 2.5 3 4 5

Even blastomeres, no fragmentation Even blastomeres, ⬍10% fragmentation 10–30% fragmentation ⬎30% fragmentation Only one intact blastomere No viable blastomeres

thawing, an aliquot (2 µl) was removed and diluted 1:12.5 in high performance liquid chromatography (HPLC) grade water. Embryofree control drops were incubated alongside the embryo-containing drops to allow for any non-specific amino acid degradation/ appearance. The amino acids were analysed by reverse-phase HPLC as previously described (Lamb and Leese, 1994), but using a Kontron 500 series automated HPLC system fitted with a Jasco F920 fluorescence detector and a 4.5⫻250 mm Hypersil ODS-16 column (Jones Chromatography, Hengoed, Mid Glamorgan, UK). Derivatization was achieved by the automated reaction of a 25 µl sample with an equal volume of reagent [10 µl 2-mercaptoethanol and 5 ml ophthaldialdehyde (OPA) reagent]. The elution gradient operated at a flow rate of 1.3 ml/min. Solvent A consisted of 18 ml tetrahydrofuran (Fisher Scientific, Loughborough, Leics, UK), 200 ml methanol and 800 ml sodium acetate (83 mmol/l, pH 5.9). Solvent B consisted of 800 ml methanol and 200 ml sodium acetate (83 mmol/l, pH 5.9). Using this method, it was not possible to detect proline and cysteine. Results were expressed as amino acid depletion/appearance in pmol/embryo/h ⫾ SEM. The term ‘turnover’ has been used to describe the sum, in pmol/embryo/h of amino acid depletion from, and appearance in, the culture medium. Embryos received on day 2 varied in developmental stage from 2- to 4-cell. After 24 h incubation they ranged from 4-cell to compacting 8-cell (c8-cell). Moreover, embryos were of variable grade; from 1–4. The data were therefore expressed in terms of amino acid depletion/appearance from day 2–3. Due to variation in the rate of human preimplantation development, amino acid profiles for the later stage embryos were best grouped according to embryo stage; c8-cell to morula and morula to blastocyst, rather than by day post-insemination. Approximately equal numbers of embryos were at the c8-cell stage on day 3 and 4 in both the arresting and developing groups. Developing embryos progressed from the c8-cell stage to the morula stage within the 24 h culture period and subsequently developed from the morula to the blastocyst stage in the next 24 h period. Each amino acid value was tested for significance from zero depletion/appearance using Student’s t-test. Differences between amino acid turnover for arresting and developing embryos were also analysed by Student’s t-test, as were differences between embryo grade.

Results The average grades of embryos which subsequently developed to the blastocyst stage and those that arrested prior to blastocyst formation are shown in Table II. There were no significant differences in grade between arresting and developing embryos at either stage of development. Metabolic profiling in terms of amino acid depletion/appearance could readily be carried out on single human embryos incubated for 24 h, from as early as day 2 post-insemination. Profiles for the embryos

Amino acids predict embryo developmental capacity

Figure 1. Amino acid depletion (negative values) and appearance (positive values) ⫾ SEM by individual, spare human preimplantation embryos which subsequently formed blastocysts (n ⫽ 14), or which arrested in culture prior to blastocyst formation (n ⫽ 28), measured non-invasively from day 2–3 of development. *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001 significance from zero. Bars with the same superscript are significantly different: a, P ⫽ 0.0004; b, P ⫽ 0.023; c, P ⫽ 0.026; d, P ⫽ 0.033; e, P ⫽ 0.044.

Table II. Mean grade ⫾ SEM for day 2 and compacting 8-cell embryos which either developed to the blastocyst or arrested prior to blastocyst formation Stage of development

Day 2 Compacting 8-cell

Embryo grade Developing

Arresting

2.50 ⫾ 0.18 1.80 ⫾ 0.14

2.95 ⫾ 0.15 2.10 ⫾ 0.10

incubated from day 2–3, during the c8-cell to morula and morula to blastocyst transitions were as follows: (i) Amino acid turnover by embryos from day 2–3 postinsemination Leucine was the only amino acid significantly depleted (P ⬍ 0.001) between day 2 and 3 of development by embryos that subsequently developed to the blastocyst stage (Figure 1) while glutamate (P ⬍ 0.01) and alanine (P ⬍ 0.001) appeared in significant quantities in the culture medium. The other amino acids were not significantly different from zero, i.e. there was no net disappearance from the medium, nor did they appear. Embryos that were morphologically indistinguishable (P ⫽ 0.067; Table II) from any other in this cohort, but which subsequently failed to develop to the blastocyst stage displayed a different pattern of amino acid depletion/appearance. Asparagine (P ⬍ 0.05), glutamine (P ⬍ 0.001), arginine (P ⬍ 0.001), methionine (P ⬍ 0.01), valine (P ⬍ 0.0001), isoleucine (P ⬍ 0.001) and leucine (P ⬍ 0.001) were all depleted while aspartate (P ⬍ 0.05), glutamate (P ⬍ 0.001), glycine (P ⬍ 0.001), alanine (P ⬍ 0.001) and lysine (P ⬍ 0.05) appeared. The amino acid profiles of asparagine (P ⫽ 0.044),

Figure 2. Amino acid turnover for those embryos which subsequently developed to the blastocyst stage compared with those which arrested prior to blastocyst formation. Values are the sum of the amino acids that significantly appeared/disappeared from the medium ⫾ SEM. ***P ⬍ 0.001 significantly different from embryos that developed to the blastocyst stage. C8-cell ⫽ compacting 8-cell.

glutamine (P ⫽ 0.026), arginine (P ⫽ 0.023), alanine (P ⫽ 0.0004) and methionine (P ⫽ 0.033) of those day 2–3 embryos which went on to form blastocysts differed from those which subsequently arrested (Figure 1). In addition, when the values for the significantly depleted and produced amino acids were summed, there was a 7.2- and 2.1-fold increase in amino acid uptake and production respectively for day 2–3 embryos which subsequently arrested prior to the blastocyst stage compared with those that continued to develop. In other words, the amino acid turnover (i.e. the sum of depletion and appearance) for embryos which arrested was 3.7-fold greater (P ⬍ 0.001) than those which developed to the blastocyst stage (Figure 2). 1001

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Figure 3. Amino acid depletion/appearance ⫾ SEM by individual human embryos over the compacting 8-cell to morula transition which subsequently formed blastocysts (n ⫽ 23), or which arrested in culture prior to cavitation (n ⫽ 32). *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001 significance from zero. Bars with the same superscripts are significantly different: a, P ⫽ 0.015; b, P ⫽ 0.019; c, P ⫽ 0.037. C8-cell ⫽ compacting 8-cell.

(ii) Amino acid turnover during the compacting 8-cell to morula transition Human embryos incubated from the c8-cell to morula stage of development depleted leucine (P ⬍ 0.001), arginine (P ⬍ 0.01) and serine (P ⬍ 0.01) significantly from the medium (Figure 3), while aspartate (P ⬍ 0.01), glutamate (P ⬍ 0.05), alanine (P ⬍ 0.001) and tryptophan (P ⬍ 0.05) were significantly produced. Embryos over this developmental period which failed to develop to the blastocyst stage gave greater amino acid appearance and disappearance values than those which developed normally [significance values were: asparagine (P ⬍ 0.05), glutamine (P ⬍ 0.01), arginine (P ⬍ 0.001), valine (P ⬍ 0.05), isoleucine (P ⬍ 0.05) and leucine (P ⬍ 0.001) in terms of depletion, and aspartate (P ⬍ 0.01), glutamate (P ⬍ 0.001), glycine (P ⬍ 0.01) and alanine (P ⬍ 0.001) in terms of appearance]. There was no significant difference in morphology at the c8-cell stage between embryos that developed to the blastocyst stage and those that arrested (Table II). However, the developmental potential of c8-cell embryos was reflected in the amino acid profiles for specific amino acids (Figure 3). Thus, serine (P ⫽ 0.019), glycine (P ⫽ 0.015) and alanine (P ⫽ 0.037) depletion/appearance values differed for developing versus arrested embryos. Compacting 8-cell to morula stage embryos which arrested prior to blastocyst formation displayed a 2.2and 1.6-fold increase in amino acid uptake and production respectively compared with those that continued to develop. In total, amino acid turnover for c8-cell embryos that arrested during development was 2-fold greater (P ⬍ 0.001) than those which developed to the blastocyst stage (Figure 2). (iii) Amino acid turnover during the morula to blastocyst transition Serine (P ⬍ 0.001), arginine (P ⬍ 0.001), methionine (P ⬍ 0.001), valine (P ⬍ 0.001) and leucine (P ⬍ 0.01) were significantly depleted while aspartate (P ⬍ 0.001), glutamate 1002

(P ⬍ 0.001) and alanine (P ⬍ 0.001) appeared in the medium (Figure 4). Amino acid turnover throughout development Table III summarizes the amino acids whose depletion or appearance was statistically significant from zero for the three developmental transitions: day 2–3; c8-cell to morula; morula to blastocyst. For embryos which developed to the blastocyst stage, there was an increase in uptake; from one amino acid (leucine) from day 2–3, to three amino acids (serine, arginine and leucine) during the compacting 8-cell to morula transition, to five amino acids (serine, arginine, methionine, valine and leucine) during the morula to blastocyst transition. By contrast, embryos which arrested had a greater amino acid uptake; for seven amino acids during the day 2–3 transition, and six amino acids during the compacting 8-cell to morula stage. Total amino acid depletion was also shown to increase in absolute terms, from 4.3 ⫾ 0.4 pmol/embryo/h from day 2–3 to 14.6 ⫾ 1.9 pmol/embryo/h over the morula to blastocyst transition (P ⫽ 0.01; Figure 5). Discussion Non-invasive assays have been used to measure amino acid depletion/appearance (i.e. turnover) by single human embryos throughout development to the blastocyst stage. Although the data are expressed as rates of depletion or appearance of given amino acids, it should be emphasised that these were not measured under conditions where initial rates of uptake or release could be determined; rather, the data represent net, embryo-mediated fluxes of amino acids, in an inward or outward direction. Because of this, the data cannot be interpreted in kinetic terms, but do provide an indication of the nutritional requirements of early human embryos and the possible relationship with embryo physiology and development. Thus, it is likely that the major quantitative use of amino acids, as in somatic cells, is in protein synthesis, which, in other


Figure 4. Amino acid depletion/appearance ⫾ SEM by individual human embryos over the morula to blastocyst transition (n ⫽ 42). **P ⬍ 0.01; ***P ⬍ 0.001 significance from zero.

Table III. Amino acid uptake/production by developing and arresting embryos Developmental stage

Amino acids taken up by the embryo

Amino acids produced by the embryo

Developing embryos

Arresting embryos

Developing embryos

Arresting embryos

Day 2–3

LeuE

GluNE AlaNE

AspNE GluNE GlyC AlaNE LysE

Compact 8-cell to morula

SerC ArgC LeuE

AsnNE GlnNE ArgC MetE ValE IsoE LeuE AsnNE GlnNE ArgC ValE IsoE LeuE

AspNE GluNE AlaNE TrpE

AspNE GluNE GlyC AlaNE

Morula to blastocyst

SerC ArgC MetE ValE LeuE

AspNE GluNE AlaNE

Superscripts depict amino acid category. E ⫽ essential, NE ⫽ non-essential and C ⫽ conditional amino acid.

species, increases during the later stages of preimplantation development (Leese, 1991). This was reflected by an increase in amino acid depletion as the embryos developed from day 2 through to the blastocyst stage (Figure 5). Curiously, amino acid depletion by embryos which arrested were 7.2 and 2.2 times greater on day 2–3 than during the c8-cell to morula transition respectively than for those which developed normally. In other words, the data indicate that preimplantation embryos competent to develop into blastocysts have a lower amino acid turnover than those which arrest. These results are intriguing and suggest that arresting embryos are more metabolically active than developmentally competent embryos. The conclusion that viable human embryos are less active metabolically was also reached by Conaghan et al. who measured the consumption of pyruvate by 590 individual invitro produced human embryos from the pronucleate stage

following fertilization, up to the day of transfer on day 2 or 3 post-insemination (Conaghan et al., 1993). For those embryos which gave rise to a successful pregnancy after transfer, the pyruvate consumption was significantly lower than those which failed to implant. The same pattern was apparent in a related study carried out on human embryos conceived through natural cycle IVF (Turner et al., 1994). The amino acid most consistently depleted throughout development by those embryos which formed blastocysts was leucine. This was an interesting finding; firstly because leucine is an ‘essential amino acid’ (see below) and also since it has an important role as a signalling molecule which stimulates protein synthesis in a variety of cell types (Kimball et al., 1999; Anthony et al., 2000; Lynch et al., 2000). An interesting observation in the present study was that glutamine was not significantly depleted from the medium at 1003

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Figure 5. Total depletion of amino acids from the medium ⫾ SEM by developing and arresting human embryos from day 2 through to the blastocyst stage. *P ⬍ 0.05; ***P ⬍ 0.001 significantly different from embryos that developed to the blastocyst stage. Bars with the same superscript are significantly different: a, P ⫽ 0.01. C8-cell ⫽ compacting 8-cell.

any stage by those embryos which developed successfully. However, there was a significant depletion of glutamine from day 2–3 (18.4 pmol/embryo/h) and from the c8-cell to morula stage (13.7 pmol/embryo/h) in embryos that subsequently arrested prior to the blastocyst stage. It has been shown that glutamine, when administered as the sole amino acid, increases the proportion of human embryos that reach both the morula and blastocyst stages (Devreker et al., 1998). While single amino acid substrate experiments are obviously required for detailed kinetic studies (Van Winkle, 2001), our data suggest that use of such an approach to address questions related to embryo development and metabolism is unlikely to be physiological. Of the amino acids for which there was a net appearance in the culture medium, the most striking data were provided by alanine, which appeared in increasing amounts throughout development. This result was similar to that obtained for bovine preimplantation embryos (Partridge and Leese, 1996). The most likely explanation is that alanine provides a means of disposing of ammonia, via the enzymes glutamate dehydrogenase and alanine transaminase, since the amounts of alanine appearing in the medium were reduced to 32 and 16% of controls in the absence of pyruvate and amino acids respectively (Donnay et al., 1999). In the present study, the alanine produced by arresting embryos was significantly greater than for developing embryos. These results indirectly support the proposition that alanine production provides a means of disposing of toxic ammonium ions in human as well as bovine embryos since amino acid turnover, which will lead to ammonia production, was greater for arrested than developing embryos. Essential, non-essential and conditional amino acids. Culture media for preimplantation mammalian embryos are widely supplemented with amino acids, which are most commonly divided into ‘essential’ and ‘non-essential’. For human embryos, non-essential amino acids are recommended for early cleavage, with a mixture of non-essential and essential for the 8-cell to blastocyst stages (Lane and 1004

Gardner, 1997). This regime has to be questioned on two grounds. Firstly, the traditional division of amino acids into ‘essential’ and ‘non-essential’ has now been replaced, albeit in terms of human nutrition, by the addition of a third category of ‘conditionally essential’ amino acids (Frayn, 1997); those which may be synthesized from other essential amino acids, but may be required in the diet under some circumstances to satisfy nutritional requirements. On this basis, all the amino acids showing a net depletion from day 2 through to the blastocyst stage, are either ‘essential’ or ‘conditional’ (Table III); none fall into the non-essential category. This requirement would obviously not be fulfilled by culture media which included only non-essential amino acids during the early preimplantation phase. Moreover, the division of amino acids into even three groups may mask considerable subtleties in their requirements (Jackson, 1999). Our data lead us to favour including all 20 amino acids in human embryo culture media, at concentrations believed to be physiological, and to let the embryos choose which to consume or release, and in what quantities. Metabolic profiling and embryo selection. Our data have revealed quite marked differences in amino acid turnover between embryos which develop to the blastocyst stage and those which arrest. Such differences are apparent in embryos incubated from day 2–3 postinsemination. This implies that oocyte quality must play a major role in determining embryo viability since zygotic genome activation is not detected in the human embryo until the 4- to 8-cell stage (Braude et al., 1988). Zygotes incubated for 24 h from day 1 have not been available for this study, since decisions on which embryos to transfer, freeze or release for research purposes were not made until day 2. An alternative method to detect differences in amino acid turnover between viable and non-viable embryos was to group the sum of selected amino acids. From day 2–3 of development, the sum of lysine, alanine and glycine gave the greatest significant difference between embryos that reached the blastocyst stage and those that arrested prior to cavitation. During the c8-cell to morula transition, the sum of glycine, alanine and lysine gave the same degree of significance between developmentally competent and arrested embryos as the sum of serine, glycine, alanine, leucine and lysine. Thus, there are a number of ways of using amino acid profiling data to predict non-invasively the ability of a human embryo to reach the blastocyst stage. These results have implications for the selection of human embryos for transfer following IVF; if the ability to cavitate can be determined as early as day 2 of development, this would permit the selection of single, developmentally competent embryos and obviate the need for prolonged culture to the blastocyst stage as a means of selection prior to transfer.

Acknowledgements We would like to thank the embryologists at the Leeds General Infirmary for their continued support. This research was carried


out as part of the MRC Co-operative Group on the Development of the Early Human Embryo. The work is the subject of a patent application.

References Alexiou, M. and Leese, H.J. (1992) Purine utilisation, de novo synthesis and degradation in mouse preimplantation embryos. Development, 114, 185–192. Anthony, J.C., Yoshizawa, F., Anthony, T.C., Vary, T.C., Jefferson, L.S. and Kimball, S.R. (2000) Leucine stimulates translation initiation in skeletal muscle of postabsorptive rats via a rapamycin-sensitive pathway. J. Nutr., 130, 2413–2419. Balen, A. (2001) GnRH agonists and superovulation for assisted conception. Infert. Reprod. Med. Clinics North Am., 12, 89–104. Bavister, B.D. and McKiernan, S.H. (1993) Regulation of hamster embryo development in vitro by amino acids. In Bavister B.D. (ed.) Preimplantation Embryo Development, Springer Verlag, New York, pp. 57–72. Bolton, V.N., Hawes, S.M., Taylor, C.T. and Parsons, J.H. (1989) Development of spare human preimplantation embryos in vitro: an analysis of the correlations among gross morphology, cleavage rates and development to the blastocyst stage. J. In vitro Fert. Embryo Transf., 6, 30–35. Braude, P.R., Bolton, V.N. and Moore, S. (1988) Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature, 332, 459–461. Conaghan, J., Hardy, K., Handyside, A.H., Winston, R.M. and Leese, H.J. (1993) Selection criteria for human embryo transfer: a comparison of pyruvate uptake and morphology. J. Assist. Reprod. Genet., 10, 21–30. Conaghan, J., Hardy, K., Leese, H.J., Winston, R.M. and Handyside, A.H. (1998) Culture of human preimplantation embryos to the blastocyst stage: a comparison of 3 media. Int. J. Dev. Biol., 42, 885–893. Dawson, K.M., Collins, J.L. and Baltz, J.M. (1998) Osmolarity dependent glycine accumulation indicates a role for glycine as an organic osmolyte in early preimplantation mouse embryos. Biol. Reprod., 59, 225–232. Devreker, F., Winston, R.M.L. and Hardy, K. (1998) Glutamine improves human preimplantation development in vitro. Fertil. Steril., 69, 293–299. Devreker, F., Van den Bergh, M., Biramane, J., Winston, R.L., Englert, Y. and Hardy, K. (1999) Effects of taurine on human embryo development in vitro. Hum. Reprod., 14, 2350–2356. Devereker, F., Hardy, K., Van den Bergh, M., Vannin, A.S., Emiliani, S. and Englert, Y. (2001) Amino acids promote human blastocyst development. Hum. Reprod., 16, 749–756. Donnay, I., Partridge, R.J. and Leese, H.J. (1999) Can embryo metabolism be used for selecting bovine embryos before transfer? Reprod. Nutr. Dev., 39, 523–533. Dumoulin, J.C.M., Van Wissen, L.C.P., Menheere, P.P.C.A., Michiels, A.H., Geraedts, J.P. and Evers, J.L. (1997) Taurine acts as an osmolyte in human and mouse oocytes and embryos. Biol. Reprod., 56, 739–744. Edwards, L.J., Williams, D.A. and Gardner, D.K. (1998) Intracellular pH of the mouse preimplantation embryo: amino acids act as buffers of intracellular pH. Hum. Reprod., 13, 3441–3448. Epstein, C.J. and Smith, S.A. (1973) Amino acid uptake and protein synthesis in preimplantation mouse embryos. Dev. Biol., 33, 171–184. Frayn, K.N. (1997) Metabolic regulation. In Snell, K. (ed.) Frontiers of Metabolism 1. Portland Press Ltd, London. Gardner, D.K., and Lane, M. (1997) Culture and selection of viable blastocysts: a feasible proposition for human IVF. Hum. Reprod. Update, 3, 367–382. Hammer, M.A., Kolajova, M., Le´ veille´ , M-C., Claman, P. and Baltz, J.M. (2000) Glycine transport by single human and mouse embryos. Hum. Reprod., 15, 419–426.

Jackson, A.A. (1999) Maternal and fetal demands for nutrients: significance of protein metabolism. In O’Brien, P.M.S., Wheeler, T. and Barker, D.J.P. (eds) Fetal Programming Influences on Development and Disease in Later Life. ROCG Press, London, pp. 246–270. Jung, Y.G., Sakata, T., Lee, E.S. and Fukui, Y. (1998) Amino acid metabolism of bovine blastocysts derived from parthenogenetically activated or in vitro fertilised oocytes. Reprod. Fertil. Dev., 10, 279–287. Kimball, S.R., Shantz, L.M., Horetsky, R.L. and Jefferson, L.S. (1999) Leucine regulates translation of specific mRNAs in L6 myoblasts through mTORmediated changes in availability of eIF4E and phosphorylation of ribosomal protein S6. J. Biol. Chem., 274, 11647–11652. Lane, M. and Gardner, D.K. (1997) Differential regulation of mouse embryo development and viability by amino acids. J. Reprod. Fertil., 109, 153–164. Lane, M. and Gardner, D.K. (1998) Amino acids and vitamins prevent cultureinduced metabolic perturbations and associated loss of viability of mouse blastocysts. Hum. Reprod., 13, 991–997. Lamb, V.K. and Leese, H.J. (1994) Uptake of a mixture of amino acids by mouse blastocysts. J. Reprod. Fertil., 102, 169–175. Leese, H.J. (1991) Metabolism of the preimplantation mammalian embryo. Oxf. Rev. Reprod. Biol., 13, 35–72. Leese, H.J., Aldridge, S. and Jeffries, K.S. (1979) The movement of amino acids into rabbit oviductal fluid. J. Reprod. Fertil., 56, 623–626. Lynch, C.J., Fox, H.L., Vary, T.C., Jefferson, L.S. and Kimball, S.R. (2000) Regulation of amino acid-sensitive TOR signaling by leucine analogues in adipocytes. J. Cell. Biochem., 77, 234–251. Miller, J.G.O. and Schultz, G.A. (1987) Amino acid content of preimplantation rabbit embryos and fluids of the reproductive tract. Biol. Reprod., 36, 125–129. Nasr-Esfahani, M.H., Winston, N.J. and Johnson, M.H. (1992) Effects of glucose, glutamine, ethylenediaminetetraacetic acid and oxygen tension on the concentration of reactive oxygen species and on development of the mouse preimplantation embryo in vitro. J. Reprod. Fertil., 96, 219–231. Partridge, R.J. and Leese, H.J. (1996) Consumption of amino acids by bovine preimplantation embryos. Reprod. Fertil. Dev., 8, 945–950. Schoolcraft, W.B., Gardner, D.K., Lane, M., Schlenker, T., Hamilton, F. and Meldrum, D.R. (1999) Blastocyst culture and transfer: analysis of results and parameters affecting outcome in two in vitro fertilization programs. Fertil. Steril., 72, 604–609. Tasca, R.J. (2001) The cell biology of preimplantation development. In Gardner, D.K. and Lane, M. (eds) ART and the Human Blastocyst. Serono Symposia USA, Springer Verlag, New York, pp. 51–60. Tay, J.I., Rutherford, A.J., Killick, S.R., Maguiness, S.D., Partridge, R.J. and Leese, H.J. (1997) Human tubal fluid: production, nutrient composition and response to adrenergic agents. Hum. Reprod., 12, 2451–2456. Turner, K., Martin, K.L., Woodward, B.J., Lenton, E.A. and Leese, H.J. (1994) Comparison of pyruvate uptake by embryos derived from conception and non-conception natural cycles. Hum. Reprod., 9, 2362–2366. Van Winkle, L.J. (2001) Amino acid transport regulation and early embryo development. Biol. Reprod., 64, 1–12. Van Winkle, L.J. and Campione, A.L. (1996) Amino acid transport regulation in preimplantation mouse embryos: effects of amino acid content and preand peri-implantation development. Theriogenology, 45, 69–80. Van Winkle, L.J., Haghighat, N. and Campione, A.L. (1990) Glycine protects preimplantation mouse conceptuses from a detrimental effect on development of the inorganic ions in oviductal fluid. J. Exp. Zool., 253, 215–219. Wu, F., Cholewa, B. and Mattson, D.L. (2000) Characterisation of L-arginine transporters in rat renal inner medullary collecting duct. Am. J. Physiol. Regul. Integr. Comp. Physiol., 278, R1506-R1512. Submitted on July 23, 2001; resubmitted on September 28, 2001; accepted on November 13, 2001

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