1 Department of Biology, University of York, PO Box 373, York YO10 5YW and 2 Assisted Conception Unit, Clarendon Wing, Leeds General Infirmary, Leeds LS1 9NS, UK
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Abstract |
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Key words: amino acids/developmental potential/human preimplantation embryo culture/IVF
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Introduction |
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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 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 23 post-insemination and during the compacting 8-cell 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.
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Materials and methods |
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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 thawing, an aliquot (2 µl) was removed and diluted 1:12.5 in high performance liquid chromatography (HPLC) grade water. Embryo-free control drops were incubated alongside the embryo-containing drops to allow for any non-specific amino acid degradation/appearance.
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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 14. The data were therefore expressed in terms of amino acid depletion/appearance from day 23. 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.
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Results |
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Discussion |
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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 any stage by those embryos which developed successfully. However, there was a significant depletion of glutamine from day 23 (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 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 23 post-insemination. 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 23 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.
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Acknowledgements |
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Notes |
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References |
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Submitted on July 23, 2001; resubmitted on September 28, 2001; accepted on November 13, 2001.