Amino Acids and ART

For most people, amino acids are synonymous with protein and DNA synthesis and their ability to act as substrates for energy production, and relatively little else. Over the past two decades however we have developed a very different appreciation for amino acids and the highly diverse roles they play in regulating embryo physiology. Here their functions (beyond body building!) are considered, and how their supplementation to embryo culture media has contributed significantly to the increase in IVF success rates worldwide.

A turning point in embryo culture

For the first two decades of human IVF, amino acids (typically found in all tissue culture media) were curiously absent from the media used for IVF and embryo culture. Media were largely based on the formulations initially developed for mouse embryos in the 1960s and comprised of a balanced salt solution supplemented with the carbohydrates glucose, pyruvate and lactate. Embryos of the mouse and human were able to grow in such media, but at a greatly reduced rate. Furthermore, if a blastocyst was formed it was typically delayed by up to 24h, and subsequent viability after transfer was low. A turning point in embryo culture came after the seminal work of Miller and Schultz in 1987¹, who analysed the amino acid composition of the rabbit oviduct and uterus, and determined that specific amino acids such as glycine, glutamate, alanine, serine and taurine, were present at relatively high levels. This, in itself, was an interesting and significant observation as these amino acids are not amongst those required by somatic cells, so why are they present at high levels and what are they doing? Questions that we will come back to later.

Reaching the blastocyst stage at the correct time

During the 1990s there was a burst of research into the role of amino acids on embryo development in the rabbit, hamster, mouse, sheep and cattle, all of which went on to show a highly beneficial effect in culture (reviewed in ^{2, 3}). With regards to our own initial studies on the mouse embryo back in the early 1990s, embryos were cultured from the 1-cell stage in the media of the day, i.e. balanced salt solutions and carbohydrates, and it took 5 days of culture to reach the blastocyst stage. Hence zygotes placed into culture on Monday formed blastocysts by Friday. While this fitted in nicely with the working week, blastocyst development was delayed by 24h, indeed in vivo mouse blastocysts would have implanted on Thursday evening, i.e. day 4. In our initial experiments, we supplemented culture media with those amino acids present at high levels in the oviduct⁴. Imagine our surprise when the embryos were scored on a Thursday afternoon and every single embryo in the amino acid group had developed into a fully expanded blastocyst, while the control group were still morulae. I can still feel the hairs standing up on the back of my neck as I looked down the microscope (part of me wondering whether it was really Friday, and I had my days mixed up!). The blastocysts had developed on time (that was a good day at the office!).

A double-edged sword

Like all good scientific stories, this one got complicated very quickly, as it became apparent that at certain concentrations those amino acids that were of greatest benefit to the cleavage stages were not the same as those that stimulated blastocyst differentiation. Conversely, those amino acids which did stimulate the inner cell mass, had a detrimental effect on the cleavage stages. This could be attributed to the different functions of different amino acids (discussed below). Further, the beneficial effect of the added amino acids appeared to be diminished over time, but when the medium was renewed, the improvement in embryo development continued. This was subsequently shown to be due to the accumulation of ammonium in the medium caused by both the spontaneous deamination of amino acids at 37oC and through the metabolism of amino acids by the embryo⁴. Ammonium accumulation in the medium was a much more sinister problem than we had first envisaged, for if embryos were chronically exposed to it, adverse outcomes were found post-transfer, and neural tube defects were detected in ~20% of the resultant foetuses⁵. However, this was readily alleviated by making two changes; first the most labile amino acid glutamine was substituted with a stable dipeptide form (alanyl-glutamine), and secondly the medium was renewed every 48h. Once this was all addressed ammonium did not accumulate and amino acids increased fetal development after blastocyst transfer, with no abnormal outcomes⁵. Indeed, in today’s Vitrolife media formulations, the levels of ammonium never reach toxic levels even after continuous culture to the blastocyst stage.

A warning on cumulative stress

It is not uncommon in science for others to challenge new findings, and so it was with the negative impacts of ammonium on fetal development. While other groups found similar issues with birth effects, others could not reproduce them⁶. This discrepancy remained unresolved for several years, until it was revealed that the use of atmospheric (20%) oxygen for culture altered the metabolic function of the preimplantation embryo, and resulted in their inability to sequester ammonium, and consequently they could not detoxify it (7). The initial studies in which neural tube defects were observed were performed after embryo culture in 20% oxygen⁴, while the later studies which failed to reproduce these findings were performed in 5% oxygen⁶. As such the ammonium story highlights the complexities of embryo culture; in this case when two stresses are present, severe and often unexpected adverse outcomes can be the result ^{8, 9.}

Amino acids function to regulate both cellular and metabolic homeostasis within the embryo

So back to the question, what are the different amino acids doing? Well it transpires it is a lot more than we first thought ^{2, 3.} Data support the following roles (at a minimum) for amino acids during embryo development: biosynthetic precursors, energy sources, regulators of carbohydrate metabolism, regulators of intracellular pH, osmolytes, antioxidants, chelators, and signalling molecules in tissue differentiation. With regards to some of these roles, which you may not have considered (such as osmolytes and pH regulators), they are particularly important for the cleavage stage embryo, which are really a collection of single cells which lack several sophisticated mechanisms for regulating cell function. Interestingly when considering how unicellular organisms regulate their homeostasis, they typically employ amino acids such as glycine. Hence, it is plausible that the high levels of glycine observed in oviduct fluid serves a similar role for the oocyte and cleavage stages.

A long history

In summary, amino acids function to regulate both cellular and metabolic homeostasis within the embryo, and without them present in the culture media, the embryo undergoes considerable stress. Needless to say, 25 years after the initial studies we have amassed considerable data regarding the significance of amino acids in early embryo development, and as a result we have learned to never expose oocytes or embryos to media lacking them.

Wait, their story has not ended yet! With the dawn of Metaboloepigenetics¹⁰, and the discovery that the Malate-Aspartate shuttle regulates intracellular NAD+ ¹¹(itself a master regulator of cellular epigenetic state), future research will reveal the mechanisms through which aspartate and glutamate help program development.

So, please enjoy your protein shake, but next time ponder the wonder of those amino acids within!

References

1. Miller JG, Schultz GA. Amino acid content of preimplantation rabbit embryos and fluids of the reproductive tract. Biol Reprod 1987;36:125-9.
2. Gardner DK. Dissection of culture media for embryos: the most important and less important components and characteristics. Reprod Fertil Dev 2008;20:9-18.
3. Gardner DK, Lane M. Embryo culture systems. In: Gardner DK, Simon C, eds. Handbook of in vitro fertilization, 4th Edition. Boca Raton: CRC press, 2017:205-44.
4. Gardner DK, Lane M. Amino acids and ammonium regulate mouse embryo development in culture. Biol Reprod 1993;48:377-85.
5. Lane M, Gardner DK. 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;102:305-12.
6. Biggers JD, McGinnis LK, Summers MC. Discrepancies between the effects of glutamine in cultures of preimplantation mouse embryos. Reprod Biomed Online 2004;9:70-3.
7. Wale PL, Gardner DK. Oxygen affects the ability of mouse blastocysts to regulate ammonium. Biol Reprod 2013;89:75.
8. Gardner DK, Kelley RL. Impact of the IVF laboratory environment on human preimplantation embryo phenotype. J Dev Orig Health Dis 2017;8:418-35.
9. Wale PL, Gardner DK. The effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human reproduction. Hum Reprod Update 2016;22;2-22.
10. Donohoe DR, Bultman SJ. Metaboloepigenetics: interrelationships between energy metabolism and epigenetic control of gene expression. J Cell Physiol 2012;227:3169-77.
11. Gardner DK, Harvey AJ. Blastocyst metabolism. Reprod Fertil Dev 2015;27:638-54.