BIOLOGY OF REPRODUCTION (2013) 89(3):74, 1–2 Published online before print 14 August 2013. DOI 10.1095/biolreprod.113.113084
Commentary When Stresses Collide1 Awoniyi O. Awonuga,3,5 Yu Yang,3,4,5 and Daniel A. Rappolee2,3,6,7 3
CS Mott Center for Human Growth and Development of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, Michigan 4 Reproductive Sciences Concentration, Physiology and Obstetrics and Gynecology Departments, Wayne State University School of Medicine, Detroit, Michigan 5 Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, Michigan 6 Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 7 Institute of Environmental Health Sciences, Wayne State University School of Medicine, Detroit, Michigan
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Supported by National Institutes of Health grant R03HD061431. Correspondence: Daniel A. Rappolee, CS Mott Center for Human Growth, Wayne State University School of Medicine, 275 East Hancock, Detroit, MI, 48201. E-mail:
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Ó 2013 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363
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growth and higher stress. There is interest in understanding how embryos adapt to 20% oxygen after coming from a 5% oxygen environment; what stress mechanisms are used by embryos that produced IVF offspring? The study by Wale and Gardner [16] elucidates the molecular mechanisms controlling pathways in embryos and aids in understanding embryo requirements during development. Ammonium is a normal product of the metabolism of amino acids by embryos. However it is deleterious to cultured mouse embryos [20], fetuses from these embryos [9], and the progression of human embryos to the blastocyst stage [21]. In the organism, ammonia is excreted as urea, but the embryo sequesters ammonium by using it as a substrate to produce the amino acids alanine and glutamine. Wales and Gardner [16] found that glutamine synthetase transaminates glutamate to sequester an ammonium ion in glutamine in embryos cultured at 5% oxygen. However, at 20% oxygen, this reaction proceeds at low rates; glutamine is consumed and ammonium is produced instead. The glutamine synthetase knockout mouse dies at Embryonic Day 3.5, and failure of tetraploid rescue and chimera experiments suggest that this is due to failure to detoxify ammonium by the embryo and not by other glutamine synthetase functions [22]. Glutamine synthesis improves embryo anabolism but also acts as an osmolyte to counteract osmotic stress [23]. Data from Wales and Gardner [16] extends the necessary function of glutamine synthetase in detoxifying ammonia from knockout embryos in vivo to cultured embryos during IVF/ART. In this report, multiple lines of evidence are presented that demonstrate that 20% oxygen is stressful and suppresses amino acid anabolism, a predictor of embryo health [24], and suppresses the glutamine synthesis induced at 5% oxygen by ammonium. Transamination of glutamate to produce glutamine and the sequestering of ammonium is more important than transamination of other amino acids. Conversion from glutamate to glutamine and the consequent decrease in ammonium is stoichiometric. Ammonium increases amino acid turnover at 5% oxygen. But, at 20% oxygen, amino acid turnover with or without added ammonia is similar. Thus, oxygen stress is dominant in suppressing amino acid synthesis. This report defines stress mechanisms activated at 20% oxygen and producing maladaptive responses in cultured embryos. This study identifies additional hypothetical patho-
In vitro fertilization/assisted reproductive technologies (IVF/ART) is a relatively new area of medicine with such a high impact that its originator, Dr. Robert Edwards, was awarded the Nobel Prize in medicine in 2010 [1]. About 10% of the world population is infertile and ;50% are from maternal or paternal defects [2]. The first IVF human baby was born in 1978, and more than five million babies since have been conceived through IVF. In 2011, nearly 62 000 infants were born using ART, comprising ;1% of births in the United States [3]. Advances in IVF have produced better hormones for increasing oocyte number, better culture media, and better methods for avoiding the implantation of aneuploid embryos as well as predicting which embryos are best to reimplant [2]. During IVF, embryos are subject to many stresses such as 20% oxygen [4–7], pipetting [8], ammonium [9], changes in osmolarity [10–12], culture media [13], transplantation [14], and genetic differences underlying stress responses [15]. The interactive and additive effects of stresses have not been addressed until the report from Wale and Gardner [16] published in this issue of Biology of Reproduction. From the beginning, oxygen levels used for embryo culture have been approximately equivalent to atmospheric concentrations. Nevertheless, 20% oxygen is abnormal and arterialvenous oxygen levels from 8% to 5% are closer to the oxygen environment that most cells face. Over time, oxygen levels in ex vivo embryo incubators have been moved from 20% to 5%. A current longitudinal study is aimed at testing the hypothesis that 5% oxygen produces higher quality offspring [17]. Placental and fetal growth in mice is suboptimal after embryo culture at 20% oxygen [6]. The implanting embryo is made up of precursors of embryonic and placental trophoblast stem cells (ESCs and TSCs, respectively). However stem cell potency of both human ESC [18] and mouse TSC [19] is best at 2%–3% oxygen and lower or higher levels of oxygen promote poorer
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genic mechanisms: methionine deficiency that may lead to methylation deficiencies and arginine deficiency that may lead to deficiencies in nitric oxide and DNA synthesis. These warrant additional studies. Future studies will provide insight into what stress responses embryos develop during IVF and how they may affect offspring. These insights will help ameliorate the long-term health effects that arise as IVF offspring age. The transition from 20% to 5% oxygen was robust. The 20% oxygen concentration clearly induces global changes in mRNA expression [25], and incorrect oxygen levels and other stresses induce loss of potency at all stages of embryo culture and in stem cells derived from the blastocyst [12, 19]. The next challenges will be determining a dose response to oxygen concentrations because this study focused on historical changes in oxygen concentrations but not the niche for the stem cells in the implanting embryo. Key improvements in future studies will relate to confirming in embryos the inverted U-shaped curve for stem cell potency and growth that are highest at 2%– 3% oxygen [18, 19]. This information will allow for definition of an oxygen optimum that has the least stress and highest growth and potency. These studies will differ from the investigation of the transition from 20% to 5% oxygen; it is evident that undertaking such studies with human embryos is unethical as these would require defining toxicity at oxygen concentrations below the optimum level.
