Analysis of metabolism to select viable human embryos for transfer

Analysis of metabolism to select viable human embryos for transfer David K. Gardner, D.Phil. and Petra L. Wale, B.Sc. Department of Zoology, University of Melbourne, Parkville, Victoria, Australia

As we move to reducing the number of embryos transferred in a given IVF cycle, ideally down to one, there is an ever-increasing need for noninvasive quantitative markers of embryo viability. Although stage-specific morphologic markers and grading systems have been developed, such an approach is unable to assess the physiological status of the embryo. Analysis of metabolism has proved to be a valuable marker of embryo viability after transfer in animal models. We therefore reviewed what is known about human embryo metabolism, how media systems can affect the patterns of nutrient utilization and the activities of metabolic pathways, and how this relates to the developmental competence of the embryo. It is proposed that a unifying hypothesis of metabolism for the entire preimplantation period is not realistic, given the dramatic changes in embryo physiology that occur from fertilization to blastocyst development, and that the concept of a ‘‘quiet metabolism’’ can be interpreted as stress induced by the presence of high oxygen in the embryo culture/analysis system. Further research is required to fully understand the origins of metabolic stress in embryos for it to be alleviated and to develop a comprehensive range of markers that Use your smartphone not only reflect embryo viability, but also sex-specific differences in physiology. (Fertil SterilÒ to scan this QR code 2013;99:1062–72. Ó2013 by American Society for Reproductive Medicine.) and connect to the Key Words: Amino acids, embryo selection, metabolism, oxygen, viability Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/gardnerd-metabolism-viability-embryo-selection/

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he goal of human assisted conception is the birth of a healthy singleton child following the transfer of a single embryo, thereby alleviating the complications associated with multiple gestations. The move to single-embryo transfer has numerous significant advantages to the patient and child, as well as to the medical system and society as a whole (1). Consequently, countries around the world are moving toward achieving the goal of routine single-embryo transfer. To facilitate this, there have been major advances in embryo culture systems, which can support the generation of viable human embryos, resulting in an increase in implantation and pregnancy rates over the past 15 years. In parallel, we have witnessed improvements in cryopreservation technolo-

gies, so that vitrification can now be used to great efficacy at all stages of development. Concomitantly, improvements in slow-freezing have demonstrated that it can also work very effectively for embryos. Given that human embryo culture and cryopreservation have become very effective, it has been proposed by Mastenbroek et al. that there is no longer a need to worry about embryo selection, rather that one can transfer embryos in successive cycles so that embryo selection is not necessary (2). However, such an approach does not take into consideration the time to pregnancy, which is not inconsequential to all patients receiving infertility treatment. By using novel technologies for embryo selection we can ensure that they have a family as soon as possible.

Received September 7, 2012; revised and accepted December 5, 2012; published online January 8, 2013. D.K.G. reports a grant from Vitrolife. P.L.W. reports a grant from the Alfred Nicholas Fellowship, University of Melbourne. Reprint requests: David K. Gardner, D.Phil., Department of Zoology, University of Melbourne, Parkville, Victoria 3010, Australia (E-mail: [email protected]). Fertility and Sterility® Vol. 99, No. 4, March 15, 2013 0015-0282/$36.00 Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2012.12.004 1062

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Reducing the number of transfers a patient must undergo also reduces the financial and emotional burden for the patient. Every time a patient undergoes a transfer and subsequent pregnancy test she experiences a dramatic rollercoaster of emotions that takes an unparalleled toll on her ‘‘infertility’’ psychology. Furthermore, the Mastenbroek et al. approach does not take into account any possible genetic abnormalities in the embryos and the potential for adverse outcomes as a result of these abnormalities, which can now be effectively analyzed through novel screening procedures (3, 4). It is the basis of the present paper that embryo selection procedures do have a role to play in the modern clinical IVF laboratory, and that through noninvasive analysis of viability markers it will be feasible to identify those embryos within a cohort with the highest probability of establishing a healthy pregnancy. This in turn will greatly reduce the time it takes a couple to conceive a family. Ensuring endometrial receptivity of the patient is also essential, and its significance is well established (5, 6). VOL. 99 NO. 4 / MARCH 15, 2013

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MORPHOLOGY AND BEYOND Since the birth of Louise Brown in 1978, analysis of embryo morphology remains the most common method of embryo selection. Over the intervening years, grading systems have been successfully developed and applied to all stages of embryonic development, from the pronucleate oocyte (7) through the cleavage stages (8) to the blastocyst (9). Although such systems have been of value, and have been recently reviewed and published in a consensus document (10), the analysis of morphology alone tells us very little about the physiology of the developing conceptus. Using morphology alone is analogous to going for an annual check-up with your doctor and they simply gauge your health by your appearance. The inclusion of time as a variable in the analysis of human embryo developmental potential was first documented by Edwards et al. in 1984 (11), and further studies have confirmed the value of including rate of development as a marker of implantation potential (12, 13). Furthermore, there exists the possibility to quantify differences in the developmental rate of male and female embryos, where male embryos of other species have been shown to develop faster than female embryos under specific culture conditions (14). More recently, the development of commercial time-lapse systems has facilitated the creation of new data sets to support the application of temporal analysis (15, 16). However, even this dynamic approach does not directly quantify any aspect of embryo physiology.

WHAT SHOULD BE MEASURED? Several specific factors have been proposed as suitable biomarkers of implantation potential in embryos. The first of these was the phospholipid 1-o-alkyl-2-acetyl-sn-gylcero3-phosphocholine (Paf), which was shown to be produced by human embryos as early as the zygote (17). Other factors measured in human embryo culture media include leptin (18), soluble human leukocyte antigen G (19), and ubiquitin (20). However, to date there is a lack of compelling data indicating that any of the above factors can be used to select prospectively human embryos for transfer. The biomarker with the most animal data to support its use in embryo selection is embryonic metabolism, quantified through the analysis of the uptake and utilization both of carbohydrates and amino acids from spent medium. It is implicit that regulation of energy production is fundamental to the survival and propagation of any cell type. Of note, the preimplantation mammalian embryo undergoes major changes in its physiology and gene expression profile during development. As the fertilized oocyte develops and differentiates into the blastocyst, embryonic genes are successively turned on (with the concomitant destruction of maternally derived mRNAs) (21, 22). Subsequently, there is a growing energy demand as mitoses and biosynthesis increases after embryonic-genome activation and with the subsequent formation of the blastocele (through the activity of Naþ/Kþ ATPase in the trophectoderm). The dynamics and regulation of embryonic metabolism are shown in Figures 1 and 2 and reviewed in greater detail elsewhere (23–25). VOL. 99 NO. 4 / MARCH 15, 2013

Of significant clinical relevance is the fact that should an embryo at any stage of development lose control of its energy production, i.e., if the flux of a specific nutrient through a metabolic pathway is altered to a significant degree, even for a brief period, then this is associated with significantly impaired development in culture and reduction of viability after transfer (26). With the use of animal models, it is possible to analyze embryos that have developed in vivo. Consequently it is possible to determine how similar, or otherwise, embryos are when maintained in the laboratory. When mouse blastocysts are flushed from the uterus and their metabolism analyzed immediately, it has been determined that 50% of glucose consumed is released into the surrounding medium as lactate (27). When mouse embryos are cultured in simple culture conditions (i.e., a culture medium lacking amino acids and vitamins, and with only glucose, pyruvate, and lactate as energy sources, typical of the early human embryo culture media, such as human tubal fluid medium [28]), resultant blastocysts display a massive increase in lactate production (27, 29) and a concomitant decrease in glucose oxidation (30). Notably, this perturbation in metabolism was found to be associated with a significant decline in viability (31). It was later determined that placing mouse blastocysts developed in vivo into a simple culture medium (i.e., lacking amino acids) induced abnormal metabolism within just 3 hours of incubation (32). However, this metabolic stress could be greatly reduced by incubating mouse blastocysts in the presence of amino acids and vitamins. Furthermore, when in vivo–developed blastocysts were incubated for just 6 hours in a simple medium before transfer, they showed a significant decrease in implantation rate and subsequent fetal development. Incubating such blastocysts in a medium containing amino acids and vitamins overcame the negative effects of culture on implantation and fetal development. Therefore, it is evident that culture-induced metabolic stress, i.e., incubation in a medium lacking amino acids and vitamins, resulted in a rapid loss of metabolic regulation, and that this metabolic stress not only decreased implantation but its effects were seen throughout fetal development, culminating in lighterweight fetuses. Thus, an incubation of just a few hours in a stressful environment can induce metabolic changes that have downstream effects leading to compromised fetal development. Consequently, several studies have used this model to develop culture conditions which ensure that embryos in vitro exhibit a metabolic profile similar to those embryos developed in vivo (33). Such data firmly establish that quantifying embryo metabolism is an appropriate physiologic parameter to relate to subsequent viability after transfer.

CARBOHYDRATE UTILIZATION RELATES TO EMBRYO VIABILITY The relationship between embryo viability and metabolic activity was first reported in 1980 by Renard et al. (34). In a study on glucose uptake by day 10 cattle blastocysts, it was shown that embryos with a glucose uptake of >5 mg/h developed better both in vitro and in utero than those embryos with a lower uptake. Measurement of glucose uptake was achieved 1063

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FIGURE 1

Metabolism of the pronucleate oocyte and cleavage-stage embryo. Before compaction, the embryo has a metabolism based on low levels of oxidation of pyruvate, lactate, and specific amino acids. The ovulated oocyte is surrounded by, and is directly connected to, cumulus cells which actively produce pyruvate and lactate from glucose (88, 89). This creates a high concentration of pyruvate and lactate and a low concentration of glucose around the fertilized oocyte. Upon dispersal of the cumulus cells, the human zygote and cleavage-stage embryo still find themselves in relatively high concentrations of pyruvate (0.32 mmol/L) and lactate (10.5 mmol/L), and low levels of glucose (0.5 mmol/L) within the ampulla (89). The early embryo is characterized by a high ATP:ADP level (90), which in turn allosterically inhibits phosphofructokinase (PFK), thereby limiting the flux of glucose through the glycolytic pathway before compaction. Significantly, the relative abundance of nutrients affects the metabolism of the embryo. For example, the pyruvate:lactate ratio in the surrounding environment directly affects the NADH:NADþ ratio in the embryo, which in turn controls the redox state of the cells and thus the flux of nutrients through specific energy-generating pathways (91). Lactate dehydrogenase comprises 5% of the total protein of the mouse oocyte (92). The oocyte, pronucleate oocyte and all stages of development to the blastocyst exhibit lactate dehydrogenase (LDH) isoform I (93), which then changes to predominantly isoform V at the late blastocyst stage upon outgrowth (94). Isoform I favors pyruvate formation, whereas isoform V favors lactate formation. This switch in isoforms is consistent with the changes in patterns of energy metabolism as the embryo develops, but does not explain the significant production of lactate by the blastocyst (see Fig. 2). Amino acids fill several niches in embryo physiology, such as the use of glycine as buffer of intracellular pH (pHi). Several amino acids are also used as energy sources by the early embryo, such as glutamine and aspartate. Aspartate can be utilized through the malate-aspartate shuttle (53), the significance of which during embryo development we are only beginning to understand. The thickness of the lines in the figures represents the relative flux of metabolites through that pathway. GLUTs ¼ glucose transporters; GSH ¼ reduced glutathione; OAA ¼ oxaloacetate; PDC ¼ pyruvate dehydrogenase complex; PK ¼ pyruvate kinase; PPP ¼ pentose phosphate pathway. Gardner. Embryo selection by metabolic analysis. Fertil Steril 2013.

with the use of spectrophotometry. Although that technology was suitable for day 10 bovine blastocysts, with a diameter of 1,000 mm, it is not sensitive enough to detect nutrient uptake of earlier stages or smaller blastocysts such as those of the mouse or the human. Gardner and Leese (35) subsequently used ultramicrofluorescence to analyze the glucose uptake of individual mouse blastocysts before uterine transfer. Blastocysts that implanted and developed into a fetus had a significantly higher rate of glucose consumption in vitro than those 1064

that failed to implant (35). Interestingly, there was a tendency for female mouse embryos to be more metabolically active even though male embryos formed blastocysts earlier. Both of these studies were retrospective, so it was not known whether quantifying metabolism could be used prospectively to select a viable embryo for transfer. Consequently, a study was undertaken in which the metabolism of mouse blastocysts (of the same morphology and diameter) was used to classify prospectively embryos as either ‘‘viable’’ VOL. 99 NO. 4 / MARCH 15, 2013

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FIGURE 2

Metabolism of the blastocyst. After compaction, the embryo exhibits greatly increased oxygen consumption (95–97) and an increased capacity to use glucose as an energy source. The increase in oxygen consumption possibly reflects the considerable energy required for the formation and maintenance of the blastocele, and the increase in glucose utilization reflects an increased demand for biosynthetic precursors (98). Consequently, there is a reduction in the ATP:ADP ratio (90), and a concomitant increase in AMP, which has a positive allosteric effect on phosphofructokinase (PFK), thereby facilitating a higher flux of glucose through glycolysis. Rather than oxidize the glucose consumed, the blastocyst exhibits high levels of aerobic glycolysis (27). Although this may appear to be energetically unfavorable, it ensures that the biosynthetic arm of the pentose phosphate pathway (PPP) has maximum substrate availability at all times. Activity of the PPP ensures that reducing equivalents are available for biosynthesis and ensure production of reduced glutathione (GSH), a key intracellular antioxidant. For high levels of glycolysis to proceed, the blastomeres need to regenerate cytosolic NADþ. This can be achieved through the generation of lactate from pyruvate. A second means of generating cytosolic NADþ is through the activity of the malate-aspartate shuttle. Although it is evident that blastocysts use aerobic glycolysis, it is here proposed that the significant increase in oxygen utilization at this stage of development could be largely attributed to the activity of the malate-aspartate shuttle and the resulting demand for oxygen to convert intramitochondrial NADH to ATP. Indeed, many tumors that exhibit aerobic glycolysis also have high levels of malate-aspartate shuttle activity (99). Furthermore, inhibition of this shuttle has dire consequences for subsequent fetal development (100, 101). A further key regulatory enzyme in glycolysis is pyruvate kinase (PK). In proliferating cells and in cancer cells, a specific isoform is present, PKM2 (102, 103). This particular isoform of PK has been shown to promote aerobic glycolysis and anabolic metabolism (104), and recently has been identified in the mammalian blastocyst (105). Further work is warranted on establishing the regulation of PKM2 in the embryo, with specific reference to its control by exogenous factors and signaling pathways involved. The pyruvate dehydrogenase complex (PDC) catalyzes the irreversible conversion of pyruvate to acetyl-CoA, and consequently functionally links glycolysis to the activity of the oxidative tricarboxylic acid (TCA) cycle. The activity of this enzyme complex is tightly regulated through three main mechanisms: 1) phosphorylation/dephosphorylation; 2) the redox state (NADþ:NADH, ATP:ADP, and acetyl-CoA:CoA ratios); and 3) transcriptional regulation. However, relatively little is known about the regulation of this complex in mammalian embryos during the preimplantation period. Citrate, formed from either mitochondrial metabolism, or provided in the culture medium, could serve as a precursor in lipid synthesis, required for membrane generation associated with proliferation. As the embryo develops, it exhibits a growing number of receptors for specific growth factors (106). Given that growth factor signaling can reorganize metabolic fluxes independently from traditional allosteric means, it will be important to determine how exogenous factors affect key metabolic processes within the embryo. Analysis of blastocyst metabolism is a measurement of the relative activity of two cell types, the trophectoderm and inner cell mass (ICM). In the mouse blastocyst it has been determined that whereas the trophectoderm converts about one-half of the glucose consumed to lactate, the ICM is almost exclusively glycolytic (107). It is not known what the relative activities of the two cell types are in the human blastocyst, but given that human embryonic stem cells are dependent on high glycolytic activity (108), it would be prudent to assume that the Gardner. Embryo selection by metabolic analysis. Fertil Steril 2013.

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FIGURE 2 Continued human ICM also exhibits low levels of glucose oxidation. Although the number of trophectoderm cells far outnumbers those of the ICM, it remains important to consider these cell-specific differences when analyzing metabolic data. The thickness of the lines in the figure represents the relative flux of metabolites through that pathway. ACL ¼ acetyl-citrate lyase; GLUTs ¼ glucose transporters; LDH ¼ lactate dehydrogenase; OAA ¼ oxaloacetate; PDC ¼ pyruvate dehydrogenase complex; pHi ¼ intracellular pH. Gardner. Embryo selection by metabolic analysis. Fertil Steril 2013.

or ‘‘nonviable’’ before transfer (31). As well as quantifying glucose consumption by individual mouse embryos, lactate was measured to obtain an indirect measure of glycolytic activity. An initial distribution of glycolytic activity was obtained from 79 blastocysts with the range of glycolytic activities varying widely from 41% to 280%. It was postulated that those embryos with a glycolytic activity close to that of blastocysts developed in vivo (i.e., 50%) would be more viable than those embryos that had clearly lost the ability to regulate their metabolism. It was also proposed that those blastocysts whose glycolytic rate was >100% were presumably utilizing endogenous glycogen stores to generate glucose to metabolize, because there were no other energy sources available to the embryo in the culture medium, which in turn could adversely affect the ability of the blastocyst to implant. When mouse blastocysts were selected prospectively on such criteria, it was found that the viability of blastocysts with a metabolism similar to that of in vivo–developed embryos was 80%, and only 6% of those embryos that were classified as ‘‘metabolically stressed’’ implanted. When embryos were selected solely for transfer based on the their morphology alone, 20% implanted. Measuring metabolic activity proved to be a valuable and quantitative means of selecting embryos for transfer (31). A question remaining was whether viable blastocysts were simply more quiescent than those embryos that failed to develop after transfer? In other words, was a high metabolic rate in some way detrimental to the embryo? Subsequent analysis of the glucose consumed by those mouse blastocysts predicted to be viable revealed that they actually had the highest glucose consumption (31), consistent with the previous data (35), and that female embryos appeared to have a higher rate of glucose utilization than male embryos (subsequently confirmed by Gardner et al. [14]). Furthermore, because the conversion of glucose to lactate was lower in viable blastocysts, this indicated that viable embryos had a much higher oxidative metabolism and more efficient energy production. Together, these observations led the development of the ‘‘Rate and Fate’’ hypothesis, which predicts that embryo viability is determined not only by the amount (or rate) of nutrient(s) consumed, but also by which metabolic pathway the embryo uses to metabolize it (i.e., fate). The Rate and Fate hypothesis also indicates that the measurement of more than one parameter gives greater power to the metrics on which the decision to select an embryo for transfer is based. In the late 1980s and into the 1990s there were few reports on nutrient utilization by human embryos conceived by IVF (36–38). In 2001, Gardner et al. demonstrated that the utilization of pyruvate and glucose on day 4 was predictive of blastocyst development in human embryos (39). 1066

Importantly, two other significant findings were reported in the same paper. First was the observation that the distribution of glucose consumption by human blastocysts of the same morphologic score and from the same patient was vast, signifying that morphology and metabolic activity were not closely related. Such observations are consistent with work on the mouse blastocyst (31) and led to the proposition that the uptake and metabolism of glucose, but not pyruvate, may serve as a suitable marker of viability for the human embryo after compaction. Second, the human embryo produced significant amounts of ammonium at the blastocyst stage, >25 pmol/embryo/h. This production of ammonium by the embryo, plausibly reflecting amino acid turnover, indicated the possibility that either ammonium production and/or amino acid turnover could be used to quantify embryonic health. To date only a handful of studies have been performed on carbohydrate utilization and subsequent viability of the human embryo. In a retrospective analysis, Conaghan et al. (40) observed an inverse relationship between pyruvate uptake by 2–8-cell embryos cultured in the presence of serum and 20% oxygen and subsequent pregnancy. In a study on human morulas and blastocysts of different degrees of expansion, no conclusive data were generated on the ability of nutrient consumption or utilization to predict pregnancy outcome (41). However, in both of these studies the medium used to assess embryo metabolism was a simple one, lacking lactate, amino acids, and vitamins. Consequently, embryos analyzed under such conditions could be expected to be experiencing considerable metabolic stress. In the following decade, little work was reported on carbohydrate utilization by human embryos, with attention being transferred to amino acids. However, in 2011 an analysis of glucose uptake by individual human embryos before single-embryo transfer was reported (42). It was observed that when embryos were cultured in a media system containing amino acids and vitamins (i.e., conditions that had previously been shown to reduce metabolic stress in the embryo [32]), and in the presence of 5% oxygen, a clear relationship between glucose consumption and resultant pregnancy was established for embryos analyzed on both day 4 and day 5 and subsequently transferred individually on day 5 (Fig. 3). As with earlier reports, human embryo morphology was not closely related to metabolic activity, where embryos that gave rise to pregnancies having the highest glucose consumption were not necessarily those with the highest morphologic grade. Furthermore, and similarly to data on the mouse, human female embryos exhibited a significantly higher glucose uptake than male embryos (Fig. 3). Detection of differences in the physiologies of male and female embryos is consistent VOL. 99 NO. 4 / MARCH 15, 2013

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FIGURE 3

(A) Glucose uptake on day 4 of human embryonic development and pregnancy outcome (positive fetal heart beat). Boxes represent the interquartile range (middle 50% of the data), whiskers represent the 5th and 95th percentiles. The line across each box is the median glucose consumption. **Significantly different from pregnant; P<.01. (B) Glucose uptake by male and female embryos on day 4 of development. Boxes represent the interquartile range (middle 50% of the data), whiskers represent the 5th and 95th percentiles. The line across each box is the median glucose consumption. *Significantly different from male embryos; P<.05. From Gardner et al. (42) with permission. Gardner. Embryo selection by metabolic analysis. Fertil Steril 2013.

with documented differences in gene expression (43, 44) and proteome (45) between the sexes before implantation. Further work is required to characterize and confirm the extent of the sex differences between human preimplantation embryos.

AMINO ACID TURNOVER RELATES TO EMBRYO VIABILITY Amino acids have several key functions during the preimplantation period of embryo development. As well as their documented role as biosynthetic precursors (46), buffers of intracellular pH in the embryo (47), as antioxidants (48) and in signaling and differentiation (49–51), amino acids serve as energy sources (52) and regulators of metabolic function (23, 32, 53). Consequently, amino acid utilization is a logical parameter for quantifying embryo viability. Houghton et al. (54) used high-performance liquid chromatography to quantify amino acid turnover by individual donated human embryos. Different patterns of amino acid utilization between embryos that went on to form a blastocyst and those that failed to develop were observed. It was determined that embryos that went on to develop consumed more leucine from the culture medium. The profiles of the amino acids alanine, arginine, glutamine, methionine, and asparagine flux were related to blastocyst formation (although blastocyst quality was not disclosed). Subsequently, Brison et al. (55) reported changes in concentration of amino acids in the spent medium of human zygotes cultured for 24 hours to the 2-cell stage. It was found that asparagine, glycine, and leucine were all significantly associated with cliniVOL. 99 NO. 4 / MARCH 15, 2013

cal pregnancy and live birth. More recent reports have revealed that there appears to be sex differences relating to amino acid utilization (56, 57), which is consistent with data on glucose consumption and with differences in the proteomes of male and female embryos (14, 45).

CARBOHYDRATE UTILIZATION TOGETHER WITH AMINO ACID TURNOVER RELATE TO EMBRYO VIABILITY The potential advantage of measuring a greater number of parameters is the possibility to create weighted algorithms associated with pregnancy outcome. We recently analyzed the amino acid turnover of individual human blastocysts together with their glucose consumption to increase the number of parameters assessed. In that analysis it was evident that distinct patterns of amino acid utilization were determined between viable and nonviable blastocysts, and that glucose utilization by viable blastocysts was significantly greater than by those embryos that did not result in pregnancy. Furthermore, glucose uptake and the utilization of specific amino acids were correlated (Sheedy et al., unpublished data).

METABOLOMICS: DRIFT NET FISHING IN A SEA OF METABOLITES With the relative abundance of data showing the relationship between embryo development and viability with carbohydrate and amino acid metabolism, one may stop to consider why there has not been more clinical application of such 1067

THE EMBRYO work? One of the drawbacks to this type of analysis has been the relative technical complexities involved and the requirement for highly specialized equipment. Furthermore, the work reported to date has for the most part been essential basic research on human embryo physiology. With the advent of emerging technologies, it is feasible that there will be a greater adoption of these kinds of analyses especially those assays that can be based on microfluidic platforms (58). An emerging technology in this area is that of metabolomics, in which an overall metabolic footprint of the surrounding medium is determined, rather than measuring known nutrients and metabolites (59, 60). Using platforms such as Raman and near-infrared (NIR) spectrometry, it is possible to obtain a spectral profile of the culture medium in which an embryo has developed (61, 62). Although it is not possible to identify specific components that an embryo is using through such spectroscopies, it is possible to attribute specific changes to the spectrum as due to the presence of a viable embryo. The potential advantage of such an approach is that one is making an overall analysis of the culture environment. Both Raman and NIR spectroscopic analyses of spent culture media of human embryos with proven reproductive potential demonstrated significantly higher viability indices than those that failed to implant (63). Interestingly, when human embryos of similar morphology were examined with the use of the same NIR spectral profile, their viability scores varied remarkably, indicating that the metabolome of embryos that look similar differ significantly. This observation is in agreement with a study on glucose consumption by individual human blastocysts (39) and one by Katz-Jaffe et al. (64) who revealed that the proteome of individual human blastocysts of the same grade differed between embryos. Such data reinforce that embryo morphology is not necessarily indicative of viability or linked to its physiology, and that there is a precedent for determining which biomarkers best reflect pregnancy potential. However, to date, NIR-based platforms have not been successfully validated in prospective trials (65, 66), which may reflect the limits of sensitivity associated with this particular technology.

IMPACT OF THE CULTURE ENVIRONMENT ON EMBRYONIC METABOLISM The relative concentrations of nutrients present in media directly affects embryo metabolism (23). Furthermore, the embryo itself, through its metabolism, is constantly changing the composition of the culture medium, which therefore can not be considered as a static system (67). Significantly, other factors of the culture system, such as pH (68) and the concentration of oxygen used in the incubator, can regulate metabolic function. Historically, atmospheric oxygen (20%) has been used in human IVF laboratories for embryo culture. However, the physiologic concentration of oxygen within the female reproductive tract is well below atmospheric levels, being reported to be <10% (69–71). In all mammalian species studied to date, embryo development is significantly improved by culture in an oxygen concentration of 5%–7% compared with 20% (72–75). Furthermore, atmospheric 1068

oxygen (like all stresses) has its most detrimental effects at the cleavage stages (15). Similar data sets are now being documented for the human embryo (76–79). A significant body of work has accumulated on the effects of oxygen on mammalian embryo development. It emerges that atmospheric oxygen has a significant negative effect on blastocyst gene expression (80, 81), the embryonic proteome (82), and metabolism, affecting the utilization of both amino acids and carbohydrates (83) (Fig. 4). Of interest, the effects of oxygen on metabolism are stage specific. During the cleavage stages, 20% oxygen was associated with an overall increase in amino acid turnover and pyruvate uptake by mouse embryos. In contrast, after compaction 20% oxygen was related to a decrease in amino acid turnover and glucose uptake (83). Given that it has been established that glucose uptake by the mouse and human embryo after compaction is positively correlated with viability, such findings are consistent with reports of lower pregnancy rates following culture in 20% oxygen, i.e., the decrease in glucose utilization induced by 20% oxygen is related to the loss in viability. Given the documented harm atmospheric oxygen imparts on the developing embryo and its physiology, its continued use in human IVF can no longer be condoned.

WHAT ARE THE ACCEPTED PATTERNS OF METABOLIC ACTIVITY? QUIETNESS REVISITED An interesting observation from the analysis of amino acid utilization by human embryos was that overall those embryos that developed exhibited a lower turnover of amino acids than ‘‘nonviable’’ embryos, leading to the hypothesis that a ‘‘quiet metabolism’’ is optimal (84). In essence, it was proposed that embryos with a low metabolic activity reflected a less stressed physiology, and consequently those embryos classified as viable would be those that had low nutrient uptake and turnover (85). However, a growing number of recent studies have generated data that do not provide support for this hypothesis (31, 42, 86), rather indicating that viability is associated with increased metabolic activity. So how can this apparent paradox be resolved? An examination of the studies upon which the quietness hypothesis was built reveals that a common factor among them is the use of 20% oxygen, either for embryo culture and analysis or during the actual analysis of metabolism. Given the documented negative impact of 20% oxygen on gene expression, embryonic proteome, and metabolism, as described above, the significance of the quietness hypothesis for embryos cultured under physiologic oxygen conditions must be carefully reviewed and studies performed to determine what is the optimal nutrient utilization profile under physiological conditions. We know that when mammalian embryos are stressed, especially by 20% oxygen, their metabolic profile changes significantly (83). Data to date indicate that those embryos cultured in 20% oxygen that are able to develop do have a lower turnover of nutrients (85). However, does this support a quietness hypothesis or reflect the fact that those embryos that are capable of developing in 20% oxygen are simply VOL. 99 NO. 4 / MARCH 15, 2013

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FIGURE 4 3

pmol/embryo/hour

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Effect of oxygen concentration on amino acid utilization by mouse postcompaction embryos from day 4–5. Significant differences in the utilization of asparagine, glutamate, tryptophan, and lysine (P<.05), with greater significance for utilization of threonine, tyrosine, methionine, valine, isoleucine, leucine, and phenylalanine (P<.01) between the two oxygen concentrations. Consumption for each of these amino acids was greater in 5% oxygen (white boxes) than in atmospheric (20%, shaded boxes) oxygen, and overall amino acid utilization was higher at 5% compared with 20% (P<.05; 25 replicates per treatment, groups of 3; n ¼ 75 embryos). The line across each box is the median uptake or release, the notch represents the confidence interval of the median, the box represents the interquartile range (middle 50% of the data), and whiskers represent the 5th and 95th percentiles. Significantly different from embryos cultured in 5% oxygen: *P<.05; **P<.01. From Wale and Gardner (83) with permission. Gardner. Embryo selection by metabolic analysis. Fertil Steril 2013.

behaving like those that develop in 5% oxygen? Perhaps another explanation for this finding may be related to the inability of unhealthy or developmentally compromised embryos to tolerate oxidative stress. Suboptimal embryos may have lower endowments of antioxidants or less robust systems for responding to oxidizing agents and consequently may have to resort to other energetically consuming mechanisms to preserve their redox status. In this context, the quietness hypothesis would pertain more to the embryo's ability to respond to oxidative stress than to its innate metabolic characteristics under physiological conditions. Recent data on oxygen consumption by cleavage-stage human embryos indicate that viability is associated with an increased oxygen consumption rate, reflecting an increase in respiration rate (87), which is difficult to interpret in terms of a ‘‘quiet’’ physiology. Furthermore, it is evident that when a 5% oxygen concentration is used during culture, glucose uptake of human embryos on days 4 and 5 at the morula and blastocyst stages is significantly higher in those embryos that give rise to pregnancies compared with those that fail to develop after transfer. Such data, therefore, do not support the quietness hypothesis for embryos beyond the 8-cell stage. At present, we do not know if there is a value for nutrient uptake that is too high to be consistent with viability, although it is plausible. There will be upper and lower values of metabolic normality, outside which embryos will show a decline in viaVOL. 99 NO. 4 / MARCH 15, 2013

bility. Animal data to date indicate that a metabolism similar to that of the embryo developed in vivo would reflect the highest viability, and that metabolic adaptations to inappropriate conditions in vitro carry a cost culminating in compromised development after transfer (32). Furthermore, because glucose uptake (14, 42) and amino acid utilization (56) are related to the sex of embryo, such data need to be factored into further hypotheses on embryo metabolism and in identifying the optimal rates of metabolic activity for embryo selection.

CONCLUSION This review has considered the dynamics of embryo metabolism during the preimplantation period and how culture conditions can affect metabolic function leading to compromised development and loss of viability. It is evident that atmospheric oxygen has a significant detrimental affect on the metabolic capacity of the embryo, and that broad hypotheses cannot be made for embryos maintained under non-physiological conditions. The next phase of research is to understand the origins of these differences and the impact of culture conditions (such as medium composition and concentration of oxygen), in inducing metabolic stress. Also, sex-specific differences in embryo metabolism need to be further explored. An increased understanding of human embryo physiology 1069

THE EMBRYO will facilitate further improvements in in vitro culture environments and the development of metabolic algorithms for embryo selection. In closing, through novel technologies (whose capabilities and costs are moving in opposing directions), fast and highly accurate quantification of embryonic metabolism is an ever-approaching reality.

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Acknowledgments: The authors thank Dr. Alexandra Harvey for valuable comments on this paper.

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