Hum. Reprod. Advance Access originally published online on October 3, 2008
Human Reproduction 2009 24(1):81-91; doi:10.1093/humrep/den346
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DNA damage and metabolic activity in the preimplantation embryo
Biology Department (Area 3), University of York, York YO10 5YW, UK
1 Correspondence address. E-mail: r.g.sturmey{at}gmail.com
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Abstract |
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BACKGROUND: Embryos with greater viability have a lower or ‘quieter’ amino acid metabolism than those which arrest. We have hypothesized this is due to non-viable embryos possessing greater cellular/molecular damage and consuming more nutrients, such as amino acids for repair processes. We have tested this proposition by measuring physical damage to DNA in bovine, porcine and human embryos at the blastocyst stage and relating the data to amino acid profiles during embryo development.
METHODS: Amino acid profiles of in vitro-derived porcine and bovine blastocysts were measured by high-performance liquid chromatography and the data related retrospectively to DNA damage in each individual blastomere using a modified alkaline comet assay. Amino acid profiles of spare human embryos on Day 2–3 were related to DNA damage at the blastocyst stage.
RESULTS: A positive correlation between amino acid turnover and DNA damage was apparent when each embryo was examined individually; a relationship exhibited by all three species. There was no relationship between DNA damage and embryo grade.
CONCLUSIONS: Amino acid profiling of single embryos can provide a non-invasive marker of DNA damage at the blastocyst stage. The data are consistent with the quiet embryo hypothesis with viable embryos (lowest DNA damage) having the lowest amino acid turnover. Moreover, these data support the notion that metabolic profiling, in terms of amino acids, might be used to select single embryos for transfer in clinical IVF.
Key words: DNA damage/IVF/comet assay/amino acid profile/blastocyst
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Introduction |
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The production of embryos in vitro is widely used in the treatment of subfertility. However, animal studies have shown that in vitro-produced embryos differ in numerous ways from those conceived in vivo, notably, in gene expression profile (Ennight et al., 2000
; Knijn et al., 2005; Corcoran et al., 2006; Lonergan and Fair, 2008; McHughes et al., 2008), morphology and ultrastructure (Holm et al., 2002; Bhojwani et al., 2007), metabolic activity (Lopes et al., 2007) and kinetics of development (Van Soom et al., 1992; Holm et al., 2002). These differences combine in a manner which is little understood to reduce the viability of in vitro-produced embryos in terms of their ability to give rise to live offspring following transfer.
Whether derived in vivo or in vitro, early mammalian embryos exhibit a broadly similar pattern of metabolic activity during preimplantation development. Overall metabolism, as assessed by oxygen consumption (Houghton et al., 1996; Thompson et al., 1996; Sturmey and Leese, 2003), is low during the cleavage stages of development, before rising sharply at the blastocyst stage. Moreover, metabolic activity has been shown to relate to developmental potential. Thus, Houghton et al. (2002) reported that the pattern of amino acid appearance and depletion by surplus human embryos on Day 2–3 of development predicted their capacity to develop to the blastocyst stage. Stokes et al. (2007) confirmed these findings using a cohort of frozen–thawed human embryos and in a small clinical trial, Brison et al. (2004) found that such ‘amino acid profiling’ predicted the capacity of early embryos conceived in clinical IVF to give rise to an ongoing pregnancy. In each of these studies, the embryos with the highest developmental capacity were characterized by a low amino acid turnover. On the basis of these and other data, Leese (2002) put forward the ‘Quiet Embryo Hypothesis’, which proposed that embryo viability was best served by a metabolism that was ‘quiet’ rather than ‘active’. This hypothesis was developed further by Baumann et al. (2007), who speculated that the origin of this relationship was linked to molecular and cellular ‘damage and challenges’; that those embryos with a lower metabolism were either exposed to fewer insults, or better equipped to deal with such insults when they occurred, and thus had to devote fewer resources to repair and maintenance processes. In contrast, embryos with higher levels of damage to the genome, proteome and transcriptome had a larger demand for nutrients and were therefore metabolically more active.
Other data suggest that metabolic activity might act as an appropriate marker of embryo viability. For example, Vergouw et al. (2008) have recently shown an association between the metabolomic profile of spent culture medium and embryo viability and Scott et al. (2008) and Seli et al. (2007) has considered extensively the promise of a metabolic or metabolomic test of embryo viability. This attractive idea is not new (Donnay et al., 1999); there have been numerous attempts to discover biomarkers of viability in spent culture media (Gardner and Leese, 1987; Conaghan et al., 1993). While early attempts were unsuccessful, progress in technology and understanding of early embryo biochemistry mean that these ideas are closer to being realized.
While oxidative metabolism represents the major pathway for the production of ATP, one undesired by-product is the generation of reactive oxygen species (ROS), which are characterized by the presence of an unpaired electron (Noda et al., 1991) and free-radical intermediaries (Burton et al., 2003). ROS can be generated in situ by electron ‘leakage’ from the electron transport chain (Burton et al., 2003), the rate of which is increased by elevated oxygen concentration (Halliwell and Gutteridge, 2007). It is therefore notable that the oxygen concentration in the reproductive tract is between 2 and 9% (data from non-human primates; Fischer and Bavister, 1993) significantly lower than that in ambient air (20%) and more conducive to embryogenesis, as confirmed by numerous in vitro studies (Thompson et al., 1990; Gardner and Lane, 1996; Dumoulin et al., 1999; Balasubramanian et al., 2007; Rho et al., 2007). Superoxide and other ROS induce damage in various molecules and cell components including nucleic acids, lipids and proteins within the cytoplasm and in membranes (Halliwell and Gutteridge, 2007).
Damage to DNA can have severe cellular consequences. It can broadly be categorized into two types; lesions and strand breaks. Lesions; alterations to base structures, result in a change in the chemical and/or physical structure of the DNA, which can produce point mutations or physical distortions, preventing transcription and replication. These can be formed by endogenous agents, ROS, or occur spontaneously. Strand breaks are physical breaks in the DNA strand. They can be single strand breaks (SSB), or the more severe, double strand breaks (DSB), the presence of a single one of which is sufficient to trigger cell death (Doherty and Jackson, 2001). Generally, this type of damage results from ionizing radiation, environmental stresses, stalling of the DNA replication fork (van Attikum and Gasser, 2005) and, in certain circumstances, ROS (Bilsland and Downs, 2005). This may be particularly relevant for the oocyte and early embryo in vitro, which will be exposed to a range of potential DNA damaging agents, such as ambient light (Hirao and Yanagimachi, 1978), airborne toxins and mutagens (Claxton et al., 2004), from which they would normally be shielded by the reproductive tract.
A variety of strategies exist to protect against, or repair such damage when it occurs. For example, superoxide dismutase, catalase and glutathione reductase are examples of enzymes which remove radical species before they can cause significant damage. The presence of scavengers such as ascorbic acid, 
-tocopherol and albumin also minimize ROS damage to nucleic acids and other cellular components. Moreover, cells have evolved extensive pathways to repair damaged DNA. For example, in a recent transcriptomic study in rhesus monkey oocytes and embryos, Zheng et al. (2005) reported the expression of genes involved in the repair of DSBs and SSBs as well as genes involved in the detection and repair of DNA lesions and adducts and those required to arrest the cell cycle to enable DNA repair to occur. The pathways and strategies for DNA repair in early embryos have been reviewed by Jaroudi and SenGupta (2007).
There are a variety of ways to measure DNA damage in cells, including high-performance liquid chromatography (HPLC) combined with electrochemical detection, single or tandem mass spectrometry, gas chromatography linked with mass spectrometry, 32P post-labelling, immunoassay and single-cell gel electrophoresis; the so-called comet assay (Cooke et al., 2008). The alkaline comet assay can be applied to measure DNA damage at the single cell level since it requires only a relatively small cell population (Tice et al., 2000). In addition, it is simple, robust, capable of detecting low levels of DNA damage (Tice et al., 2000) and can semi-quantify the amount of DNA damage, in terms of strand breaks and alkali labile sites. This comet assay has been used extensively to assess DNA damage in sperm (Ahmad et al., 2007; Lewis and Agbaje, 2008; Shamsi et al., 2008) but, its application to oocytes (Jebelli et al., 2001; Chung et al., 2007) and early embryos (Muller et al., 1996; Takahashi et al., 1999b; Harrouk et al., 2000; Kitagawa et al., 2004) has been limited to the zygote, or early stages of cleavage; presumably due to technical difficulties associated with the tight association of the blastomeres post-compaction. In the present work, we have modified the comet assay to measure the blastomeres of individual embryos, thus allowing the study of DNA damage in each cell of a given blastocyst.
In this paper we have asked whether non-invasive measurement of metabolism—amino acid depletion/appearance; a marker of developmental competence—is related to DNA damage in preimplantation embryos produced in vitro. Specifically, we have measured the amino acid metabolism of individual bovine, porcine and human blastocysts and determined DNA damage in the individual blastomeres of these embryos. We report a significant positive correlation between overall amino acid turnover and levels of DNA damage, independent of embryo morphology. A preliminary account of part of this work was presented at the Fertility 2007, The Biennial Meeting of the UK Fertility Societies (Sturmey et al., 2007).
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Unless otherwise stated, all chemicals were supplied by Sigma-Aldrich (Poole, UK). Solvents used for chromatography were purchased from Fisher Scientific (Loughborough, UK).
Production of animal embryos
Porcine embryos were produced as described previously (Sturmey and Leese, 2003). Briefly, oocyte–cumulus complexes (OCCs) were aspirated from 3–6 mm follicles of abattoir-derived ovaries and matured in a chemically defined maturation medium for 40–44 h. They were fertilized in modified Tris-buffered medium with frozen–thawed semen from a boar of proven fertility and the resulting putative zygotes cultured to the blastocyst stage in medium NCSU23aa (Petters et al., 1990; Petters and Reed, 1991; Humpherson et al., 2005) under 5% CO2 in air. Bovine embryos were generated as described (Orsi and Leese, 2004a). Briefly, OCCs were aspirated from 3–8 mm follicles of abattoir-derived ovaries and matured in M199 (Invitrogen, Paisley, UK), supplemented with 10% fetal calf serum, 0.025 IU FSH/LH (Ferring Pharmaceutical, Langley, UK) and 0.47 µg ml–1 epidermal growth factor for 24 h. Matured OCCs and sperm from a bull of proven fertility were co-incubated in ‘Fert Tyrodes-Albumin-Lactate-Pyruvate’ for 18 h and the putative zygotes cultured in Synthetic Oviduct Fluid + 0.4% bovine serum albumin (BSA), modified by inclusion of amino acids at close-to-physiological concentrations (Tay et al., 1997) designated SOFmaaBSA. Bovine embryos were cultured from zygote to blastocyst in 5% O2/5% CO2/90% N2. All animal blastocysts used in this study were Day 6 of development at equivalent stages of development.
Human embryos
Surplus human embryos, donated with patient informed-consent, were obtained from the Assisted Conception Unit, Leeds General Infirmary. The work was done under Research Licence (No. R0067) from the Human Fertilisation and Embryology Authority and with Local Ethics Committee approval. Ovarian stimulation, oocyte collection, IVF, transfer to the York laboratory, culture and grading were as described elsewhere (Houghton et al., 2002). In brief, human embryos were transported to the York laboratory and cultured in Earle’s Balanced Salt Solution supplemented with 1 mM glucose, 5 mM lactate, 0.47 mM pyruvate, 0.5% human serum albumin (Baxter Healthcare, Norfolk, UK; Bioproducts Lab, UK) and amino acids at close-to-physiological concentrations; designated Human Embryo Culture Medium (HCM). Embryo grade was recorded every 24 h according to Houghton et al. (2002).
Amino acid profiling
Porcine and bovine blastocysts at Day 6 of development were placed in 4 µl fresh, pre-equilibrated medium NSCU23aa or SOFmaaBSA, respectively, overlaid with mineral oil and cultured for 
24 h alongside blank medium control droplets; the precise time of incubation was recorded. The embryos were then removed for DNA damage assay (see later) while the spent culture drops were frozen at –80°C until analysis for amino acid content, which was carried out within 21 days. Human embryos were similarly profiled; however, in this case, embryos at Day 2 of development were cultured in 4 µl of fresh HCM. Following incubation, the embryos were placed into individual 10 µl culture drops and their development to blastocyst recorded. Those embryos which successfully developed to blastocysts were assayed for DNA damage when expanded and the culture medium analysed for amino acid content.
Amino acid analysis
The amino acid content of spent embryo culture media was determined by reverse-phase HPLC. This method relies on the derivitization of amino acids with O-phthaldialdehye. SOFmaaBSA and HCM medium were analysed on a Kontron 500 series HPLC, fitted with a Jasco F920 fluorescence detector. Chromatography was performed using a Phenomenox HyperClone 5 µm C18 ODS column, 250 x 4.6 mm column. Two eluants were used; Eluant A (80% 83 mM sodium acetate/19.5% methanol/0.5% tetrahydrofuran) and Eluent B (80% methanol/20% sodium acetate). Chromatography was performed for 60 min at 30°C at a flow rate of 1.3 ml/min. Chromatography for the NCSUaa medium was modified due to the presence of taurine and hypotaurine in the embryo culture medium; tetrahydrofuran was omitted from Eluant A, total chromatography time was 70 min, the column was an Agilent Zorbax AAA 3.5 µm 4.6 x 75 mm and samples were analysed on a Waters Alliance 2695 system, fitted with a Waters 2475 multi 
fluorescence detector. Full details of this method can be found in Humpherson et al. (2005).
DNA damage assay
DNA damage was determined in terms of SSBs and DSBs and alkali labile sites by the alkaline comet assay (McKelvey-Martin et al., 1997; Gedik and Collins, 2005; Moller, 2005), modified for use on single embryos (Sturmey et al., 2007). Embryos selected for comet assay were incubated in 0.5% (w/v) Pronase in 0.9% NaCl for 30 s to remove the zona pellucida. They were then moved to a droplet of Enzyme-Free Cell Dissociation Buffer (Sigma), supplemented with 10% v/v trypsin and incubated for 1–3 min at 39°C in reduced lighting to isolate the individual blastomeres. A 4 µl droplet of 0.8% [w/v in phosphate-buffered saline (PBS)] low melting point (LMP) agarose was placed onto a slide, which had been pre-coated with 1% (w/v in PBS) normal agarose. The blastomeres were collected, added to the droplet of LMP, and a coverslip placed onto the droplet, spreading the LMP, which contained the disrupted embryo, into a thin layer. The slide was incubated on ice for 30 s to allow the LMP agarose to set and then immediately placed into ice-cold lysis buffer (2.5 M NaCl; 1 mM EDTA; 10 mM Tris; 10% dimethylsulphoxide; 1% Triton X-100; pH 10), protected from light and incubated at 4°C overnight. All subsequent stages were performed in the dark. The lysis buffer was decanted and the slides were incubated with Unwinding/Electrophoresis Buffer (0.3 M NaOH; 1 mM EDTA; pH 13) at 4°C for 40 min, before electrophoresis was performed at 23 V, 300 mA for 20 min. Slides were neutralized by rinsing in ice-cold neutralization buffer (0.4 M Tris; pH 7.5) for 10 min, and stained with SYBR-Gold (1x staining solution as per manufacturer’s instructions; Invitrogen). Comets were scored from images taken on an Olympus BX-60 inverted microscope with a 20 x 0.5 NA lens. Acquired images were analysed using Scion Image with a macro for comet assay analysis. The percentage DNA damage in each individual blastomere identified was recorded. One embryo per slide was analysed and the DNA damage for each embryo is presented as the mean amount of damage per embryo. This method cannot distinguish between inner cell mass and trophectoderm cells; the damage presented is therefore of all blastomeres. DNA damage was measured in expanded blastocysts for all species.
Data presentation and statistical analysis
Amino acid profiles are presented as mean ± SEM. The data were confirmed as non-parametric by the Anderson–Darling test for normality. Significant uptake and depletion (i.e. difference from 0) was tested by 1-sample Wilcoxon, and differences between groups were tested by the Mann–Whitney U-test. ‘Amino acid turnover’ represents the sum of depletion and appearance of every amino acid. DNA damage is expressed as the median percentage value for all comets scored per individual embryo. Percentage data were subjected to arcsin transformation and compared by Mann–Whitney U-test. A Pearson’s correlation test was used to identify relationships between amino acid turnover and DNA damage. A P-value <0.05 was considered significant.
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Amino acid metabolism of bovine and porcine blastocysts
Fig. 1 shows the mean amino acid profiles of the porcine (n = 34) and bovine (n = 31) embryos assayed in this study. Fig. 1a shows the overall mean amino acid production (above the x-axis) and consumption (below the x-axis) of each of the 18 amino acids measured. Amino acids that were produced or consumed in amounts that differed significantly to 0 are indicated by the presence of stars. Comparisons between the species of the depletion/appearance of amino acids are shown in the inset table of Fig. 1a, along with P-values. The amino acid profiles for the two species show qualitative similarities, however quantitatively, bovine blastocysts tended to be more metabolically active. Fig. 1b shows the amino acid ‘turnover’ as calculated by summing all amino acids produced and consumed on a per embryo basis. Bovine blastocysts show significantly higher overall consumption (P = 0.0014), production (P = 0.0046) and turnover (P = 0.0016) of amino acids than porcine.
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Fig. 2 shows an example ‘comet’ from an individual blastomere from a human blastocyst. DNA migration to the right of the image can clearly be seen. In this particular image, the DNA damage was measured as 18.7%, indicating that 18.7% of the fluorescence had migrated away from the nucleoid ‘head’ of the comet. Only broken DNA can migrate in this assay, and so the data are expressed as percentage DNA damage, based on the amount of DNA migrating away from the nucleoid.
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DNA damage in bovine and porcine blastocysts
Fig. 3a shows the overall mean DNA damage as measured in bovine and porcine blastocysts; bovine blastocysts tended to have higher mean levels of DNA damage than porcine embryos (27.87 versus 19.01%) although this difference was not significant (P = 0.098). Fig. 3b shows the proportion of cells within blastocysts of both species with a given amount of DNA damage; classified as ‘minor’ (i.e. <10% DNA migration), ‘intermediate’ (between 10 and 25% DNA migration), ‘major’ (25–50% of the DNA migrating) and ‘severe’ (>50% DNA migration). In general, porcine blastocysts tended to have less cells with ‘minor’ DNA damage, and a significantly lower proportion of blastomeres with ‘severe’ DNA damage when compared with bovine blastocysts; on average, 16.3% of bovine blastomeres had severe DNA damage, whereas the corresponding figure for porcine blastomeres was only 5.8% (P < 0.001).
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In Fig. 4, the mean DNA damage in a given embryo has been plotted against total amino acid turnover (i.e., the sum of total amino acid consumption and production). Fig. 4a shows a positive correlation between DNA damage and amino acid turnover for porcine blastocysts (P < 0.001), while Fig. 4b shows a similar relationship for bovine blastocysts (P < 0.001).
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Amino acid metabolism of human embryos on Day 2–3 of development
Fig. 5a shows the mean consumption and production of amino acids of those human embryos during cleavage (Day 2–3) that subsequently developed to blastocysts (n = 21). Arginine (P < 0.001), valine (P < 0.05), isoleucine (P < 0.01) and leucine (P < 0.01) were significantly depleted from the medium, while only alanine showed significant accumulation in the medium (P < 0.01). Fig. 5b shows the mean amino acid production, consumption and turnover by individual human blastocysts that developed to blastocysts.
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DNA damage and amino acid metabolism by human embryos
Fig. 6 shows a positive correlation between the mean DNA damage in each blastocyst and overall amino acid turnover of the same embryo at Day 2 of development (P = 0.028, Pearson value 0.479, n = 21); embryos with a higher mean amount of DNA damage tended to be more active metabolically, in terms of amino acid turnover. In Fig. 7, embryo morphological grade of the blastocyst immediately before DNA damage assay has been plotted against observed DNA damage. There was no significant correlation between morphology and mean DNA damage for a given embryo. Grade 1 embryos (i.e. the highest quality, in terms of morphology) had mean DNA damage levels between 15 and 38%, whereas Grade 2 embryos had DNA damage levels between 9 and 47%.
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In the human embryos used in this study, DNA damage was measured at the blastocyst stage with values which varied between 9 and 47%. The amino acid profile of these human embryos had been determined during early cleavage, from Day 2 to 3. The data were then compared retrospectively to the mean measurement of DNA damage for each embryo. When the data were plotted as the sum of amino acid depletion and appearance (amino acid ‘turnover’) for each individual embryo measured on Day 2–3, there was a strong correlation with the proportion of cells exhibiting DNA damage at the blastocyst stage (Pearson value 0.470, P = 0.028). Similarly, strong correlations were obtained between amino acid turnover (blastocyst stage) and DNA damage (blastocyst stage) for bovine (Pearson value 0.58, P < 0.001) and porcine embryos (Pearson value 0.736, P < 0.001).
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Discussion |
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We report a correlation between the amount of DNA damage and the metabolic activity, measured as ‘amino acid profile’, of mammalian embryos produced in vitro; the greater the DNA damage, the higher the overall amino acid turnover, a finding consistent with the ‘Quiet Embryo Hypothesis’ (Leese, 2002
; Baumann et al., 2007), which proposes that ‘quiet’ embryos are more viable than ‘active’. The correlation was apparent in all three species studied; bovine, porcine and human strongly suggesting it to be a general feature of early mammalian embryos.
The amino acid profiles of embryos of each species were broadly similar to those previously reported (Houghton et al., 2002; Gopichandran and Leese, 2003; Brison et al., 2004; Booth et al., 2005, 2007; Humpherson et al., 2005; Stokes et al., 2007). Although the data varied between the three species, general features of amino acid metabolism were apparent; aspartate asparagine, threonine and arginine were all consumed, whereas alanine was consistently produced. This is striking when one considers that the amino acid profiles of cattle and pig embryos were obtained at the blastocyst stage (and are, therefore, more directly comparable), whereas the profiles of the human embryos represent Days 2–3 of development. These similarities in profiles across species and developmental stage suggest there are common pathways of amino acid metabolism operating during preimplantation embryo development. We have previously postulated that the consistent arginine consumption may be linked to a need for nitric oxide (Manser et al., 2004; Humpherson et al., 2005) and that the alanine production may play a key role in ammonium detoxification (Humpherson et al., 2005), as the preimplantation embryo has no functional urea cycle (Orsi and Leese, 2004b). The potential roles and interactions between amino acids during embryo development are discussed in detail elsewhere (Sturmey et al., 2008).
To the best of our knowledge, this is the first report of DNA damage in in vitro-derived bovine, porcine and human blastocysts. The collection of these data involved disaggregating the blastocysts into composite blastomeres and assaying the DNA damage in each individual blastomere, by the conventional comet assay. The extent of DNA damage was broadly comparable between the bovine and porcine embryos, at around 25% of the total blastomere number. However, while porcine blastocysts had a significantly lower proportion of cells with ‘severe’ DNA damage than the bovine, the latter tended to have more cells with ‘minor’ and ‘intermediate’ amounts of damage (i.e. <10%), although this was not statistically significant. In other words, the majority of blastomeres in the porcine blastocysts had either intermediate or major damage, whereas in the bovine, the majority of cells had ‘minor’ DNA damage. This observation of more cells with greater damage in the porcine embryo may, in part, contribute to the lower success rates seen with porcine embryos compared with bovine systems; typical zygote to blastocyst rates in our laboratory are 40% for the bovine system, but only 20% for the porcine (data not shown); values broadly comparable to those reported elsewhere. In addition, care needs to be taken when comparing these data for porcine and bovine embryos as the production systems are different; culture media, timing of oocyte maturation and fertilization, and gas phase all differ and may contribute to differences in DNA damage load. For example, bovine embryos are typically grown under 5% O2, with porcine embryos under 20% O2. The reduced O2 phase has been shown to alter gene expression (Kind et al., 2005; Harvey et al., 2007a, b; Balasubramanian et al., 2007), heat-shock protein levels (DE Castro E Paula and Hansen, 2007) and embryo viability in terms of generating offspring (Cebral et al., 2007; Rho et al., 2007b), demonstrating that exposure to ROS compromised embryo viability. The reduced proportion of blastomeres with large amounts of DNA damage seen in bovine embryos may be a function of species differences, but more likely is related to their development under reduced O2 tension, thus minimizing ROS formation (Halliwell and Gutteridge, 2007) and damage to DNA, as reported by Kitagawa et al. (2004) working on porcine cleavage stage embryos.
DNA damage may also be as a result of damaged sperm penetrating the egg. It has previously been shown that sperm with damaged DNA are capable of fertilizing the egg (Fatehi et al., 2006) and that there is some capacity to repair this damage (Fatehi et al., 2006), presumably by the translation of transcripts of DNA repair genes known to be expressed in early mammalian embryo development (Zheng et al., 2005; Jaroudi and SenGupta, 2007).
The observed differences in DNA damage between cattle and pig embryos may relate to variation in the timing of embryonic genome activation. Bovine major genome activation occurs at the 8–16-cell stage, whereas in the porcine and human this is at the 4-cell stage (Maddox-Hyttel et al., 2007), i.e. the third cell cycle post-fertilization. Assuming that the genes for repair of damaged DNA (Zheng et al., 2005) are expressed at the protein level after genome activation the difference in timing of genome activation might mean that damaged DNA in the porcine and, by inference, the human embryo has been subjected to an extra round of replication-coupled repair relative to the bovine. In terms of the present work, one could speculate that this contributes to the observation that bovine embryos have more blastomeres with ‘severe’ levels of damage, whereas porcine and human embryos, in which genome activation occurs one cell cycle earlier, may have expressed the necessary DNA repair enzymes to cope with cells with severe levels of DNA damage.
DNA damage has previously been measured in zygotes and early cleavage mammalian embryos. For example, the interaction between radiation and caffeine in the processes of DNA repair was examined by Muller et al. (1996) using 2-cell mouse embryos. It was reported that damage load was low in these embryos (<5%); significantly below the values in the present study. It should be noted that these mouse embryos were in vivo-fertilized and harvested and exposed to in vitro conditions for a relatively short time and may not have accrued the same amount of damage as true in vitro-fertilized embryos. Interestingly, these authors also reported DNA repair activity in early mouse embryos. Similarly, Takahashi et al. (1999a) measured DNA damage in 1- and 2-cell hamster embryos, exposed to light at different wavelengths and for different times. They reported that a short direct exposure to UV or visible light was sufficient to increase the proportion of DNA damage and that DNA damage load was significantly elevated in embryos after a period of exposure to the in vitro environment. Due to the way in which these data were presented, direct comparison with the findings of the present work is not possible; however, it is clear that the early embryos exposed to the in vitro environment have elevated DNA damage. Our work supports and extends these findings by quantifying the DNA damage load in blastocysts; i.e. embryos that have been exposed to the in vitro environment for up to 7 days. DNA damage has also been assessed by means of the terminal transferase dUTP nick end labelling (TUNEL) assay in mouse (Rausell et al., 2007), bovine (Park et al., 2006; Brad et al., 2007) and porcine (Rajaei et al., 2005) embryos. These studies have provided valuable data on DNA fragmentation levels in embryos, however, the comet assay gives more precise information on specific types of DNA damage; strand breaks and alkali labile sites. Due to the nature of apoptosis, fragmentation as measured in terms of nick-end labelling from terminal transferase can indicate controlled fragmentation arising from apoptotic events, whereas the comet assay measures spontaneously occurring DNA damage.
Each human embryo was graded on the basis of its morphology, as carried out in IVF clinics to select embryos for transfer, with Grade 1 representing the highest quality embryos. However, there was no relationship between DNA damage and embryo grade (Fig. 7), in line with our previous data showing that the ability of amino acid profiling to predict embryo viability was independent of embryo grade (Brison et al., 2004) and confirming the unreliability of morphological assessment in determining embryo quality. In agreement with this conclusion, Vergouw et al. (2008) recently showed how metabolomic observations, assessed by near infrared spectroscopy, related to human embryo viability. In particular, they demonstrated that embryo viability based on metabolomic data did not correlate with conventional morphological characteristics, such as blastomere number and fragmentation.
The biological reasons behind the observed links between DNA damage and metabolic activity can at this stage only be speculated upon. The basis of the Quiet Embryo Hypothesis is that a ‘compromised’ embryo has a greater requirement for exogenous nutrients in order to correct errors and damage and maintain development (Leese, 2002; Baumann et al., 2007; Leese et al., 2007). Moreover, a large proportion of damage may result in apoptosis. In either situation, there is likely to be an increased demand for nutrients. For example, human embryos show a direct correlation between the amount of DNA damage and glutamine uptake (data not shown); those embryos with the greatest levels of damage take up the highest amounts of glutamine. Glutamine is a key amino acid in purine and pyrimidine synthesis, where it provides carbon atoms for de novo synthesis (Leese, 1993) and has other roles (Sturmey et al., 2008) such as provision of ATP (Wu et al., 2000), which may point to an increase in overall energy demand. It is well established that amino acids supplied to the early embryo in culture contribute to protein biosynthesis (Epstein and Smith, 1973, reviewed by Sturmey et al. 2008), and may, as we have hypothesized, be as a result of the need to increase protein synthesis, due to premature genome activation in response to DNA damage (Baumann et al., 2007).
Our data provide strong evidence that amino acid profiling of single human embryos can act as a non-invasive marker of DNA damage at the blastocyst stage, with viable embryos (lowest DNA damage) having the lowest amino acid turnover, consistent with the quiet embryo hypothesis (Leese, 2002). Such information is not provided by current methods of embryo selection and we suggest that amino acid profiling, which is highly predictive of DNA damage, could provide an appropriate marker with which to select embryos for replacement in clinical IVF. Moreover, there is good evidence that this assay can be performed, at least in the human, at Day 2–3 of development, in a timely manner to enable selection of embryos for transfer on the basis of metabolic activity (Brison et al., 2004). The possible value of such a test in the production of domestic animal embryos is less clear; however, the method may have value for breeding stock with high genetic merit. The data presented in this paper provide a biological rationale for the observed association between metabolic activity and viability. Further validation is required to enable metabolic activity to be used as a biomarker of embryo viability and selection in clinical IVF.
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R.G.S. and H.J.L. wish to acknowledge the financial support from the Department of Health Sciences University of York, The Medical Research Council, and The Leverhulme Trust.
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Acknowledgements |
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The authors gratefully acknowledge the constructive comments from Dr C.G. Baumann, University of York. In addition, the work would not have been possible without the support of the nurses and embryologists at Leeds General Infirmary Assisted Conception Unit and Mr C. Bingham of the University of York. Ovaries for animal studies were provided by ABP, Murton, UK, (cattle) and Grampian Country Foods, Malton, UK (pig). H.J.L. is a shareholder and scientific advisor to Novocellus, a company working to develop non-invasive assays of embryo viability.
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References |
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Ahmad L, Jalali S, Shami SA, Akram Z. Sperm preparation: DNA damage by comet assay in normo- and teratozoospermics. Arch Androl (2007) 53:325–338.[Web of Science][Medline]
Balasubramanian S, Son WJ, Kumar BM, Ock SA, Yoo JG, Im GS, Choe SY, Rho GJ. Expression pattern of oxygen and stress-responsive gene transcripts at various developmental stages of in vitro and in vivo preimplantation bovine embryos. Theriogenology (2007) 68:265–275.[CrossRef][Web of Science][Medline]
Baumann CG, Morris DG, Sreenan JM, Leese HJ. The quiet embryo hypothesis: molecular characteristics favoring viability. Mol Reprod Dev (2007) 74:1345–1353.[CrossRef][Web of Science][Medline]
Bhojwani S, Alm H, Torner H, Kanitz W, Poehland R. Selection of developmentally competent oocytes through brilliant cresyl blue stain enhances blastocyst development rate after bovine nuclear transfer. Theriogenology (2007) 67:341–345.[CrossRef][Web of Science][Medline]
Bilsland E, Downs JJ. Tails of histones in DNA double-strand break repair. Mutagenesis (2005) 20:153–163.
Booth PJ, Humpherson PG, Watson TJ, Leese HJ. Amino acid depletion and appearance during porcine preimplantation embryo development in vitro. Reproduction (2005) 130:655–668.
Booth PJ, Watson TJ, Leese HJ. Prediction of porcine blastocyst formation using morphological, kinetic, and amino acid depletion and appearance criteria determined during the early cleavage of in vitro-produced embryos. Biol Reprod (2007) 77:765–779.
Brad AM, Hendricks KE, Hansen PJ. The block to apoptosis in bovine two-cell embryos involves inhibition of caspase-9 activation and caspase-mediated DNA damage. Reproduction (2007) 134:789–797.
Brison DR, Houghton FD, Falconer D, Roberts SA, Hawkhead J, Humpherson PG, Lieberman BA, Leese HJ. Identification of viable embryos in IVF by non-invasive measurement of amino acid turnover. Hum Reprod (2004) 19:2319–2324.
Burton GJ, Hempstock J, Jauniaux E. Oxygen, early embryonic metabolism and free radical-mediated embryopathies. Reprod Biomed Online (2003) 6:84–96.[Medline]
Cebral E, Carrasco I, Vantman D, Smith R. Preimplantation embryotoxicity after mouse embryo exposition to reactive oxygen species. Biocell (2007) 31:51–59.[Web of Science][Medline]
Chung JT, Tosca L, Huang TH, Xu L, Niwa K, Chian RC. Effect of polyvinylpyrrolidone on bovine oocyte maturation in vitro and subsequent fertilization and embryonic development. Reprod Biomed Online (2007) 15:198–207.[Web of Science][Medline]
Claxton LD, Matthews PP, Warren SH. The genotoxicity of ambient outdoor air, a review: Salmonella mutagenicity. Mutat Res (2004) 567:347–399.[CrossRef][Web of Science][Medline]
Conaghan J, Hardy K, Handyside AH, Winston RM, Leese HJ. Selection criteria for human embryo transfer: a comparison of pyruvate uptake and morphology. J Assist Reprod Genet (1993) 10:21–30.[CrossRef][Web of Science][Medline]
Cooke MS, Olinski R, Loft S. Measurement and meaning of oxidatively modified DNA lesions in urine. Cancer Epidemiol Biomarkers Prev (2008) 17:3–14.
Corcoran D, Fair T, Park S, Rizos D, et al. Suppressed expression of genes involved in transcrition and translation in in vitro compared with in vivo cultured bovine embryos. Soc Reprod Fertil (2006) 651–660.
de Castro E, Paula LA, Hansen PJ. Interactions between oxygen tension and glucose concentration that modulate actions of heat shock on bovine oocytes during in vitro maturation. Theriogenology (2007) 68:763–770.[CrossRef][Web of Science][Medline]
Doherty AJ, Jackson SP. DNA repair: how Ku makes ends meet. Curr Biol (2001) 11:R920–R924.[CrossRef][Web of Science][Medline]
Donnay I, Partridge RJ, Leese HJ. Can embryo metabolism be used for selecting bovine embryos before transfer? Reprod Nutr Dev (1999) 39:523–533.[CrossRef][Web of Science][Medline]
Dumoulin JCM, Meijers JJ, Bras M, Cooner E, Geraedts JPM, Evers JLH. Effects of oxygen concentration on human in-vitro fertilization and embryo culture. Hum Reprod (1999) 14:465–469.
Ennight BP, Lonergan P, Dinnyes A, Fair T, Ward FA, Yang X, Boland MP. Culture of in vitro produced bovine zygotes in vitro vs in vivo implications for early embryo development and quality. Theriogenology (2000) 54:659–673.[CrossRef][Web of Science][Medline]
Epstein CJ, Smith SA. Amino acid uptake and protein synthesis in preimplantation mouse embryos. Dev Biol (1973) 33:171–184.[CrossRef][Web of Science][Medline]
Fatehi AN, Bevers MM, Schoevers E, Roelen BA, Colenbrander B, Gadella BM. DNA damage in bovine sperm does not block fertilization and early embryonic development but induces apoptosis after the first cleavages. J Androl (2006) 27:176–188.
Fischer B, Bavister BD. Oxygen tension in the oviduct and uterus of rhesus monkeys, hamsters and rabbits. J Reprod Fertil (1993) 99:673–679.
Gardner DK, Lane M. Alleviation of the ‘2-cell block’ and development to the blastocyst of CF1 mouse embryos: role of amino acids, EDTA and physical parameters. Hum Reprod (1996) 11:2703–2712.
Gardner DK, Leese HJ. Assessment of embryo viability prior to transfer by the noninvasive measurement of glucose uptake. J Exp Zool (1987) 242:103–105.[CrossRef][Web of Science][Medline]
Gedik CM, Collins A. Establishing the background level of base oxidation in human lymphocyte DNA: results of an interlaboratory validation study. FASEB J (2005) 19:82–84.
Gopichandran N, Leese HJ. Metabolic characterization of the bovine blastocyst, inner cell mass, trophectoderm and blastocoel fluid. Reproduction (2003) 126:299–308.[Abstract]
Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine (2007) 4th edn. Oxford: Oxford University Press.
Harrouk W, Codrington A, Vinson R, Robaire B, Hales BF. Paternal exposure to cyclophosphamide induces DNA damage and alters the expression of DNA repair genes in the rat preimplantation embryo. Mutat Res (2000) 461:229–241.[Web of Science][Medline]
Harvey AJ, Kind KL, Thompson JG. Regulation of gene expression in bovine blastocysts in response to oxygen and the iron chelator desferrioxamine. Biol Reprod (2007) a 77:93–101.
Harvey AJ, Navarrete SA, Kirstein M, Kind KL, Fischer B, Thompson JG. Differential expression of oxygen-regulated genes in bovine blastocysts. Mol Reprod Dev (2007) b 74:290–299.[CrossRef][Web of Science][Medline]
Hirao Y, Yanagimachi R. Detrimental effect of visible light on meiosis of mammalian eggs in vitro. J Exp Zool (1978) 206:365–369.[CrossRef][Web of Science][Medline]
Holm P, Booth PJ, Callesen H. Kinetics of early in vitro development of bovine in vivo- and in vitro-derived zygotes produced and/or cultured in chemically defined or serum-containing media. Reproduction (2002) 123:553–565.[Abstract]
Houghton F, Thompson J, Kennedy C, Leese HJ. Oxygen consumption and energy metabolism in the early mouse embryo. Mol Reprod Dev (1996) 476–485.
Houghton FD, Hawkhead JA, Humpherson PG, Hogg JE, Balen AH, Rutherford AJ, Leese HJ. Non-invasive amino acid turnover predicts human embryo developmental capacity. Hum Reprod (2002) 17:999–1005.
Humpherson PG, Leese HJ, Sturmey RG. Amino acid metabolism of the porcine blastocyst. Theriogenology (2005) 64:1852–1866.[Web of Science][Medline]
Jaroudi S, SenGupta S. DNA repair in mammalian embryos. Mutat Res (2007) 635:53–77.[CrossRef][Web of Science][Medline]
Jebelli B, Chan PJ, Corselli J, Patton WC, King A. Oocyte comet assay of luteal phase sera from nonpregnant patients after assisted reproductive procedures. J Assist Reprod Genet (2001) 18:421–425.[CrossRef][Web of Science][Medline]
Kind KL, Collett RA, Harvey AJ, Thompson JG. Oxygen-regulated expression of GLUT-1, GLUT-3, and VEGF in the mouse blastocyst. Mol Reprod Dev (2005) 70:37–44.[CrossRef][Web of Science][Medline]
Kitagawa Y, Suzuki K, Yoneda A, Watanabe T. Effects of oxygen concentration and antioxidants on the in vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentation in porcine embryos. Theriogenology (2004) 62:1186–1197.[CrossRef][Web of Science][Medline]
Knijn HM, Wrenzycki C, Hendriksen PJ, Vos PL, Zeinstra EC, van der Weijden GC, Niemann H, Dieleman SJ. In vitro and in vivo culture effects on mRNA expression of genes involved in metabolism and apoptosis in bovine embryos. Reprod Fertil Dev (2005) 17:775–784.[CrossRef][Medline]
Leese HJ. Energy Metabolism in Preimplantation Development Serono Symposia USA (1993) New York: Springer-Verlag.
Leese HJ. Quiet please, do not disturb: a hypothesis of embryo metabolism and viability. Bioessays (2002) 24:845–849.[CrossRef][Web of Science][Medline]
Leese HJ, Sturmey RG, Baumann CG, McEvoy TG. Embryo viability and metabolism: obeying the quiet rules. Hum Reprod (2007) 22:3047–3050.
Lewis SE, Agbaje IM. Using the alkaline comet assay in prognostic tests for male infertility and assisted reproductive technology outcomes. Mutagenesis (2008) 23:163–170.
Lonergan P, Fair T. In vitro-produced bovine embryos: dealing with the warts. Theriogenology (2008) 69:17–22.[CrossRef][Web of Science][Medline]
Lopes AS, Madsen SE, Ramsing NB, Lovendahl P, Greve T, Callesen H. Investigation of respiration of individual bovine embryos produced in vivo and in vitro and correlation with viability following transfer. Hum Reprod (2007) 22:558–566.
Maddox-Hyttel P, Svarcova O, Laurincik J. Ribosomal RNA and nucleolar proteins from the oocyte are to some degree used for embryonic nucleolar formation in cattle and pig. Theriogenology (2007) 68(Suppl 1):S63–S70.[CrossRef][Web of Science][Medline]
Manser RC, Leese HJ, Houghton FD. Effect of inhibiting nitric oxide production on mouse preimplantation embryo development and metabolism. Biol Reprod (2004) 71:528–533.
McHughes CE, Springer GK, Spate LD, Li R, Woods R, Green MP, Korte SW, Murphy CN, Green JA, Prather RS. Identification and quantification of differentially represented transcripts in in vitro and in vivo derived preimplantation bovine embryos. Mol Reprod Dev (2008).
McKelvey-Martin VJ, Melia N, Walsh IK, Johnston SR, Hughes CM, Lewis SE, Thompson W. Two potential clinical applications of the alkaline single-cell gel electrophoresis assay: (1). Human bladder washings and transitional cell carcinoma of the bladder; and (2). Human sperm and male infertility. Mutat Res (1997) 375:93–104.[Web of Science][Medline]
Moller P. Genotoxicity of environmental agents assessed by the alkaline comet assay. Basic Clin Pharmacol Toxicol (2005) 96(Suppl 1):1–42.[CrossRef][Medline]
Muller WU, Bauch T, Wojcik A, Bocker W, Streffer C. Comet assay studies indicate that caffeine-mediated increase in radiation risk of embryos is due to inhibition of DNA repair. Mutagenesis (1996) 11:57–60.
Noda Y, Matsumoto H, Umaoka Y, Tatsumi K, Kishi J, Mori T. Involvement of superoxide radicals in the mouse two-cell block. Mol Reprod Dev (1991) 28:356–360.[CrossRef][Web of Science][Medline]
Orsi NM, Leese HJ. Amino acid metabolism of preimplantation bovine embryos cultured with bovine serum albumin or polyvinyl alcohol. Theriogenology (2004) a 61:561–572.[CrossRef][Web of Science][Medline]
Orsi NM, Leese HJ. Ammonium exposure and pyruvate affect the amino acid metabolism of bovine blastocysts in vitro. Reproduction (2004) b 127:131–140.
Park SY, Kim EY, Cui XS, Tae JC, Lee WD, Kim NH, Park SP, Lim JH. Increase in DNA fragmentation and apoptosis-related gene expression in frozen-thawed bovine blastocysts. Zygote (2006) 14:125–131.[CrossRef][Web of Science][Medline]
Petters RM, Reed ML. Addition of taurine or hypotaurine to culture medium improves development of one- and two-cell pig embryos in vitro. Theriogenology (1991) 35:253.[CrossRef]
Petters RM, Johnson BH, Reed ML, Archibong AE. Glucose, glutamate and inorganic phosphate in early developmet of the pig embryo in vitro. J Reprod Fertil (1990) 89:269–275.
Rajaei F, Karja NW, Agung B, Wongsrikeao P, Taniguchi M, Murakami M, Sambuu R, Nii M, Otoi T. Analysis of DNA fragmentation of porcine embryos exposed to cryoprotectants. Reprod Domest Anim (2005) 40:429–432.[CrossRef][Web of Science][Medline]
Rausell F, Pertusa JF, Gomez-Piquer V, Hermenegildo C, Garcia-Perez MA, Cano A, Tarin JJ. Beneficial effects of dithiothreitol on relative levels of glutathione S-transferase activity and thiols in oocytes, and cell number, DNA fragmentation and allocation at the blastocyst stage in the mouse. Mol Reprod Dev (2007) 74:860–869.[CrossRef][Web of Science][Medline]
Rho GJ, Balasubramanian S, Kim DS, Son WJ, Cho SR, Kim JG, MK B, Choe SY. Influence of in vitro oxygen concentrations on preimplantation embryo development, gene expression and production of Hanwoo calves following embryo transfer. Mol Reprod Dev (2007) a 74:486–496.[CrossRef][Web of Science][Medline]
Scott R, Seli E, Miller K, Sakkas D, Scott K, Burns DH. Noninvasive metabolomic profiling of human embryo culture media using Raman spectroscopy predicts embryonic reproductive potential: a prospective blinded pilot study. Fertil Steril (2008).
Seli E, Sakkas D, Scott R, Kwok SC, Rosendahl SM, Burns DH. Noninvasive metabolomic profiling of embryo culture media using Raman and near-infrared spectroscopy correlates with reproductive potential of embryos in women undergoing in vitro fertilization. Fertil Steril (2007) 88:1350–1357.[CrossRef][Web of Science][Medline]
Shamsi MB, Kumar R, Dada R. Evaluation of nuclear DNA damage in human spermatozoa in men opting for assisted reproduction. Indian J Med Res (2008) 127:115–123.[Web of Science][Medline]
Stokes PJ, Hawkhead JA, Fawthrop RK, Picton HM, Sharma V, Leese HJ, Houghton FD. Metabolism of human embryos following cryopreservation: implications for the safety and selection of embryos for transfer in clinical IVF. Hum Reprod (2007) 22:829–835.
Sturmey RG, Leese HJ. Energy metabolism in pig oocytes and early embryos. Reproduction (2003) 126:197–204.[Abstract]
Sturmey RG, Barker EA, Leese HJ. DNA damage and amino acid metabolism in the pig blastocyst. Hum Fertil (2007) 10:264.
Sturmey RG, Brison DR, Leese HJ. Assessing embryo viability by measurement of amino acid turnover. RBMOnline (2008) 17:486–496.
Takahashi M, Keicho K, Takahashi H, Ogawa H, Schultz R, Okano A. Effect of oxidative stress on development and DNA damage in in vitro cultured bovine embryos by comet assay. Theriogenology (1999) a 54:137–145.[CrossRef][Web of Science]
Takahashi M, Saka N, Takahashi H, Kanai Y, Schultz RM, Okano A. Assessment of DNA damage in individual hamster embryos by comet assay. Mol Reprod Dev (1999) b 54:1–7.[CrossRef][Web of Science][Medline]
Tay JI, Rutherford AJ, Killick SR, Maguiness SD, Partridge RJ, Leese HJ. Human tubal fluid: production, nutrient composition and response to adrenergic agents. Hum Reprod (1997) 12:2451–2456.
Thompson JGE, Simpson AC, Pugh PA, Donnelly PE, Tervit. Effect of oxygen concentration on in vitro development of preimplantation sheep and cattle embryos. J Reprod Fertil (1990) 89:573–578.
Thompson JG, Partridge RJ, Houghton FD, Cox CI, Leese HJ. Oxygen uptake and carbohydrate metabolism by in vitro derived bovine embryos. J Reprod Fertil (1996) 106:299–306.
Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen (2000) 35:206–221.[CrossRef][Web of Science][Medline]
van Attikum H, Gasser SM. The histone code at DNA breaks: a guide to repair? Nat Rev Mol Cell Biol (2005) 6:757–765.[CrossRef][Web of Science][Medline]
Van Soom A, Van Vlaenderen I, Mahmoudzadeh AR, Deluyker H, de Kruif A. Compaction rate of in vitro fertilized bovine embryos related to the interval from insemination to first cleavage. Theriogenology (1992) 38:905–919.[CrossRef][Web of Science][Medline]
Vergouw CG, Botros LL, Roos P, Lens JW, Schats R, Hompes PG, Burns DH, Lambalk CB. Metabolomic profiling by near-infrared spectroscopy as a tool to assess embryo viability: a novel, non-invasive method for embryo selection. Hum Reprod (2008).
Wu F, Orlefors H, Bergstrom M, Antoni G, Omura H, Eriksson B, Watanabe Y, Langstrom B. Uptake of 14C- and 11C-labeled glutamate, glutamine and aspartate in vitro and in vivo. Anticancer Res (2000) 20:251–256.[Web of Science][Medline]
Zheng P, Schramm RD, Latham KE. Developmental regulation and in vitro culture effects on expression of DNA repair and cell cycle checkpoint control genes in rhesus monkey oocytes and embryos. Biol Reprod (2005) 72:1359–1369.
Submitted on May 28, 2008; resubmitted on July 8, 2008; accepted on August 8, 2008.
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