Hum. Reprod. Advance Access originally published online on November 30, 2006
Human Reproduction 2007 22(3):829-835; doi:10.1093/humrep/del447
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Metabolism of human embryos following cryopreservation: Implications for the safety and selection of embryos for transfer in clinical IVF
1 Department of Biology, University of York, York, UK 2 Assisted Conception Unit, St James's University Hospital, Leeds, UK 3 Reproduction and Early Development Research Group, Department of Obstetrics and Gynaecology, University of Leeds, Leeds, UK 4 Current address: Human Genetics Division, University of Southampton, Duthie Building, Mailpoint 808, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, UK
5 To whom correspondence should be addressed at: Human Genetics Division, University of Southampton, Duthie Building (Mailpoint 808), Southampton General Hospital, Tremona Road, Southampton, SO16 6YD, UK. E-mail: fdh1{at}soton.ac.uk
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Abstract |
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BACKGROUND: Cryopreservation of supernumerary embryos is routinely performed in human-assisted reproduction, providing a source of embryos which can be thawed for use in subsequent treatment cycles. However, the viability of cryopreserved embryos has traditionally relied on morphological assessment, which is a poor predictor of embryo health since freezing leads to a significant overall reduction in implantation potential, and its long-term efficacy is unknown. This study describes how the post-thaw metabolism of human embryos can be used to predict future development to the blastocyst stage.
METHODS: HPLC was used to analyse the post-thaw amino acid metabolism of human embryos from day 2 to day 3 of development.
RESULTS: It was possible to predict with 87% accuracy which frozen–thawed embryo would develop to the blastocyst stage. Developmentally competent embryos were more metabolically quiescent than their arresting counterparts. Amino acid turnover was also capable of distinguishing between the developmental potential of the best, Grade I embryos P < 0.05.
CONCLUSIONS: The data suggests that cryopreservation in IVF is a safe procedure and that amino acid turnover can be used to select which cryopreserved embryo will develop to the blastocyst stage, irrespective of their post-thaw grade.
Key words: amino acid turnover/cryopreservation/developmental competency/embryo viability
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Introduction |
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Cryopreservation of pronucleate, or early cleavage stage embryos, is routinely offered in most assisted conception units. This allows good quality, supernumerary embryos to be frozen for use in subsequent treatment cycles, thereby increasing the cumulative pregnancy rate while decreasing the risk of ovarian hyperstimulation syndrome (Trounson, 1986
). One of the most significant factors limiting the success of cryopreservation is the post-thaw embryo survival due to the formation of ice crystals during the freezing and thawing processes (Liebermann et al. 2002), which may damage cell membranes and lead to blastomere lysis (Pal et al., 2004). Although successful pregnancies can be achieved following transfer of embryos with <50% of the blastomeres intact post-thaw (Veiga et al., 1987), pregnancy rates are higher when all blastomeres survive (Burns et al., 1999; Edgar et al., 2000). Indeed, if embryos survive the freeze–thaw process with all the blastomeres intact, then the pregnancy rate is comparable with that of fresh IVF cycles (Edgar et al. 2000; Guerif et al., 2002). However, overall, cryopreservation leads to a 30–40% reduction in the implantation potential (Edgar et al., 2000; El-Toukhy et al., 2003).
Embryo freezing has no effect on early (<20 weeks of gestation) or late (
20 weeks of gestation) obstetric outcome after transfer, in comparison with fresh embryos (Aytoz et al., 1999). Similarly, there is no significant effect of cryopreservation on the mean gestational age, birth weight of singleton, twin and triplet births or the perinatal mortality rates (Wada et al., 1994). Although embryo cryopreservation is generally considered a safe procedure (Wood, 1997) controversy still remains regarding the long-term, post-natal safety of this technique (Winston and Hardy, 2002). This is largely a result of studies performed in the mouse, where it has been suggested that embryo freezing may affect metabolism in late preimplantation development (Emiliani et al., 2000) and have more subtle long-term as well post-natal effects which are only manifested late in development (Dulioust et al., 1995). In human, Tachataki et al. 2003 reported that the freezing of embryos altered gene expression profiles and that day 2 frozen–thawed embryos contained less mRNA for the tuberous sclerosis gene, TSC2, than fresh day 2 embryos.
Metabolism is intrinsic to early embryo health and is immediately perturbed when embryos are stressed (Houghton and Leese, 2004; Lane and Gardner, 2005). Embryos are able to adapt to stresses due to the variations in culture conditions, but with cryopreservation, there is the extra stress of the freeze–thaw process, which may alter homeostasis, metabolism, cell integrity and developmental potential. We have previously shown that amino acid depletion/appearance (turnover) during cleavage predicts the ability of spare human embryos to develop to the blastocyst stage in vitro (Houghton et al., 2002) and give rise to a pregnancy and live offspring following transfer (Brison et al., 2004). This suggests that embryo developmental competence is determined early in development and that perturbations during the early stages of preimplantation development may have long-term implications. There is currently a lack of information on the metabolism of frozen–thawed human embryos, which is surprising since metabolic factors are likely to be central in understanding the reduced success rates of embryos transferred in frozen cycles. In this study, the post-thaw metabolism of single human embryos has been measured non-invasively in terms of amino acid turnover and used to predict development to the blastocyst stage. The value of morphological scoring systems to differentiate between high-grade embryos post-thaw was also examined.
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Materials and methods |
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Spare, frozen human embryos donated to research with informed patient consent were obtained from 28 patients undergoing IVF at the Assisted Conception Unit at St James's University Hospital, Leeds. Full ethical approval for this work was granted by the Human Fertilization and Embryology Authority (HFEA) as well as the Local Research Ethics Committees of the collaborating institutions.
Ovarian stimulation and oocyte collection were performed as previously described (Balen, 2001). In general, a long pituitary desensitization protocol was used, with intranasal nafarelin followed by gonadotrophin stimulation with either HMG (Menogon, Ferring Pharmaceuticals Ltd) or recombinant FSH (Puregon, Organon Laboratories Ltd). Briefly, oocytes were collected by follicular aspiration 36–38 h after HCG administration and cultured at 37°C in 5% CO2 in Medi-Cult IVF Medium under oil (Medi-Cult, Copenhagen, Denmark). The oocyte-cumulus complexes were inseminated with a final concentration of 25 000 motile sperm per millilitre at 
40–42 h post-HCG and incubated overnight until fertilization was confirmed by the presence of two pronuclei some 16–17 h later (day 1 post-insemination). Zygotes were cultured in 70 µl drops of Medi-Cult IVF medium under oil as above. A maximum of three embryos were transferred on day 2 post-insemination, and suitable spare embryos were frozen in straws according to the established slow freezing protocols using the Sydney IVF Cryopreservation Kit (Cook, Queensland, Australia). The freezing protocol began at a temperature of 15°C before dropping to –7°C at a rate of –2.0°C min–1. This temperature was maintained for 5 min to allow the straws to be seeded. Cooling continued at a rate of 0.3°C min–1 until –30°C was reached. This temperature was maintained for 5 min before decreasing at a rate of –50°C min–1 until –180°C was reached. The straws containing the embryos were removed from the freezer and stored in liquid nitrogen for potential future use. Cryopreservation was performed only if a patient had two or more embryos at the 2- to 4-cell stages which were Grade I or II. According to HFEA guidelines, embryos may only be stored for 5 years, and hence patients were contacted on a yearly basis to assess whether they wished their embryos to remain in storage, be discarded or donated to research with informed patient consent. Embryos thus donated to research were transported to York in a dry shipper for use in this study.
Embryos were thawed using the Sydney IVF thawing kit (Cook, Queensland, Australia) and placed into 10 µl drops of Earle's balanced salt solution based culture medium supplemented with 0.5% (v/v) human serum albumin, 1 mM glucose, 0.47 mM pyruvate, 5 mM lactate and a close to physiological mixture of amino acids (Tay et al., 1997) under mineral oil for 3 h. The developmental grade and stage of each embryo were assessed (Houghton et al., 2002) and cultured individually for 24 h, from day 2 to day 3 in 4 µl drops of the same medium in a humidified atmosphere containing 5% CO2, in air at 37°C. After incubation, the spent medium was stored at –80°C for amino acid analysis and the embryos transferred into fresh 10 µl drops of medium and cultured to day 6 of development, where their morphological stage and grade were assessed.
Amino acid analysis
Embryo-free control drops were incubated in the same dish as those containing embryos to allow for any non-specific changes in amino acid concentration throughout the culture period. The spent media were thawed and 2 µl aliquots diluted with 23 µl high-performance liquid chromatography (HPLC) grade water. Amino acid analysis was performed by reverse-phase HPLC using a Kontron 500 attached to a Jasco F920 fluorescence detector and a 4.5 x 250 mm Hypersil ODS-16 column (Jones Chromatography) as previously described (Houghton et al., 2002). The elution gradient operated at a flow rate of 1.3 ml min–1. Solvent A consisted of 18 ml tetrahydrofuran (Fisher Chemicals), 200 ml methanol and 800 ml sodium acetate (83 mmol/l, pH 5.9). Solvent B consisted of 800 ml methanol and 200 ml sodium acetate (83 mmol/l, pH 5.9). Using this method, it was not possible to detect proline and cysteine.
Statistical analysis
All data were analysed to determine whether they were normally distributed, using the Anderson–Darling normality test. Data for amino acid depletion/appearance were tested for significance from zero using either a 1-sample t-test or 1-sample Wilcoxon test, depending on whether the data were normally distributed. Differences between amino acid profiles for arresting and developing embryos were analysed using either a Student's t-test or Mann–Whitney U-test. Differences between amino acid depletion, appearance, turnover and balance were analysed by Student's t-test. A value of P < 0.05 was considered significant. Data are presented as mean ± SEM.
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Results |
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The amino acid profiles of thawed embryos, which subsequently developed to the blastocyst stage, were similar to those obtained for fresh embryos (Houghton et al., 2002
). Serine, glutamine, arginine, valine, isoleucine and leucine were significantly depleted from the medium (Figure 1), whereas aspartate, glutamate, alanine, tryptophan and phenylalanine appeared. Thawed embryos, which subsequently failed to develop to the blastocyst stage, consumed histidine, glutamine, arginine, valine, isoleucine and leucine and produced aspartate, glutamate, glycine, alanine, tryptophan, phenylalanine and lysine. There was a significant difference in the utilization of glutamine (P = 0.001), alanine (P = 0.001), glycine (P = 0.0025), glutamate (P = 0.016), arginine (P = 0.032) and lysine (P = 0.0448) between the thawed embryos which developed to the blastocyst stage and those which arrested prior to blastocyst formation (Figure 1). Surprisingly, the cohort of frozen–thawed day 2 to day 3 embryos that subsequently failed to develop to the blastocyst stage contained more Grade I embryos than those that developed (Table I). However, there was no difference in the average number of blastomeres per embryo between Grade I embryos which subsequently developed to the blastocyst and those that arrested prior to the blastocyst formation.
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Overall, embryos that arrested prior to the blastocyst stage were metabolically more active in terms of amino acid turnover (P < 0.001), depleting (P < 0.05) and producing (P < 0.01) significantly more amino acids than their developing counterparts (Figure 2). Interestingly, it was found that both developing and arresting embryos were balanced in terms of their amino acid turnover i.e. the amount depleted was not significantly different from that produced. There was a significant difference (P < 0.001) in the sum of glutamine, glycine and alanine from day 2 to day 3 between the embryos that developed to the blastocyst stage and those which arrested prior to the blastocyst formation (Figure 3). Using the sum of glutamine, glycine and alanine for individual embryos, it was possible to predict with 87% accuracy which embryos would develop to the blastocyst stage (Figure 4).
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When the amino acid profile of Grade I embryos was determined, there was a significant difference in amino acid depletion/appearance between those embryos which subsequently developed to the blastocyst stage compared with those Grade I embryos which arrested prior to the blastocyst formation (Figure 5). Specific differences between the two groups were in the utilization of lysine (P = 0.014), glycine (P = 0.024), tryptophan (P = 0.029), arginine (P = 0.035), glutamate (P = 0.0493) and glutamine (P = 0.050). Grade I embryos displayed no significant difference in total amino acid depletion, appearance or in the balance of amino acids (Figure 6). However, arresting Grade I embryos were metabolically more active in terms of amino acid turnover than those that developed to the blastocyst stage (P < 0.05).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConflict of interest statementAcknowledgementsReferences |
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The metabolism of spare, frozen–thawed human embryos has been determined from day 2 to day 3 post-insemination in terms of amino acid depletion from, and appearance in, the culture medium. To the best of our knowledge, this is the first study to investigate the amino acid metabolism of cryopreserved human embryos. The amino acid profiles obtained were similar to those for fresh embryos (Houghton et al., 2002
) and may be used to predict which embryo will develop to the blastocyst stage; a predictive capacity which is independent of embryo grade and blastomere number on day 2. Similar to fresh embryos, frozen–thawed embryos with the capacity to develop to the blastocyst stage had a lower rate of amino acid depletion, appearance and turnover than arresting embryos. This effect is consistent with the ‘quiet embryo hypothesis’ (Leese, 2002) and suggests that there is some inherent loss of metabolic homeostasis in arresting embryos. It is likely that amino acid profiling of cryopreserved human embryos will also be able to predict the potential to implant and give rise to live offspring, as in the study by Brison et al. 2004. These investigators used retrospective analysis of amino acid profiles of ICSI embryos measured from day 1 to day 2 of development. The embryos were selected for transfer on day 2 on the basis of morphology alone, and it was found that the turnover of asparagine, glycine and leucine correlated with a subsequent clinical pregnancy. Moreover, this predictive ability of amino acid profiling was independent of known predictors such as female age, basal FSH levels, embryo cell number and grade.
Success in IVF is dependent on the selection of the best embryo(s) being chosen for transfer into the uterus. Embryos are currently selected using morphological criteria, which are subjective and do not provide a robust test of developmental potential, contributing to low success rates per fresh treatment cycle started (28.2% for women below 35 decreasing to 3.2% for women over the age of 42) according to the HFEA guide to infertility 2006–7. Possible reasons for this low rate, despite the transfer of morphologically good embryos, include poor endometrial receptivity (Bourgain and Devroey, 2003; Devroey et al., 2004; Lukassen et al., 2004) and a traumatic embryo transfer leading to increased junctional zone contractions (reviewed by Lesny and Killick, 2004). However, the ability of amino acid profiling to differentiate between embryos of the same grade, which subsequently develop to the blastocyst stage, or arrest, indicates that morphological scoring is limited in its capacity to select embryos for transfer, especially when differentiating between embryos of the highest quality. This is not surprising since there is a large amount of data from a number of species demonstrating altered gene expression patterns of embryos cultured in different media but which appear morphologically normal (Ho et al., 1995; Rizos et al., 2003; Rinaudo and Schultz, 2004). We have shown that amino acid profiling is efficient in the retrospective identification of the capacity of frozen–thawed embryos to develop to the blastocyst stage. Moreover, to the best of our knowledge, this is the first study to have distinguished between viable and non-viable Grade 1 embryos.
In conclusion, the data suggest that cryopreservation in IVF is a safe procedure and that measurement of amino acid turnover can be used to select which frozen–thawed embryo will develop to the blastocyst stage, irrespective of their grade post-thaw.
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Conflict of interest statement |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConflict of interest statementAcknowledgementsReferences |
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F.D.H. and H.J.L. are shareholders in Novocellus, a company which is developing embryo culture and diagnostic systems for use in clinical IVF. Data in this article have been protected by patent no. GB 0601746.1.
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Acknowledgements |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConflict of interest statementAcknowledgementsReferences |
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This work was funded by a Wellcome Trust Research Career Development Fellowship to F.D.H and the UK Medical Research Council.
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Submitted on June 15, 2006; resubmitted on October 13, 2006; accepted on October 19, 2006.
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