DNA damage and metabolic activity in the preimplantation embryo

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

Non-invasive amino acid turnover predicts human embryo developmental capacity

Background: IVF is limited by low success rates and a confounding high multiple birth rate contributing to prematurity, increased neonatal mortality and child handicap. These problems could be overcome if single embryos of known developmental competence could be selected for transfer on day 2/3 of development, but current methods, which rely on morphological appearance, are poor predictors of viability. Methods: We have measured non-invasively the depletion/appearance (i.e. turnover) of a physiological mixture of 18 amino acids by single human embryos during in-vitro culture using high performance liquid chromatography. Results: From the time of transfer (day 2/3), embryos with future competence to develop to the blastocyst stage (day 5/6) exhibit amino acid flux patterns distinct from those of embryos with similar morphological appearance which arrest. Significantly, the profiles of Ala, Arg, Gln, Met and Asn flux predict blastocyst potentiality at >95%. The amino acid most consistently depleted throughout development by those embryos which form blastocysts was leucine. Of the amino acids which were produced, the most striking was alanine, which appeared in increasing amounts throughout development. Conclusions: Non-invasive amino acid profiling has the potential to select developmentally competent single embryos for transfer, thereby increasing the success rate and eliminating multiple births in IVF

Metabolism of human embryos following cryopreservation: implications for the safety and selection of embryos for transfer in clinical IVF

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

Association between amino acid turnover and chromosome aneuploidy during human preimplantation embryo development in vitro

This study investigated the relationship between human preimplantation embryo metabolism and aneuploidy rates during development in vitro. 188 fresh and cryopreserved embryos from 59 patients (33.9±0.6 years) were cultured for 2-5 days. The turnover of 18 amino acids was measured in spent media by high performance liquid chromatography (HPLC). Embryos were either fixed for interphase fluorescent in situ hybridisation (FISH) analysis of chromosomes 13, 18, 19, 21, X or Y, or were assayed for mitochondrial activity. Amino acid turnover was different (P&lt;0.05) between stage-matched fresh and cryopreserved embryos due to blastomere loss following warming. The proportion of embryos with aneuploid cells increased as cell division progressed from pronucleate- (23%) to late cleavage-stages (50-70%). Asparagine, glycine and valine turnover was significantly different between uniformly genetically normal and uniformly abnormal embryos on day 2-3 of culture. By day 3-4 the profiles of serine, leucine and lysine differed between uniformly euploid vs. aneuploid embryos. Gender significantly (P&lt;0.05) affected the metabolism of tryptophan, leucine and asparagine by cleavage-staged embryos. Pronucleate zygotes had a significantly higher proportion of active:inactive mitochondria compared to cleavage-staged embryos. Furthermore, mitochondrial activity was correlated (P&lt;0.05) with altered aspartate and glutamine turnover. These results demonstrate the association between the metabolism, cytogenetic composition and health of human embryos in vitro. <br/

Characterization of a novel sialic acid transporter of the sodium solute symporter (SSS) family and in vivo comparison with known bacterial sialic acid transporters.

The function of sialic acids in the biology of bacterial pathogens is reflected by the diverse range of solute transporters that can recognize these sugar acids. Here, we use an Escherichia coliDeltananT strain to characterize the function of known and proposed bacterial sialic acid transporters. We discover that the STM1128 gene from Salmonella enterica serovar Typhimurium, which encodes a member of the sodium solute symporter family, is able to restore growth on sialic acid to the DeltananT strain and is able to transport [(14)C]-sialic acid. Using the DeltananT genetic background, we performed a direct in vivo comparison of the transport properties of the STM1128 protein with those of sialic acid transporters of the major facilitator superfamily and tripartite ATP-independent periplasmic families, E. coli NanT and Haemophilus influenzae SiaPQM, respectively. This revealed that both STM1128 and SiaPQM are sodium-dependent and, unlike SiaPQM, both STM1128 and NanT are reversible secondary carriers, demonstrating qualitative functional differences in the properties of sialic acid transporters used by bacteria that colonize humans

Human embryos developing in vitro are susceptible to impaired epithelial junction biogenesis correlating with abnormal metabolic activity

BACKGROUND: Blastocyst biogenesis occurs over several cell cycles during the preimplantation period comprising the gradual expression and membrane assembly of junctional protein complexes which distinguish the outer epithelial trophectoderm (TE) cells from the inner cell mass (ICM). In the human, TE integrity and the formation of a junctional seal can often be impaired. Embryos likely to result in a successful pregnancy after transfer are mostly selected according to morphological criteria. Recent data suggest that non-invasive measurement of amino acid turnover may be useful to complement such morphological scores. Whether morphological and metabolic criteria can be linked to poor TE differentiation thereby underpinning developmental predictions mechanistically remains unknown. METHODS: We examined TE intercellular junction formation in human embryos by immunofluorescence and confocal microscopy and correlated this process with morphological criteria and amino acid turnover during late cleavage. RESULTS: Our results show that TE differentiation may be compromised by failure of membrane assembly of specific junction constituents. This abnormality relates more closely to metabolic profiles than morphological criteria. CONCLUSION: Our data identify that amino acid turnover can predict TE differentiation. These findings are the first to link two mechanisms, metabolism and junction membrane assembly, which contribute to early embryo development