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Amino acids are now recognised as having multiple cellular functions in addition to their traditional role as constituents of proteins. This is well-illustrated in the early mammalian embryo where amino acids are now known to be involved in intermediary metabolism, as energy substrates, in signal transduction, osmoregulation and as intermediaries in numerous pathways which involve nitrogen metabolism, e.g., the biosynthesis of purines, pyrimidines, creatine and glutathione. The amino acid derivative S-adenosylmethionine has emerged as a universal methylating agent with a fundamental role in epigenetic regulation. Amino acids are now added routinely to preimplantation embryo culture media. This review examines the routes by which amino acids are supplied to the early embryo, focusing on the role of the oviduct epithelium, followed by an outline of their general fate and function within the embryo. Functions specific to individual amino acids are then considered. The importance of amino acids during the preimplantation period for maternal health and that of the conceptus long term, which has come from the developmental origins of health and disease concept of David Barker, is discussed and the review concludes by considering the potential utility of amino acid profiles as diagnostic of embryo health.

Aneurysmal bone cyst (ABC) is a non-malignant, locally destructive, blood-filled lesion in the bone that tends to grow aggressively. A young girl presented with a rapid recurrence after aggressive surgery of a large symptomatic sacral-spinal ABC. After a multidisciplinary tumour board, she was successfully treated with sclerotherapy and monthly intravenous denosumab. The patient has maintained asymptomatic for over 36 months now and has returned to full activity and strength. She never required surgery and has had radiologic resolution of the lesions. Treatment of recurrent ABC requires a multidisciplinary team approach. We believe this to be the first report to use this combined therapy to provide an alternative to morbid surgery for children with ABCs.

Triclosan (TCS) is an antimicrobial agent in a wide range of health care products. It has been found in various human bodily fluids and is a potential reproductive toxicant. However, the effect of TCS on early embryo development in mammalian species is limited. We therefore asked whether exposure to TCS affects mammalian cumulus–oocyte complexes (COCs), and if so, whether the effects persist into the early embryo. COCs, isolated from abattoir-derived bovine ovaries, were exposed to two environmentally relevant doses of TCS (1 and 10 nM) during in vitro maturation. When exposed to 1 nM TCS during in vitro maturation, progesterone release from bovine oocytes was elevated. Furthermore, altered pyruvate metabolism and mitochondrial dysfunction were also observed; specifically, O2 consumption coupled to ATP production was significantly decreased in COCs after acute exposure to TCS prior to maturation, whereas proton leak from the respiratory chain was increased. Subsequently, TCS-exposed COCs were fertilised. Fewer oocytes were able to develop to blastocyst when exposed to 1 nM TCS during maturation compared to the Control group, and those that did reach the blastocyst displayed impaired glycolytic and amino acid metabolic activity. These findings indicate for the first time that oocytes exposed to TCS during the final stages of maturation give rise to embryos with impaired mitochondrial function, altered steroidogenesis, and disrupted metabolic activity.

We report a novel method to profile intrcellular oxygen concentration (icO 2 ) during in vitro mammalian oocyte and preimplantation embryo development using a commercially available multimodal phosphorescent nanosensor (MM2). Abattoir-derived bovine oocytes and embryos were incubated with MM2 in vitro. A series of inhibitors were applied during live-cell multiphoton imaging to record changes in icO 2 associated with mitochondrial processes. The uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) uncouples mitochondrial oxygen consumption to its maximum, while antimycin inhibits complex III to ablate mitochondrial oxygen consumption. Increasing oxygen consumption was expected to reduce icO 2 and decreasing oxygen consumption to increase icO 2 . Use of these inhibitors quantifies how much oxygen is consumed at basal in comparison to the upper and lower limits of mitochondrial function. icO 2 measurements were compared to mitochondrial DNA copy number analysed by qPCR. Antimycin treatment increased icO 2 for all stages tested, suggesting significant mitochondrial oxygen consumption at basal. icO 2 of oocytes and preimplantation embryos were unaffected by FCCP treatment. Inner cell mass icO 2 was lower than trophectoderm, perhaps reflecting limitations of diffusion. Mitochondrial DNA copy numbers were similar between stages in the range 0.9–4 × 10 6 copies and did not correlate with icO 2 . These results validate the MM2 probe as a sensitive, non-toxic probe of intracellular oxygen concentration in mammalian oocytes and preimplantation embryos.

Tissue and whole organ regeneration is a dramatic biological response to injury that occurs across different plant and animal phyla. It frequently requires the dedifferentiation of mature cells to a condensed mesenchymal blastema, from which replacement tissues develop. Human somatic cells cannot regenerate in this way and differentiation is considered irreversible under normal developmental conditions. Here, we sought to establish in vitro conditions to mimic blastema formation by generating different three-dimensional (3D) condensates of human mesenchymal stromal cells (MSCs). We identified specific 3D growth environments that were sufficient to dedifferentiate aged human MSCs to an early mesendoderm-like state with reversal of age-associated cell hypertrophy and restoration of organized tissue regenerating capacity in vivo . An optimal auophagic response was required to promote cytoplasmic remodeling, mitochondrial regression and a bioenergetic shift from oxidative phosphorylation to anaerobic metabolism. Our evidence suggests that human cell dedifferentiation can be achieved through autonomously controlled autophagic flux.

Non-invasive assay of the consumption and release of metabolites by individual human embryos could allow selection at the cleavage stage of development and facilitate Single Embryo Transfer in clinical IVF but will require simple, high throughput, sensitive methods applicable to small volume samples. A rapid, simple, non-invasive method has therefore been devised using a standard fluorescence plate reader, and used to measure the consumption of pyruvate and glucose, and release of lactate by single bovine embryos at all stages of preimplantation development in culture; amino acid profiles have been determined using HPLC. Early embryos with an 'intermediate' level (6.14±0.27 pmol/embryo/h) of pyruvate uptake were associated with the highest rate (68.3%) of blastocyst development indicating that a mid \"optimum\" range of pyruvate consumption correlates with high viability in this bovine model.

Myocardial bridging (MB) is a congenital variant in which a segment of a coronary artery follows an atypical intramural course under a “bridge” of myocardium and is notably common in hypertrophic cardiomyopathy (HCM). This systematic review and meta-analysis explored the clinical consequences of MB in patients with HCM. A total of 3 outcome domains were investigated: cardiovascular mortality, nonfatal adverse cardiac events, and investigative indicators of myocardial ischemia. A meta-analysis was performed on 10 observational studies comparing outcomes in patients with HCM with and without MB. Studies were identified through a systematic search of 4 databases (PubMed, Scopus, Medline Complete, and Web of Science). The quality of the studies was assessed using a modified version of the Downs and Black tool, from which studies could score a maximum of 23 points. The mean score was 17.5 ± 1.3 (good). The meta-analysis showed that MB was not associated with cardiovascular mortality (odds ratio [OR] 1.70, 95% confidence interval [CI] 0.56 to 5.15, p = 0.35) or nonfatal adverse cardiac events (OR 1.80, 95% CI 0.98 to 3.28, p = 0.06) but was associated with myocardial ischemia (OR 1.89, 95% CI 1.03 to 3.44, p = 0.04). In conclusion, the potential prognostic implications of MB in HCM, especially in those with hemodynamically significant bridges and/or severe underlying disease, should not be ignored. The focus of future studies should be to establish functional and morphologic thresholds, by which MB may adversely influence prognosis by corroborating imaging findings with clinical outcome data.

Non-invasive assay of the consumption and release of metabolites by individual human embryos could allow selection at the cleavage stage of development and facilitate Single Embryo Transfer in clinical IVF but will require simple, high throughput, sensitive methods applicable to small volume samples.

The preimplantation embryo must satisfy dynamic changes in energy demand during development to the blastocyst stage. Energy is provided through regulated metabolic pathways including glycolysis, β-oxidation and oxidative phosphorylation. Oxygen consumption rate (OCR), representing overall oxidative metabolism, has been reported in several species but few studies have examined the bioenergetics of embryo development. Several methods were optimised to measure components of OCR by individual embryos. On average, 66% of blastocyst OCR was coupled to ATP synthesis, the majority being complex I-dependent. A further 13% was of non-mitochondrial origin, while maximal OCR was 189% of basal, providing a spare respiratory capacity of +89%. This profile allows re-interpretation of existing data to estimate ATP production by the bovine embryo. The endogenous triglyceride store of the oocyte is increasingly considered a vital energy source in preimplantation development. In the present study, β-oxidation was manipulated during embryo culture. Inhibition of β-oxidation led to i) increased OCR ii) increased lipid content, iii) increased pyruvate uptake and iv) decreased lactate release at the blastocyst stage. Enhancing β-oxidation caused i) OCR at blastocyst stage to fall, ii) decreased lipid content during early cleavage, iii) decreased pyruvate consumption and iv) increased lactate release. Neither treatment affected blastocyst development rate or differential cell count, while both led to mitochondrial depolarisation. These metabolic observations were hypothesised to have legacy effects on gene expression. Groups of 10 blastocysts with similar metabolic profiles were analysed using transcription and DNA methylation microarray platforms. Following manipulation of β-oxidation, gene transcripts involved in mitochondrial function, metabolism, key signalling cascades, recognition of pregnancy, stress response, protein modification and transcription were differentially expressed. Genes involved in transcription, protein modification, key signalling cascades and disease were differentially methylated, potentially linking dysregulated β-oxidation to deleterious conditions in later development. These data highlight the plasticity of metabolic regulation in the embryo, allowing successful preimplantation development despite an apparently deleterious phenotype, yet indicate that metabolic activity has subtle effects on development.

Triclosan (TCS) is an anti-microbial agent in a wide range of health care products. It has been found in various human bodily fluids and is a potential reproductive toxicant. However, the effect of TCS on early embryo development in mammalian species is limited. We therefore asked whether exposure to TCS affects mammalian cumulus-oocyte-complexes (COCs), and if so, whether the effects persist into the early embryo. COCs, isolated from abattoir-derived bovine ovaries were exposed to two environmentally relevant doses of TCS (1 nM and 10 nM) during in vitrao maturation. When exposed to 1 nM TCS during in vitro maturation, progesterone release from bovine oocytes was elevated. Furthermore, altered pyruvate metabolism and mitochondrial dysfunction also observed; specifically, O2 consumption coupled to ATP production was significantly decreased in COCs after acute exposure of TCS prior to maturation, whereas proton leak from the respiratory chain was increased. Subsequently, TCS-exposed COCs were fertilised. Fewer oocytes were able to develop to blastocyst when exposed to 1 nM TCS during maturation compared to the Control group (12.22±% vs 29.11±% n=5; p=0.02) and those that did reach the blastocyst displayed impaired glycolytic and amino acid metabolic activity. These findings indicate for the first time that oocytes exposed to TCS during the final stages of maturation give rise to embryos with impaired mitochondria function, altered steroidogenesis and disrupted metabolic activity.