Henry J. Leese

Metabolism of the viable mammalian embryo: quietness revisited

This review examines the ‘Quiet Embryo Hypothesis’ which proposes that viable preimplantation embryos operate at metabolite or nutrient turnover rates distributed within lower ranges than those of their less viable counterparts. The ‘quieter’ metabolism consistent with this hypothesis is considered in terms of (i) ‘functional’ quietness; the contrasting levels of intrinsic metabolic activity in different cell types as a consequence of their specialized functions, (ii) inter-individual embryo/cell differences in metabolism and (iii) loss of quietness in response to environmental stress. Data are reviewed which indicate that gametes and early embryos function in vivo at a lower temperature than core body temperature, which could encourage the expression of a quiet metabolism. We call for research to determine the optimum temperature for mammalian gamete/embryo culture. The review concludes by examining the key role of reactive oxygen species, which can induce molecular damage, trigger a cellular stress response and lead to a loss of quietness.

Elevated non-esterified fatty acid concentrations during bovine oocyte maturation compromise early embryo physiology.

Elevated concentrations of serum non-esterified fatty acids (NEFA), associated with maternal disorders such as obesity and type II diabetes, alter the ovarian follicular micro-environment and have been associated with subfertility arising from reduced oocyte developmental competence. We have asked whether elevated NEFA concentrations during oocyte maturation affect the development and physiology of zygotes formed from such oocytes, using the cow as a model. The zygotes were grown to blastocysts, which were evaluated for their quality in terms of cell number, apoptosis, expression of key genes, amino acid turnover and oxidative metabolism. Oocyte maturation under elevated NEFA concentrations resulted in blastocysts with significantly lower cell number, increased apoptotic cell ratio and altered mRNA abundance of DNMT3A, IGF2R and SLC2A1. In addition, the blastocysts displayed reduced oxygen, pyruvate and glucose consumption, up-regulated lactate consumption and higher amino acid metabolism. These data indicate that exposure of maturing oocytes to elevated NEFA concentrations has a negative impact on fertility not only through a reduction in oocyte developmental capacity but through compromised early embryo quality, viability and metabolism.

Metabolic induction and early responses of mouse blastocyst developmental programming following maternal low protein diet affecting life-long health

Previously, we have shown that a maternal low protein diet, fed exclusively during the preimplantation period of mouse development (Emb-LPD), is sufficient to induce by the blastocyst stage a compensatory growth phenotype in late gestation and postnatally, correlating with increased risk of adult onset cardiovascular disease and behavioural dysfunction. Here, we examine mechanisms of induction of maternal Emb-LPD programming and early compensatory responses by the embryo. Emb-LPD induced changes in maternal serum metabolites at the time of blastocyst formation (E3.5), notably reduced insulin and increased glucose, together with reduced levels of free amino acids (AAs) including branched chain AAs leucine, isoleucine and valine. Emb-LPD also caused reduction in the branched chain AAs within uterine fluid at the blastocyst stage. These maternal changes coincided with an altered content of blastocyst AAs and reduced mTORC1 signalling within blastocysts evident in reduced phosphorylation of effector S6 ribosomal protein and its ratio to total S6 protein but no change in effector 4E-BP1 phosphorylated and total pools. These changes were accompanied by increased proliferation of blastocyst trophectoderm and total cells and subsequent increased spreading of trophoblast cells in blastocyst outgrowths. We propose that induction of metabolic programming following Emb-LPD is achieved through mTORC1signalling which acts as a sensor for preimplantation embryos to detect maternal nutrient levels via branched chain AAs and/or insulin availability. Moreover, this induction step associates with changes in extra-embryonic trophectoderm behaviour occurring as early compensatory responses leading to later nutrient recovery.

Human embryos from overweight and obese women display phenotypic and metabolic abnormalities

STUDY QUESTIONnIs the developmental timing and metabolic regulation disrupted in embryos from overweight or obese women?nnnSUMMARY ANSWERnOocytes from overweight or obese women are smaller than those from women of healthy weight, yet post-fertilization they reach the morula stage faster and, as blastocysts, show reduced glucose consumption and elevated endogenous triglyceride levels.nnnWHAT IS KNOWN ALREADYnFemale overweight and obesity is associated with infertility. Moreover, being overweight or obese around conception may have significant consequences for the unborn child, since there are widely acknowledged links between events occurring during early development and the incidence of a number of adult disorders.nnnSTUDY DESIGN, SIZE, DURATIONnWe have performed a retrospective, observational analysis of oocyte size and the subsequent developmental kinetics of 218 oocytes from 29 consecutive women attending for ICSI treatment and have related time to reach key developmental stages to maternal bodyweight. In addition, we have measured non-invasively the metabolic activity of 150 IVF/ICSI embryos from a further 29 consecutive women who donated their surplus embryos to research, and have related the data retrospectively to their body mass index (BMI).nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnIn a clinical IVF setting, we compared oocyte morphology and developmental kinetics of supernumerary embryos collected from overweight and obese women, with a BMI in excess of 25 kg/m(2) to those from women of healthy weight. A Primovision Time-Lapse system was used to measure developmental kinetics and the non-invasive COnsumption/RElese of glucose, pyruvate, amino acids and lactate were measured on spent droplets of culture medium. Total triglyceride levels within individual embryos were also determined.nnnMAIN RESULTS AND THE ROLE OF CHANCEnHuman oocytes from women presenting for fertility treatment with a BMI exceeding 25 kg/m(2) are smaller (R(2) = -0.45; P = 0.001) and therefore less likely to complete development post-fertilization (P < 0.001). Those embryos that do develop reach the morula stage faster than embryos from women of a BMI < 25 kg/m(2) (<0.001) and the resulting blastocysts contain fewer cells notably in the trophectoderm (P = 0.01). The resulting blastocysts also have reduced glucose consumption (R(2) = -0.61; P = 0.001), modified amino acid metabolism and increased levels of endogenous triglyceride (t = 4.11, P < 0.001). Our data further indicate that these differences are independent of male BMI.nnnLIMITATIONS, REASONS FOR CAUTIONnAlthough statistical power has been achieved, this is a retrospective study and relatively small due to the scarcity of human embryos available for research. Consequently, subanalysis of overweight and obese was not possible based on the sample size. The analysis has been performed on supernumerary embryos, originating from a single IVF unit and not selected for use in treatment. Thus, it was not possible to speculate how representative the findings would be of the better quality embryos transferred or frozen for each patient.nnnWIDER IMPLICATIONS OF THE FINDINGSnThe data indicate that a high BMI of women at conception is associated with distinct phenotypic changes in the embryo during the preimplantation period, highlighting the importance of prepregnancy body weight in optimizing the chances of fertility and safeguarding maternal and offspring health. These changes to the metabolic fingerprint of human embryos which are most likely a legacy of the ovarian conditions under which the oocyte has matured may reduce the chances of conception for overweight women and provide good evidence that the metabolic profile of the early embryo is set by sub-optimal conditions around the time of conception. The observed changes could indicate long-term implications for the health of the offspring of overweight and obese women.nnnSTUDY FUNDING/COMPETING INTERESTSnThis study was funded by the Hull IVF Unit Charitable Trust and the Hull York Medical School. There are no conflict of interests.

Amino Acid Turnover by Bovine Oocytes Provides an Index of Oocyte Developmental Competence In Vitro

ABSTRACT Amino acid profiling has been used to distinguish between human embryos of differing developmental competence. We sought to determine whether amino acid profiling could be used to distinguish between metaphase II (MII) bovine oocytes with different developmental capabilities in vitro. Amino acid turnover was assayed during the final 6 h of in vitro maturation prior to oocytes undergoing individual fertilization in vitro. Following insemination, zygotes were immobilized in groups of 16 on the base of a Petri dish using Cell-Tak tissue adhesive to enable the developmental progress of each to be tracked to the blastocyst stage. Spent droplets of in vitro maturation medium were analyzed by high performance liquid chromatography, which revealed glutamine, arginine, and asparagine were depleted in the greatest quantities. Incompetent MII oocytes that failed to cleave by 72 h postfertilization depleted significantly more glutamine from (Pu2009=u20090.0006) and released more alanine (Pu2009=u20090.0001) into the medium than oocytes that cleaved. When cutoff values were selected for the turnover of alanine, arginine, glutamine, leucine, and tryptophan and modeled to predict fertilization and cleavage potential, oocytes that did not exceed the cutoff values for ≥2 of these key amino acids were more likely to cleave. The sensitivity, specificity, accuracy, and positive predictive value of this model were 60.5%, 76.8%, 63.5%, and 92.0%, respectively. Significant differences (P ≤ 0.015) in the consumption/production of alanine and glutamine were also observed when comparing uncleaved oocytes with those that produced blastocysts. The data show that noninvasive amino acid profiling can be used to measure oocyte developmental competence.

A simple approach for COnsumption and RElease (CORE) analysis of metabolic activity in single mammalian embryos.

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.

Metabolic heterogeneity during preimplantation development: the missing link?

BACKGROUNDnMost tissues in the body rely on the presence of gap junctions in order to couple their component cells electrically and metabolically via intercellular transport of ions, metabolites and signalling agents. As a result, cells within tissues achieve a high degree of, metabolic homogeneity which enables them to develop in an integrated way and co-ordinate their response to physiological signals and environmental cues. Unusually, the developing mammalian preimplantation embryo does not form functional gap junctions until it has divided into 8 or more cells. We discuss the implications of this missing link during the first few days of development for the maintenance of homogeneity between embryonic cells and for the co-ordination of the embryonic response to intrinsic genetic damage and external environmental signals.nnnMETHODSnNo systematic review has been carried out. The physiology of preimplantation development and the general nature of gap junctions have been reviewed briefly before examining experimental evidence which addresses the following points: (i) whether there are functional differences between early blastomeres; (ii) when during preimplantation development the embryo is most sensitive to environmental perturbation and (iii) the consequences for early embryos of ablating gap junction formation and function.nnnRESULTS AND CONCLUSIONSnGeneral conclusions are confounded by species differences, especially in the timing of embryonic genome activation (EGA) and the extent of intrinsic genotypic and phenotypic variation (low in embryos from inbred mice; high in human embryos). Nevertheless, we propose that the absence of gap junctions requires cleavage stage mammalian embryos to behave cell autonomously in a metabolic sense, contributes to their heightened sensitivity to environmental perturbation compared with the later stages of preimplantation development and poses more problems in the early human embryo, where there is a high degree of heterogeneity between the blastomeres. We argue that the legacy of metabolic heterogeneity, in part generated by the absence of gap junctions, is rescued by the onset of apoptosis following EGA. In the context of human-assisted conception, since early embryos lacking gap junctions are more sensitive to environmental stress during cleavage, this would support transfer to the natural environment as early as possible after fertilization.

Amino acid turnover by human oocytes is influenced by gamete developmental competence, patient characteristics and gonadotrophin treatment

STUDY QUESTION Can amino acid profiling differentiate between human oocytes with differing competence to mature to metaphase II (MII) in vitro? SUMMARY ANSWER Oocytes which remained arrested at the germinal vesicle (GV) stage after 24 h of in vitro maturation (IVM) displayed differences in the depletion/appearance of amino acids compared with oocytes which progressed to MII and patient age, infertile diagnosis and ovarian stimulation regime significantly affected oocyte amino acid turnover during IVM. WHAT IS KNOWN ALREADY Amino acid profiling has been proposed as a technique which can distinguish between human pronucleate zygotes and cleavage stage embryos with the potential to develop to the blastocyst stage and implant to produce a pregnancy and those that arrest. Most recently, the amino acid turnover by individual bovine oocytes has been shown to be predictive of oocyte developmental competence as indicated by the gametes capacity to undergo fertilization and early cleavage divisions in vitro. STUDY DESIGN, SIZE, DURATION The study was conducted between March 2005 and March 2010. A total of 216 oocytes which were at the GV or metaphase I (MI) stages at the time of ICSI were donated by 67 patients. PARTICIPANTS/MATERIALS, SETTINGS, METHODS The research was conducted in university research laboratories affiliated to a hospital-based infertility clinic. Oocytes were cultured for 24 h and the depletion/appearance of amino acids was measured during the final 6 h of IVM. Amino acid turnover was analysed in relation to oocyte meiotic progression, patient age, disease aetiology and controlled ovarian stimulation regime. MAIN RESULTS AND THE ROLE OF CHANCE The depletion/appearance of key amino acids was linked to the maturation potential of human oocytes in vitro. Oocytes which arrested at the GV stage (n = 9) depleted significantly more valine and isoleucine than those which progressed to MI (n = 32) or MII (n = 107) (P < 0.05). Glutamate, glutamine, arginine and valine depletion or appearance differed in MII versus degenerating oocytes (n = 20) (P < 0.05). Glutamine, arginine, methionine, phenylalanine, total depletion and total turnover all differed in oocytes from patients aged < 35 years versus patients ≥35 years (P < 0.05). MII oocytes obtained following ovarian stimulation with recombinant FSH depleted more isoleucine (P < 0.05) and more alanine and lysine (P < 0.05) appeared than oocytes from hMG-stimulated cycles. MII oocytes from patients with a polycystic ovary (PCO) morphology (n = 33) depleted more serine (P < 0.05) than oocytes from women with normal ovaries (n = 61). LIMITATIONS, REASONS FOR CAUTION Immature oocytes collected at the time of ICSI were used as the model for human oocyte maturation. These oocytes have therefore failed to respond to the ovulatory hCG trigger in vivo (they are meiotically incompetent), and have limited capacity to support embryo development in vitro. The lack of cumulus cells and stress of the conditions in vitro may have influenced turnover of amino acids, and owing to the small sample sizes further studies are required to confirm these findings. WIDER IMPLICATIONS OF THE FINDINGS The findings provide support for the hypothesis that oocyte metabolism reflects oocyte quality. Longitudinal studies are required to link these functional metabolic indices of human oocyte quality with embryo developmental competence. Oocyte amino acid profiling may be a useful tool to quantify the impact of new assisted reproduction technologies (ART) on oocyte quality. STUDY FUNDING/COMPETING INTERESTS This project was funded by the UK Biology and Biotechnology Research Council (BB/C007395/1) and the Medical Research Council (G 0800250). K.E.H was in receipt of a British Fertility Society/Merck Serono studentship. H.J.L. is a shareholder in Novocellus Ltd, a company which seeks to devise a non-invasive biochemical test of embryo health.

History of oocyte and embryo metabolism.

The basic pattern of metabolism in mammalian oocytes and early embryos was established in the 1960s and 1970s, largely in terms of the consumption of oxygen and the utilisation of nutrients present in culture media at the time, mainly glucose, pyruvate and lactate. The potential importance of endogenous fuels was also recognised but was largely ignored, only to be rediscovered quite recently. The 1980s and 1990s saw the arrival of a new generation of culture media, characterised metabolically by the addition of amino acids, an initiative driven strongly by the need to improve embryo culture and selection methods in assisted reproductive technologies. This trend has continued alongside some basic metabolic studies and the general recognition of the importance of metabolism in all aspects of biology. A framework for future studies on oocyte and early embryo metabolism has been provided by: (1) the developmental origins of health and disease concept and recognition of the relationship between development, epigenetics and metabolism; (2) the need to understand cell signalling within, and between the cells of, the early embryo; and (3) the importance of identifying the mechanisms underlying dialogue between the oocyte and early embryo and the female reproductive tract.

Modelling aspects of oviduct fluid formation in vitro

© 2017 Society for Reproduction and Fertility. Oviduct fluid is the microenvironment that supports early reproductive processes including fertilisation, embryo cleavage and genome activation. However, the composition and regulation of this critical environment remain rather poorly defined. This study uses an in vitro preparation of the bovine oviduct epithelium to investigate the formation and composition of in vitro-derived oviduct fluid (ivDOF) within a controlled environment. We confirm the presence of oviduct-specific glycoprotein 1 in ivDOF and show that the amino acid and carbohydrate content resembles that of previously reported in vivo data. In parallel, using a different culture system, a panel of oviduct epithelial solute carrier genes and the corresponding flux of amino acids within ivDOF in response to steroid hormones were investigated. We next incorporated fibroblasts directly beneath the epithelium. This dual culture arrangement represents more faithfully the in vivo environment and impacts on ivDOF composition. Lastly, physiological and pathophysiological endocrine states were modelled and their impact on the in vitro oviduct preparation was evaluated. These experiments help clarify the dynamic function of the oviduct in vitro and suggest a number of future research avenues, such as investigating epithelial-fibroblast interactions, probing the molecular aetiologies of subfertility and optimising embryo culture media.