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Human Fallopian tubal epithelial cells in culture lose morphological features associated with the epithelium in situ and the extent to which they retain their in-vivo phenotype or function is unknown. In order to address this question, immunocytochemical markers were identified which distinguish secretory (HMFG2+, LhS28-) from ciliated (HMFG2-, LhS28+) epithelial cells in tissue sections of Fallopian tube. These markers were used to analyse the phenotype of tubal cells in vitro. Primary cultures of human tubal epithelial cells were seeded onto glass and grown to confluence before addition of oestradiol-17β. In the absence of hormone, tubal epithelial cells expressed cytokeratins and nuclear receptors for oestrogen and progesterone and adopted a homogeneous (HMFG2+, LhS28-) secretory cell phenotype. Following the addition of oestradiol-17β, a proportion of cells became positive for LhS28. The induction of a ciliated epithelial cell phenotype was confirmed by scanning electron microscopy, where on permeable collagen membranes, approximately one-third of tubal epithelial cells became ciliated in the presence of oestradiol-17β. We suggest that in vitro, tubal epithelial cells adopt an immature secretory-like phenotype and that oestrogen can induce differentiation to a ciliated epithelial cell phenotype. [ABSTRACT FROM PUBLISHER]

A number of factors may impinge on thermal homeostasis in the early embryo. The most obvious is the ambient temperature in which development occurs. Physiologically, the temperature in the lumen of the female tract is typically lower than the core body temperature, yet rises at ovulation in the human, while in an IVF setting, embryos are usually maintained at core body temperature. However, internal cellular developmental processes may modulate thermal control within the embryo itself, especially those occurring in the mitochondria which generate intracellular heat through proton leak and provide the embryo with its own 'central heating system'. Moreover, mitochondrial movements may serve to buffer high local intracellular temperatures. It is also notable that the preimplantation stages of development would generate proportionally little heat within their mitochondria until the blastocyst stage as mitochondrial metabolism is comparatively low during the cleavage stages. Despite these data, the specific notion of thermal control of preimplantation development has received remarkably scant consideration. This opinion paper illustrates the lack of reliable quantitative data on these markers and identifies a major research agenda which needs to be addressed with urgency in view of laboratory conditions in which embryos are maintained as well as climate change-derived heat stress which has a negative effect on numerous clinical markers of early human embryo development., (© 2024. The Author(s).)

This article revisits the hypothesis, proposed in 2002, that the successful development of oocytes and preimplantation mammalian embryos is associated with a metabolism which is "quiet" rather than "active", within limits which had yet to be defined. A distinction was drawn between Functional Quietness, Loss of quietness in response to stress and Inter-individual differences in embryo metabolism and here we document applications of the hypothesis to other areas of reproductive biology. In order to encompass the requirement for "limits" and replace the simple distinction between "quiet" and "active", evidence is presented which led to a re-working of the hypothesis by proposing the existence of an optimal range of metabolic activity, termed a "Goldilocks zone", within which oocytes and embryos with maximum developmental potential will be located. General and specific mechanisms which may underlie the Goldilocks phenomenon are proposed and the added value that may be derived by expressing data on individual embryos as distributions rather than mean values is emphasised especially in the context of the response of early embryos to stress and to the concept of the Developmental Origins of Health and Disease. The article concludes with a cautionary note that being "quietly efficient" may not always ensure optimal embryo survival., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Leese, Brison and Sturmey.)

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.

The use of in vitro embryo production in the horse is increasing in clinical and research settings; however, protocols are yet to be optimised. Notably, the two most commonly used base media for in vitro maturation (IVM) supply glucose at markedly different concentrations: physiological (5.6 mM, M199) or supraphysiological (17 mM, DMEM/F-12). Exposure to high glucose has detrimental effects on oocytes and early embryos in many mammalian species, but the impact has not yet been examined in the horse. To address this, we compared the energy metabolism of equine COCs matured in M199-based maturation medium containing either 5.6 or 17 mM glucose, as well as expression of key genes in oocytes and cumulus cells. Oocytes were fertilised by ICSI and cultured. Analysis of spent medium revealed that COC glucose consumption and production of lactate and pyruvate were similar between treatments. However, the glycolytic index was decreased at 17 mM and analysis of mitochondrial function of COCs revealed that IVM in 17 mM glucose was associated with decreased ATP-coupled respiration and increased non-mitochondrial respiration compared to that for 5.6 mM glucose. We also found that the metabolic enzyme lactate dehydrogenase-A (LDHA) was downregulated in cumulus cells of oocytes that completed IVM in 17 mM glucose. There was no difference in maturation or blastocyst rates. These data indicate that COC mitochondrial function and gene expression are altered by high glucose concentration during IVM. Further work is needed to determine if these changes are associated with developmental changes in the resulting offspring.

Horses are one of the few species, beside humans, in which assisted reproductive technology has important clinical applications. Furthermore, the horse can serve as a valuable model for the study of comparative reproductive biology. Here we present the first comprehensive characterisation of energy metabolism and mitochondrial efficiency in equine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM), as determined using a combination of non-invasive consumption and release assays and mitochondrial function analysis. These data reveal notable species-specific differences in the rate and kinetics of glucose consumption and glycolysis throughout IVM. Approximately 95% of glucose consumed was accounted for by lactate production; however, high concurrent oxygen consumption indicated a comparatively increased role for non-glycolytic oxidative phosphorylation. Up to 38% of equine COC oxygen consumption could be attributed to non-mitochondrial activities and there was a significant loss of spare respiratory capacity over the course of IVM. Notably, our data also revealed that current IVM protocols may be failing to satisfy the metabolic demands of the equine COC. Our findings constitute the first report on mitochondrial efficiency in the equine COC and provide new insight into comparative gamete biology as well as metabolism of the COC during in vitro maturation.

Numerical data in biology and medicine are commonly presented as mean or median with error or confidence limits, to the exclusion of individual values. Analysis of our own and others' data indicates that this practice risks excluding 'Goldilocks' effects in which a biological variable falls within a range between 'too much' and 'too little' with a region between where its function is 'just right'; a concept captured by the Swedish term 'Lagom'. This was confirmed by a narrative search of the literature using the PubMed database, which revealed numerous relationships of biological and clinical phenomena of the Goldilocks/Lagom form including quantitative and qualitative examples from the health and social sciences. Some possible mechanisms underlying these phenomena are considered. We conclude that retrospective analysis of existing data will most likely reveal a vast number of such distributions to the benefit of medical understanding and clinical care and that a transparent approach of presenting each value within a dataset individually should be adopted to ensure a more complete evaluation of research studies in future., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Mitochondria provide the major source of ATP for mammalian oocyte maturation and early embryo development. Oxygen Consumption Rate (OCR) is an established measure of mitochondrial function. OCR by mammalian oocytes and embryos has generally been restricted to overall uptake and detailed understanding of the components of OCR dedicated to specific molecular events remains lacking. Here, extracellular flux analysis (EFA) was applied to small groups of bovine, equine, mouse and human oocytes and bovine early embryos to measure OCR and its components. Using EFA, we report the changes in mitochondrial activity during the processes of oocyte maturation, fertilisation, and pre-implantation development to blastocyst stage in response to physiological demands in mammalian embryos. Crucially, we describe the real time partitioning of overall OCR to spare capacity, proton leak, non-mitochondrial and coupled respiration - showing that while activity changes over the course of development in response to physiological demand, the overall efficiency is unchanged. EFA is shown to be able to measure mitochondrial function in small groups of mammalian oocytes and embryos in a manner which is robust, rapid and easy to use. EFA is non-invasive and allows real-time determination of the impact of compounds on OCR, facilitating an assessment of the components of mitochondrial activity. This provides proof-of-concept for EFA as an accessible system with which to study mammalian oocyte and embryo metabolism.

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 remains 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 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.

The quiet embryo hypothesis postulates that early embryo viability is associated with a relatively low metabolism (Leese, 2002 BioEssays 24: 845-849). This proposal is re-visited here using retrospective and prospective data on the metabolic activity and kinetics of preimplantation development alongside the concept that an optimal range of such indices and of energetic efficiency influences embryogenesis. It is concluded that these considerations may be rationalized by proposing the existence of a "Goldilocks zone," or as it is known in Sweden, of lagom-meaning "just the right amount"-within which embryos with maximum developmental potential can be categorized. Mol. Reprod. Dev. 83: 748-754, 2016 © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The epithelium lining the oviduct is critical for early reproductive events, many of which are mediated via intracellular calcium ions. Despite this, little is known about the regulation of calcium homeostasis in the oviductal epithelium. Epithelial transient receptor potential channels (TRPCs) modulate calcium flux in other tissues, and their expression and functional regulation have therefore been examined using the bovine oviduct as a model for the human. The effects of FSH, LH, 17β-estradiol, and progesterone on TRPCs expression and intracellular calcium flux were determined. Transient receptor potential channels 1, 2, 3, 4, and 6 were expressed in the bovine reproductive tract, and their gene expression varied throughout the estrous cycle. In more detailed studies undertaken on TRPC1 and 6, we show that protein expression varied through the estrus cycle; specifically, 17β-estradiol, FSH, and LH individually and in combination upregulated TRPC1 and 6 expression in cultured bovine oviduct epithelial cells although progesterone antagonized these effects. Functional studies showed changes in calcium mobilization in bovine oviduct epithelial cells were dependent on TRPCs. In conclusion, TRPC1, 2, 3, 4, and 6 are present in the epithelium lining the bovine oviduct, and TRPC1 and 6 vary through the estrous cycle suggesting an important role in early reproductive function., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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.

Study Question: Is the developmental timing and metabolic regulation disrupted in embryos from overweight or obese women?, Summary Answer: Oocytes 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., What Is Known Already: Female 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., Study Design, Size, Duration: We 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)., Participants/materials, Setting, Methods: In 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., Main Results and the Role of Chance: Human 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., Limitations, Reasons for Caution: Although 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., Wider Implications of the Findings: The 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., Study Funding/competing Interests: This study was funded by the Hull IVF Unit Charitable Trust and the Hull York Medical School. There are no conflict of interests., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

This article summarises presentations at the plenary session of the Annual Meeting of the British Fertility Society, on Effective nutrition from conception to adulthood, held in Sheffield, UK in January 2014. It highlights the pivotal role of the late David Barker (1938-2014) in revolutionising our understanding of the influence of maternal, gamete, embryo, foetal and infant nutrition on the health of the offspring in later life.

Background: Most 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., Methods: No 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., Results and Conclusions: General 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., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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.

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 gamete's 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.

Creatine Kinase (CK) catalyses the "creatine shuttle," the reversible conversion of creatine phosphate to creatine with the liberation of ATP. This article examines the potential role of the creatine shuttle in the provision of ATP during mouse preimplantation embryo development. Using quantitative PCR, transcripts of four subunit isoforms of CK--CKM, CKB, CKMT1, and CKMT2--were detectable at all developmental stages, from the presumptive zygote to late blastocyst, but there was no obvious pattern in gene expression. By contrast, total CK biochemical activity, measured by a novel method, was relatively constant from the 2- to 8-cell stage, before exhibiting a significant decrease in activity at the blastocyst stage. Immunocytochemical studies revealed a marked association of CKB with the mitotic spindle in 2- and 4-cell mouse embryos, consistent with the proposition that the creatine shuttle plays a key role in local delivery of ATP during cytokinesis. Endogenous creatine was detected in the blastocyst at a level of 0.53 pmol/embryo. In conclusion, we believe that creatine phosphate can now be added to the list of potential sources of ATP during preimplantation development., (Copyright © 2013 Wiley Periodicals, Inc.)

Elevated plasma nonesterified fatty acid (NEFA) concentrations are associated with negative energy balance and metabolic disorders such as obesity and type II diabetes. Such increased plasma NEFA concentrations induce changes in the microenvironment of the ovarian follicle, which can compromise oocyte competence. Exposing oocytes to elevated NEFA concentrations during maturation affects the gene expression and phenotype of the subsequent embryo, notably prompting a disrupted oxidative metabolism. We hypothesized that these changes in the embryo are a consequence of modified energy metabolism in the oocyte. To investigate this, bovine cumulus oocyte complexes were matured under elevated NEFA conditions, and energy metabolism-related gene expression, mitochondrial function, and ultrastructure evaluated. It was found that expression of genes related to REDOX maintenance was modified in NEFA-exposed oocytes, cumulus cells, and resultant blastocysts. Moreover, the expression of genes related to fatty acid synthesis in embryos that developed from NEFA-exposed oocytes was upregulated. From a functional perspective, inhibition of fatty acid β-oxidation in maturing oocytes exposed to elevated NEFA concentrations restored developmental competence. There were no clear differences in mitochondrial morphology or oxygen consumption between treatments, although there was a trend for a higher mitochondrial membrane potential in zygotes derived from NEFA-exposed oocytes. These data show that the degree of mitochondrial fatty acid β-oxidation has a decisive impact on the development of NEFA-exposed oocytes. Furthermore, the gene expression data suggest that the resulting embryos adapt through altered metabolic strategies, which might explain the aberrant energy metabolism previously observed in these embryos originating from NEFA-exposed maturing oocytes.

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 (P = 0.0006) and released more alanine (P = 0.0001) 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.

This review considers how our understanding of preimplantation embryo metabolism has progressed since the pioneering work on this topic in the late 1960s and early 1970s. Research has been stimulated by a desire to understand how metabolic events contribute to the development of the zygote into the blastocyst, the need for biomarkers of embryo health with which to improve the success of assisted conception technologies, and latterly by the 'Developmental Origins of Health and Disease' (DOHaD) concept. However, arguably, progress has not been as great as it might have been due to methodological difficulties in working with tiny amounts of tissue and the low priority assigned to fundamental research on fertility and infertility, with developments driven more by technical than scientific advances. Nevertheless, considerable progress has been made in defining the roles of the traditional nutrients: pyruvate, glucose, lactate, and amino acids; originally considered as energy sources and biosynthetic precursors, but now recognized as having multiple, overlapping functions. Other nutrients; notably lipids, are beginning to attract the attention they deserve. The pivotal role of mitochondria in early embryo development and the DOHaD concept, and in providing a cellular focus for metabolic events is now recognized. Some unifying ideas are discussed; namely 'stress-response models' and the 'quiet embryo hypothesis'; the latter aiming to relate the metabolism of individual preimplantation embryos to their subsequent viability. The review concludes by updating the state of knowledge of preimplantation embryo metabolism in the early 1970s and listing some future research questions.

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.

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.

This study investigated the relationship between human preimplantation embryo metabolism and aneuploidy rates during development in vitro. One hundred and eighty-eight 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. Embryos were either fixed for interphase fluorescent in situ hybridization analysis of chromosomes 13, 18, 19, 21, X or Y, or were assayed for mitochondrial activity. Amino acid turnover was different (P < 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 Days 2-3 of culture. By Days 3-4, the profiles of serine, leucine and lysine differed between uniformly euploid versus aneuploid embryos. Gender significantly (P < 0.05) affected the metabolism of tryptophan, leucine and asparagine by cleavage-stage embryos. Pronucleate zygotes had a significantly higher proportion of active:inactive mitochondria compared with cleavage-stage embryos. Furthermore, mitochondrial activity was correlated (P < 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.

The ratio of male/female embryos may be modified by environmental factors such as maternal diet in vivo and the composition of embryo culture media in vitro. We have used amino acid profiling, a noninvasive marker of developmental potential to compare the effect of sex on the metabolism of bovine blastocysts conceived in vivo and in vitro. Blastocysts were incubated individually for 24 hr in a close-to-physiological mixture of amino acids and the depletion or appearance of 18 amino acids measured using HPLC. Blastocysts were then sexed by PCR. Amino acid depletion by in vitro-produced blastocysts and expanded blastocysts was higher than in embryos conceived in vivo (P = 0.02). When cultured in vitro, female embryos exhibited increased depletion of arginine, glutamate, and methionine and appearance of glycine, while male embryos displayed increased depletion of phenylalanine, tyrosine, and valine. Overall, in vitro-produced blastocysts exhibited sex-specific differences in metabolic profiles of 7 out of 18 amino acids; in vivo-produced, in 2 out of 18. These differences had disappeared by the expanded blastocyst stages. We have also shown that amino acid metabolism can predict the ability of bovine zygotes to develop to the blastocyst stage, providing "proof of principle" for the use of this technology in clinical IVF to select single embryos for transfer and thereby avoid the problem of multiple births.

Insight into the normal and abnormal function of an interphase nucleus can be revealed by using fluorescence in situ hybridization (FISH) to determine chromosome copy number and/or the nuclear position of loci or chromosome territories. FISH has been used extensively in studies of mouse and human early embryos, however, translation of such methods to domestic species have been hindered by the presence of high levels of intracytoplasmic lipid in these embryos which can impede the efficiency of FISH. This chapter describes in detail a FISH protocol for overcoming this problem. Following extensive technical development, the protocol was derived and optimized for IVF porcine embryos to enable investigation of whole chromosome and subchromosomal regions by FISH during these early stages of development. Porcine embryos can be generated in-vitro using semen samples from commercial companies and oocytes retrieved from discarded abattoir material. According to our method, porcine embryos are lyzed and immobilized on slides using Hydrochloric acid and "Tween 20" detergent, prior to pretreatment with RNase A and pepsin before FISH. The method described has been optimized for subsequent analysis of FISH in two dimensions since organic solvents, which are necessary to remove the lipid, have the effect of flattening the nuclear structure. The work in this chapter has focussed on the pig; however, such methods could be applied to bovine, ovine, and canine embryos, all of which are rich in lipid.

Early embryo loss is a major factor affecting the conception rate in cattle. Up to 40% of cattle embryos die within 3 weeks of fertilisation while they are nutritionally dependent on oviduct and uterine fluids for their survival. Inadequate systemic progesterone is one of the factors contributing to this loss. We have characterised the effects of changes in systemic progesterone on amino acid, ion and energy substrate composition of oviduct and uterine fluids on Days 3 and 6, respectively, of the oestrus cycle in cattle. Oviduct and uterine fluids were collected in situ following infusion of progesterone. There was no effect of progesterone on oviduct fluid secretion rate; however, uterine fluid secretion rate was lowered. Progesterone increased uterine glucose, decreased oviduct sulfate and, to a lesser degree, oviduct sodium, but had no effect on any of the ions in the uterus. The most marked effect of progesterone was on oviducal amino acid concentrations, with a twofold increase in glycine, whereas in the uterus only valine was increased. These results provide novel information on the maternal environment of the early cattle embryo and provide further evidence of progesterone regulation of oviduct amino acid concentrations in cattle.

Growing oocytes in vitro from the most immature stages until they are developmentally competent is a major goal of reproductive technology, requiring fundamental knowledge of metabolic processes. Carbohydrate metabolism and oxygen consumption have been analysed in a series of experiments designed to investigate important energy substrates for mouse oocytes and to reveal any qualitative or quantitative changes between the primordial and ovulatory follicle stages. Primordial follicles were incubated in groups in modified-KSOM medium, whereas growing or ovulated oocytes were studied singly and, in both cases, the depletion or accumulation of metabolites in spent medium were analysed using ultramicrofluorometric assays. The rates of glucose (0.014 +/- 0.006 pmol/hr) and pyruvate (0.028 +/- 0.009 pmol/hr) consumption and l-lactate (0.058 +/- 0.023 pmol/hr) production by primordial follicles suggested that energy production was supported by a combination of metabolic pathways, including glycolysis. Pyruvate and oxygen consumption per oocyte increased two- and ninefold, respectively, between the primary and pre-ovulatory stages (0.82 +/- 0.1 and 1.67 +/- 0.1 pmol pyruvate/hr, respectively and 1.4 +/- 0.3 and 7 +/- 0.6 pmol oxygen/hr) after which oxygen (12.7 +/- 1.1 pmol/hr) utilisation nearly doubled. Oxygen consumption by fully grown oocytes was in excess of oxidation requirements for pyruvate. When pyruvate and oxygen consumption rates were normalised for oocyte cellular volume, which increased over 130-fold during growth, oocyte metabolism was higher in primary follicles than at any subsequent stage, indicating that energy needs are greater during a developmental transition. To conclude, pyruvate and oxygen were consumed throughout oocyte development at increasing rates. When oocyte cellular volume was accounted for, oocytes from primary follicles displayed greatest metabolic rates.

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.

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.

This review assesses the ability of non-invasive 'amino acid profiling' to predict early embryo viability. The history of amino acid supplementation of embryo culture media and the role of amino acids in early embryo development are first considered and these are followed by a review of methods to quantify amino acid depletion and production by single embryos. Data on amino acid profiling of embryos from a number of species are then discussed. It is concluded that this technology has excellent potential to improve the selection of single embryos for transfer in clinical IVF.

This article's objectives are to investigate the relationship between adenosine triphosphate (ATP) production (oxidative phosphorylation and glycolysis) and fertility of bovine spermatozoa, determine the proportion of oxygen consumption devoted to proton leak and that due to nonmitochondrial processes, and discover whether freeze/thawing affects sperm oxygen consumption. Oxygen consumption of bovine spermatozoa was measured using a standard Clark electrode and, for the first time, in an Oxygen Biosensor System (OBS). Total ATP formation by bovine spermatozoa was calculated from the oxygen consumption and lactate production (glycolysis) by the same spermatozoa sample. ATP production varied from 1.99 to 8.09 mumol ATP per 10(8) spermatozoa per hour; glycolysis accounted for 16% to 38% of ATP. Nonmitochondrial oxygen consumption could not be detected in bovine spermatozoa using these methods. A significant proportion (16%-43%) of oxygen consumption was insensitive to oligomycin and was due to "proton leak." There was no significant difference between oxygen consumption of frozen/thawed and fresh spermatozoa for 2 of the 3 bulls tested. However, oxygen consumption of frozen/thawed spermatozoa was significantly higher (P < .05) than fresh spermatozoa for the third bull. When ZO(2) of frozen/thawed spermatozoa from 20 bulls was compared with their 49 day nonreturn rates (NRRs), oxygen consumption was correlated positively with NRR (ie, fresh spermatozoa with a higher ZO(2) were more fertile). Moreover, total ATP production correlated with NNR better than ZO(2). Bulls with a lower NRR produce spermatozoa that are susceptible to damage during the freeze/thawing process, causing an increase in ZO(2), possibly due to mitochondrial membrane damage resulting in more energy being expended in maintaining the proton gradient, or capacitation-like changes causing hyperactivation. Oxygen consumption measured in the OBS may be useful in assessing bovine sperm fertility.

Little is understood about the regulation of gene expression in human preimplantation embryos. We set out to examine the expression in human preimplantation embryos of a number of genes known to be critical for early development of the murine embryo. The expression profile of these genes was analysed throughout preimplantation development and in response to growth factor (GF) stimulation. Developmental expression of a number of genes was similar to that seen in murine embryos (OCT3B/4, CDX2, NANOG). However, GATA6 is expressed throughout preimplantation development in the human. Embryos were cultured in IGF-I, leukaemia inhibitory factor (LIF) or heparin-binding EGF-like growth factor (HBEGF), all of which are known to stimulate the development of human embryos. Our data show that culture in HBEGF and LIF appears to facilitate human embryo expression of a number of genes: ERBB4 (LIF) and LIFR and DSC2 (HBEGF) while in the presence of HBEGF no blastocysts expressed EOMES and when cultured with LIF only two out of nine blastocysts expressed TBN. These data improve our knowledge of the similarities between human and murine embryos and the influence of GFs on human embryo gene expression. Results from this study will improve the understanding of cell fate decisions in early human embryos, which has important implications for both IVF treatment and the derivation of human embryonic stem cells.

Up to 40 percent of cattle embryos die within 3 weeks of fertilization but there is little or no published information on the composition of the oviduct and uterine fluids essential for their survival during this time. We have measured the concentrations of the energy substrates, glucose, lactate, and pyruvate in cattle oviduct fluid on Days 0, 2, 4, and 6 and uterine fluid on Days 6, 8, and 14 of the oestrous cycle and corresponding blood samples. Oviduct and uterine fluids were collected in situ. Glucose concentrations in oviduct and uterine fluids were similar on all days and lower than in plasma (P < 0.05). Oviduct lactate concentration was up to eightfold higher than uterine or plasma concentration (P < 0.01). Oviduct pyruvate concentrations were similar on all days and lower than plasma concentrations on Days 0 and 2 (P < 0.005). Pyruvate concentrations were similar in the uterus and in plasma except on Day 14 when the concentration in plasma was higher (P < 0.05). There were no associations between systemic progesterone or oestradiol and glucose, lactate or pyruvate. There was a linear positive relationship (P < 0.001) between oviduct fluid secretion rate and oviduct glucose concentration and a linear negative relationship (P < 0.001) between oviduct fluid secretion rate and oviduct lactate, but no association between uterine fluid secretion rate and energy substrates. The different concentrations and associations between the energy substrates in oviduct and uterine fluids and blood plasma indicate a differential regulation of the secretion of these energy substrates by the oviduct and uterine epithelium., (Copyright 2007 Wiley-Liss, Inc.)

The oviduct and uterus provide the environments for the earliest stages of mammalian embryo development. However, little is known about the mechanisms that underlie the formation of oviduct and uterine fluids, or the extent to which the supply of nutrients via these reproductive tract tissues matches the nutrient requirements of early embryos. After reviewing our limited knowledge of these phenomena, a new experimental paradigm is proposed in which the epithelia lining the endosalpinx and endometrium are seen as the final components in a supply line that links maternal diet at one end and embryo uptake of nutrients at the other. When considered in this way, the oviduct and uterine epithelia become, for a few days, potentially the most critical maternal tissues in the establishment of a healthy pregnancy. In fulfilling this 'gatekeeper' role, female reproductive tract fluids have a key role in the 'developmental origins of health and disease' concept.

Background: It has been proposed that preimplantation embryo viability during culture and following embryo transfer is associated with a 'quiet' metabolism. Viable embryos may be better equipped to contend with damage to the genome, transcriptome and proteome, or they may possess less damage than non-viable embryos., Methods: Much of the data for the quiet embryo hypothesis was obtained in the human and mouse. In this article, evidence is reviewed suggesting that the quiet hypothesis may equally be applied to reproduction in livestock, which can provide good models for the human., Results: Data, particularly for the sheep and cow, suggest that a quiet metabolism during early embryo development is consistent with successful embryo development. Conversely, an 'active' metabolism is associated with sub-optimal outcomes in later life., Conclusions: The challenge is to identify the range of values for a given marker within which an embryo has a high chance of giving rise to healthy offspring. We also speculate on the ways in which such a metabolic profile might be encouraged and the implications for weight loss in obese women prior to conception.

The determination for early cleavage-stage embryos of noninvasive morphologic and metabolic criteria that are predictive of blastocyst development and/or full-term viability remains an important research target. We describe the derivation of a logistic regression model that predicts the probability of porcine blastocyst formation in vitro. Pig zygotes, derived by in vitro maturation and fertilization of slaughterhouse oocytes, were cultured in NCSU-23 medium that was supplemented with a mixture of 20 amino acids (NCSU-23(aa)). On Day 1, at 21, 23, 25, 27, 29 and 31 h postinsemination, cleaving embryos were evaluated morphologically in terms of the: i) number of blastomeres, ii) evenness of division, and iii) degree of fragmentation. These embryos were then placed in 1.5-microl drops of NCSU-23(aa) for 24 h, after which time the three morphologic criteria were re-evaluated and 1.2 microl of spent medium were removed for analysis by HPLC, in order to determine the net rates of amino acid depletion and appearance. Embryos were then cultured singly in NCSU-23(aa) by placing them between the filaments of a woven polyester mesh until Day 6, in order to permit the identification of individual embryos. Of 256 cleaved embryos, 28.7 +/- 6.2% (n = 5 replicates) developed into blastocysts. Discriminant analysis was used to select a subset of amino acids (threonine, valine, lysine, and phenylalanine) that discriminated optimally between embryos that became blastocysts or degenerated. These discriminant scores were entered into the logistic regression. Significant univariate relationships were established between the probability of blastocyst development and amino acid score (odds ratio [OR] 0.53, 95% confidence interval [CI] 0.40-0.69, P < 0.001), cleavage time (OR 0.79, 95% CI 0.71-0.87, P < 0.001), degree of fragmentation on Day 1 (OR 0.55, 95% CI 0.35-0.84, P = 0.009) and Day 2 (OR 0.53, 95% CI 0.35-0.78, P = 0.002), evenness of division on Day 2 (OR 0.66, 95% CI 0.46-0.96, P = 0.028), and categorical values of blastomere number on Day 2 (all P < 0.02), although no single variate could accurately predict blastocyst formation. However, multivariate analysis of the cell numbers on Day 1 and Day 2 correctly classified 51.9% of the predicted blastocysts. The inclusion of cleavage time in the regression analysis raised this rate to 63.5%, which was increased to 66.2% by the addition of evenness of division and degree of fragmentation. Finally, the full logistic regression model, which incorporated amino acid score together with all the other morphologic and kinetic variables, correctly classified 80.8% of the predicted blastocysts. This represented 51.2% of the observed blastocysts. Our data are novel in that they not only define in a quantitative manner the influence of previously undescribed predictors of porcine blastocyst formation, but they also provide a simple model of preimplantation development with reasonable predictive accuracy. The present study also provides a basic model for the examination and incorporation of additional early morphologic and metabolic correlates of developmental competence and could potentially be applied to the selection of human embryos for transfer in clinical IVF.

It has been proposed that the viability of early mammalian embryos is associated with a metabolism that is "quiet" rather than "active" (Leese HJ, 2002:BioEssays 24:845-849). The data on which this hypothesis was based were largely drawn from measurements on the depletion and appearance of amino acids from the culture medium. Data on the de novo synthesis of protein in in vivo- and in vitro-derived bovine embryos, as determined from the flux of radiolabeled methionine, have provided further support of the hypothesis and are interpreted to provide a new set of testable propositions that could illuminate the molecular basis of the quiet metabolism phenotype. The propositions are based on the premise that the extent of DNA damage, and the RNA and protein content of the immature oocyte, are key factors in determining whether the zygote progresses to the blastocyst stage. We propose that stochastic events and environmental stresses determine whether the condition of the genome, transcriptome, and proteome of the zygote will support development. Several molecular components are identified that may determine the viability of a zygote, and we speculate that the cellular response to unfavorable events or excessive DNA damage may be the premature activation of the embryonic genome and of apoptosis., ((c) 2007 Wiley-Liss, Inc.)