Nutritional and metabolic requirements of early cleavage stage embryos and blastocysts

During preimplantation human embryo development there is an increase in the synthesis of macromolecules and a demand for energy. Consequently, the metabolic requirements of the human embryo change as development proceeds from the zygote to the blastocyst stage. Evidence from a number of species indicates that before activation of the embryonic genome, human and other mammalian embryos have a preference for oxidizable energy substrates, particularly pyruvate, non-essential amino acids and glutamine. After embryonic genome activation, glucose and essential amino acids become increasingly important. As such, there is a switch in energy metabolism during preimplantation development from one based principally on aerobic respiration, to another based on oxidative metabolism and aerobic glycolysis.     

Nutritional and metabolic requirements of early cleavage stage embryos and blastocysts

During preimplantation human embryo development there is an increase in the synthesis of macromolecules and a demand for energy. Consequently, the metabolic requirements of the human embryo change as development proceeds from the zygote to the blastocyst stage. Evidence from a number of species indicates that before activation of the embryonic genome, human and other mammalian embryos have a preference for oxidizable energy substrates, particularly pyruvate, non-essential amino acids and glutamine. After embryonic genome activation, glucose and essential amino acids become increasingly important. As such, there is a switch in energy metabolism during preimplantation development from one based principally on aerobic respiration, to another based on oxidative metabolism and aerobic glycolysis.     

Embryo nutrition and energy metabolism and its relationship to embryo growth, differentiation, and viability

Over the past decade there has been a resurgence of interest in the culture media used in clinical in vitro fertilization. Unfortunately, during this time more confusion than consensus appears to have developed regarding the composition of these media. In order to facilitate a clearer understanding of this field, it is important to understand the role of specific medium components and how their use is regulated by the embryo. The roles of the key nutrients glucose, pyruvate, lactate, and amino acids during the preimplantation period have therefore been presented. Analysis of how the embryo regulates the utilization of such nutrients has led to a clearer understanding of the embryo's requirements during the dynamic period of preimplantation development. From such information, sequential culture media have been developed along with novel noninvasive tests of embryonic viability. It is proposed that continued studies on the human embryo will lead to further improvements in embryo culture conditions and the optimization of viability assays, culminating in the ability to transfer single embryos for the majority of, if not all patients.     

Human follicular fluid and mouse cumulus cells act synergistically to enhance preimplantation mouse Balb/cJ embryo development

Problem The development of preimplantation mammalian embryos in vitro is less than optimal. Follicular fluid and cumulus cells have both been used, independently, to improve preimplantation embryo quality in culture.Method To determine the ability of mouse cumulus cell coculture in the presence of human follicular fluid to support preimplantation mouse Balb/cJ embryo development in vitro.Results Culture of preimplantation mouse Balb/cJ embryos independently in human follicular fluid or on mouse cumulus cells had no significant affect on blastocyst development or total cell number per blastocyst. The coculture of mouse Balb/cJ preimplantation-stage embryos on mouse cumulus cells in the presence of human follicular fluid significantly (P<0.07) improved blastocyst development and the total number of cells per blastocyst.Conclusions Cumulus cells and follicular fluid have a positive synergistic affect on preimplantation mouse Balb/cJ embryo development and formation in vitro.     

Autophagic activity as an indicator for selecting good quality embryos

Is it possible to predict the quality of embryos that appear to be morphologically identical when viewed under a microscope? Thirty‐five years have passed since the world's first human birth from in vitro fertilization. While the dissemination of assisted reproduction technologies during this time has been remarkable, the evaluation of embryo quality in both humans and mice currently relies entirely on morphological observation. More efficient infertility treatments will likely be possible if high‐quality embryos can be selected by screening. To develop a novel quality evaluation method that does not rely on morphology, we focused on autophagy, one of the molecular mechanisms essential for the early embryonic development. Autophagy is a massive cytoplasmic degradation pathway mediated by the lysosome. Our previous studies have demonstrated that fertilization‐induced autophagy is essential for preimplantation embryonic development. This autophagy is thought to supply the nutrients and amino acids necessary for maintaining subsequent embryo development, through the bulk degradation of maternal cytoplasmic factors that are accumulated during oogenesis. Here, we briefly summarize autophagy and its physiological function, and describe a recently developed method for using autophagic activity as an indicator to predict embryo quality.     

Maternal enzyme masks the phenotype of mouse embryos lacking dihydrolipoamide dehydrogenase

During early embryogenesis, the phenotype reflecting the embryonic genotype emerges only as maternal proteins are replaced by embryonically encoded forms, a process known as the maternal-to-embryonic transition (MET). Little is understood about MET for most proteins. This study investigates how complete deficiency of the murine dihydrolipoamide dehydrogenase gene (Dld), a gene that encodes an enzyme of mitochondrial energy metabolism, affects the phenotype of the early embryo and how the MET of the DLD protein affects the phenotype. Dld-deficient (?/?) embryos were found to develop similarly to wild-type (+/+) or heterozygous (+/?) embryos throughout the preimplantation period. These three genotypic classes also have comparable rates of glucose uptake (4.9–5.0 pmoles/embryo/h) and lactate production (0.97–1.0 pmoles/embryo/h). Dld-deficient embryos at the end of the preimplantation stage have 44% of DLD enzyme present in oocytes, a proportion similar to that found in +/+ or +/? embryos. This study demonstrates that Dld-deficient preimplantation embryos are phenotypically normal, as the MET for the DLD enzyme is only partially complete by the end of the preimplantation period. These findings have implications for phenotype- or enzyme-based approaches to identify mutations in Dld and other genes that encode proteins with similar MET kinetic profiles.     

The preimplantation embryo: handle with care

The past decade has seen considerable advances in our understanding of intrinsic developmental mechanisms associated with gametogenesis and embryogenesis and accompanying applications in the fields of reproductive medicine, embryonic stem cell biology, and nuclear reprogramming. However, a new focus has recently emerged concerning the homeostatic regulation of embryonic cells, how this is set, and how it may influence the longitudinal progression and optimization of the developmental program and indeed the phenotype of the offspring. Attention has been drawn to the preimplantation stage of development as a sensitive "window" when in vitro and in vivo manipulations, such as culture conditions or maternal diet, may have critical consequences. In this article, we review how changes in environmental conditions, mediated via a range of epigenetic, cellular, and metabolic mechanisms in the preimplantation embryo, may alter the pattern of cell division, gene expression, morphology, and potential. We consider how fetal and postnatal phenotype may become susceptible to the plasticity of the preimplantation embryo and the risks for adult health and physiology.     

Individual culture and atmospheric oxygen during culture affect mouse preimplantation embryo metabolism and post-implantation development

Research questionDoes single embryo culture under atmospheric or reduced oxygen alter preimplantation metabolism and post-implantation development compared with culture in groups?DesignMouse embryos were cultured under 5% or 20% oxygen, individually or in groups of 10. Spent media were analysed after 48, 72 and 96 h of culture. Blastocysts were assessed by outgrowth assay or transferred to pseudo-pregnant recipients, and fetal and placental weight, length and morphology were assessed.ResultsCompared with group culture, individually cultured blastocysts had lower net consumption of glucose and aspartate and higher glutamate production. Atmospheric oxygen reduced uptake of glucose and aspartate and increased production of glutamate and ornithine compared with 5% oxygen. Combining 20% oxygen and single culture resulted in further metabolic changes: decreased leucine, methionine and threonine consumption. Under 5% oxygen, individual culture decreased placental labyrinth area but had no other effects on fetal and placental development or outgrowth size compared with group culture. Under 20% oxygen, however, individual culture reduced outgrowth size and fetal and placental weight compared with group-cultured embryos.ConclusionsPreimplantation metabolism of glucose and amino acids is altered by both oxygen and individual culture, and fetal weight is reduced by individual culture under atmospheric oxygen but not 5% oxygen. This study raises concerns regarding the increasing prevalence of single embryo culture in human IVF and adds to the existing evidence regarding the detrimental effects of atmospheric oxygen during embryo culture. Furthermore, these data demonstrate the cumulative nature of stress during embryo culture and highlight the importance of optimizing each element of the culture system.     

Preimplantation embryo metabolism and culture systems: experience from domestic animals and clinical implications

Despite advantages of in vitro embryo production in many species, widespread use of this technology is limited by generally lower developmental competence of in vitro derived embryos compared to in vivo counterparts. Regardless, in vivo or in vitro gametes and embryos face and must adjust to multiple microenvironments especially at preimplantation stages. Moreover, the embryo has to be able to further adapt to environmental cues in utero to result in the birth of live and healthy offspring. Enormous strides have been made in understanding and meeting stage-specific requirements of preimplantation embryos, but interpretation of the data is made difficult due to the complexity of the wide array of culture systems and the remarkable plasticity of developing embryos that seem able to develop under a variety of conditions. Nevertheless, a primary objective remains meeting, as closely as possible, the preimplantation embryo requirements as provided in vivo. In general, oocytes and embryos develop more satisfactorily when cultured in groups. However, optimization of individual culture of oocytes and embryos is an important goal and area of intensive current research for both animal and human clinical application. Successful culture of individual embryos is of primary importance in order to avoid ovarian superstimulation and the associated physiological and psychological disadvantages for patients. This review emphasizes stage specific shifts in embryo metabolism and requirements and research to optimize in vitro embryo culture conditions and supplementation, with a view to optimizing embryo culture in general, and culture of single embryos in particular.     

Quality control in human in vitro fertilization

The implementation of suitable quality control (QC) is not only required for the accreditation of a human in vitro fertilization (IVF) laboratory, but is also fundamental to its success. Several assays have been employed to screen culture media and contact supplies. The suitability of one assay in particular, the mouse embryo assay (MEA), has been questioned over the years. Here we discuss how the conditions of such an assay, together with the stage of embryonic development used, have a profound effect on the outcome of the assay. Furthermore, by assessing embryos at multiple time points during the preimplantation period (rather than simply determining blastocyst formation), together with quantitating key parameters such as blastocyst cell number, it is possible to identify suboptimal components of a culture system. As well as identifying those components that result in outright embryonic demise, under the appropriate conditions the MEA can detect components that lead to impaired development. It is proposed that under the appropriate conditions, the MEA is a useful adjunct to quality control in human IVF, but several assays used in concert are better than a single test.     

Soluble HLA-G and embryo implantation: frequently asked questions

Two major criteria are currently used for optimal human embryo selection in in vitro fertilization or intracytoplasmic sperm injection: overall morphology-based grading system and rate of fragmentation. The measurement of soluble HLA-G (sHLA-G) in human preimplantation embryo culture supernatants gives great hope that addition of such a biochemical quantitative criterion would further improve the rates of implantation. In this short review, we will examine the functional significance of this latter approach by discussing the following frequently asked questions: (1) Which sHLA-G isoform(s) might be present in the human preimplantation embryo culture supernatants? (2) Where do these sHLA-G molecules come from? (3) Why are some preimplantation embryos 'HLA-G secretors' and others not? (4) How might the functions exerted by sHLA-G molecules influence embryonic implantation?     

Regulation of embryonic implantation

The preimplantation embryo produces several factors during its development to signal its presence to the maternal organism. This paper will focus on the role of two distinctive cytokine and growth factor systems (interleukin-1 (IL-1) system and the vascular endothelial growth factor (VEGF) system) during early embryonic development and implantation. IL-1 receptor is expressed in the endometrium of various species and antagonising the biological effects of IL-1 leads to implantation failure in mice. We could show that this is due to an endometrial, not an embryonic effect. Furthermore, we could detect the expression of all components of the IL-1 system in preimplantation embryos from mice and humans. We could show a possible influence of IL-1 on other systems involved in embryonic implantation, including invasion (MMPs/TIMPs) and angiogenesis (VEGF), therefore suggesting a role of this cytokine family during early embryonic development. Immediately after contact to the endometrium, the embryo must induce angiogenesis to ensure its survival, VEGF is a potent angiogenetic growth factor. We have shown a cyclic regulation of the soluble VEGF-receptor, sflt, in human endometrium and have detected the expression of the transmembraneous VEGF-receptors, Flt-1 and kinase insert domain containing receptor (KDR) throughout the menstrual cycle. Furthermore, we have shown that the VEGF gene is one of the earliest genes activated during human preimplantation embryo development, giving rise to the assumption that VEGF is crucial for embryonic development.     

Towards a single embryo transfer

The delivery of a single, healthy child is the desired outcome of human assisted reproduction techniques. To attain this goal, there is an increasing movement toward single embryo transfer. The question is, therefore, at what stage to transfer the human embryo back to the uterus? Maximal implantation rates reported to date have come from the transfer of blastocysts (70% fetal heart rate). In any given cycle of treatment the probability of conceiving a child will be further increased by the ability to cryopreserve those embryos not transferred. It is therefore proposed that the transfer of a single blastocyst is the best treatment for most patients, given the high implantation rates of fresh transfers, and that it is now possible to cryopreserve supernumerary blastocysts effectively. The next decision is how to culture the human embryo to the blastocyst stage. The use of sequential culture media, designed not only to allow for changes in nutrient requirements and metabolism as development proceeds, but also to minimize intracellular trauma, can facilitate the development of highly viable blastocysts. Sequential culture media have been evaluated against a single-step culture system. It has been shown that sequential media (G1/G2) produce more viable blastocysts than those embryos cultured in a single medium formulation (simplex optimized medium with elevated potassium and with amino acids, KSOM(AA)) throughout the preimplantation period. Furthermore, even if KSOM(AA) is used for embryo culture, it is essential that the medium be renewed after 48 h to alleviate the toxicity associated with ammonium build-up. Of great significance, embryos cultured in sequential media G1 and G2 have the same rate of development as embryos developed in vivo.     

Künftige Entwicklungen in der Reproduktionsmedizin

The possibilities of assisted reproduction have rapidly developed over the 35 years since the birth of the first child after in vitro fertilization (IvF) and embryo transfer in the year 1978. New possibilities have been implemented, such as intracytoplasmic sperm injection (ICSI), preimplantation diagnostics, ovarian stimulation with gonadotropin-releasing hormone (GnRH) antagonists and gonadotropins. This has led to the fact that in 2010 worldwide 5.2 million children were born after IvF and embryo transfer, to couples whose desire for children would have been very difficult or even impossible to fulfil without this option. In this article new possibilities for improving the pregnancy rates after assisted reproduction will be presented and critically assessed, such as ovarian stimulation, single embryo transfer, vitrification, blastocyst transfer, preimplantation diagnostics and genetic screening. Furthermore, developments in the preservation of fertility for cancer patients and in the field of time-lapse imaging of early embryonic development will be discussed.     

Human implantation: the last barrier in assisted reproduction technologies?

Implantation processes are highly complex involving the actions of numerous hormones, immunoglobulins, cytokines and other factors in the endometrium. They are also essential matters for the success of assisted reproduction. The nature of early embryonic development is of equal significance. It involves ovarian follicle growth, ovulation, fertilization and preimplantation growth. These processes are affected by imbalanced chromosomal constitutions or slow developmental periods. Post-implantation death is also a significant factor in cases of placental insufficiency or recurrent abortion. Clearly, many of these matters can significantly affect birth rates. This review is concerned primarily with the oocyte, the early embryo and its chromosomal anomalies, and the nature of factors involved in implantation. These are clearly among the most important features in determining successful embryonic and fetal growth. Successive sections cover the endocrine stimulation of follicle growth in mice and humans, growth of human embryos in vitro, their apposition and attachment to the uterus, factors involved in embryo attachment to uterine epithelium and later stages of implantation, and understanding the gene control of polarities and other aspects of preimplantation embryo differentiation. New aspects of knowledge include the use of human oocyte maturation in vitro as an approach to simpler forms of IVF, and new concepts in developmental genetics.     

Coculture cells that express leukemia inhibitory factor (LIF) enhance mouse blastocyst developmentin vitro

Purpose Leukemia inhibitory factor is a cytokine that plays an important role in implantation and enhances mouse preimplantation embryo development in vitro.Since leukemia inhibitory factor enhances early embryo development, we tested the hypothesis that coculture cells that express leukemia inhibitory factor would enhance mouse blastocyst development in vitro.In this study, Northern analysis for leukemia inhibitory factor was performed on total RNA extracted from Vero cells, human embryonic, fibroblasts, and human placental fibroblasts.Methods Two-cell mouse embryos were cultured to blastocyst in Ham's F-10 with 15% human cord serum (control) or with the coculture cells. Northern analysis demonstrated expression of LIF mRNA in Vero cells and embryonic fibroblasts but not in placental fibroblasts. Development to blastocyst was significantly enhanced in two-cell embryos cultured with Vero cells (86%, 140/163) and embryonic fibroblasts (83%, 118/142) when compared to controls (71%, 67/94, P<0.05) or placental fibroblasts (71%, 95/134), P<0.05).Conclusion These data suggest that coculture cells that express leukemia inhibitory factor may be superior to nonleukemia inhibitory factor expressing cells for early preimplantation embryo development.Presented at the 50th Annual Meeting of the American Fertility Society, San Antonio, Texas, November 5–10, 1994.     

The importance of growth factors for preimplantation embryo development and in-vitro culture

PURPOSE OF REVIEW: The present paper reviews evidence that preimplantation embryos are naturally exposed and designed to respond to growth factors during preimplantation development. RECENT FINDINGS: Recent studies have demonstrated that in-vivo human preimplantation embryos are exposed to a mixture of many different growth factors, expressed by the follicles, oviducts and endometrium. Receptors for many of these growth factors have also been shown to be expressed by preimplantation embryos, suggesting a functional role during preimplantation development. Studies of in-vitro fertilization in both animals and humans indicate that in-vitro culture of embryos in conventional media lacking growth factors can result in suboptimal growth and a variety of short-term and long-term developmental abnormalities. Studies of embryo coculture indirectly suggest that growth factors can improve in-vitro development. Many studies of defined growth factor supplements demonstrate that their inclusion in culture media can substantially improve preimplantation development and efficacy of in-vitro fertilization, and may reduce long-term developmental abnormalities as well. SUMMARY: Embryos are naturally exposed to a complex mixture of growth factors that play an important role in preimplantation embryo development and that are likely to be of substantial benefit if added to in-vitro culture media.     

Embryo-maternal interactions at the implantation site: a delicate equilibrium

Blastocyst implantation and successful establishment of pregnancy require delicate interactions between the embryo and the maternal environment. During preimplantation, maternal/embryo communication is mediated by the trophectoderm. In the late luteal phase, physiological changes occur in the endometrium to allow blastocyst implantation. The "window of implantation" represents the period of maximum uterine receptivity for implantation. In response to signals from the embryo, pregnancy-specific proteins are released in maternal serum and a series of morphological, biochemical and immunological changes occur in the uterine environment. These systemic and local modifications can be considered to constitute "the maternal recognition of pregnancy". The human hemochorial placenta arises primarily through proliferation, migration and invasion of the endometrium and its vasculature by the embryonic trophoblast. The complex invasive processes accompanying implantation of the embryo are controlled at the embryo-maternal interface by factors from decidualized endometrium and the trophoblast itself. An inflammatory reaction and a proper maternal immune response allow survival and development of the feto-placental unit. In this review, we focus on interactions between trophoblast and uterine tissues and on cellular mechanisms and molecular signals involved in the closely regulated process of implantation.     

Physiology and culture of the human blastocyst

The human embryo undergoes many changes in physiology during the first 4 days of life as it develops and differentiates from a fertilized oocyte to the blastocyst stage. Concomitantly, the embryo is exposed to gradients of nutrients within the female reproductive tract and exhibits changes in its own nutrient requirements and utilization. Determining the nature of such nutrient gradients in the female tract and the changing requirements of the embryo has facilitated the formulation of stage-specific culture media designed to support embryo development throughout the preimplantation period. Resultant implantation rates attained with the culture and transfer of human blastocysts are higher than those associated with the transfer of cleavage stage embryos to the uterus. Such increases in implantation rates have facilitated the establishment of high pregnancy rates while reducing the number of embryos transferred. With the introduction of new scoring systems for the blastocyst and the non-invasive assessment of metabolic activity of individual embryos, it should be possible to move to single blastocyst transfer for the majority of patients.