The embryo is a dynamic structure that can be affected by the interaction with the surrounding environment. During its journey through the female reproductive tract from fertilization to implantation, the embryo undergoes numerous biochemical and physiological changes which are essential for a successful reproductive outcome. During successive cleavage rounds, the embryo increases in cell number, switches from maternal to embryonic genome control (embryonic genome activation; EGA) and forms cell–cell junctions. This coincides with the cells flattening and compacting at the morula stage (Coticchio et al., 2019). At the final stage of the preimplantation period, the blastomeres differentiate to form the trophectoderm and the inner cell mass cell lineages. The blastocyst undergoes remarkable events in preparation for implantation and establishment of pregnancy, including initiation of overall growth, significant rise in transcriptional activity, increased protein synthesis, and active Na+/K+ ATPase activity in the trophectoderm leading to the formation of the blastocoel cavity (reviewed by Smith & Sturmey, 2013). The blastocyst also improves homeostatic regulatory mechanisms, including defense against oxidative damage (Lane & Gardner, 2000). These changes are energy dependent, and therefore underpinned by specific metabolic pathways. Disruptions in energy production during the preimplantation period are related to embryonic developmental impairment and reduced fetal viability post-transfer (Gardner, ; Lane & Gardner, 2005b). For these reasons, metabolism is considered a key determinant of embryo competence and viability.
4.1 Embryo Metabolism: An Overview
The embryo is a dynamic structure that can be affected by the interaction with the surrounding environment. During its journey through the female reproductive tract from fertilization to implantation, the embryo undergoes numerous biochemical and physiological changes which are essential for a successful reproductive outcome. During successive cleavage rounds, the embryo increases in cell number, switches from maternal to embryonic genome control (embryonic genome activation; EGA) and forms cell–cell junctions. This coincides with the cells flattening and compacting at the morula stage (Coticchio et al., 2019). At the final stage of the preimplantation period, the blastomeres differentiate to form the trophectoderm and the inner cell mass cell lineages. The blastocyst undergoes remarkable events in preparation for implantation and establishment of pregnancy, including initiation of overall growth, significant rise in transcriptional activity, increased protein synthesis, and active Na+/K+ ATPase activity in the trophectoderm leading to the formation of the blastocoel cavity (reviewed by Smith & Sturmey, 2013). The blastocyst also improves homeostatic regulatory mechanisms, including defense against oxidative damage (Lane & Gardner, 2000). These changes are energy dependent, and therefore underpinned by specific metabolic pathways. Disruptions in energy production during the preimplantation period are related to embryonic developmental impairment and reduced fetal viability post-transfer (Gardner, 1998; Lane & Gardner, 2005b). For these reasons, metabolism is considered a key determinant of embryo competence and viability.
As in other cells, metabolism in the embryo aims to provide energy (mainly by producing ATP, NADH, and FADH2) to maintain normal cellular function as well as to provide precursors for the macromolecule synthesis (Figure 4.1). Embryo energy metabolism follows a generally accepted pattern of progressing from being relatively quiescent during cleavage to an overall boost of oxygen and glucose consumption as well as lactate production at the blastocyst stage (Leese, 2012). The cleavage stage embryo uses pyruvate as a major source of energy, which is obtained from glycolysis or the external environment and consumed at all stages of preimplantation development (Lewis & Sturmey, 2015).
Figure 4.1 Simplified overview of energy metabolism. Glucose can enter the cell and take part in a variety of pathways; however, only glycolysis can generate ATP. The end product of glycolysis is pyruvate. This may be converted into lactate and excreted by the cell, or the pyruvate may be taken into the mitochondria and converted into acetyl Co A. Pyruvate itself may be taken up by the cell and used directly to make acetyl Co A. Acetyl Co A can also by synthesized by fatty acid catabolism or conversion of a number of amino acids. Acetyl Co A feeds into the TCA cycle which occurs in the mitochondrial matrix and produces the electronic carriers NADH and FADH2. These transport electrons to Complex 1 and Complex 2 respectively of the electron transport chain (ETC). The final reaction of the electron transport chain is the phosphorylation of ADP to make ATP and the reduction of molecular oxygen to water. This is the major cellular consumer of oxygen and is the reason that measurement of oxygen consumption is a good marker of mitochondrial activity.
After compaction and blastocyst formation, glycolysis becomes prevalent, leading to rising glucose consumption rates (Leese, 2012).
Carbohydrates and energy metabolism have long been the focus of attention while studying embryo development, but there is now compelling evidence that other components, including amino acids and lipids, are crucial for correct development of the embryo during the preimplantation period.
Embryo metabolism is mostly quantified as the uptake and release of different compounds into the culture medium (Leese & Barton, 1984; Guerif et al., 2013). Despite the inherent variability in metabolism between embryos, viable embryos appear to share the feature of “lower” levels of metabolic activity compared to those that are nonviable. This was elegantly summarized by Leese in 2002 (Leese, 2002) which lead to the “Quiet Embryo Hypothesis.” This was refined further to include molecular determinants leading to these distinct phenotypes (Baumann et al., 2007), the concept of a “quiet range” (Leese et al., 2007) and different categories of quietness (Leese et al., 2008a). The most recent update of the initial hypothesis includes the concept of a “Goldilocks zone” or “lagom,” which refers to the “just-right” range within which those embryos with maximum developmental potential are located. This would imply a minimum threshold to maintain energy homeostasis and an upper limit to maximize cellular metabolism performance (Leese et al., 2016, Leese et al., 2019).
4.2 What Does an Embryo Need, and Why?
As previously described, the embryo changes dramatically from fertilization to implantation as it moves from the fallopian tube to the uterus. Those changes are critical for embryo development and are driven largely by the energy requirements at each stage, which mirror ATP demands. In order to satisfy these needs, the embryo adapts its energy metabolism and uses a variety of sources from those present in the physiological environment to which it is sequentially exposed (Gardner et al., 1996). The mammalian oviduct and uterine fluids are composed of a myriad of factors secreted by epithelial cells or derived from blood plasma, including energy substrates traditionally added to culture media (i.e., glucose, lactate, and pyruvate) as well as albumin, glycoproteins, amino acids, growth factors, and electrolytes (e.g., potassium and bicarbonate) (Leese, 1988; Aguilar & Reyley, 2005; Leese et al., 2008b; Cheong et al., 2013; Kermack et al., 2015). In vitro culture must replicate those conditions as closely as possible in order to reduce the stress that the embryo suffers when taken from its physiological environment (Gardner & Leese, 1990).
Glucose, lactate, and pyruvate are major nutrients for ATP production and normal cellular development. Apart from energy sources, these molecules play a variety of important roles: glucose is involved in biosynthetic processes (e.g., nucleic acid and protein synthesis) and acts as a cell signaling factor. Glucose is also required for embryos to express specific nutrient transporters, whose activity, in turn, regulates intracellular pH and the reactive oxygen species-mediated stress response (Leese, 2012). It has been suggested that at time of implantation, lactate facilitates trophoblast invasion by lowering the pH and promoting uterine tissue degradation as well as by acting as a signaling factor to induce endometrial immune and vascular responses (Gardner, 2015). Pyruvate appears to be essential to overcome the so-called “2-cell block” in mice and must be present in a specific time frame, as shown by Nagaraj et al., (2017). They demonstrated that EGA occurs only when the enzyme pyruvate dehydrogenase locates to the nucleus at the 2-cell stage in mice or 4-cell stage in human, for which the presence of pyruvate is required (Nagaraj et al., 2017). When pyruvate is not present, the normal phenotype can be rescued by precursors of pyruvate, a-ketoglutarate, and arginine. This key observation expands on the traditional view of metabolism beyond the provision of energy and indicates molecular regulatory roles for metabolic processes.
Protein synthesis is low during early stages of embryo development and increases at the blastocyst stage, concomitant with embryo growth. However, exogenous amino acids supplied by culture media are incorporated into proteins during the early preimplantation stages, which justifies their wide presence in current culture media (Epstein & Smith, 1973; Summers & Biggers, 2003). Often, amino acids are classified into two groups – so called essential and non-essential. The origin of these terms come from whole body physiology, and relates to whether they can be synthesized de novo or whether they need to be taken up from dietary sources – see Box 4.1 for further explanation. The aspects of amino acid turnover in relation with embryo development and supplementation in culture media have been comprehensively reviewed by Sturmey et al. (2008). Apart from their classical function as building blocks for protein synthesis (Crosby et al., 1988), amino acids play a variety of key roles in the preimplantation embryo. They may act as chelators and antioxidants (Suzuki et al., 2007), regulate the intracellular pH (Edwards et al., 1998a), and serve as organic osmolytes (Dawson & Baltz, 1997). Amino acids also mediate important signaling processes for development and implantation (reviewed by Van Winkle et al., 2006); e.g. it has been suggested, that leucine and arginine regulate trophoblast motility at the time of implantation in mice (Gonzalez et al., 2012). Furthermore, amino acids provide precursors for nucleotide, GTP, and NAD+ synthesis (Leese et al., 1993) and can regulate carbohydrate metabolism (Lane & Gardner, 2005a; Mitchell et al., 2009).
The 20 common protein-encoding amino acids are commonly placed into two convenient groups: essential or non-essential. This categorization is based on whether amino acids can be made by the body from other substrates (i.e., nonessential) or whether they must be provided from the diet, because they cannot be synthesized de novo in sufficient quantities to satisfy needs (i.e., essential). This is an unfortunate terminology since it implies that some amino acids are not essential; however, all of the 20 amino acids are components of protein.
Typically, eight amino acids are considered essential – leucine, isoleucine, valine, threonine, methionine, phenylalanine, tryptophan, and lysine. These distinctions are based on knowledge of whole body nutrition of adults.
To complicate matters further, a further seven amino acids – arginine, histidine, glycine, tyrosine, glutamine, cysteine, and proline – are essential for children, since de novo synthesis is insufficient to satisfy demand. These are often termed conditionally essential.
Thus, applying the concept of essential and non-essential amino acids to the embryo may not be truly representative of the needs of the developing embryo in vitro.
Human embryos show stage-specific patterns of amino acid release and consumption (Houghton et al., 2002; Brison et al., 2004; Stokes et al., 2007) and display general patterns of glutamine, leucine, and arginine consumption as well as alanine release throughout the preimplantation period (Houghton et al., 2002; Brison et al., 2004; Stokes et al., 2007). Given the importance of amino acid functions in embryo development, many studies have researched the possibility of using them as predictors of IVF success. Viable embryos showed a different metabolic profile from that of nonviable embryos in different species, including humans (Houghton et al., 2002; Brison et al., 2004; Orsi & Leese, 2004a; Humpherson et al., 2005; Sturmey et al., 2009). Interestingly, the metabolic signature has also been related to embryo sex (Sturmey et al., 2010; Gardner et al., 2011) as well as to DNA integrity, genetic health and assembly of trophectoderm cell junctions (Eckert et al., 2007; Picton et al., 2010; Sturmey et al., 2010). The amino acid profile is therefore an indirect evidence of the roles mentioned above, giving a snapshot of the physiological status of the embryo.
In addition to exogenous nutrients, embryos also utilize some endogenous energy stores, such as fatty acids. Fatty acids are constituents of lipids and part of all cell membranes. In addition, fatty acids are stored in cells as cytoplasmic hydrophobic lipid droplets, essentially formed by a neutral core of triglyceride and cholesterol esters coated by a phospholipid monolayer and proteins (Walther & Farese, 2012). The amount and distribution of lipid droplets is species specific. Pig, cattle, and sheep oocytes and embryos show significant levels of fatty acids (Coull et al., 1998; Ferguson & Leese, 1999; McEvoy et al., 2000; Sturmey & Leese, 2003; Leroy et al., 2005; Sturmey et al., 2006; Sudano et al., 2011), which are responsible for their characteristic dark appearance compared to those of mice or human, which have low and moderate lipid content, respectively (Matorras et al., 1998; Haggarty et al., 2006; Bradley et al., 2016). Fatty acids are important sources of energy for preimplantation embryo development, and the importance of fatty acid for embryo development has been demonstrated in species with lower lipid content, such as mice and human. Using a forced selective autophagy of lipid droplets system, it has been demonstrated that mouse embryos require endogenous lipids to develop, showing decreased triglyceride levels and developmental impairment when the number of lipid droplets was reduced (Tatsumi et al., 2018).
Energy metabolism is compartmentalized in the cytosol (glycolysis) or the mitochondria (TCA cycle, β-oxidation, and oxidative phosphorylation) of living cells (Figure 4.1). In the egg, pyruvate and lactate are produced from glucose by the cumulus cells (Leese & Barton, 1985; Gardner & Leese, 1990; Gardner et al., 1996). In the cleavage stage embryo, before compaction, the majority of ATP is produced through mitochondrial oxidation of pyruvate, as well as low levels of lactate (from the 2-cell stage) and certain amino acids. Around 85–90% of ATP in pre-compaction embryos is derived through oxidative phosphorylation (Thompson et al., 1996; Sturmey & Leese, 2003).
It is noteworthy that despite oxidative metabolism being the main source of ATP during the pre-compaction period, overall oxygen consumption rates remain low until the blastocyst stage, when energy demands increase (Houghton et al., 1996; Goto et al., 2018). Indeed, postimplantation oxygen consumption rates of human embryos are similar to those of pre-compaction stages, highlighting the high energy demands accompanying blastocyst formation and largely derived from the Na+/K+ ATPase activity (Houghton et al., 2003). Oxidation of pyruvate through the TCA cycle is the main source of energy production, but other substrates, such as amino acids, can be incorporated into this metabolic pathway. In particular, glutamine appears important for embryo development in cattle, mice, pig, and human (Devreker et al., 1998; Steeves & Gardner, 1999; Houghton et al., 2002; Brison et al., 2004; Rezk et al., 2004; Chen et al., 2018).
Accompanying the rise in energy demands at the blastocyst stage is a fall in ATP levels as it is rapidly consumed, and a simultaneous increase in AMP levels. An important aspect of glucose metabolism in the blastocyst is that, in the presence of oxygen, they do not oxidize all the glucose but rather convert part of it to lactate. This phenomenon is known as aerobic glycolysis and mirrors the so-called Warburg effect, which appears to be a feature of rapidly dividing cells, such as cancer cells (Warburg, 1956; Redel et al., 2012). The parallels between the metabolism of embryos and cancer cells have been reviewed by Smith and Sturmey (2013). The conversion of glucose to lactate is a rapid method of generating ATP, but it is rather inefficient at producing cellular energy. Consequently, the high levels of aerobic glycolysis may not be intended solely to respond to the increasing energy requirements of the blastocyst. Other reasons could be the provision of precursors for biosynthetic processes (e.g., synthesis of proteins, lipids, complex sugars, and moieties for nucleic acid synthesis as well as NADPH from metabolizing glucose though the pentose phosphate pathway (Lewis & Sturmey, 2015) and the adaptation of the blastocyst to the hypoxic environment during implantation (Leese, 1995). One intriguing idea to explain the rise in glycolysis is the increase in production of lactate, which when excreted will cause a local fall in pH. It was postulated by Gardner that this might be important in facilitating invasive implantation (Gardner, 2015).
From fertilization to implantation in vivo, the embryo is exposed to dynamic conditions of pH, nutrient availability, and oxygen levels through the female genital tract. These physiological conditions differ markedly compared to the environment in vitro, which compromise their homeostasis and elicit stress responses. As in vivo studies in human are largely impractical, animal models have provided valuable knowledge about the different regulation of metabolism occurring between in vivo vs. in vitro embryos. The mouse is considered an appropriate animal model to study this comparison due to the ease and speed of collecting in vivo-derived embryos. Thus, it has been reported that the glycolytic rates of in vitro-derived blastocysts are higher than those of in vivo blastocysts and this is associated with decreased implantation and viability after transfer (Gardner & Leese, 1990; Lane & Gardner, 1996; 1998). Exposing in vivo-developed mice blastocysts to in vitro culture conditions induces metabolic adaptation in just 3 hours. Such effects are milder when in vivo-derived blastocysts are cultured in the presence of amino acids and vitamins, underlining the importance of culture media composition for correct metabolic status (Lane & Gardner, 1998). The impact of in vitro culture on metabolism can also be seen in cattle embryos, which exhibit reduced glucose oxidation – and therefore higher glycolysis, in agreement with data from mice – and higher amino acid turnover compared to their in vivo-derived counterparts (Thompson, 1997; Sturmey et al., 2010). The extent of this impact in embryo development is time-dependent: the longer the exposure to in vitro conditions, the higher the impact (Merton et al., 2003). In vitro culture also induces changes in gene expression related to lipid metabolism and oxidative stress, as shown when in vitro vs. in vivo generated cattle or porcine blastocysts were compared (Bauer et al., 2010; Gad et al., 2012). Moreover, DNA methylation patterns are also affected by in vitro conditions, leading to the transmission of hypomethylated marks on imprinted genes in in vivo-derived cattle blastocysts exposed to in vitro culture before the EGA (Salilew-Wondim et al., 2018).
4.5 How Are Stress and Metabolism Linked?
Gamete and embryo manipulation in vitro cause cellular stress, which is linked to altered gene expression patterns. This occurs through stress pathways, such as altered metabolic status, in response to environmental perturbations (Leese et al., 1998; Thompson et al., 2002). Whether there are particularly sensitive windows of development remains unclear. However, one view is that the embryo is more sensitive to environmental stress before EGA occurs (Brison et al., 2014).
Intracellular pH undoubtedly affects metabolic activity by altering the activity of different enzymes and disruption of specific molecular pathways, including glycolytic and oxidative metabolism (Edwards et al., 1998b; Lane & Gardner, 2000). Human embryos have the ability to buffer their own intracellular pH to a certain extent (Phillips et al., 2000). In mice, buffering capacity is stronger from compaction and blastocyst formation, when the number of cell–cell junctions increases (Edwards et al., 1998a). This plasticity makes sense, considering the physiological conditions as the embryo is successively exposed to change from alkaline pH in the fallopian tube (pH 7.1–8.4) to a more acidic pH (pH 7.3–7.9) in the uterine endometrium (Ng et al., 2018). However, this plasticity can be modified by extracellular pH in culture conditions; slight variations in the extracellular pH during embryo manipulation in mice led to developmental and genetic disorders (Koustas & Sjoblom, 2011). The aspects of pH in the culture environment have been reviewed in detail in Swain, 2010 and Swain, 2012. Concomitant with pH is the effect of temperature in culture systems, as any variation is reflected by a change in pH (Wale & Gardner, 2016). This is usually solved by using buffering systems (e.g., bicarbonate/carbon dioxide).
The concentration of ambient oxygen can further impact embryo development and reproductive outcome. In all mammalian species including humans, atmospheric oxygen levels (20%) have proved inferior compared to low oxygen levels (5%) at supporting both cleavage and post-compaction stages of development (Thompson et al., 1990; Batt et al., 1991; Catt & Henman, 2000; Kovacic & Vlaisavljevic, 2008; Kovacic et al., 2010; Wale & Gardner, 2010; Bontekoe et al., 2012; Zaninovic et al., 2013). This is unsurprising, given that the physiological oxygen levels in the mammalian oviduct range from 5 to 8.7% (Fischer & Bavister, 1993). Indeed, oxygen levels in the female reproductive tract drop from 5 to 7% in the fallopian tube to around 2% in the uterus, where implantation occurs (Fischer & Bavister, 1993). Based on that, it has been recently suggested that in vitro culture systems with ultra-low levels of oxygen (2%) from the blastocyst stage were advantageous for embryo development (Morin et al., 2017; Kaser et al., 2018). However, other studies did not find any effect after sequential in vitro culture from low to ultra-low oxygen levels (De Munck et al., 2019).
The metabolism of oxygen is important for embryo development, but it can be a source of oxidative stress via the production of reactive oxygen species (ROS). Low levels of ROS, which are produced as a result of redox reactions and oxidative phosphorylation, are needed to ensure correct signaling pathways in physiological conditions (Hancock et al., 2001). However, rising levels of ROS can cause ATP depletion, mitochondrial damage, DNA damage, and alterations of cell constituents, such as lipids and proteins (Guerin et al., 2001), as well as developmental arrest (Balaban et al., 2005; Favetta et al., 2007). Although the optimal metabolic conditions to ensure a proper oxidative balance have not been established, the inclusion of compounds such as metal chelators, thioredoxin, and certain vitamins can help regulate embryonic ROS production (Guerin et al., 2001). Results from experiments in mice suggest that the addition of antioxidants to the culture media may be beneficial (Truong et al., 2016; Truong & Gardner, 2017).
Ammonium, which is toxic for mammalian embryos, is spontaneously built up from amino acid deamination at 37°C. Under stress, embryos release increased amounts of ammonium and lactate to the culture medium, modifying the intracellular pH, and unbalancing the glycolytic activity in order to restore homeostasis (Lane & Gardner, 2000; Lane & Gardner, 2003; Dagilgan et al., 2015). High levels of ammonium in culture medium can result in the differential expression of genes involved in metabolism, cell communication, and development, among others (Gardner et al., 2013). Of note, it has been reported that ammonium levels can accumulate in IVF culture medium, both following prolonged storage and also at 37°C when supplemented with glutamine and proteins (Kleijkers et al., 2016). This is enhanced by culture at atmospheric oxygen levels (Wale & Gardner, 2013). Importantly, both oxygen and ammonium alter the overall amino acid metabolism in an independent manner, high oxygen being the most detrimental (Orsi & Leese, 2004b; Wale & Gardner, 2013).
Each of the stress-derived effects described above relies on the disturbance of embryo metabolic homeostasis. These effects are likely to persist throughout the post-natal life and subsequent generations by changing transcriptional expression and epigenetic patterns according to the Developmental Origins of Health and Disease (DOHaD) hypothesis (Barker, 2007; Reid et al., 2017).
Stress-related effects in in vitro culture systems clearly influence the outcome of in vitro embryo technologies, but is it possible to reverse those effects? The idea of manipulating metabolism in vitro to improve IVF outcomes is tempting and seems feasible, especially given that the metabolic profile of embryos can be modified comparatively simply. For example, studies in mice have shown that maternal low protein diet during the preimplantation period induce metabolic changes in the maternal serum and uterine secretions, showing reduced insulin and amino acids and increased glucose levels in serum, as well as reduced amino acid availability in the uterine fluid. Those changes were concomitant with abnormal blastocyst growth and metabolic signaling, which the authors interpreted as compensatory responses to the environmental challenges sensed by the embryo (Eckert et al., 2012). The transfer of blastocysts cultured in insulin and amino acid-depleted media in a follow-up study resulted in higher weight (both at birth and early postnatal life), increased blood pressure in males, and decreased heart to body weight ratio in females compared to controls (Velazquez et al., 2018). In another example, it was recently reported that the administration of a mitochondrial targeted antioxidant can rescue the developmental competence and quality of cattle embryos exposed to lipotoxicity and oxidative stress during oocyte maturation (Marei et al., 2019).
In mice, it has been reported that advanced maternal age induces altered programming in blastocysts leading to abnormal postnatal cardiometabolic profiles, i.e., body weight, blood pressure, and glucose metabolism (Velazquez et al., 2016). Supplementation of the culture medium with dichloroacetic acid, a stimulator of pyruvate dehydrogenase, boosted the mitochondrial activity and improved the development of embryos derived from aged mice (McPherson et al., 2014). Whether such responses would be apparent in human embryos is unknown, but negative effects of maternal ageing include increased aneuploidy rates (Capalbo et al., 2017; Shi et al., 2019), preterm birth (Kenny et al., 2013) and low birth weight (Fall et al., 2015). The idea of adapting the composition of culture medium to influence metabolism with the intention of minimizing negative responses to stress is therefore a fascinating avenue for future study. However, like any new advances in reproductive medicine, any research into this area must be fully supported by well conducted randomized control trials and subsequent follow-up before made widely available to patients.
4.7 Summary, Conclusion, and Future Prospects
In summary, the embryo undergoes dynamic changes during preimplantation development showing concordant utilization patterns of substrates such as glucose, lactate, pyruvate, amino acids, and fatty acids – summarized in Figure 4.2. These nutrients play important roles beyond energy production. The embryo is responsive to the external environment and this response is driven by its metabolic activity, which ultimately also determines its genetic status. Metabolic profiling may have some utility as a noninvasive approach to give us a snapshot of embryo physiology. However, there are some methodological limitations hampering translation of metabolic studies. In the way it has been done so far, measuring metabolism does not directly focus on specific pathways or the flux of metabolites across them, but only the end products of metabolism at specific and static time points. Furthermore, all current knowledge about human embryos is derived from either in vitro studies or animal studies. Many aspects of the physiological preimplantation environment remain to be discovered and could allow us to improve on current IVF outcomes; an example of this is the fact that adding low doses of oviduct and uterine fluid to the culture medium improves embryo quality and development in cattle (Hamdi et al., 2018). Apart from the variety of molecules mentioned in this chapter which are present in the oviduct and the uterus, other components such as miRNA and extracellular vesicles remain comparatively unexplored; the effects of these compounds in embryo development are unknown.
Figure 4.2 Summary ‘heatmap’ of embryo metabolism. Increasing color intensity indicates increased activity, with red indicating substrate depletion and green indicating release. Blue represents ‘activity’.
Emerging data suggest that IVF might affect the health of the offspring and in vitro culture systems are in some aspects being questioned (Sunde et al., 2016). Over 8 million babies have been born from IVF worldwide (De Geyter et al., 2018). These data highlight the importance of developing controlled in vitro culture systems in order to ensure the safety of IVF children. Culture media, pH, temperature, and oxygen levels are some of the factors that should be carefully considered. In conclusion, culture systems should fulfil the energy demands of the embryo but must also balance their metabolic homeostasis. Optimization of media formulations must be based on evidence, and the application of any change in their composition as well as any new technology to the IVF clinical context must be carefully evaluated (Harper et al., 2012).