Connections between preimplantation embryo physiology and culture

Purpose

To review the history of experimental embryo culture and how culture media that permitted complete preimplantation development in vitro were first discovered, and the physiological insights gained.

Methods

This article reviews the history of in vitro mammalian embryo culture, in particular the efforts that led to the current generation of successful culture media and how these reflect embryo physiology, highlighting the contributions of Dr. John D. Biggers and his colleagues and students.

Results

The culture of mammalian embryos began about a century ago. However, defined media without biological fluids were only developed in the late 1950s, and the first live young born from cultured embryos, using these media, were produced by McLaren and Biggers in 1958. It wasn’t until the late 1980s, however, that preimplantation mammalian embryos could generally be cultured in vitro from fertilized eggs to blastocysts. These new media led to insights into embryo physiology, including the importance of cell volume homeostasis to early embryo viability.

Conclusions

The development of successful preimplantation embryo culture media has had a profound effect on assisted reproduction technologies and on research into early embryo physiology.     

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.     

In vitro development and metabolism of the human embryo up to the blastocyst stage

The preimplantation period begins with the fertilisation of the oocyte and ends with the formation of the blastocyst. During this period, several major events occur resulting in an embryo composed of pluripotent cells. These morphological changes correspond to changes in the embryonic metabolism. The cleavage stages are characterised by a low metabolism and the inability of the embryo to metabolise glucose. After the activation of the embryonic genome, there is a surge in the embryonic metabolism with increased demand for ATP. The embryo is then able to metabolise glucose. Recently, the importance of amino acids has been highlighted by experiments with mouse, hamster and bovine embryos. Amino acids have also been reported to benefit human embryo development in vitro. Some growth factors have been shown to play a role in human embryo development too. The importance of lipids or vitamins, however, is poorly investigated. Culture media have been developed to improve preimplantation development, but more information is required for adapting culture condition to embryonic requirements which hopefully will improve the outcome of in vitro fertilisation (IVF).     

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.     

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.     

Assessment of Embryo Viability: The Ability to Select a Single Embryo for Transfer—a Review

By being able to select the most viable embryo(s) within a given cohort it will be possible to reduce the number of embryos transferred in a given IVF procedure. Several morphological scoring systems have been proposed for the successive stages of human embryo development. Other indicators of embryo viability include rate of cleavage. Finally, non-invasive methods of assessing nutrient uptake and utilization have been developed that can be used to measure the health of individual embryos. A sequential scoring system has therefore been proposed that uses the above parameters in order to create a history for each embryo during the preimplantation period. Such systems will help lead to single embryo transfers for the majority of IVF patients.     

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.     

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.     

Advances in embryo culture systems

The growth of mammalian embryos in the laboratory has progressed nicely over the last 60 to 70 years. This has been important for basic studies of preimplantation embryo regulation of growth, development, and differentiation. It has also been critical for the establishment of assisted reproductive technologies in the clinical setting to treat infertility. During the first 40 to 50 years, the primary focus was on defining soluble media components, which fortunately has yielded improved success in the culture of embryos. More recently, attention has shifted to other aspects of culture beyond media composition. In this review we discuss recent advances in preimplantation embryo culture that deal with modifying the insoluble culture environment, producing culture environments that are dynamic and not static, systems that allow more precise gas phase and/or media pH regulation/monitoring, and platforms that integrate with culture systems to allow analysis of embryos and thus assist in selection of the embryos with the greatest implantation potential.     

Alterations in mouse embryo intracellular pH by DMO during culture impair implantation and fetal growth

The preimplantation embryo is highly susceptible to in-vitro stress, and although this does not necessarily perturb blastocyst development, it can significantly affect embryo physiology and the ability to form a viable pregnancy. This study determined that the preimplantation mouse embryo is highly sensitive to a small decrease in intracellular pH (<0.2 pH units). Embryos cultured in media containing a weak acid (5,5-dimethyl-2,4-oxazolidinedione; DMO) formed blastocysts with decreased cell number and inner cell mass number, as well as increased apoptosis, even though blastocyst development and morphology were unchanged. Interestingly, the effects were similar regardless of whether the pH stress was present for a short-term ‘acute’ exposure (during the zygote to 2-cell, or 2-cell to 8-cell division) or an extended ‘chronic’ period of time (continually from the zygote to the blastocyst stage). Exposure to DMO during the first cleavage division did not alter implantation; however, fetal weight and crown–rump length were significantly decreased (P < 0.05). In contrast, continuous exposure to DMO throughout preimplantation development reduced not only implantation but also fetal weight and crown–rump length. This study highlights the importance of correct intracellular pH and demonstrates that slight deviations can significantly impact embryo development and viability.The early embryo is known to be sensitive to its environment. This study determined the effect of a small alteration to pH of the culture environment on the ability of the embryo to grow and develop into a successful pregnancy. We found that the younger the embryo was the less capable it was to cope with the stress. Also embryos that appeared normal by observation were often not and by closer examination were showing signs of stress within the cells of the embryo resulting in poor pregnancy outcomes.     

Maternal blood platelet physiology and luteal-phase endocrinology as a means of monitoring pre- and postimplantation embryo viability following in vitro fertilization

The discovery that the fertilized mouse ovum triggers an increased demand for platelets and results in thrombocytopenia during the preimplantation phase of pregnancy provides a monitor for embryo survival and viability. This paper reports a study in which the platelet count was significantly reduced throughout the human preimplantation phase of pregnancy and returned to normal following embryo implantation. The human embryo was shown to produce a platelet activating factor in vitro which caused the reduction in platelet count after embryo transfer. This factor in the embryo culture medium could be measured using a bioassay which provided a means of assessing embryo viability prior to transfer. Some women showed no reduction in platelets after transfer. These embryos failed to produce a platelet activating factor in vitro and pregnancy was not established. Other women displayed a reduction in platelets following transfer but failed to become pregnant. All of these women had elevated luteal-phase plasma E₂ levels compared to pregnant patients, which may have interfered with the implantation process. Our observations provide a possible rapid and simple means for monitoring the viability of human embryos cultured in vitro and the survival of embryos in utero.     

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.     

Discrepancies between the effects of glutamine in cultures of preimplantation mouse embryos

A review of the literature shows divergent differences between laboratories of the effects of glutamine in mouse preimplantation embryo culture media. One laboratory reported several cases of exencephaly, which was attributed to ammonia produced by the breakdown of glutamine. Two other laboratories have found no such effects. It is suggested, but not proved, that the differences in results may have a genetic basis. Further, it is argued that studies on the toxicological actions of exogenous ammonium chloride on preimplantation development provide a biased model of the effects of glutamine as used in embryo culture protocols. The finding that ammonium can also cause exencephaly thus fosters undue concern about the teratological effects of glutamine.     

Blastocyst quality criteria

Embryo culture to the blastocyst stage has progressed enormously with the new generation of acellular culture media, or sequential media. The main advantages of embryo transfer at the blastocyst stage are that it provides a natural embryo selection during culture, or a selection using preimplantation diagnosis, it provides better conditions for SET, avoiding multiple pregnancies and overcoming repeated IVF failures by improving embryo selection. The risks are failing to select a single blastocyst or obtaining a limited number of frozen blastocysts, with reduced survival after thawing. However, prolonged embryo culture seems to be good practice for day 3 embryos with delayed development, and blastocyst transfer. More effective at day 5 than at day 6.     

Should every embryo undergo preimplantation genetic testing for aneuploidy? A review of the modern approach to in vitro fertilization

Aneuploid conceptions constitute the majority of pregnancy failures in women of advanced maternal age. The best way to combat age-related decline in fertility is through preimplantation genetic testing for aneuploidy (PGT-A). PGT-A allows for better embryo selection, which improves implantation rates with single embryo transfer and reduces miscarriage rates. Single embryo transfers decrease multiple gestations and adverse pregnancy outcomes such as preterm or low birth weight infants. Advancements in extended embryo culture, blastocyst biopsy techniques, and 24-chromosome aneuploidy screening platforms have made PGT-A safe and accessible for all patients who undergo in vitro fertilization. Improved genomic coverage of new sequencing platforms, such as next-generation sequencing, has increased the identification and diagnosis of mosaicism and partial aneuploidies in preimplantation embryos. Mosaic embryos have decreased viability compared to euploid embryos when transferred, but some mosaic embryos result in normal live births. Whole genome amplification artifacts may contribute to a misdiagnosis of mosaicism, or some mosaic embryos may self-correct to euploid after implantation. For this reason, patients without euploid embryos should be given the option of transferring mosaic embryos after genetic counseling. Further research is needed to characterize which mosaic embryos may be viable.     

Embryo-derived platelet activating factor,a marker of embryo quality and viability following ovarian stimulation for in vitro fertilization

Embryo-derived platelet-activating factor (PAF) may be an important mediator of early maternal recognition of pregnancy. PAF was higher in media associated with clinical pregnancies when compared to preclinical pregnancies but not higher in pregnant vs nonpregnant groups. The production of PAF by the preimplantation embryo was not related to follicle size or embryo morphology. However, differences in PAF concentrations in the culture media were related to the age of the embryo culture medium and the developmental stage of the embryo.     

Is there still a place for co-cultures in the era of sequential media?

Co-cultures have been advocated in assisted reproduction owing to the inadequacy of simple media to support embryo development beyond the cleavage stage. Different human and non-human cells and cell lines have been used for co-cultures. High rates of blastocyst formation have been reported with the use of co-cultures, and they have been proposed as a salvage treatment option in couples with repeated implantation failures. Since the advent of complex sequential media, which yield very high blastocyst formation and blastocyst implantation rates, the need for co-cultures has been questioned. Upon review of the literature, it is evident that well-designed randomized studies that compare co-cultures with simple or sequential media do not exist. Progression to the blastocyst stage for cleavage stage embryos appears to be similar, if not better, for embryos that are cultured in modern sequential media, rendering the use of co-cultures obsolete. Furthermore, there is no consensus regarding the necessity of sequential media, as similar results have been obtained with a single medium formulation that supports all stages of the preimplantation period. Whether co-cultures are beneficial in patients with repeated implantation failures, however, should be investigated in randomized trials. Co-cultures still serve as powerful tools for understanding embryo metabolism. Furthermore, co-cultures may be instrumental in studying expression of implantation-related genes and embryo-endometrium interaction.     

Developmental consequences of cryopreservation of mammalian oocytes and embryos

During the last three decades, significant advances have been made in successful cryopreservation of mammalian preimplantation embryos, and more recently oocytes. The ability to cryopreserve, thaw, and establish pregnancies with supernumerary preimplantation embryos has become an important tool in fertility treatment. Human oocyte cryopreservation has practical application in preserving fertility for individuals at risk of compromised egg quality due to cancer treatments or advanced maternal age. While oocyte/embryo cryopreservation success has increased over time, there is still room for improvement. Oocytes and embryos are susceptible to cryo-damage, which collectively entails cellular damage caused by mechanical, chemical, or thermal forces during the freeze-thaw process. Basic studies focused on understanding cellular structures, their composition, and more importantly their functions, in normal cell developments will continue to be critical in assessing, understanding, and correcting oocyte/embryo cryo-damage. This review will delineate many of the oocyte/embryo intracellular and extracellular structures that are or may be compromised during cryopreservation. A global theme presented throughout this review is that many structural components of the oocyte/embryo also have essential functional roles in development. Compromising these cellular structures, and thus their cellular homeostatic functions, can deleteriously influence initial cryo-survival or compromise subsequent normal development through effects on the oocyte and/or early embryo.     

Gamete/embryo - oviduct interactions: implications on in vitro culture

Fertilization and development of mouse embryos occur in the oviduct. Accumulating data suggested that embryo-maternal communication exists in the preimplantation period, with the female reproductive tract providing the optimal microenvironment conducive to the development of embryos. Signals produced from the developing embryos not only affect their own transport in the oviduct, but the physiology and gene expression patterns of the oviduct. As a step towards understanding the action of embryos on oviductal physiology, both genomics and proteomics approaches are being used to unveil the underlying mechanism of embryo-maternal interaction at the preimplantation stage. Results from recent studies allow us to better understand the roles and the use of oviductal secretory proteins or factors that affect embryo development in vivo and in vitro. It has been shown that in vitro culture alters gene expression of the cultured embryos and may predispose the embryo to certain disease. Therefore, the interaction between gamete/embryo and oviduct in vitro and in vivo, and the long-term effects of embryo culture on foetal development warrant further investigation.