The effect of light on embryos and embryo culture
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Maintaining consistent and reliably high success rates is a daily challenge for every IVF laboratory. This step-by-step guide is an essential aid in navigating the complex maze of physical, chemical, biological, and logistic parameters that underpin successful gamete and embryo culture: temperature, pH, osmolality, gas supplies, air quality, light exposure, infections, managing supplies, personnel, as well as overall quality control. Numerous real-life troubleshooting case reports are presented, identifying all aspects necessary for troubleshooting. Process maps and flow charts accompanying each chapter offer a logical and systematic approach to problem solving in the laboratory. This is an essential resource for scientists in assisted reproductive technology and specialists in reproductive biology and medicine, helping IVF clinics to achieve the dream of every infertile couple: the birth of a healthy child.
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... Because light generates reactive oxygen species (ROS), oxidative stress is considered one of the plausible causes at the origin of the embryonic lesion. The harmful effects of light are associated with the generation of H2O2 in peroxisomes and mitochondria [103], activation of stress genes, or direct DNA damage via ionization [100]. The importance of ROS is decreased at the blastocyst stage because the embryo shifts from oxidative phosphorylation to aerobic glycolysis for sustenance protein synthesis and ion transport systems [97,98]. ...
... The reproductive tract not only produces oocytes, but also protects gametes and the embryo from visible light exposure. During the processes of assisted reproduction, (retrieval of oocytes, preparation of the sperm, IVF or ICSI procedure, incubation and microscopic examination of formed embryos, embryo transfer), gametes, zygotes, and embryos are subjected to a variable spectrum of light from different sources, including safety cabinets, microscopes, or time-lapse imaging cameras [100,101]. ...
... Because light generates reactive oxygen species (ROS), oxidative stress is considered one of the plausible causes at the origin of the embryonic lesion. The harmful effects of light are associated with the generation of H 2 O 2 in peroxisomes and mitochondria [103], activation of stress genes, or direct DNA damage via ionization [100]. ...
Based on current findings, the presence of oxidative stress has a significant impact on the quality of gametes and embryos when performing assisted reproductive techniques (ART). Unfortunately, in vitro manipulation of these cells exposes them to a higher level of reactive oxygen species (ROS). The primary goal of this review is to provide a comprehensive overview of the development of oxidative stress in female and male reproductive systems, as well as in the case of the pre-implantation embryo and its environment. This review also focuses on the origins of ROS and the mechanisms of oxidative stress-induced damage during ART procedures. A well-known but underestimated hazard, light exposure-related photo-oxidation, is particularly concerning. The effect of oxidative stress on ART outcomes, as well as the various strategies for preventing it, are also discussed. We emphasize the role and significance of antioxidants and light protection including forms, functions, and mechanisms in the development of gametes and embryos in vivo and in vitro.
... In parallel with the above findings, both the intracytoplasmic concentration of reactive oxygen species (ROS) and the incidence of embryonic cell apoptosis were increased [53]. Similar findings were reported in several cases [54][55][56][57][58]. ...
Recently, infertility has become one of the most important endemic conditions, affecting approximately 15–20 % of couples worldwide. Among others, the careerist lifestyle, the increasing maternal age and the parallel increment in the aneuploidy rate of embryos play a crucial role in this phenomenon. In this study, embryological parameters and pregnancy outcomes were investigated in IVF cycles using either sequential embryo culture or a single step culture system. By sequential media, oocytes/embryos are needlessly exposed to the potentially negative effects of light exposure, temperature decrement and altered oxygen tension. In comparison with sequential media, single step media induced 1.28, 1.21 and 1.21-fold increments in implantation, biochemical pregnancy and clinical pregnancy rates, respectively. Pregnancy outcomes showed strong maternal age-dependency, so the difference between the two investigated culture systems was equalized by the increasing maternal ages (35–44 years) and the supposed incidence of embryo aneuploidy. Nevertheless, the significant enlargements in the outcomes of the younger ages (25–34) induced by the single step cultures suggest that, beside the resultant maternal aneuploidy, aneuploidy (reduced pregnancy rates) may evolve from exposure to the mentioned environmental stress factors.
The process of embryonic development is crucial and radically influences preimplantation embryo competence. It involves oocyte maturation, fertilization, cell division and blastulation and is characterized by different key phases that have major influences on embryo quality. Each stage of the process of preimplantation embryonic development is led by important signalling pathways that include very many regulatory molecules, such as primary and secondary messengers. Many studies, both in vivo and in vitro , have shown the importance of the contribution of reactive oxygen species (ROS) as important second messengers in embryo development. ROS may originate from embryo metabolism and/or oocyte/embryo surroundings, and their effect on embryonic development is highly variable, depending on the needs of the embryo at each stage of development and on their environment ( in vivo or under in vitro culture conditions). Other studies have also shown the deleterious effects of ROS in embryo development, when cellular tissue production overwhelms antioxidant production, leading to oxidative stress. This stress is known to be the cause of many cellular alterations, such as protein, lipid, and DNA damage. Considering that the same ROS level can have a deleterious effect on the fertilizing oocyte or embryo at certain stages, and a positive effect at another stage of the development process, further studies need to be carried out to determine the rate of ROS that benefits the embryo and from what rate it starts to be harmful, this measured at each key phase of embryonic development.
Introduction The 2010 Nobel Prize in Medicine was awarded to Dr. Robert Edwards in recognition of his efforts toward development of human in vitro fertilization (IVF); which gave rise to the birth of the world's first IVF-baby Louise Brown in 1978 [1] and millions of births since. In fact, laboratory growth of mammalian embryos in culture media with defined components was described almost 30 years before the first successful human IVF cycle [2-4]. These studies set foundations for refinements of media composition based on mammalian embryo requirements [5-12] and they were important for the determination of IVF conditions in various species. Several culture media were developed and have been in use since the establishment of modern embryo culture; however, the static nature of systems for embryo culture remains unaltered. Usually, media for embryo culture are restrained in a culture dish placed into an incubator with a controlled atmosphere [13]. The static environment clearly sustains mammalian embryo development; nevertheless, it does not mirror the environment experienced by oocytes and preimplantation embryos within the body.
Assisted reproductive technologies have had a profound impact on biomedical research through transgenic animals, food supply and production, as wells as genetic gain of domestic species, and treatment of human infertility. While significant advances in embryo culture have occurred over the last few decades. In Embryo Culture: Methods and Protocols, expert researchers in the field detail many of the methods which are now commonly used to study human embryo culture. These include emerging methods and the impact of embryo culture on epigenetics and offspring health is presented to set the stage for future research and laboratory application involving embryo culture. Written in the highly successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Embryo Culture: Methods and Protocols seeks to aid scientists in the further study of this crucially important field of embryology and assisted reproductive technologies.
Background/Aim: Spermatozoa from inbred mice provide a useful model to study male infertility as they produce a low number of zygotes with conventional IVF. This study aimed to assess whether the use of zona laser dissection might improve the number of zygotes developing after conventional IVF with inbred spermatozoa. Materials & Methods: Mouse oocytes from an inbred C57BL/6J strain and a hybrid B6D2F1 strain were allocated to either conventional IVF or laser assisted IVF, using spermatozoa from the inbred strain C57BL/6J. Laser assisted IVF involved use of a 1.48 diode microsurgical laser device to ablate three holes into each zona immediately prior to insemination. Results: Use of a laser significantly improved the zygote production rate compared with zona intact oocytes (88.4% vs. 18.3% for inbred oocytes, and 92.3% vs. 1.6% for hybrid oocytes, P Conclusion: Zona laser dissection is a simple technique that avoids the need for ICSI, significantly reduces operator time and allows numerous oocytes to be treated in a short period. Since high numbers of zygotes can develop using laser assisted IVF for this animal model where the spermatozoa are of poor quality, one might speculate that laser assisted IVF may have a possible role in the treatment male infertility in a clinical setting.
This fully updated new edition of a successful and popular practical guide is an indispensable account of modern in-vitro fertilization practice. Initial chapters cover theoretical aspects of gametogenesis and embryo development at the cellular and molecular level, while the latter half of the book describes the requisites for a successful IVF laboratory and the basic technologies in ART. Advanced techniques, including pre-implantation genetic diagnosis, vitrification and stem-cell technology, are comprehensively covered, providing up-to-date analyses of these groundbreaking technologies. This edition includes:• New practical techniques, including preservation of fertility for cancer patients, stem-cell biology/technology, vitrification and in-vitro maturation• A 'refresher' study review of fundamental principles of cell and molecular biology• The latest information available from animal and human research in reproductive biologyPacked with a wealth of practical and scientific detail, this is a must for all IVF practitioners.
Legislation on ART varies considerably in Europe. Rules and regulations regarding procedures such as gamete donation, embryo cryopreservation, and other aspects of ART are not identical between the European countries. The ESHRE Web site gives an excellent illustration of the situation in the various countries in Europe in this respect. Details regarding specific requirements for laboratories vary as well; examples of this are the requirement of ISO 15189 accreditation of ART laboratories in some countries and ISO 9001 certification in several other countries, etc. Therefore, we have decided to focus here on common requirements with reference to the EU Tissues and Cells Directive and give further details in a summary at the end of each section of the chapter. Although some laboratories in Europe may currently be operating without fulfilling these standards in all details, they will in the coming years be expected to do so.
Significantly more mouse zygotes developed to blastocysts in culture in a medium formulated on the composition of human tubal fluid (HTF) than in modified Tyrode's medium (T6). In a randomized 2×2 factorial trial of human in vitro fertilization that compared the two media and culture under oil versus culture in loosely capped tubes, significantly more clinical pregnancies (30% of 60 transfers) were obtained with HTF medium than with T6 medium (11% of 53 transfers). Decreasing the K + content of HTF medium to that present in T6 medium significantly decreased the number of mouse zygotes that developed in culture. Modifying Ca + + levels had no effect. It is therefore likely that the higher K + content in HTF medium is primarily responsible for the superiority of HTF medium over T6 medium, but other differences in the composition of the two media could contribute to the results observed.
This volume describes culture media and solutions used in human ART; how they have been developed for in vitro human pre-implantation embryo development, the function and importance of the various components in media and solutions and how they interact, and how the systems in which these are used can influence outcomes. Chapters discuss inorganic solutes, energy substrates, amino acids, macromolecules, cytokines, growth factors, buffers, pH, osmolality, and the interaction of these parameters. The role of incubators and other physical factors are reviewed, along with the relevance and prospects of emerging technologies: morphokinetic analysis using time-lapse imaging and dynamic fluid incubation systems. Results of prospective randomized trials are emphasized to ascertain the added value of these techniques for selecting viable embryos. This comprehensive guide will be invaluable for embryologists, physicians and all personnel involved in the fluid products used in human ART seeking to optimize their successful use of these components.
Alberto Monroy, together with Kao, Grundfest and Tyler, were amongst the first to recognize the importance of ion fluxes through the plasma membrane in the process of egg activation (Tyler et. al. 1956). In the 1970’s the electrical events at activation in sea urchin eggs were recorded (Steinhardt et al 1971; Ito and Yoshioka, 1973; Jaffe, 1976 and Chambers and de Armendi, 1979), and shortly afterwards measurements were extended to fish, amphibia and mammals (see Igusa et al., 1983; McCulloh et al., 1983; Nuccitelli, 1980; Talevi et al., 1985 for references). The sea urchin response was noted to be a biphasic depolarization (Ito and Yoshioka, 1973). However, neither the biophysical origin of this depolarization, nor the mechanism by which it was triggered, were known. Here we follow the progress made in this field and describe how electrical measurements have been utilized to elucidate the mechanism of egg activation.
Introduction The main goal of cryopreservation of cells, gametes, or embryos is to preserve the specimen in a state of suspended animation, with the hope that it can be reanimated after a certain period of time to continue its normal development. Cryopreservation procedures are designed to minimize damages caused by formation and growth of ice crystals [1]; however, the challenge of a successful cryopreservation is to be able to cool and recover cells from the ultra-low temperatures at which no changes in metabolism and structures are stable over time. Cryopreservation occurs when cryoprotectants or antifreeze solutions are added to the solution surrounding the cell, and then cooled at a certain rate. The biophysical changes caused by the transition of water to ice during cooling are the main cause of damage rather than the low temperature per se. As ice crystals grow, there is a significant osmotic stress, this “freeze-dehydration” was one of the first harmful consequences identified in cell cryobiology that causes several hazardous events including changes in ultrastructure of cell membranes, loss or fusion of membranes, and organelle disruption. In order to avoid cryo-damages, the cooling and warming protocol must be tailored according to the cell characteristics. Therefore, the compositions and modifications of cryomedia are very important to the success of cryopreservation.