The effect of low and ultra-low oxygen tensions on mammalian embryo culture and development in experimental and clinical IVF

ABSTRACT

Over the last forty years, many trials have been performed using mammalian embryo cultures with reduced oxygen tension (O₂) to encourage proper embryo development and increase the success rate for in vitro fertilization (IVF) outcome. Even if the use of atmospheric O₂ (20%) affects in vitro embryo development and intracellular redox balance, the use of low (5% O₂, physiologic) and ultra-low (close or less to 5% O₂) O₂ applied to in vitro embryo culture is still under debate. Numerous studies in various mammalian species have shown that embryo development improves when culturing embryos under low O₂, although culture conditions are not the only factors involved in the success of IVF. This article reviews the literature data of the last four decades and discusses the current evidence on the use of low and ultra-low O₂ in embryo culture, and examines the impact of multiple factors on IVF outcomes.     

Mammalian diversity: gametes, embryos and reproduction

The class Mammalia is composed of approximately 4800 extant species. These mammalian species are divided into three subclasses that include the monotremes, marsupials and eutherians. Monotremes are remarkable because these mammals are born from eggs laid outside of the mother's body. Marsupial mammals have relatively short gestation periods and give birth to highly altricial young that continue a significant amount of 'fetal' development after birth, supported by a highly sophisticated lactation. Less than 10% of mammalian species are monotremes or marsupials, so the great majority of mammals are grouped into the subclass Eutheria, including mouse and human. Mammals exhibit great variety in morphology, physiology and reproduction. In the present article, we highlight some of this remarkable diversity relative to the mouse, one of the most widely used mammalian model organisms, and human. This diversity creates challenges and opportunities for gamete and embryo collection, culture and transfer technologies.     

LIF and TNF alpha concentrations in embryo culture media are predictive for embryo implantation in IVF

ObjectiveThere is strong evidence that the cytokines leucemia inhibitory factor (LIF) and tumor necrosis factor (TNF) alpha are related to embryo development and implantation. The aim of this study was to determine the levels of LIF and TNF alpha in embryo culture media and to assess its relationship to the outcome of in-vitro fertilization and embryo transfer.MethodsA total of 99 patients were included in this prospective trial and underwent either IVF or ICSI procedure. A total of 865 oocytes were collected. Embryos were cultured in sequential media until day 5. A standardized morphology evaluation of all embryos, including a detailed pronuclear scoring, was performed daily during this period followed by the replacement of one or two selected embryos. Collected embryo culture fluids of days 3 and 5 were analysed for LIF and TNF alpha on days 3 and 5.ResultsMean TNF alpha concentration in culture media on day 3 was 0.54 and 0.37 pg/mL on day 5 and was significantly lower in women conceiving than in not conceiving (0.43 pg/mL versus 0.59 pg/mL on day 3). Mean LIF concentration on day 3 was 31.5 pg/mL and 35.5 pg/mL on day 5 and was significantly higher in women conceiving (56.2 pg/mL versus 22.2 pg/mL on day 3).ConclusionsThe results indicate that LIF could have a function in early embryogenesis and as a factor required for embryo implantation. High TNF alpha concentrations seem to be predictive of implantation failure.     

Toxicity of Heavy Metals on Embryogenesis and Larvae of the Marine Sedentary Polychaete <Emphasis Type="Italic">Hydroides elegans</Emphasis>

The toxicity of heavy metals to marine invertebrates has been widely investigated; however, the effects on marine sedentary polychaetes have largely been ignored. The toxicity of copper, aluminium, lead, nickel, and zinc on fertilization, embryogenesis, and larvae of Hydroides elegans was examined in laboratory acute-toxicity tests. Exposure to metal during fertilization or early developmental stages leads to fertilization block and arrested development, which resulted in morphologic abnormalities in embryo and larvae. Fertilization rate showed a drastic decrease at the highest metal concentration tested. Embryos of H. elegans showed a differential response to metals, and the responses were stage-specific. The different morphologic effects of heavy metals reflect differentiation of the early embryonic cells. For individual metals, the toxicity ranking for 24−hour trochophore larvae was Cu > Al > Pb > Ni > Zn, with EC₅₀ values of 0.122, 0.210, 0.231, 0.316, and 0.391 mg l⁻¹, respectively. Rate of larval development and embryogenesis were the most sensitive end points, although the latter is more advisable for routine assessment of seawater quality because of its greater simplicity. In addition to bivalves and sea urchins, polychaete embryos can provide biologic criteria for seawater quality taking into account the sensitivity of a polychaete and contributing to the detection of harmful chemicals with no marked effect on the species currently in use in seawater quality bioassays.     

Developmental complexity of early mammalian pluripotent cell populations in vivo and in vitro

Early mammalian embryogenesis is characterised by the coordinated proliferation, differentiation, migration and apoptosis of a pluripotent cell pool that is able to give rise to extraembryonic lineages and all the cell types of the embryo proper. These cells retain pluripotent differentiation capability, defined in this paper as the ability to form all cell types of the embryo and adult, until differentiation into the three embryonic germ layers at gastrulation. Our understanding of pluripotent cell biology and molecular regulation has been hampered by the difficulties associated with experimental manipulation of these cells in vivo. However, a more detailed understanding of pluripotent cell behaviour is emerging from the application of molecular technologies to early mouse embryogenesis. The construction of mouse mutants by gene targeting, mapping of gene expression in vivo, and modelling of cell decisions in vitro are providing insight into the cellular origin, identity and action of key developmental regulators, and the nature of pluripotent cells themselves. In this review we discuss the properties of early embryonic pluripotent cells in vitro and in vivo, focusing on progression from inner cell mass (ICM) cells in the blastocyst to the onset of gastrulation.     

Female Germ Cell Loss From Radiation and Chemical Exposures

Female germ cell in some mammals are extremely sensitive to killing by ionizing radiation, especially during development. Primordial oocytes in juvenile mice have an LD₃₀ of only 6-7 rad, and the germ cell pool in squirrel monkeys is destroyed by prenatal exposure of 0.7 rad/day. Sensitivity varies greatly with species and germ cell stage. Unusually high sensitivity has not been found in macaques and may not occur in man, but this has not been established for all developmental stages. The exquisite oocyte radiosensitivity in mice apparently reflects vulnerability of the plasma membrane, not DNA, which may have implications fur estimating human genetic risks. Germ cells can be killed also by chemicals. Such oocyte loss, with similarities to radiation effects, is under increasing study, including chemotherapy observations in women. More than 75 compounds have been tested in mice, with in vivo toxicity quantified by oocyte loss; certain chemicals apparently act on the membrane.     

Does genome organization matter in spermatozoa? A refined hypothesis to awaken the silent vessel

ABSTRACT

The spermatozoon is considered by many to be a silent vessel whose only function is to safely deliver the paternal genome to the maternal oocyte. As a result, the paternal contribution to fertilization and embryogenesis is frequently overlooked. However, the spermatozoon is a highly elaborate and specialized cell that is formed through the process of spermatogenesis. Spermatogenesis is a complex cellular program of differentiation that produces mature spermatozoa, which are essential for reproduction, fertilization, and normal embryonic development. The sperm cell is unique in morphology, chromatin structure, and function. Increasing evidence demonstrates that perturbations in chromatin integrity and organization could have a significant clinical impact on fertilization and embryogenesis. In this article we will review the evidence that demonstrates the paternal genome to be highly packaged and uniquely organized. We will postulate how the integrity and organization of the paternal genome likely has functional consequences that are critical for the establishment and maintenance of a viable pregnancy. In doing so, we hope to dispel the common myth that the sperm cell is a silent vessel; instead we will demonstrate the sperm cell to be a highly segmentally organized, epigenetically primed cell.

Abbreviations: 2D: two-dimension; 3C: chromosome conformation capture; 3D: three-dimension; 4D: four-dimension; CTs: chromosome territories; FISH: fluorescence in situ hybridization; IMSI: intra cytoplasmic morphologically selected sperm injection; ICSI: intracytoplasmic sperm injection; IVF: in-vitro fertilization; mESCs: mouse embryonic stem cells; NORs: nuclear organizing regions; TADs: topologically associated domain     

Off to a Rough Start: Environmental Exposures May Alter Germ Cell Development

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Normal development of germ cells—precursors of egg and sperm cells—is integral for the survival of a species. Prenatal exposure to toxic environmental agents can have profound effects on reproductive health decades after birth,¹ yet our understanding of germ cell development has long been hampered by technical challenges.² In an in vitro study recently published in Environmental Health Perspectives, researchers show for the first time how toxicant exposures at environmentally relevant concentrations can influence the earliest stages of germ cell specification.³ The results add to mounting evidence that the timing of exposure is critical when assessing chemical toxicities.Multicellular life is made possible by interactions between two cell types: somatic cells that carry out the essential tasks required for life, and germ cells that transfer genetic information to future generations.² In mammals, germ cell development starts in the epiblast (also known as the primitive ectoderm), a layer of cells that gives rise to the embryo and also contributes to extraembryonic tissues, such as the future amniotic sac. The newly formed primordial germ cells (PGCs) move into extraembryonic tissues, where, guided by cues from neighboring somatic cells, they proliferate and are folded into the embryo.⁴ Finally, they migrate to their final destination: the developing gonads.^5,6Open in a separate windowScanning electron micrograph of a primordial germ cell undergoing division in the ovary of a 7-week-old embryo. Eventually these cells will become oocytes (eggs). Magnification: ×1920 at 6 × 7 cm size. Image: © Professors Pietro M. Motta and Sayoko Makabe/Science Source.During proliferation and relocation, PGCs undergo a complex reprogramming process known as epigenetic erasure, which involves removal of biochemical modifications to DNA and some of its associated proteins.^7,8 Errors that are introduced during this sensitive window can bridge generations and lead to both early-life^9,10 and adult-onset outcomes such as infertility,¹¹ cancer,¹² and other diseases.Examining the impact of early environmental exposures on germ cell development has been hampered by its intrinsic complexity: PGCs exist in very limited numbers during embryogenesis and move from one site to another, whereas toxic exposures can span multiple developmental stages from conception through childhood.² Past studies have therefore been limited to evaluating exposure effects long after PGCs have moved to the developing ovary or testis and differentiated into oocytes or spermatocytes.² Patricia Hunt, Edward Meyer Distinguished Professor at Washington State University, who was not involved in the new study, notes that the work provides insight into a period of germ cell development that has remained virtually inaccessible until now.To capture these key developmental events, the investigators used an established mouse embryonic stem cell model of in vitro differentiation into epiblast-like cells (EpiLCs), followed by PGC-like cells.^13,14 “We’re standing on the shoulders of [germ cell biology] giants,” says Patrick Allard, senior author of the study. “They have shown that this technology is highly representative of the mammalian in vivo context, so it can now be applied to the question of environmental health.”The researchers studied the effects of exposure to bisphenol A (BPA), a pervasive environmental contaminant and known endocrine disruptor used primarily in the production of plastics.¹⁵ Exposure of EpiLCs to environmentally relevant concentrations of BPA led to greater proliferation, compared with unexposed EpiLCs. However, this effect was also associated with increased DNA damage in BPA-treated cells. Although PGC differentiation was not affected, gene expression analyses revealed BPA-specific alterations to the cells’ gene expression landscape—changes that the authors suggested may affect gamete function later in life. “While one might expect [BPA exposure] to have global effects on the different types of cell lineages, the more subtle perturbations observed are more relevant to the biology of humans or infertility overall,” says Renee Reijo Pera, director of the McLaughlin Research Institute for Biomedical Sciences, who was not involved in the study.These effects were not observed in a cell line that is genetically male, suggesting that responsiveness to BPA at this stage of germ cell development may be sex specific. “Even though developmental biologists have long thought of PGCs as [an] asexual stage, the differences in gene expression profiles observed highlights the need to pay attention to X-linked gene dosage effects on environmental toxicant sensitivity,” says Steen Ooi, first author of the new paper.Hunt believes this system can also open the door for more in-depth mechanistic studies. Ooi thinks it would be particularly useful to dissect the epigenetic phenomena underlying how the “memory” of exposure is transmitted to later stages in development. Allard adds that a long-term goal would be to replicate some of these findings in human cells, saying, “We hope to one day develop in vitro systems to recapitulate the entire reproductive cycle in a dish, characterizing every single stage of germ cell development and reproduction.”     

Comparative Effects of Di(n-Butyl) Phthalate Exposure on Fetal Germ Cell Development in the Rat and in Human Fetal Testis Xenografts

Background

Phthalate exposure induces germ cell effects in the fetal rat testis. Although experimental models have shown that the human fetal testis is insensitive to the steroidogenic effects of phthalates, the effects on germ cells have been less explored.

Objectives

We sought to identify the effects of phthalate exposure on human fetal germ cells in a dynamic model and to establish whether the rat is an appropriate model for investigating such effects.

Methods

We used immunohistochemistry, immunofluorescence, and quantitative real-time polymerase chain reaction to examine Sertoli and germ cell markers on rat testes and human fetal testis xenografts after exposure to vehicle or di(n-butyl) phthalate (DBP). Our study included analysis of germ cell differentiation markers, proliferation markers, and cell adhesion proteins.

Results

In both rat and human fetal testes, DBP exposure induced similar germ cell effects, namely, germ cell loss (predominantly undifferentiated), induction of multinucleated gonocytes (MNGs), and aggregation of differentiated germ cells, although the latter occurred rarely in the human testes. The mechanism for germ cell aggregation and MNG induction appears to be loss of Sertoli cell–germ cell membrane adhesion, probably due to Sertoli cell microfilament redistribution.

Conclusions

Our findings provide the first comparison of DBP effects on germ cell number, differentiation, and aggregation in human testis xenografts and in vivo in rats. We observed comparable effects on germ cells in both species, but the effects in the human were muted compared with those in the rat. Nevertheless, phthalate effects on germ cells have potential implications for the next generation, which merits further study. Our results indicate that the rat is a human-relevant model in which to explore the mechanisms for germ cell effects.

Citation

van den Driesche S, McKinnell C, Calarrão A, Kennedy L, Hutchison GR, Hrabalkova L, Jobling MS, Macpherson S, Anderson RA, Sharpe RM, Mitchell RT. 2015. Comparative effects of di(n-butyl) phthalate exposure on fetal germ cell development in the rat and in human fetal testis xenografts. Environ Health Perspect 123:223–230; http://dx.doi.org/10.1289/ehp.1408248     

Histidine-rich glycoprotein derived peptides affect endometrial angiogenesis in vitro but has no effect on embryo development

Histidine-rich glycoprotein (HRG) is an abundant plasma protein involved in multiple biological processes including immunology, vascularisation, and coagulation. These processes are of importance in regulating embryo development and implantation. A specific polymorphism in the HRG gene, HRG C633T, has an impact on various aspects of fertility, such as oocyte quality, endometrial receptivity, and possibly the capacity of the embryo itself to implant. To further examine the potential role of the HRG C633T polymorphism in regulating endometrial angiogenesis and on embryo development, two HRG peptides were constructed. These HRG peptides correspond to the amino acids 169-203 of the protein which, in turn, reflects the C633T polymorphism in the gene. The HRG proline or serine peptides were added to cultures of primary human endometrial endothelial (HEE) cells and to human embryos in vitro. The HRG peptides inhibited vascular endothelial growth factor (VEGF) induced proliferation and migration and promoted tube formation of HEE cells. The embryos were monitored using a time-lapse system (EmbryoScope®). Except for a prolonged time from first cleavage after thawing to development of the morula, no difference in embryo morphokinetics or embryo quality was noted in human embryos cultured in the presence of the HRG proline peptide. Taken together, these results suggest that treatment with a specific HRG peptide might prime the endometrium for implantation and be beneficial for adequate placentation. However, addition of a specific HRG proline peptide to human embryos has no beneficial effects in terms of embryo development.

Abbreviations: HRG: histidine-rich glycoprotein; HEE: human endometrial endothelial; VEGF: vascular endothelial growth factor; TSP: thrombospondin; SNP; single nucleotide polymorphism; IVF: in vitro fertilization; CLESH-1: CD36 LIMPII Emp structural homology domain-1; ECM: endothelial cell medium; FBS: fetal bovine serum; cDNA: complementary DNA     

Critical windows of exposure for children's health: the reproductive system in animals and humans

Drugs and environmental chemicals can adversely affect the reproductive system. Currently, available data indicate that the consequences of exposure depend on the nature of the chemical, its target, and the timing of exposure relative to critical windows in development of the reproductive system. The reproductive system is designed to produce gametes in far greater excess than would seem to be necessary for the survival of species. Ten to hundreds of millions of spermatozoa are generated daily by most adult male mammals, yet very few of these germ cells succeed in transmitting their genetic material to the next generation. Although the number of oocytes produced in mammalian females is more limited, and their production occurs only during fetal life, most ovaries contain several orders of magnitude more oocytes than ever will be fertilized. Toxicant exposures may affect critical events in the development of the reproductive system, ranging from early primordial germ cell determination to gonadal differentiation, gametogenesis, external genitalia, or signaling events regulating sexual behavior. Although there are differences between the human reproductive system and that of the usual animal models, such models have been extremely useful in assessing risks for key human reproductive and developmental processes. The objectives for future studies should include the elucidation of the specific cellular and molecular targets of known toxicants; the design of a systematic approach to the identification of reproductive toxicants; and the development of sensitive, specific, and predictive animal models, minimally invasive surrogate markers, or in vitro tests to assess reproductive system function during embryonic, postnatal, and adult life.     

Correlation between embryo morphology and development and chromosomal complement

ObjectiveTo analyze the correlation between embryo morphology and the chromosomal status using the array comparative genomic hybridization [array comparative genomic hybridization (a-CGH)] technique for screening 23 chromosome pairs in a single blastomere biopsy from Day 3 embryos.MethodsOne thousand five hundred and fifty seven embryos were included from 203 cycle ICSI patients undergoing preimplantation genetic screening. The 23 chromosome pairs were analyzed by blastomere biopsy from day 3 embryos using a-CGH array method. Embryo development rate, fragmentation rate and chromosome status of the analyzed blastomeres were recorded and correlated with the aCGH results.ResultsThe incidence of chromosomal abnormalities was significantly higher in slow-and fast cleaving embryos at day 3 after insemination. The incidence of fragmentation and the type of fragmentation was associated with an increased incidence of chromosomal abnormalities. The symmetry of the blastomeres also correlated with the aneuploidy rates.ConclusionsEmbryo development rate and morphological parameter such as degree, type of fragmentation and the symmetry of the blastomeres to a large extent reflect the cytogenetic status of the embryo and thus are important in the selection of embryos with the highest implantation potential.     

Responses of embryonic germ cells of the radiation-sensitive Medaka mutant to gamma-irradiation

The radiation-sensitive mutant "ric1" has a defect in the repair mechanism of DNA double strand breaks induced by gamma-rays in early embryogenesis. In this study, the new radiation-sensitive Medaka (Oryzias latipes) strain, ric1olvas-GFP was established to monitor the development of germ cells in vivo. The development of germ cells was normal in ric1olvas-GFP, but embryonic germ cells at Stage 7 (32-cell stage) and Stage 33 (extensive proliferating stage of PGCs) showed higher radiosensitivity. There was no sex difference in germ cell radiosensitivity at Stage 7, but female embryos showed higher radiosensitivity than male at Stage 33. In embryos obtained by crossing ric1 female with olvas-GFP male, germ cells showed similar radiosensitivity to ric1olvas-GFP and increased sensitivity compared to embryos obtained from crossing wild-type female with olvas-GFP male at Stage 7. These results suggest that germ cells have the ric1 dependent DNA repair system during embryogenesis and the maternal ric1gene factor may play a critical role in radiosensitivity at an early developmental stage.     

Nutritional Status Impacts Epigenetic Regulation in Early Embryo Development: A Scoping Review

With the increasing maternal age and the use of assisted reproductive technology in various countries worldwide, the influence of epigenetic modification on embryonic development is increasingly notable and prominent. Epigenetic modification disorders caused by various nutritional imbalance would cause embryonic development abnormalities and even have an indelible impact on health in adulthood. In this scoping review, we summarize the main epigenetic modifications in mammals and the synergies among different epigenetic modifications, especially DNA methylation, histone acetylation, and histone methylation. We performed an in-depth analysis of the regulation of various epigenetic modifications on mammals from zygote formation to cleavage stage and blastocyst stage, and reviewed the modifications of key sites and their potential molecular mechanisms. In addition, we discuss the effects of nutrition (protein, lipids, and one-carbon metabolism) on epigenetic modification in embryos and emphasize the importance of various nutrients in embryonic development and epigenetics during pregnancy. Failures in epigenetic regulation have been implicated in mammalian and human early embryo loss and disease. With the use of reproductive technologies, it is becoming even more important to establish developmentally competent embryos. Therefore, it is essential to evaluate the extent to which embryos are sensitive to these epigenetic modifications and nutrition status. Understanding the epigenetic regulation of early embryo development will help us make better use of reproductive technologies and nutrition regulation to improve reproductive health in mammals.     

Early mammalian embryonic development

This report examines the major events occurring during the first month of development in the human embryo. Developmental events are detailed, beginning with cleavage of the zygote and ending with the formation of the three primary germ layers. Certain aspects of early human development are compared with that in lower forms of vertebrates to emphasize species differences in these basic embryological processes. A discussion of the formation of identical twins is included, since it has its inception during the first month of development.     

Sodium Fluoride as Potential Mutagen in Mammalian Eggs

Evidence from other forms demonstrating mitotic chromosome damage has suggested that sodium fluoride (NaF), a primary air pollutant, be explored as a mutagenic compound in female mammalian germ cells. In vitro experiments with mouse, sheep, and cow oocytes have disclosed a low incidence of anaphase lags, suppression of polar body I, and fragmentation with rearrangement. In vivo experiments showed only a minor effect on oocyte meiotic maturation, but species’ variations of sensitivity to NaF would suggest further investigations of progeny of the ewe and cow in contaminated areas.     

卵泡晚期血清孕酮水平对体外受精、胚胎发育及妊娠结局的影响

目的:探讨控制性卵巢刺激中,卵泡晚期血清孕酮水平与体外受精、胚胎发育及妊娠结局的关系。方法:2008年1月～2009年4月在遵义医学院附属医院生殖中心接受体外受精助孕的不孕症患者88例,根据hCG注射日血清孕酮水平的不同分为两组,血清孕酮5.5nmol/L者67例(A组),血清孕酮≥5.5nmol/L者21例(B组),回顾性分析卵泡晚期血清孕酮水平对体外受精、胚胎发育及妊娠结局的影响。结果:A组的总受精率、正常受精率、早期卵裂率、优质胚胎率、胚胎种植率和临床妊娠率明显高于B组(P0.01)。结论:卵泡晚期血清孕酮水平升高对体外受精和胚胎发育具有负面影响,并导致胚胎种植率和临床妊娠率明显降低。     

The politics and ethics of human embryo and stem cell research.

There is great promise for stem cell research to develop cells and tissues for transplantation and treatment of diseases such as Alzheimer and Parkinson disease, diabetes, and heart problems. There is also promise to advance understanding and treatment of cancer and congenital defects. Human embryo research is fundamentally the only way to understand human fertilization, implantation, and early development. For years, federal funding of human embryo research has been held hostage to a congressional prolife agenda. Any reasonable solution to these political disputes that so greatly affect women's reproductive interests and the promise of health benefits from embryo and stem cell research should mandate that governmental sponsorship proceed.