Staging of porcine embryos: Comparison of Standard Event System-based statistical clusters with a Carnegie-based staging system.

BACKGROUND: Methods to compare events defined as newly occurring characters in development has advanced vertebrate developmental research but events are not easily extrapolated into traditional staging systems used in biomedical research. RESULTS: First, we scored 95 porcine embryos in the age range of 15 to 33 days post conception by stereomicroscopy using to a slightly modified version of the Standard Event System (SES). Subsequent statistical clustering allowed the embryos to be grouped into 15 clusters. Staging of the same embryos in a way that generally follow the description of external features of human embryos in the Carnegie stages 10 to 23 allowed us to describe 14 stages of porcine embryonic development that correlate to the Carnegie stages of human development with minor species differences. When arranged by average age, the statistic clusters had a distribution that correlated well with the stages produced by the Carnegie-based staging system. CONCLUSIONS: Statistical analysis of developmental events allow grouping of porcine embryos into clusters that can be extrapolated into a Carnegie-based staging system, thus serving the dual purpose of facilitating the use of the pig as a biomedical model animal and providing data for integrating porcine developmental events into a phylogenetic context.

Guidelines and definitions for research on epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) encompasses dynamic changes in cellular organization from epithelial to mesenchymal phenotypes, which leads to functional changes in cell migration and invasion. EMT occurs in a diverse range of physiological and pathological conditions and is driven by a conserved set of inducing signals, transcriptional regulators and downstream effectors. With over 5,700 publications indexed by Web of Science in 2019 alone, research on EMT is expanding rapidly. This growing interest warrants the need for a consensus among researchers when referring to and undertaking research on EMT. This Consensus Statement, mediated by 'the EMT International Association' (TEMTIA), is the outcome of a 2-year-long discussion among EMT researchers and aims to both clarify the nomenclature and provide definitions and guidelines for EMT research in future publications. We trust that these guidelines will help to reduce misunderstanding and misinterpretation of research data generated in various experimental models and to promote cross-disciplinary collaboration to identify and address key open questions in this research field. While recognizing the importance of maintaining diversity in experimental approaches and conceptual frameworks, we emphasize that lasting contributions of EMT research to increasing our understanding of developmental processes and combatting cancer and other diseases depend on the adoption of a unified terminology to describe EMT.

Strength of nonhuman primate studies of developmental programming: review of sample sizes, challenges, and steps for future work.

Nonhuman primate (NHP) studies are crucial to biomedical research. NHPs are the species most similar to humans in lifespan, body size, and hormonal profiles. Planning research requires statistical power evaluation, which is difficult to perform when lacking directly relevant preliminary data. This is especially true for NHP developmental programming studies, which are scarce. We review the sample sizes reported, challenges, areas needing further work, and goals of NHP maternal nutritional programming studies. The literature search included 27 keywords, for example, maternal obesity, intrauterine growth restriction, maternal high-fat diet, and maternal nutrient reduction. Only fetal and postnatal offspring studies involving tissue collection or imaging were included. Twenty-eight studies investigated maternal over-nutrition and 33 under-nutrition; 23 involved macaques and 38 baboons. Analysis by sex was performed in 19; minimum group size ranged from 1 to 8 (mean 4.7 ± 0.52, median 4, mode 3) and maximum group size from 3 to 16 (8.3 ± 0.93, 8, 8). Sexes were pooled in 42 studies; minimum group size ranged from 2 to 16 (mean 5.3 ± 0.35, median 6, mode 6) and maximum group size from 4 to 26 (10.2 ± 0.92, 8, 8). A typical study with sex-based analyses had group size minimum 4 and maximum 8 per sex. Among studies with sexes pooled, minimum group size averaged 6 and maximum 8. All studies reported some significant differences between groups. Therefore, studies with group sizes 3-8 can detect significance between groups. To address deficiencies in the literature, goals include increasing age range, more frequently considering sex as a biological variable, expanding topics, replicating studies, exploring intergenerational effects, and examining interventions.

Micro-CT imaging: Developing criteria for examining fetal skeletons in regulatory developmental toxicology studies - A workshop report.

During the past two decades the use and refinements of imaging modalities have markedly increased making it possible to image embryos and fetuses used in pivotal nonclinical studies submitted to regulatory agencies. Implementing these technologies into the Good Laboratory Practice environment requires rigorous testing, validation, and documentation to ensure the reproducibility of data. A workshop on current practices and regulatory requirements was held with the goal of defining minimal criteria for the proper implementation of these technologies and subsequent submission to regulatory agencies. Micro-computed tomography (micro-CT) is especially well suited for high-throughput evaluations, and is gaining popularity to evaluate fetal skeletons to assess the potential developmental toxicity of test agents. This workshop was convened to help scientists in the developmental toxicology field understand and apply micro-CT technology to nonclinical toxicology studies and facilitate the regulatory acceptance of imaging data. Presentations and workshop discussions covered: (1) principles of micro-CT fetal imaging; (2) concordance of findings with conventional skeletal evaluations; and (3) regulatory requirements for validating the system. Establishing these requirements for micro-CT examination can provide a path forward for laboratories considering implementing this technology and provide regulatory agencies with a basis to consider the acceptability of data generated via this technology.

The POU-er of gene nomenclature.

The pluripotency factor POU5F1 (OCT4) is well known as a key regulator of stem cell fate. Homologues of POU5F1 exist throughout vertebrates, but the evolutionary and functional relationships between the various family members have been unclear. The level to which function has been conserved within this family provides insight into the evolution of early embryonic potency. Here, we seek to clarify the relationship between POU5F1 homologues in the vertebrate lineage, both phylogenetically and functionally. We resolve the confusion over the identity of the zebrafish gene, which was originally named pou2, then changed to pou5f1 and again, more recently, to pou5f3. We argue that the use of correct nomenclature is crucial when discussing the degree to which the networks regulating early embryonic differentiation are conserved.

Lucha por sobrevivir, diabetes tipo 2 y obesidad. Respuesta del autor / Fight for survival: type 2 diabetes and obesity. Author's reply

No disponible

A critical evaluation of developmental and reproductive toxicology in nonhuman primates.

The nonhuman primates (NHPs) are used in many areas of biomedical research where their similarities to humans make them exclusively valuable animal models. The use of NHPs in pre-clinical testing is expected to increase due to the increase in the development of biological compounds for therapeutic uses. The regulatory agencies around the world including Food and Drug Administration (FDA) generally requires developmental and reproductive toxicity (DART) testing of all new drugs to be used by women of childbearing age or men of reproductive potential. NHPs are most frequently used for DART testing when commonly used rodents and/or rabbits are not pharmacologically relevant species. Animal studies are unique in that assessment of reproduction and development as DART studies are not performed in controlled clinical trials; therefore, pre-clinical safety assessment forms the basis for risk assessment for marketed drug products. This paper provides a critical evaluation of developmental and reproductive toxicity studies in NHPs. The manuscript will focus on species selection, limitation of International Conference for Harmonization stages (A-F) using NHPs as a test system, study designs, logistical/technical challenges, and strength, and limitations. It will also pinpoint confounding factors inherent to the test system that may complicate the interpretation of the NHP DART data.

REACH and reproductive and developmental toxicology: still questions.

Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH) is a new chemicals regulation law in the European Union (EU). The law is supplemented by tens of thousands of pages of guidance documents, and the implementation of REACH is still a work in progress. Requirements for chemical testing are based on the annual volume of a chemical or 'substance' that is produced or imported into the EU. These requirements include reproductive and developmental toxicity testing in experimental animals for an annual volume of 10 metric tonnes or more. However, under REACH, the testing in vertebrate animals may not be performed without permission, and the law encourages the use of alternative methods of filling data gaps on the toxicological properties of chemicals. These alternatives might include in vitro and structure-activity relationship studies, but the REACH technical guidance indicates that these kinds of studies are not adequate to replace reproductive and developmental toxicity testing in whole animals. The most practical opportunity for the avoidance of whole animal testing may be 'read-across,' a process in which gaps are filled using data from related compounds. A method called 'weight of evidence' under REACH may also be used to avoid whole animal reproductive and developmental toxicity testing based on existing data in regulation and non-regulation studies and based on factors such as chemical structure and anticipated exposure. It is also possible that thresholds of toxicological concerns will be accepted under REACH as a method to avoid vertebrate animal testing.

Promoting the "3Rs" principle in developmental biology with early and convenient diagnosis of pregnancy in mice.

The 3Rs stand for Replacement of animals in experiments, Reduction in the number of experimental animals, and Refinement of experiments to minimize animal pain and stress. We propose to address Reduction and Refinement in the use of mice as experimental models in developmental research. This study focuses on the maternal percentage of weight increase at gestational day 8 (%WI(GD8)) to diagnose pregnancy early in BALB/c mice. We documented sensitivity, specificity, false positive and negative rates and probability of pregnancy associated with %WI(GD8). This predictive model of pregnancy allows for significant reduction in the number of mice to be sacrificed in developmental research. Reported observations and literature suggest that this model is independent of litter size and should be applicable to other mice strains. This procedure allows mice pregnancy detection before midgestation and proposes an ethically sound approach to experimental animal use by optimizing the number of mice used and refining animal manipulation.

Practical considerations on the design, execution and analysis of developmental neurotoxicity studies to be published in Neurotoxicology and Teratology.

The present article is an attempt to use the "ILSI Research Foundation/Risk Science Institute Reports from the Expert Working Group on Neurodevelopmental Endpoints" (2008) to help improve the quality of the manuscripts submitted to Neurotoxicology and Teratology, as well as the quality of their review. The points discussed in the present paper have been encountered during the peer-review process. A number of recommendations are proposed on the basis of general principles (clarity, full disclosure, and evidence-based interpretation) to help authors and reviewers. Clarity of language is a prerequisite, but clarity of purpose is essential. The methodology and statistical analysis for each dependent variable should be unambiguously presented and justified. Full disclosure encompasses a range of topics, such as defining the sample size for each experiment, clearly distinguishing between hypothesis-testing and hypothesis-generating (e.g., a priori vs. a posteriori analyses), clearly defining the statistical model appropriate to the study design and questions (e.g., repeated-measure approach), recognizing and addressing the multiplicity problem (e.g., conceptual unit for the error rate), identifying the appropriate unit for statistical analysis (e.g., litter), addressing the results of all analyses (e.g., "negative" results are important). Data interpretation should be evidence-based and not exceed the limits of the findings.

Multiparous species present problems (and possibilities) to developmentalists.

Multiparous species present a special set of problems and opportunities for study design and analysis. The present review reintroduces old concerns and raises new ones with empirical illustrations. Evidence is presented that litter effects are pervasive and persist into adulthood. Unaccounted for, they lead to spurious findings, inflate real effect sizes and produce false negatives. Furthermore, two-stage sampling, the practice of sampling only a subset of littermates from dams, can lower reliability, and therefore power, to unacceptable levels. In addition, the greater sensitivity offered by within-litter analyses over between-litter analyses is demonstrated. Statistical and experimental solutions are suggested and referenced. Surveys of recent developmental studies showed that the great majority do not attend to these issues, thereby casting doubt on the validity of their positive and negative findings. All developmentalists can strengthen their research by systematically addressing these concerns in study design and analysis.