Teratogenesis, Perinatal, and Neurodevelopmental Outcomes After In Utero Exposure to Antiseizure Medication: Practice Guideline From the AAN, AES, and SMFM.

This practice guideline provides updated evidence-based conclusions and recommendations regarding the effects of antiseizure medications (ASMs) and folic acid supplementation on the prevalence of major congenital malformations (MCMs), adverse perinatal outcomes, and neurodevelopmental outcomes in children born to people with epilepsy of childbearing potential (PWECP). A multidisciplinary panel conducted a systematic review and developed practice recommendations following the process outlined in the 2017 edition of the American Academy of Neurology Clinical Practice Guideline Process Manual. The systematic review includes studies through August 2022. Recommendations are supported by structured rationales that integrate evidence from the systematic review, related evidence, principles of care, and inferences from evidence. The following are some of the major recommendations. When treating PWECP, clinicians should recommend ASMs and doses that optimize both seizure control and fetal outcomes should pregnancy occur, at the earliest possible opportunity preconceptionally. Clinicians must minimize the occurrence of convulsive seizures in PWECP during pregnancy to minimize potential risks to the birth parent and to the fetus. Once a PWECP is already pregnant, clinicians should exercise caution in attempting to remove or replace an ASM that is effective in controlling generalized tonic-clonic or focal-to-bilateral tonic-clonic seizures. Clinicians must consider using lamotrigine, levetiracetam, or oxcarbazepine in PWECP when appropriate based on the patient's epilepsy syndrome, likelihood of achieving seizure control, and comorbidities, to minimize the risk of MCMs. Clinicians must avoid the use of valproic acid in PWECP to minimize the risk of MCMs or neural tube defects (NTDs), if clinically feasible. Clinicians should avoid the use of valproic acid or topiramate in PWECP to minimize the risk of offspring being born small for gestational age, if clinically feasible. To reduce the risk of poor neurodevelopmental outcomes, including autism spectrum disorder and lower IQ, in children born to PWECP, clinicians must avoid the use of valproic acid in PWECP, if clinically feasible. Clinicians should prescribe at least 0.4 mg of folic acid supplementation daily preconceptionally and during pregnancy to any PWECP treated with an ASM to decrease the risk of NTDs and possibly improve neurodevelopmental outcomes in the offspring.

New candidate genes potentially involved in Zika virus teratogenesis.

Despite efforts to elucidate Zika virus (ZIKV) teratogenesis, still several issues remain unresolved, particularly on the molecular mechanisms behind the pathogenesis of Congenital Zika Syndrome (CZS). To answer this question, we used bioinformatics tools, animal experiments and human gene expression analysis to investigate genes related to brain development potentially involved in CZS. Searches in databases for genes related to brain development and CZS were performed, and a protein interaction network was created. The expression of these genes was analyzed in a CZS animal model and secondary gene expression analysis (DGE) was performed in human cells exposed to ZIKV. A total of 2610 genes were identified in the databases, of which 1013 were connected. By applying centrality statistics of the global network, 36 candidate genes were identified, which, after selection resulted in nine genes. Gene expression analysis revealed distinctive expression patterns for PRKDC, PCNA, ATM, SMC3 as well as for FGF8 and SHH in the CZS model. Furthermore, DGE analysis altered expression of ATM, PRKDC, PCNA. In conclusion, systems biology are helpful tools to identify candidate genes to be validated in vitro and in vivo. PRKDC, PCNA, ATM, SMC3, FGF8 and SHH have altered expression in ZIKV-induced brain malformations.

Updated EUROCAT guidelines for classification of cases with congenital anomalies.

BACKGROUND: Precise and correct classification of congenital anomalies is important in epidemiological studies, not only to classify according to etiology but also to group similar congenital anomalies together, to create homogeneous subgroups for surveillance and research. This paper presents the updated EUROCAT (European surveillance of congenital anomalies) subgroups of congenital anomalies and the updated multiple congenital anomaly (MCA) algorithm and provides the underlying arguments for the revisions. METHODS: The EUROCAT methodology is described. In addition, we show how we validated the revised EUROCAT subgroups and MCA algorithm, which are both based on the International Classification of Diseases (ICD10/ICD9) codes. RESULTS: The updated EUROCAT subgroups and the updated MCA algorithm are described in detail and the updated version is compared to the previous versions. CONCLUSION: The EUROCAT subgroups and MCA algorithm provide a standardized and clear methodology for congenital anomaly research and epidemiological surveillance of congenital anomalies in order to facilitate the identification of teratogenic exposures and to assess the impact of primary prevention and prenatal screening policies. The EUROCAT subgroups and MCA algorithm are made freely available for other researchers via the EUROCAT Database Management Software.

Epidemiology studies on effects of lithium salts in pregnancy are confounded by the inability to control for other potentially teratogenic factors.

INTRODUCTION: In bipolar women who took lithium during pregnancy, several epidemiology studies have reported small increases in a rare fetal cardiac defect termed Ebstein's anomaly. METHODS: Behavioral, environmental, and lifestyle-associated risk factors associated with bipolar disorder and health insurance status were determined from an Internet search. The search was conducted from October 1, 2023, through October 14, 2023. The search terms employed included the following: bipolar, bipolar disorder, mood disorders, pregnancy, congenital heart defects, Ebstein's anomaly, diabetes, hypertension, Medicaid, Medicaid patients, alcohol use, cigarette smoking, marijuana, cocaine, methamphetamine, narcotics, nutrition, diet, obesity, body mass index, environment, environmental exposures, poverty, socioeconomic status, divorce, unemployment, and income. No quotes, special fields, truncations, etc., were used in the searches. No filters of any kind were used in the searches. RESULTS: Women who remain on lithium in the United States throughout their pregnancy are likely to be experiencing mania symptoms and/or suicidal ideation refractory to other drugs. Pregnant women administered the highest doses of lithium salts would be expected to have been insufficiently responsive to lower doses. Any small increases in the retrospectively determined risk of fetal cardiac anomalies in bipolar women taking lithium salts cannot be disentangled from potential developmental effects resulting from very high rates of cigarette smoking, poor diet, alcohol abuse, ingestion of illegal drugs like cocaine or opioids, marijuana smoking, obesity, and poverty. CONCLUSIONS: The small risks in fetal cardiac abnormalities reported in the epidemiology literature do not establish a causal association for lithium salts and Ebstein's anomaly.

The teratogenesis risk associated with antiseizure medication duotherapy in women with epilepsy.

PURPOSE: To investigate rates of occurrence of pregnancies associated with a foetal malformation (FM pregnancy rates) following simultaneous intrauterine exposure to two antiseizure medications in 524 pregnancies in women with epilepsy from the Australian Pregnancy Register who were treated simultaneously with various combinations and dosages of two antiseizure medications (duotherapy). RESULTS: FM pregnancy rates tended to be higher in those exposed simultaneously to two antiseizure medications, each of which was a statistically significant teratogen (valproate, topiramate, or carbamazepine), than when there was exposure to only one such teratogen. When there was exposure to only one such teratogen together with clonazepam or levetiracetam, for neither of which there was statistically significant evidence of heightened teratogenicity, the FM pregnancy rates also tended to be higher, but less so. When lamotrigine was the other component of the duotherapy with an established teratogen, FM pregnancy rates tended to be lower than that for the teratogen used as monotherapy. CONCLUSION: Leaving aside issues in relation to seizure control, our data suggest that it would be best to avoid using established teratogenic antiseizure medications (carbamazepine, valproate and topiramate) in combination with each other due to the increased FM risks. When combining an established teratogenic medication with a less teratogenic one, i.e. lamotrigine, levetiracetam or clonazepam, lamotrigine appears to be the safer option.

Guidelines on Developmental Toxicity Tests: Brief Insights.

Developmental toxicology is a constantly evolving research field which needs to attend to a complex underlying regulatory network. In order to ensure human health and environmental safety, new substances have to be tested for toxic effects on reproduction and development, before being commercialized. Traditional in vivo mammalian models represent the intricacy of human development and provide more adequately an assessment of the interaction of chemical compounds with the reproductive system. However, in the last years, the directives are to reduce the use of vertebrate animals, promoting their use only as a last resort. Consequently, the interest on the development and validation of alternative tests, able to cover the various aspects of the reproductive cycle, has significantly increased. Reproductive toxicity is probably the most difficult endpoint to be replaced by alternative assays, since it should provide information on mechanism interactions essential for female and male fertility and also knowledge on the animal development during the first phases of its life cycle. This complexity explains the slow progress in implementing alternative models for reproductive toxicity safety assays. Alternative test models may be based on in vitro systems and nonmammalian animal models. Many biological processes have been successfully addressed using in vitro models, opening the possibility to study the interference of teratogenic compounds. Their validation and implementation have lagged behind, in part because of difficulties in establishing their predictability. Nevertheless, the advance toward the process of validation is crucial to replace and reduce the use of living animals. Based on the present state of the art, it is not probable that such testing strategies will completely replace the need to assess reproductive toxicity in vivo in the near future, but they will contribute to reduce animal tests and will provide important information. In this chapter, the approved guidelines for standard methods and alternative methods, according to their regulatory and scientific status, are enumerated and briefly described.

The Future of the Teratogenicity Testing.

The purpose of this review is to examine the importance, possible advantages and disadvantages of teratogenicity tests, and their future. For this purpose, numerous sources have been scanned in the field of teratogenicity. Although there are many methods related to teratogenic studies and very important studies have been made in this field, there are still serious deficiencies. There are advantages and disadvantages of in vitro and in vivo classical tests that have been used so far. The current status of in vivo tests is a matter of debate, especially due to the use of experimental animals. However, in vitro tests that do not perform the distribution and metabolism of chemicals also raise doubts in determination of teratogenicity. Despite the modern approaches of molecular biology and genetics and the best diagnostic techniques, the real cause of more than half of congenital diseases is still not understood. In this sense, the importance and necessity of teratogenic tests are understood once again. It is necessary to develop faster, reliable, and inexpensive techniques to replace traditional in vivo tests. It is important to disseminate harmless and reliable imaging techniques such as micro-CT. The use of European Center for the Validation of Alternative Methods (ECVAM) scientifically validated and approved in vitro tests such as embryonic stem cell test (EST), micro mass test (MM), and whole embryo culture (WEC) tests in routine screening can provide a solution in a shorter time than the classical tests. Improving these tests and developing new tests can help to solve the problem permanently.

Virus as Teratogenic Agents.

Viral infectious diseases are important causes of reproductive disorders, as abortion, fetal mummification, embryonic mortality, stillbirth, and congenital abnormalities in animals and in humans. In this chapter, we provide an overview of some virus, as important agents in teratology.We begin by describing the Zika virus, whose infection in humans had a very significant impact in recent years and has been associated with major health problems worldwide. This virus is a teratogenic agent in humans and has been classified as a public health emergency of international concern (PHEIC).Then, some viruses associated with reproductive abnormalities on animals, which have a significant economic impact on livestock, are described, as bovine herpesvirus, bovine viral diarrhea virus, Schmallenberg virus, Akabane virus, and Aino virus.For all viruses mentioned in this chapter, the teratogenic effects and the congenital malformations associated with fetus and newborn are described, according to the most recent scientific publications.

Development Features on the Selection of Animal Models for Teratogenic Testing.

Today, the use of animal models from different species continues to represent a fundamental step in teratogenic testing, despite the increase in alternative solutions that provide an important screening to the enormous quantity of new substances that aim to enter the market every year. The maintenance of these models is due to the sharing of similar development processes with humans, and in this way they represent an important contribution to the safety in the use of the compounds tested. Furthermore, the application of advances in embryology to teratology, although hampered by the complexity of reproductive processes, continues to prove the importance of sensitivity during embryonic and fetal development to detect potential toxicity, inducing mortality/abortion and malformations.In this chapter, essential periods of development in different models are outlined, highlighting the similarities and differences between species, the advantages and disadvantages of each group, and specific sensitivities for teratogenic testing. Models can be divided into invertebrate species such as earthworms of the species Eisenia fetida/Eisenia andrei, Caenorhabditis elegans, and Drosophila melanogaster, allowing for rapid results and minor ethical concerns. Vertebrate nonmammalian species Xenopus laevis and Danio rerio are important models to assess teratogenic potential later in development with fewer ethical requirements. Finally, the mammalian species Mus musculus, Rattus norvegicus, and Oryctolagus cuniculus, phylogenetically closer to humans, are essential for the assessment of complex specialized processes, occurring later in development.Regulations for the development of toxicology tests require the use of mammalian species. Although ethical concerns and costs limit their use in large-scale screening. On the other hand, invertebrate and vertebrate nonmammalian species are increasing as alternative animal models, as these organisms combine low cost, less ethical requirements, and culture conditions compatible with large-scale screening. Their main advantage is to allow high-throughput screening in a whole-animal context, in contrast to the in vitro techniques, not dependent on the prior identification of a target. Better knowledge of the development pathways of animal models will allow to maximize human translation and reduce the number of animals used, leading to a selection of compounds with an improved safety profile and reduced time to market for new drugs.

Developmental Toxicity Using the Rat Whole Embryo Culture.

In vivo and in vitro experiments have been used to investigate the effect of drugs, chemical agents, growth factors, vitamins, etc., on embryonic development. Alternative tests have been developed to determine whether drugs or other compounds have toxic or teratogenic effects on a particular organ or tissue. The rat whole embryo culture method is useful for studying the mechanism of normal embryo development. The explanted rat conceptuses grow in the culture environment at the same rate as in utero development. Furthermore, the considerable advantage of explanting rat embryos is that it allows direct observation of embryogenesis. The rat whole embryo culture is run by explanting rat embryos on gestation day 9.5 for rats. The conceptuses are then cultured on a rotating platform in a mixture of culture medium with appropriate gassing for 48 hours. The maternal serum is used as the culture medium, and chemicals or other agents to be evaluated separately are added to this medium. At the end of the culture period, growth and development of the embryos are evaluated morphologically.

A Modified Murine Embryonic Stem Cell Test for Evaluating the Teratogenic Effects of Drugs.

Animal-based test systems have traditionally been used to screen for the potential teratogenic activity of drugs. Still, their deficits in predicting precise human-specific outcomes and ethical concerns have led to a need for alternative approaches. In vitro, teratogenicity testing using cell cultures or other in vitro systems is a potential alternative. Of the different in vitro platforms, the mouse embryonic stem cell test (mEST) is currently the most widely used and validated in vitro test for assessing the potential effects of teratogens on early embryonic development. The mEST involves exposing mouse embryonic stem cells to the test compound and monitoring their differentiation for several days.Nevertheless, its predictive ability was comparatively lower when distinguishing weak developmental toxicants from non-toxic substances. Since then, several modifications and adaptations of the mEST protocol have been developed. This chapter describes an alternative method based on molecular approaches to predict embryotoxicity. This method, originated from the mEST, analyzes the expression of differentiation genes involved in the development of mesoderm, endoderm, and stoderm and allows screening embryo-toxicants with different mechanisms of action. The hanging drops embryoid bodies used in the original mEST protocol have been replaced with monolayer culture, and thus the process has been shortened. In general, the method shows higher predictability compared with the traditional ones.

Machine Learning to Predict Teratogenicity: Theory and Practice.

Machine learning (ML) is a subfield of artificial intelligence (AI) that consists of developing algorithms that can automatically learn patterns and relationships from data, without being explicitly programmed. It continues to advance with the development of more sophisticated algorithms, increased computational power, and larger datasets, leading to significant advancements in AI technology. With the significant progress made in ML, the need to apply these systems in the area of teratogenicity is growing. It is sought as robust boosting methods to overcome many limitations and restrictions facing the experimental studies. By performing tasks such as classification, regression, clustering, anomaly detection, and decision systems, ML can be used to assess whether an agent is teratogen or not or to determine its teratogenic potential. It may also be used for the purpose of deciding on the use of medicinal products. In this chapter, we describe how ML can be used to investigate teratogenicity.

Assessment of the Teratogenic Effect of Drugs on the Chicken Embryo.

Pre-clinical trials are an essential step that underpins the drug discovery, development, and safety process. During this process, animal testing is performed to determine the safety of new compounds and any potential adverse effects. Developmental toxicity tests are carried out to verify whether the drug has potential to cause congenital anomalies to the developing embryo/fetus. Chicken embryos are very useful for these purposes and present several advantages, such as low cost of production and housing, easy handling and manipulation, and rapid development in addition to sharing similarities to the human embryo at molecular, cellular, and anatomical levels. In this chapter, we bring methods for using the chicken embryo model for testing the teratogenic effects of drugs and assessing the main outcomes of them.

Teratogenic Effects of Drugs on Primary Lymphocytes Assessed by Flow Cytometry.

Peripheral blood lymphocytes as primary cells can be isolated from human, animal, fetus, and placenta. These cells are an excellent cellular model for the assessment of cytotoxicity, genotoxicity, oxidative stress, and mitochondrial and lysosomal dysfunction induced by drug and chemicals. Moreover, peripheral blood lymphocytes are an easily available source of primary cells appropriate for basic research and in cellular studies regarding teratogenic, genotoxic, and cytotoxic effect of drugs and chemicals. Most drugs and other chemicals that produce birth defects, known as teratogenic agents, produce reactive oxygen species (ROS) formation and mitochondrial and lysosomal dysfunction. It seems that there is an important mechanistic link between oxidative stress, mitochondrial damages, lysosomal integrity, and teratogenic drug-induced birth defects. One of the most sensitive periods in the embryo is transition from an important developmental event to another such as transition from proliferation to differentiation. Mitochondria, lysosomes, and cellular ROS have an important role in proliferative, differentiative, and apoptotic activities during the development. Therefore, disruption of the function of mitochondria, lysosomes, oxidative stress, and redox imbalance leads to cellular dysfunctions and subsequently poor developmental outcomes in the fetus. In this chapter, we will focus on evaluation of mitochondrial/lysosomal functions and estimation of ROS formation using flow cytometry methods in isolated lymphocytes and their isolated mitochondria.

Exploration of Teratogenic and Genotoxic Effects on Model Organism Drosophila melanogaster.

Drosophila melanogaster is one of the crucial in vivo models in terms of analyzing the toxicity of various unknown chemicals. Every part of the fly serves as a model in metabolic and therapeutic approaches. Genotoxic and teratogenic compounds are exposed to Drosophila through the oral route. Further, the toxicity of genotoxic compounds is analyzed in Drosophila's gut, hemolymph, and phenotype. The toxicity of teratogen compounds is also analyzed using a Drosophila embryo. The current chapter summarizes several techniques that are used to detect the genotoxicity and teratogenicity of any unknown compound in this model.

Daphnia magna as a Model Organism to Predict the Teratogenic Effect of Different Compounds.

For aquatic ecosystem Daphnia magna is evolving as a model organism to check the teratogenicity of numerous compounds. D. magna can be easily cultured in the laboratory, and the teratogen effect of several compounds can be easily studied. The developmental stages are well studied in D. magna. All the developmental stages are transparent so the defect can be easily accessed. So, the postembryonic developmental changes can be easily studied after the exposure with teratogen. More importantly, D. magna also have a swimming behavioral phenotype. The behavioral defect can be easily accessed after teratogen exposure. The current chapter summarizes numerous protocols associated with embryo and adult staining and adult behavioral assays that can be used to access the teratogenicity of any unknown compound.

Caenorhabditis elegans as an In Vivo Model Organism to Elucidate Teratogenic Effects.

Exogenous teratogens contribute to approximately 10% of the human abnormality with exposure occurrence during the prenatal and fetal period. However, the assessment methods and underlying mechanism remain unclear. The nematode Caenorhabditis elegans has been recognized as one of the ideal model animals for toxicologic research as convenient culture, low cost, and complete phenotypes and genomic profiling. This chapter describes the protocols about the estimations on the teratogenic effects using nematodes as model organisms, including the growth, development, behavior, reproduction, energy balance, and transgenes.

Biochemical Studies to Understand Teratogenicity and Lethality Outcomes in Modified-FETAX.

The frog embryo teratogenesis assay-Xenopus (FETAX) is a standardized test used to assess the toxic and teratogenic effects of xenobiotics. With this test, toxic and/or teratogenic concentrations of xenobiotic substances can be determined using morphological parameters such as lethality, length, and malformations in stage 8-11 Xenopus laevis embryos after 96 h exposure. These parameters enable the determination of the median lethal and effective concentrations (LC50 and EC50), minimum concentration to inhibit growth (MCIG), and teratogenic index of the tested chemical to reveal the short-term effects of relatively high concentrations. On the other hand, although FETAX provides quantitative and qualitative data on teratogenicity and toxicity, the biochemical and molecular mechanisms of these effects cannot be explained. Recent studies have tried to elucidate the mechanisms causing malformations and to explain the underlying causes of toxicity and teratogenicity by biochemical marker analysis. This chapter describes methods to analyze modified-FETAX and some detoxification and oxidative stress-related biomarkers during the early embryonic development of X. laevis.

Bioinformatics Methods for Transcriptome Analysis on Teratogenesis Testing.

Teratogenesis testing can be challenging due to the limitations of both in vitro and in vivo models. Test-systems, based especially on human embryonic cells, have been helping to overcome the difficulties when allied to omics strategies, such as transcriptomics. In these test-systems, cells exposed to different compounds are then analyzed in microarray or RNA-seq platforms regarding the impacts of the potential teratogens in the gene expression. Nevertheless, microarray and RNA-seq dataset processing requires computational resources and bioinformatics knowledge. Here, a pipeline for microarray and RNA-seq processing is presented, aiming to help researchers from any field to interpret the main transcriptome results, such as differential gene expression, enrichment analysis, and statistical interpretation. This chapter also discusses the main difficulties that can be encountered in a transcriptome analysis and the better alternatives to overcome these issues, describing both programming codes and user-friendly tools. Finally, specific issues in the teratogenesis field, such as time-course analysis, are also described, demonstrating how the pipeline can be applied in these studies.