Meta-analysis of Transcriptomic Data from Lung Autopsy and Cellular Models of SARS-CoV-2 Infection.

Severe COVID-19 is a systemic disorder involving excessive inflammatory response, metabolic dysfunction, multi-organ damage, and several clinical features. Here, we performed a transcriptome meta-analysis investigating genes and molecular mechanisms related to COVID-19 severity and outcomes. First, transcriptomic data of cellular models of SARS-CoV-2 infection were compiled to understand the first response to the infection. Then, transcriptomic data from lung autopsies of patients deceased due to COVID-19 were compiled to analyze altered genes of damaged lung tissue. These analyses were followed by functional enrichment analyses and gene-phenotype association. A biological network was constructed using the disturbed genes in the lung autopsy meta-analysis. Central genes were defined considering closeness and betweenness centrality degrees. A sub-network phenotype-gene interaction analysis was performed. The meta-analysis of cellular models found genes mainly associated with cytokine signaling and other pathogen response pathways. The meta-analysis of lung autopsy tissue found genes associated with coagulopathy, lung fibrosis, multi-organ damage, and long COVID-19. Only genes DNAH9 and FAM216B were found perturbed in both meta-analyses. BLNK, FABP4, GRIA1, ATF3, TREM2, TPPP, TPPP3, FOS, ALB, JUNB, LMNA, ADRB2, PPARG, TNNC1, and EGR1 were identified as central elements among perturbed genes in lung autopsy and were found associated with several clinical features of severe COVID-19. Central elements were suggested as interesting targets to investigate the relation with features of COVID-19 severity, such as coagulopathy, lung fibrosis, and organ damage.

Possible New Candidates Involved to Thalidomide-Related Limbs and Cardiac Defects: A Systems Biology Approach.

Thalidomide is a known teratogen that causes malformations especially in heart and limbs. Its mechanism of teratogenicity is still not fully elucidated. Recently, a new target of thalidomide was described, TBX5, and was observed a new interaction between HAND2 and TBX5 that is disrupted in the presence of thalidomide. Therefore, our study aimed to raise potential candidates for thalidomide teratogenesis, through systems biology, evaluating HAND2 and TBX5 interaction and heart and limbs malformations of thalidomide. Genes and proteins related to TBX5 and HAND2 were selected through TF2DNA, REACTOME, Human Phenotype Ontology, and InterPro databases. Networks were assembled using STRING © database. Network analysis were performed in Cytoscape © and R v3.6.2. Differential gene expression (DGE) analysis was performed through gene expression omnibus. We constructed a network for HAND2 and TBX5 interaction; a network for heart and limbs malformations of TE; and the two joined networks. We observed that EP300 protein seemed to be important in all networks. We also looked for proteins containing C2H2 domain in the assembled networks. ZIC3, GLI1, GLI3, ZNF148, and PRDM16 were the ones present in both heart and limbs malformations of TE networks. Furthermore, in the DGE analysis after treatment with thalidomide, we observed that FANCB, ESCO2, and XRCC2 were downregulated and present both in heart and limbs networks. Through systems biology, we were able to point to different new proteins and genes, and selected specially EP300, which was important in all the analyzed networks, to be further evaluated in the TE teratogenicity.

Challenges to the effectiveness of next-generation sequencing in formalin-fixed paraffin-embedded tumor samples for non-small cell lung cancer.

INTRODUCTION: Next-generation sequencing (NGS) of Formalin-Fixed and Paraffin-Embedded (FFPE) specimens is routine in precision oncology practice. However, results are not always conclusive, and it is important to identify which factors may influence FFPE tumor sequencing success. MATERIALS AND METHODS: Here, we evaluated the influence of pre-analytical factors on 705 samples of non-small cell lung cancer specimens that underwent NGS testing. Factors such as tumor site, tumor cell percentage, fragment size, primary tumor or metastasis, presence of necrosis, DNA purity, DNA concentration, sample origin and year of testing. RESULTS: The overall NGS success rate was 84.9 % (n = 599). Bone site specimens had a very low success rate (42.1 %), differing from lung samples (79.8 %) (P < 0.05). Samples with tumor percentages <5 % (success rate of 44.4 %) represented 14.1 % of failed sequencings. Moreover, samples with tumor percentages >10 %-20 % (82 %) did not differ from those with >30 % (88.9 %) on sequencing outcomes (P = 0.086). Specimens that provided DNA concentrations >2.0 ng/uL, 1.0-2.0 ng/uL, 0.5-1.0 ng/uL and <0.5 ng/uL had success rates of 92 %, 77.1 %, 61.3 % and 20.4 %, respectively. Small fragments (≤0.2 cm2) had a success rate of 74.7 % and were more prevalent in the unsuccessful group (P < 0.05). CONCLUSIONS: Our results suggest that tumor percentage, fragment size, decalcified bone specimens, and DNA concentration are potential modifiers of NGS success rates. Interestingly, specimens with tumor percentages between 10 % and 20 % have the same sequencing outcome than specimens with >30 %. These results can strengthen the understanding of factors that lead to NGS success variability.

Brazilian women in Bioinformatics: Challenges and opportunities.

Bioinformatics is a growing research field that received great notoriety in the years of the COVID-19 pandemic. It is a very integrative area, comprising professionals from science, technology, engineering, and mathematics (STEM). In agreement with the other STEM areas, several women have greatly contributed to bioinformatics ascension; however, they had to surpass prejudice and stereotypes to achieve recognition and leadership positions, a path that studies have demonstrated to be more comfortable to their male colleagues. In this review, we discuss the several difficulties that women in STEM, including bioinformatics, surpass during their careers. First, we present a historical context on bioinformatics and the main applications for this area. Then, we discuss gender disparity in STEM and present the challenges that still contribute to women's inequality in STEM compared to their male colleagues. We also present the opportunities and the transformation that we can start, acting in academia, inside the family and school environments, and as a society, hence contributing to gender equality in STEM. Finally, we discuss specific challenges in the bioinformatics field and how we can act to overcome them, especially in low and middle-income countries, such as Brazil.

Human genetic determinants of COVID-19 in Brazil: challenges and future plans.

COVID-19 pandemic represented a worldwide major challenge in different areas, and efforts undertaken by the scientific community led to the understanding of some of the genetic determinants that influence the different COVID-19 outcomes. In this paper, we review the studies about the role of human genetics in COVID-19 severity and how Brazilian studies also contributed to those findings. Rare variants in genes related to Inborn Errors of Immunity (IEI) in the type I interferons pathway, and its phenocopies, have been described as being causative of severe outcomes. IEI and its phenocopies are present in Brazil, not only in COVID-19 patients, but also in autoimmune conditions and severe reactions to yellow fever vaccine. In addition, studies focusing on common variants and GWAS studies encompassing worldwide patients have found several loci associated with COVID-19 severity. A GWAS study including only Brazilian COVID-19 patients identified a new locus 1q32.1 associated with COVID-19 severity. Thus, more comprehensive studies considering the Brazilian genomic diversity should be performed, since they can help to reveal not only what are the genetic determinants that contribute to the different outcomes for COVID-19 in the Brazilian population, but in the understanding of human genetics in different health conditions.

Exploring the frequency of a TP53 polyadenylation signal variant in tumor DNA from patients diagnosed with lung adenocarcinomas, sarcomas and uterine leiomyomas.

The TP53 3'UTR variant rs78378222 A>C has been detected in different tumor types as a somatic alteration that reduces p53 expression through modification of polyadenylation and miRNA regulation. Its prevalence is not yet known in all tumors. Herein, we examine tumor tissue prevalence of rs7837822 in Brazilian cohorts of patients from south and southeast regions diagnosed with lung adenocarcinoma (LUAD, n=586), sarcoma (SARC, n=188) and uterine leiomyoma (ULM, n=41). The minor allele (C) was identified in heterozygosity in 6/586 LUAD tumors (prevalence = 1.02 %) and none of the SARC and ULM samples. Additionally, next generation sequencing analysis revealed that all variant-positive tumors (n=4) with sample availability had additional pathogenic or likely pathogenic somatic variants in the TP53 coding regions. Among them, 3/4 (75 %) had the same pathogenic or likely pathogenic sequence variant (allele frequency <0.05 in tumor DNA) namely c.751A>C (p.Ile251Leu). Our results indicate a low somatic prevalence of rs78378222 in LUAD, ULM and SARC tumors from Brazilian patients, which suggests that no further analysis of this variant in the specific studied regions of Brazil is warranted. However, these findings should not exclude tumor molecular testing of this TP53 3'UTR functional variant for different populations.

A New Strategy for the Old Challenge of Thalidomide: Systems Biology Prioritization of Potential Immunomodulatory Drug (IMiD)-Targeted Transcription Factors.

Several molecular mechanisms of thalidomide embryopathy (TE) have been investigated, from anti-angiogenesis to oxidative stress to cereblon binding. Recently, it was discovered that thalidomide and its analogs, named immunomodulatory drugs (IMiDs), induced the degradation of C2H2 transcription factors (TFs). This mechanism might impact the strict transcriptional regulation of the developing embryo. Hence, this study aims to evaluate the TFs altered by IMiDs, prioritizing the ones associated with embryogenesis through transcriptome and systems biology-allied analyses. This study comprises only the experimental data accessed through bioinformatics databases. First, proteins and genes reported in the literature as altered/affected by the IMiDs were annotated. A protein systems biology network was evaluated. TFs beta-catenin (CTNNB1) and SP1 play more central roles: beta-catenin is an essential protein in the network, while SP1 is a putative C2H2 candidate for IMiD-induced degradation. Separately, the differential expressions of the annotated genes were analyzed through 23 publicly available transcriptomes, presenting 8624 differentially expressed genes (2947 in two or more datasets). Seventeen C2H2 TFs were identified as related to embryonic development but not studied for IMiD exposure; these TFs are potential IMiDs degradation neosubstrates. This is the first study to suggest an integration of IMiD molecular mechanisms through C2H2 TF degradation.

Evaluation of the influence of genetic variants in Cereblon gene on the response to the treatment of erythema nodosum leprosum with thalidomide.

BACKGROUND: Erythema nodosum leprosum (ENL) is an acute and systemic inflammatory reaction of leprosy characterised by painful nodules and involvement of various organs. Thalidomide is an immunomodulatory and anti-inflammatory drug currently used to treat this condition. Cereblon (CRBN) protein is the primary target of thalidomide, and it has been pointed out as necessary for the efficacy of this drug in others therapeutics settings. OBJECTIVES: In this study, we aimed to evaluate the influence of CRBN gene variants on the dose of thalidomide as well as its adverse effects during treatment of ENL. METHODS: A total of 103 ENL patients in treatment with thalidomide were included in this study. DNA samples were obtained from saliva and molecular analysis of CRBN gene were performed to investigate the variants rs1620675, rs1672770 and rs4183. Different genotypes of CRBN variants were evaluated in relation to their influence on the dose of thalidomide and on the occurrence of adverse effects. FINDINGS: No association was found between CRBN variants and thalidomide dose variation. However, the genotypes of rs1672770 showed association with gastrointestinal effects (p = 0.040). Moreover, the haplotype DEL/C/T (rs4183/rs1672770/rs1620675) was also associated with gastrointestinal adverse effects (p = 0.015). MAIN CONCLUSIONS: Our results show that CRBN variants affect the treatment of ENH with thalidomide, especially on the adverse effects related to the drug.

COVID-19 during pregnancy and adverse outcomes: Concerns and recommendations from The Brazilian Teratology Information Service.

SARS-CoV-2 virus was first identified in the beginning of 2020 and has spread all over the world, causing the Coronavirus Disease 2019 (COVID-19) pandemic. The virus is a member of the Coronavirus family, which includes viruses that cause common cold, Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). MERS and SARS are known by causing adverse events in pregnancy. Considering that SARS-CoV-2 is a new infection agent, little is known about the risk of its infection to human embryo/fetal development. However, SARS and MERS were associated with negative outcomes, such as miscarriage, preterm birth, intrauterine growth restriction and perinatal death. Here, we raise concerns and possibilities related the harmful potential of SARS-CoV-2 and COVID-19 to pregnancy, discussing symptoms, immunological changes during pregnancy, SARS-CoV-2 mutation rate (and the risks related to it). Finally, we point out recommendations to be performed by the scientific community and health care workers in order to identify and to manage potential risks to pregnant women and their babies.

Fetal Alcohol Spectrum Disorders: Health Needs Assessment in Brazil.

BACKGROUND: Fetal alcohol syndrome (FAS) is a disorder caused by alterations in embryo-fetal development due to prenatal alcohol exposure. It is estimated that between 0.5 and 2 per 1,000 individuals are born with FAS every year. In Brazil, there are few studies addressing the extent of the problem of FAS/fetal alcohol spectrum disorders (FASD); these studies are confined to limited geographic areas. Therefore, we decided to perform a health needs assessment for FAS/FASD in Brazil. METHODS: To estimate the prevalence of FAS and FASD in Brazil, we used information from the literature, which estimates between 0.5 and 2/1,000 births per year for FAS and 10 to 50/1,000 for FASD. RESULTS: We estimated that approximately 1,500 to 6,000 children are born with FAS every year. Considering the whole population, the prevalence would be 95,377 to 380,000 affected people. However, when we consider FASD as a whole, we estimate that between 1,900,000 and 9,500,000 Brazilians might suffer the more severe consequences of alcohol exposure during pregnancy and be living with FASD. CONCLUSION: The results of the current study indicate that FAS and FASD are prevalent disorders in Brazil, and more policies targeting alcohol intake during pregnancy must be developed.

Performance of the Gail and Tyrer-Cuzick breast cancer risk assessment models in women screened in a primary care setting with the FHS-7 questionnaire.

Breast cancer (BC) risk assessment models base their estimations on different aspects of a woman's personal and familial history. The Gail and Tyrer-Cuzick models are the most commonly used, and BC risks assigned by them vary considerably especially concerning familial history. In this study, our aim was to compare the Gail and Tyrer-Cuzick models after initial screening for familial history of cancer in primary care using the FHS-7 questionnaire. We compared 846 unrelated women with at least one positive answer to any of the seven FHS-7 questions (positive group) and 892 unrelated women that answered negatively (negative group). Concordance between BC risk estimates was compared by Bland-Altman graphics. Mean BC risk estimates were higher using the Tyrer-Cuzick Model in women from the positive group, while women from the negative group had higher BC risk estimates using the Gail model. With increasing estimates, discordance also increased, mainly in the FHS-7 positive group. Our results show that in women with a familial history of cancer, the Gail model underestimates risk and the Tyrer-Cuzick seems to be more appropriate. FHS-7 can be a useful tool for the identification of women with higher breast cancer risks in the primary care setting.

From abortion-inducing medications to Zika Virus Syndrome: 27 years experience of the First Teratogen Information Service in Latin America.

In 1990, the first Teratogen Information Service in Brazil (SIAT) was implemented in the Medical Genetics Service at Hospital de Clinicas de Porto Alegre. SIAT is a free-to-use information service both to health professionals and the general population, especially to women who are pregnant or planning pregnancy. The main objective of this paper is to present the activities of SIAT in its initial years (1990-2006), compared to those in the last decade (2007-2017). In addition we review the scientific contribution of SIAT in the field of human teratogenesis. Since 1990, SIAT received 10,533 calls. Use of medications were the main reason for concern, accounting for 74% of all questions, followed by other chemical exposures (occupational, cosmetics, environmental), and maternal infectious diseases. Among its main contributions to scientific knowledge was the collaboration for the identification of two new human teratogens: misoprostol in the 1990s and Zika virus in 2015/16. In conclusion, SIAT is still evolving, as is the Medical Genetics Service that hosts it. Through its 27 years of existence more than 300 undergraduate and graduate students have rotated at SIAT. Presently, SIAT is expanding the research to experimental teratogenesis and to investigation of molecular mechanisms of teratogens.

Zika Virus as a Possible Risk Factor for Autism Spectrum Disorder: Neuroimmunological Aspects.

The recent outbreak of the Zika virus (ZIKV) and the discovery that perinatal Zika exposure can lead to the Congenital Zika Syndrome has promoted a call for prevention measures. Due to the increased number of babies born with microcephaly, structural brain abnormalities, and neurological alterations in regions affected by ZIKV, investigations were carried out in order to better understand this process. The maternal immune system directly influences the fetal central nervous system, and complications during pregnancy have been associated with neurodevelopmental disorders. Autism spectrum disorder (ASD), a neurodevelopmental disorder commonly manifested in the first years of life, is a disease with multifactorial etiology and is manifested typically by social and communication impairments, as well as stereotyped behaviors. Brain abnormalities, including both anatomically and functionally, can be observed in this disorder, suggesting delays in neuronal maturation and altered brain connectivity. It is known that some viral congenital infections, such as rubella, and cytomegalovirus can interfere with brain development, being associated with brain calcification, microcephaly, and ASD. Here, we reviewed a range of studies evaluating the aspects concerning brain development, immunological status during pregnancy, and neuroimmunomodulation in congenital viral infections, and we discuss if the fetal brain infection caused by ZIKV could predispose to ASD. Finally, we suggest a mechanism encompassing neurological and immunological pathways that could play a role in the development of ASD in infants after ZIKV infection in pregnancy.

Is intrauterine exposure to acetaminophen associated with emotional and hyperactivity problems during childhood? Findings from the 2004 Pelotas birth cohort.

BACKGROUND: Longitudinal studies have consistently reported that prenatal exposure to acetaminophen can to lead to an increased risk of attention deficit-hyperactivity disorder during childhood. This study aimed to investigate the association between intrauterine exposure to acetaminophen and the presence of emotional and behavioral problems at the ages of 6 and 11 years in a low-middle income country. METHODS: We performed a prospective longitudinal population-based study using data from the 2004 Pelotas birth cohort. From the 4231 initial cohort participants, 3722 and 3566 children were assessed at 6 and 11 years of age, respectively. The outcomes were assessed using the parent version of Strengths and Difficulties Questionnaire (SDQ). The cut-off points established for the Brazilian population were used to categorize the outcomes. Crude and adjusted odds ratio were obtained through logistic regression. RESULTS: Acetaminophen was used by 27.5% (95% confidence interval [CI]: 26.1-28.9) of the mothers at least once during pregnancy. The prevalence of emotional problems at 6 and 11 years was 13.6 and 19.9%, respectively. For hyperactivity problems, prevalence was 13.9 and 16.1%, respectively. Intrauterine exposure to acetaminophen increased the odds of having emotional (odds ratio [OR] = 1.47; 95% CI: 1.07-2.02) and hyperactivity/inattention (OR = 1.42; 95% CI: 1.06-1.92) problems in 6-year-old boys. At the age of 11, a small decrease in the effect was observed for both outcomes after adjustment: OR = 1.31 (95% CI: 0.99-1.73) for emotional problems and OR = 1.25 (95% CI: 0.95-1.65) for hyperactivity/inattention in boys. No association for any phenotypes at both ages was observed for girls. CONCLUSION: The effect of intrauterine exposure to acetaminophen in emotional and hyperactivity symptoms was dependent on sex in a Brazilian cohort. While it seemed to be important for boys, mainly at 6 years of age, for girls, no association was observed.

Angiogenesis and oxidative stress-related gene variants in recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) affects ~3-5% of couples attempting to conceive and in around 50% of cases the aetiology remains unknown. Adequate vascularisation and placental circulation are indispensable for the development of a normal pregnancy. Prostaglandin-endoperoxide synthase 2 (PTGS2), vascular endothelial growth factor (VEGF) and the nitric oxide (NO) systems play important roles in reproductive physiology, participating in several steps including implantation and apoptosis of trophoblast cells. In this study we evaluated genetic polymorphisms in the inducible nitric oxide synthase (NOS2), PTGS2 and VEGFA genes as susceptibility factors for RPL. A case-control study was conducted in 149 women having two or more miscarriages and 208 controls. Allele and genotype distributions of the polymorphisms studied in the two groups were not statistically different. However, the dominant model showed that the presence of variant T (TT/GT) of rs2779249 (-1290G>T) of NOS2 was significantly associated with RPL (OR=1.58, CI 95%=1.03-2.44; P=0.037). The increased risk remained significant when adjusted for number of pregnancies, alcohol consumption and ethnicity (OR=1.92, CI95%=1.18-3.11; P=0.008). These results suggest that the variant genotypes of the functional polymorphism rs2779249 in the NOS2 promoter are a potential risk for RPL, possibly due to oxidative stress mechanisms.

Teratogens: a public health issue - a Brazilian overview.

Congenital anomalies are already the second cause of infant mortality in Brazil, as in many other middle-income countries in Latin America. Birth defects are a result of both genetic and environmental factors, but a multifactorial etiology has been more frequently observed. Here, we address the environmental causes of birth defects - or teratogens - as a public health issue and present their mechanisms of action, categories and their respective maternal-fetal deleterious effects. We also present a survey from 2008 to 2013 of Brazilian cases involving congenital anomalies (annual average of 20,205), fetal deaths (annual average of 1,530), infant hospitalizations (annual average of 82,452), number of deaths of hospitalized infants (annual average of 2,175), and the average cost of hospitalizations (annual cost of $7,758). Moreover, we report on Brazilian cases of teratogenesis due to the recent Zika virus infection, and to the use of misoprostol, thalidomide, alcohol and illicit drugs. Special attention has been given to the Zika virus infection, now proven to be responsible for the microcephaly outbreak in Brazil, with 8,039 cases under investigation (from October 2015 to June 2016). From those cases, 1,616 were confirmed and 324 deaths occurred due to microcephaly complications or alterations on the central nervous system. Congenital anomalies impact life quality and raise costs in specialized care, justifying the classification of teratogens as a public health issue.

Music genetics research: Association with musicality of a polymorphism in the AVPR1A gene.

Musicality is defined as a natural tendency, sensibility, knowledge, or talent to create, perceive, and play music. Musical abilities involve a great range of social and cognitive behaviors, which are influenced by both environmental and genetic factors. Although a number of studies have yielded insights into music genetics research, genes and biological pathways related to these traits are not fully understood. Our hypothesis in the current study is that genes associated with different behaviors could also influence the musical phenotype. Our aim was to investigate whether polymorphisms in six genes (AVPR1A, SLC6A4, ITGB3, COMT, DRD2 and DRD4) related to social and cognitive traits are associated with musicality in a sample of children. Musicality was assessed through an individualized music therapy assessment profile (IMTAP) which has been validated in Brazil to measure musical ability. We show here that the RS1 microsatellite of the AVPR1A gene is nominally associated with musicality, corroborating previous results linking AVPR1A with musical activity. This study is one of the first to investigate musicality in a comprehensive way, and it contributes to better understand the genetic basis underlying musical ability.

Thalidomide embryopathy: Follow-up of cases born between 1959 and 2010.

BACKGROUND: Thalidomide is a known teratogen and it is estimated that more than ten thousand babies were affected by thalidomide embryopathy (TE), which is characterized mainly by limb defects, but can involve many organs and systems. Most people with TE were only evaluated at birth and it is not well established if thalidomide exposure during embryonic development leads to later effects. We analyzed the clinical history of adults with TE to better understand this gap in the clinical findings of TE. METHODS: Brazilian individuals with TE were invited to answer a clinical questionnaire which considered family history, social information, medical history, and current clinical and psychological health status. A clinical examination was also performed, including on the infant subjects to evaluate congenital anomalies. The characterization of the features was analyzed using descriptive statistics and Chi-square or Fisher's exact test. RESULTS: The congenital anomalies caused by thalidomide were reviewed in 28 Brazilian individuals, and the questionnaire was applied to the 23 adult subjects with TE (aged 19 to 55). Progressive deafness and dental loss were reported. From the comparison of TE individuals with the general Brazilian population, the early onset of cardiovascular diseases (p = 0.009) and a higher frequency of psychological disorders (p = 0.011) were observed. CONCLUSION: Although there is no sufficient evidence that thalidomide exposure caused or worsened the described events, this approach helps to better understand the TE phenotype, improves the clinical diagnosis, and can lead to adequate health support for these individuals.

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.

Caffeine intake during pregnancy and adverse outcomes: An integrative review.

Caffeine intake during pregnancy is common. Caffeine crosses the placenta, raising concerns about its possible deleterious effects on the developing embryo/fetus. Studies on this subject show conflicting results, and still there is no consensus on the recommended dose of caffeine during pregnancy. We performed an integrative review with studies from six databases, using broad MESH terms to allow the identification of publications that addressed the outcomes of caffeine use during pregnancy, with no date limit for publications, in English and Portuguese language. The research returned 16,192 articles. After removing duplicates, screening by title, abstract and full-text, we evaluated 257 and included 59 articles. We found association between caffeine intake and pregnancy loss, low birth weight, cardiac and genital anomalies, higher body mass, and neurodevelopmental and neurobehavioral outcomes. The effects were often dose dependent. No association with prematurity has been demonstrated, but one study showed a small reduction in gestational age with increasing doses of caffeine intake. Defining a safe dose for caffeine intake during pregnancy is a challenging task due to the heterogeneity in study designs and results, as well as the difficulty of reliably assessing the amount of caffeine consumed. In some studies, exposures below the recommended level of caffeine intake during pregnancy (200 mg/day), as suggested by the guidelines, were associated with pregnancy loss, low birth weight, cardiac and genital anomalies, higher body mass, and neurodevelopmental and neurobehavioral outcomes. Well-designed studies with reliable quantification of caffeine intake are needed to assess the safety of low doses during pregnancy.