Is burnout empirically distinct from depression and anxiety in intensive care unit clinicians?This cross-sectional study used baseline data from a randomized clinical trial of 715 clinicians and found that burnout was statistically distinct from anxiety and depression using both latent variable and exploratory graph analysis. Core indicators of value for inclusion in short screening instruments were identified.These findings suggest that health professionals at high risk of stress need to be screened for both burnout and clinical symptoms, such as anxiety and depression, to provide timely and efficient treatment. This cross-sectional study examines the associations of symptoms of burnout, depression, and anxiety in intensive care unit clinicians in Brazil. It is unclear whether burnout, anxiety, and depression constitute the same or different constructs. Better understanding of these constructs is important for diagnosis and treatment for intensive care unit (ICU) clinicians.To determine the associations and distinctiveness of burnout, depression, and anxiety in a sample of ICU clinicians.This cross-sectional study used baseline data from the ICU Visits Study, a cluster-randomized crossover clinical trial conducted from April 2017 to July 2018 in 36 mixed public and private nonprofit ICUs in Brazil. ICU clinicians, including day-shift physicians, nurses, nurse technicians, and physiotherapists working in an ICU at least 20 hours per week, were enrolled. Data were analyzed from December 27, 2019, to October 10, 2020.The main outcome measures were burnout, depression, and anxiety measured with the Maslach Burnout Inventory  and the Hospital Depression and Anxiety Scale . Internal consistencies were satisfactory.The total sample included 715 ICU clinicians , including 96 physicians (13.4%), 159 nurses (22.2%), 358 nurse technicians (50.1%), and 102 physiotherapists (14.3%). Clinicians reported low levels of emotional exhaustion , depersonalization , and personal accomplishment  on the MBI, and similarly low levels of depression  and anxiety  on the HADS. Confirmatory factor analyses consistently showed improved fit separating latent burnout dimensions from depression and anxiety. An exploratory graph analysis combining gaussian graphical model with clustering algorithms for weighted networks suggested 3 clusters, with distinct burnout, anxiety, and depression clusters. This structure was confirmed using a bootstrap with 1000 random samples, in which the 3-cluster solution emerged in 625 samples (62.5%). Both latent variable loadings and network statistics suggested 3 key indicators  that can be used for short screening instruments.These findings suggest that burnout and clinical symptoms of depression and anxiety were empirically distinct in a large sample of ICU clinicians, highlighting the importance of screening for burnout and clinical symptoms to allow fast access to adequate support and treatment in health professionals at high risk of burnout. Burnout is classified as an occupational syndrome13 A 2018 systematic review11 indicated that the heterogeneity of published research does not allow a reliable examination of comorbidities, raising questions about whether it is possible to clearly distinguish burnout as an occupational syndrome from potentially underlying comorbidities. Similarly, studies in nonhealth sectors come to conflicting conclusions about the burnout-depression association. Emotional exhaustion and depression often correlate at moderate to high levels, with lower-quality studies reporting higher correlations12 and few high-quality studies using appropriate statistical methods to empirically test the distinctiveness of burnout from depression.9 The uncertainty around the possible distinctiveness raises important clinical questions for assessing the health status and providing adequate treatment options.13However, there is currently a significant level of discussion and debate about the associations and distinctiveness of burnout with other mental health problems, including depression and anxiety.The objective of our study is to empirically differentiate among depression, anxiety, and burnout in a representative sample of ICU clinicians, using appropriate statistical techniques and sufficient sample sizes to overcome noted problems with previous studies.15 a cluster-randomized crossover clinical trial designed to assess the effects of a flexible ICU visiting policy on outcomes for patients, family members, and ICU clinicians. Details of trial rationale and methods have been reported previously.14 The ICU Visits study was conducted from April 2017 to July 2018 in 36 mixed Brazilian ICUs after approval by institutional review boards of all centers. All participating ICU clinicians provided written informed consent for study participation. The secondary analysis of data used in this cross-sectional study was covered by the original institutional review board protocol of all participating centers. Data were analyzed from December 27, 2019, to October 10, 2020.We performed a subanalysis of baseline data from the ICU Visits Study,19In this secondary cross-sectional study, we assessed the overlap of burnout, depressive, and anxiety symptoms among ICU clinicians using confirmatory factor analysis (CFA) and exploratory graph analysis (EGA).At the cluster level, medical-surgical ICUs from 36 hospitals in Brazil with 6 or more beds and restricted visiting hours , including 19 public hospitals (53%) and 17 private nonprofit hospitals (47%), were enrolled. The median (interquartile range [IQR]) number of ICU beds was 13.5 (10-18) beds. At the participant level, we enrolled day-shift physicians, nurses, nurse technicians, and physiotherapists. ICU clinicians working less than 20 hours per week, planning to take a leave of absence , and those with missing values for burnout, anxiety, or depression outcomes were excluded. Details regarding exclusions are shown in 20 Responses were measured on a scale from 0 (never) to 6 (every day), with higher scores indicating more burnout. A Brazilian Portuguese version was available.21 Anxiety and depression symptoms were measured with the Hospital Anxiety and Depression Scale (HADS),22 using a Brazilian version.23 Symptoms were measured on a scale from 0 (best) to 3 (worst), with higher scores indicating worse anxiety or depression. Both mental assessment tools were chosen based on their reported validity, objectivity, and reliability in previous research for appropriately assessing mental health symptoms in occupational contexts.25 In our sample, reliability estimates using ω showed good internal consistency .26 Descriptive statistics and correlations at item level are available in the eTable in the ICU clinicians were evaluated using self-administered questionnaires 2 weeks before trial interventions initiation. Burnout symptoms were assessed using the Maslach Burnout Inventory (MBI).17 in R statistical software version 4.0.0 . We used the diagonal-weighted least-squares estimator, given its superior performance with ordered data.27 Model fit was evaluated using standard fit indices, including comparative fit index, Tucker-Lewis index , root mean square error of approximation  and standardized root mean residual .29 We also report the robust χ2 value but do not interpret the significance level, given the well-known dependence on sample size.We conducted a series of CFAs using the lavaan packageWe tested a series of theoretical structures to examine the statistical independence of the key constructs as measured with these 2 instruments. We first tested a 1-factor model (M1), in which all items of the MBI and HADS were forced to load on a single factor. We then tested two 2-factor models. First, we estimated a 2-factor model in which all items from the MBI and HADS loaded on their respective instrument factors, conceptually separating a burnout factor from a combined depression and anxiety latent factor (M2). A second plausible 2-factor model is one in which the emotional exhaustion and depersonalization items from the MBI and the anxiety and depression items from the HADS are forced to load on a single emotional distress and clinical symptoms factor and the personal accomplishment items from the MBI load on a efficacy factor (M3). This permits a broad test of the distinctiveness of the clinical symptoms from core components of burnout.We then tested a series of 3 factor models that further probe the comparative distinctiveness of burnout components from clinical syndromes. First, we tested a model in which emotional exhaustion and depersonalization from the MBI and personal accomplishment from the MBI were loaded on 2 separate factors and anxiety and depression were loaded on a single factor (M4). Second, we tested a model in which the emotional exhaustion and depersonalization items from the MBI and the depression items from the HADS were loaded on a single factor (testing the depression association of burnout), personal accomplishment was loaded on a second factor, and anxiety was loaded on a third factor (M5). Third, we tested the anxiety association of emotional exhaustion and depersonalization by forcing the core components of the MBI and anxiety items from the HADS to load on a single factor, depression items and personal accomplishment items were loaded on their separate factors (M6). Fourth, we tested a model in which we forced all burnout items to load on a single burnout factor and allowed separate anxiety and depression factors (M7).4) and separate anxiety and depression factors (M8). A second 4-factor model separated the 3 MBI factors from a combined HADS anxiety and depression factor (M9).An additional model included 2 burnout factors .30 We first tested a configural model with no equality constraints across groups, in the next step constrained the factor loadings, and finally constrained the item intercepts to be equal.We also tested whether the best fitting model differed for the different professions. We ran a multigroup invariance test, with increasing restrictive equality assumptions across models.33 To overcome problems with latent confounding,34 we tested the distinctiveness of network clusters with bootstrapped EGA, using extended bayesian information criterion (EBIC) graphical least absolute shrinkage and selection operator (GLASSO) estimation.35 The GLASSO36 is a regression-based approach that shrinks coefficients to obtain a network that faithfully represents the network while also reducing near-zero correlations (correlations are represented as edges between nodes in network systems) to exact zero.32 The EBIC GLASSO method has been shown to work particularly well in retrieving a true network structure.37 To identify network communities, we used a walktrap algorithm,38 which has been shown to be superior to standard methods for identifying optimal number of clusters.19 To overcome potential instabilities and accuracy problems in sample specific solutions, we bootstrapped the EGA results using 1000 samples.32We then used a network analysis approach, which is ideally suited to uncover possible associations between symptoms reported in psychological inventories, especially in the context of comorbidities.A total of 959 clinicians were assessed for eligibility . After eOn the MBI, participants reported overall low levels of emotional exhaustion  and depersonalization  and high levels of personal accomplishment . Similarly, on the HADS, participants reported low levels depression  and anxiety .r = 0.02 to 0.13).The CFA analyses showed that across all possible comparisons, a solution combining the core burnout dimensions of emotional exhaustion and depersonalization with either anxiety or depression or both combined always fit worse compared with a model that separated burnout from anxiety and depression . The besAn EGA showed 3 distinct clusters within our network. 2R values  and 0.87 with the standardized factor loadings from M10, the 5-factor CFA model. Therefore, the analyses converge and confirm recently identified core items for short screening instruments.39Both factor loadings and network centrality parameters allow the identification of salient indicators within the burnout-depression-anxiety network . Burnout39This cross-sectional study found that burnout and depression are correlated but empirically distinct latent factors and from distinct networks and that emotional exhaustion and depersonalization form the core of the burnout construct, highlighting that the distinctions within the burnout construct might be of clinical importance. Additionally, we identified 3 central items that were core symptoms of burnout, depression, and anxiety, which could be tracked in large populations using short scales.13 leading to misdiagnosis of symptoms and inappropriate and possibly harmful interventions. Therefore, identifying whether these symptoms are overlapping or distinct has important consequences for efficient diagnosis and delivery of adequate treatment options, which impact both the well-being and effectiveness of clinicians as well as health outcomes of patients in critical care units, especially in the context of the increased demands on ICUs associated with the coronavirus disease 2019 pandemic.The differentiation of burnout from related mental health problems is clinically important because it may be less stigmatizing to classify a physician’s distress as burnout, but it “may prevent or delay appropriate treatment of MDD [major depressive disorder], a serious and sometimes life-threatening mental disorder,” as suggested by Oquendo et al,8 Furthermore, by examining the network parameters of the individual indicators, we identified core behavioral indicators that are central within the burnout and depression networks. These core indicators can be used to rapidly and easily screen both depression and burnout in health care workers, allowing fast access to adequate support and treatment, which is of utmost importance in the current pandemic.The clinical implications of our findings are noteworthy, because previous research has found inconsistent associations between burnout and depression. Using state-of-the-art latent variable and EGAs, which are ideally suited to identify associations with potential comorbidity, burnout and depression are empirically distinct in this high-risk population of clinicians working in ICUs. Burnout itself appears to have 2 distinct components, pointing to a greater need to differentiate clinical profiles of burnout. The combined emotional exhaustion and depersonalization component is more central within the larger network, pointing to the greater clinical relevance.This study has several limitations. First, the randomized clinical trial from which we extracted our data was not primarily designed to assess the associations between burnout, depression, and anxiety symptoms among ICU clinicians. Therefore, our results should be considered exploratory. Second, although the study recruited a relatively large sample of ICUs and ICU clinicians, the conclusions might be limited to the middle-income sociocultural context where the study was conducted. Thus, distinct results are possible in different sociocultural settings. All data were collected using self reports, and this strategy may have resulted in a higher proportion of missing values for MBI and HADS questionnaires than a face-to-face interview would have. Independent assessment by trained clinicians could be included in future studies. We used both theory-driven and exploratory statistical methods to empirically evaluate the overlap between the constructs. The theoretically derived 5-factor structure provided the best fit, but the exploratory EGA approach suggested that a 3-cluster solution is more parsimonious, suggesting that different choices of statistical techniques and cutoff criteria would lead to different conclusions. These findings need replication in new samples using different instruments and a wider variety of statistical techniques.These findings suggest that burnout was empirically distinct from depression and anxiety in population of ICU clinicians who were at high risk of job stress and burnout. Practitioners should screen for burnout as a work-related stress syndrome and for clinical syndromes, such as depression and anxiety, to provide appropriate diagnosis and offer appropriate treatment. Our analysis offers options for measuring core constructs for screening purposes.


Parkinson's disease (PD) is associated with a selective loss of the neurons in the midbrain area called the substantia nigra pars compacta and the loss of projecting nerve fibers in the striatum. Predominant pathological hallmarks of PD are the degeneration of discrete neuronal populations and progressive accumulation of α-synuclein–containing intracytoplasmic inclusions called Lewy bodies and dystrophic Lewy neuritis. There is currently no therapy to terminate or delay the neurodegenerative process as the exact mechanisms underlying the pathogenesis of PD require further investigation. The identification and validation of novel biomarkers for the diagnosis of PD is a great challenge using contemporary approaches and optimizing sampling handling as well as interpretation using bioinformatics analysis. In this review, recent evidences associated with multi-omic data-sets and molecular mechanisms underlying PD are examined. A combined mapping of several transcriptional evidences could establish a patient-specific signature for early diagnose of PD though eligible systems biology tools, which can also help develop effective drug-based therapeutic approaches. It is clinically manifested by resting tremor, cogwheel rigidity, bradykinesia, and postural instability, while the motor syndrome of PD is highly associated with the loss of dopaminergic (DAergic) neurons 2.Providing an established definition, a biomarker is a measured indicator of normal biological processes or pathogenic condition. Specificity, selectivity, reproducibility and easy-collection are important factors that ultimately determine the diagnostic utility of a biological marker. Focusing on PD diagnosis, the baseline evidence is the type of biomarker requested while ideal molecular markers may be premotor, prodromal or motor stage biomarkers. Appropriate biomarkers could also be classified as clinical, genetic, proteomic or biochemical along with imaging evidences. On the other hand, the identification of applicable biomarkers is fairly difficult since clinical recognition occurs after degeneration of a variety of SN neurons As it is widely known that upon a significantly increased level of substantia nigra neuronal degeneration the clinical diagnosis of PD can be performed, it is necessary to reveal characteristic diagnostic criteria for the disease as an important basis before clinical symptoms occur. Among these, premotor stage molecular evidences, susceptibility markers and undoubtedly motor stage indicators in an entire diagnostic panel or potential clinical and imaging biomarkers should be included PINK1a are associated with Parkinsonism that slowly progresses associated with limited doses of L-DOPA PARK2 (the gene that encodes parkin) are linked with Parkinsonism with daily variations whereas genomic duplications in SNCA (the gene that encodes α-synuclein) or prevalent Ala30Pro and Ala53Thr substitutions cause nigral neuronal loss LRRK2  leading to neurofibrillary tangles and predominant Lewy body abnormalities DJ1 and UCHL1  associated with either delayed progressive Parkinsonism or sporadic symptoms expression, respectively, should be emphasized here. Leu166Pro substitution related to DJ1 may lead to Parkinsonism with dementia or amyotrophy Exon-deletion mutations in 3.PTGDS, SLC25A20 and LRRN3A plethora of gene expression studied has been conducted to unravel key genes in PD. In a recent study by Jiang et al., microarray datasets of patients with PD from the Gene Expression Omnibus database were downloaded and alterations between PD and normal groups were compared. From 1229 differential expressed genes nine of them were determined that may serve as potential biomarkers of PD, among them Associations between PD and alterations in pathways that regulate lipid metabolism and mitochondrial dysregulation have been identified in an outstanding work by Chi et al. Upon meta-analysis of multiple datasets obtained from the Gene Expression Omnibus database, including substantia nigra and peripheral blood samples, the levels of MAPK8, CDC42, NDUFS1, COX4I1, and SDHC were found to be downregulated Multiple miRNAs have been associated with PD and could be potential therapeutic targets, as miRNAs are able to specifi-cally regulate the expression of known PD genes and gene products Helicobacter and Mucispirillum with NMS scopes while no genus in blood was referred to Mini Mental State Examination and Montreal Cognitive Assessment scores. In the light the observation of significant alterations of the microbial 16S rRNA gene in PD patients, this study clearly links the fundamental role of the microbial motif to the pathogenesis of PD LRRK2 arise unlike to previous studies Genome wide association studies (GWAS) were performed to determine the association between sporadic PD with variety of genes with emphasis on SNCA encoding a-synuclein, leucine- rich repeat kinase 2 and MAPT appropriate of tau 4.Bioinformatics deals with the development and validation of computational methodologies for the analysis, clustering and validation of molecular and clinical indications, with the aim of diagnostic predictors identification and treatment. The availability of high-throughput technologies indented for clinical applications substantially helps to establish bioinformatic platforms for data pre-processing that could be applied to the storage and integration of homogenous data, the extraction of updated clinical knowledge with analysis pipelines as well as developing therapeutic approaches to consulting patients when interpreting results c oxidase IV were found differentiated while GO analysis indicated that synapse and dopamine metabolic process along with biogenic amine biosynthetic pathways were implicated Many studies present extensive bioinformatics analysis either at gene level or miRNA expression to reveal important molecular markers associated with potential therapeutics for PD. In a recent study by Dong et al., the identification of global miRNA and mRNA expression profiles of normal individuals and patients with PD was carried out using Gene Expression Omnibus database A web portal which can visualize mouse transcriptomic data associated with neurological disorders has been designed, supporting also selection of the ideal mouse model for follow-up investigation. It summarizes data into a centralized pool including annotations to each study for comprehensive exploration NPTX2, DEFA3 and DEFA1. In a recent review by Glaab, it is proposed that modifications of known cellular pathways and molecular processes unraveling could explain systems-level variations in omics datasets. To answer this question, distinct pathways and methodologies can be extracted from well-known databases such as Kyoto Encyclopedia of Genes and Genomes, Gene Ontology database, BioCarta and Reactome In a recent study, a software called TRAM (Transcriptome Mapper) was used and meta-analyses was conducted on brain PD and healthy controls microarray data to ascertain DA neuronal transcription alterations by combining multiple data sets from independent studies Lastly, immunological system and neurodevelopment were enriched in PD-related genes gene set (PDgset) in a study by Hu et al., including 242 genes associated with PD through assimilating data from GO, pathway and pathway crosstalk process. WebGestalt and ToppGene were used to examine the functional characteristics of the PD-related genes 5.PD treatment begins at the onset of the disease, with more than 50% of dopaminergic neurons destroyed. An ideal therapeutic protocol would have already targeted the pre-clinical phase for reserving neurodegenerative effects Neuroprotection focuses on the inhibition of primary neurodegenerative events, providing significant improvements in many PD patients, despite the clinical, etiological, and genetic heterogeneity of the disease. Given that there is an initial loss of phenotype for midbrain dopamine neurons in Parkinson's disease (PD) rather than neuronal death, neurorestoration could also be a promising category of potential treatment presenting placement of new cells including fetal nigral neurons into the striatum of patient 6.The topological complexity of neuropathological vulnerability and transcriptional regulation in PD suggests the need to characterize in more detail the molecular mechanisms underlying disease susceptibility and progression. Gene and pathway analyses provide an effective tool for cross-disease connections and for the molecular effects of factors involved in disease risk. It is increasingly important to discover novel biomarkers at the onset of PD along with the necessity to establish new therapies to prevent increased neurodegeneration and disease progression. The availability of high-throughput technologies intended for clinical applications in combination with computational systems biology approaches and bioinformatics analyses support this valuable purpose.


BRCA1 germline mutation carrier state, which include triple-negative basal breast carcinomas and high-grade serous ovarian carcinomas (referred to as extra-uterine Müllerian carcinomas). Whereas many chronic diseases can currently be prevented , no recent tangible progress was made in cancer prevention of BRCA1 mutation carriers apart from surgical resections of at-risk organs. This lack of progress is largely due to (1) poor understanding of the initiating events triggered by known risk factors in the development of these cancers, (2) the fact that current preventive measures rely on evidence obtained from adjuvant breast cancer treatment that fail to protect against poor prognostic cancers, and (3) problems with using cancer incidence in high-risk women as an ethically justifiable endpoint in cancer prevention trials. Here, we propose that cancer predisposition in BRCA1 mutation carriers is driven, at least in part, by cell-nonautonomous mechanisms  and that biomarkers of epigenomic reprogramming, hypothesized to be a direct consequence of such cell-nonautonomous mechanisms, are attractive as intermediate surrogate endpoints to assess the efficacy of cancer risk-reducing strategies targeting these mechanisms.Women-specific cancers are a major health issue, particularly those associated with the  BRCA1 mutations. Emotional, physiological, and reproductive consequences of these procedures lead a significant proportion of mutation carriers to either delay or refuse these potentially life-saving interventions, hence a need for effective non-surgical means of risk reduction. There are currently no prospectively validated non-surgical measures to prevent cancers of the reproductive tract in BRCA1 mutation carriers. Current non-surgical measures of breast cancer prevention using tamoxifen or aromatase inhibitors rely on evidence gained from adjuvant treatment and offer poor protection against the triple-negative subtype typically associated with the BRCA1 mutation carrier state [Surgical resection of ovaries, fallopian tubes, and breasts is currently the most effective means of cancer risk reduction in individuals with germline er state .BRCA1 mutation carriers will likely come from thorough understanding of the mechanisms responsible for cancer predisposition in this population. BRCA1 is widely regarded as a classical tumor suppressor, implying that loss of its function in a given cell directly leads to increased risk of malignant transformation in that particular cell. Progress in understanding the consequences of BRCA1 mutations on DNA repair pathways evolved from this concept and had a significant impact on the treatment of cancers associated with the BRCA1 mutation carrier state [BRCA1 mutation carrier state that do not directly target tissues that are at elevated cancer risk in carriers of such mutations, but alter homeostasis in tissues that influence Müllerian and mammary epithelia from a distance (cell-nonautonomously) through hormones or cytokines released in the blood circulation  in specific segments of the reproductive tract. We hypothesize that the resulting high replicative age triggers cell-autonomous effects of this carrier state on mitotic homeostasis, resulting in aneuploidy and malignant transformation. Work with experimental animals suggests that accelerated replicative aging in the fallopian tubes of BRCA1 mutation carriers may be driven not only by increased proliferation rate, but also by prolongation of the pre-ovulatory phase of the menstrual cycle, which is clearly associated with increased proliferation in this epithelium [Cancers associated with the  lesions . Reductieuploidy ,24. Thesithelium . BRCA1 mutation carrier state may not be limited to menstrual cycle homeostasis. Other physiological processes or systems such as the immune system [BRCA1/2 mutation carriers [BRCA1 mutation carriers than in unaffected non-carriers [Brca1 mutation confined to ovarian granulosa cells on olfaction, an important driver of reproductive functions, further underscore this complexity [The elements of our hypothesis are shown in e system , fat mete system , etc., ae system ,28,29 whe system . Moreovecarriers . The procarriers . This atmplexity .BRCA1 mutation carrier state such as those illustrated in BRCA1 mutation carrier state should also lead to the identification of novel biomarkers of cancer risk predisposition, facilitating evaluation of cancer risk in germline mutation carriers. This would be especially useful in the management of individuals with germline mutations of unknown significance. Such insights should also lead to the identification of novel biomarkers that may be useful as intermediate surrogates of responsiveness to treatment and intermediate endpoints in cancer prevention trials. Using cancer incidence as an endpoint for cancer prevention trials targeting BRCA1 mutation carriers may raise ethical concerns due to unrealistically long study periods or prohibitive costs. The availability of intermediate endpoint biomarkers would alleviate this concern. Using biomarkers associated with systemic consequences of the BRCA1 mutation carrier state would also raise the possibility of measuring such biomarkers not only in risk-prone tissues, but also perhaps in more readily accessible tissues such as, for example, cervical epithelium, buccal cells, or blood, further facilitating the design and execution of cancer prevention trials [BRCA1 mutation carrier state, including how these consequences are altered as a consequence of age or menopausal status, and of their cell-nonautonomous effects on the tissues at elevated cancer risk in such carriers would accelerate progress toward these goals.Targeting cell-nonautonomous effects of the n trials . A thoroBRCA1 mutation carrier state on fimbrial epithelium [BRCA1 mutation carrier state on extra-uterine Müllerian epithelium, this panel might also represent an attractive biomarker of response to non-surgical means of cancer prevention.Epigenetic reprogramming of the fallopian tube fimbrial epithelium may be a direct consequence of cell-nonautonomous effects of the ithelium . The merithelium , led to ithelium . This efithelium . A diffeithelium . Given tBRCA1 mutation carrier state that indirectly contribute to breast and Müllerian cancer risk elevation in such carriers is a potentially attractive approach toward the important goal of developing effective non-surgical means of cancer risk reduction in this population. Mediators of such cell-nonautonomous mechanism of cancer predisposition could also represent useful biomarkers to facilitate overall evaluation and clinical management of BRCA1 mutation carriers. These biomarkers could even facilitate evaluation of cancer risk in the general population because menstrual cycle homeostasis, which is an important driver of cell-nonautonomous mechanisms of cancer predisposition in BRCA1 mutation carriers, is the greatest known risk factor for the main histological subtypes of sporadic  Müllerian carcinomas. We propose that targeting systemic effects of the


Streptococcus pyogenes and the use of nucleases from other bacteria have increased the diversity of genomic sequences that can be targeted and allow the delivery of protein cargos such as transcriptional activators and repressors. Furthermore, the direct delivery of protein–RNA complexes to plant cells and tissues has enabled the production of engineered plants without the delivery or genomic integration of foreign DNA. Here, we review toolkits derived from bacterial CRISPR systems for targeted mutagenesis, gene delivery and modulation of gene expression in plants, focusing on their composition and the strategies employed to reprogramme them for the recognition of specific genomic targets.Molecular tools adapted from bacterial CRISPR  adaptive immune systems have been demonstrated in an increasingly wide range of plant species. They have been applied for the induction of targeted mutations in one or more genes as well as for directing the integration of new DNA to specific genomic loci. The construction of molecular tools for multiplexed CRISPR-mediated editing in plants has been facilitated by cloning techniques that allow multiple sequences to be assembled together in a single cloning reaction. Modifications of the canonical Cas9 protein from  When fu spacers . The majence NGG . On recoTo direct Cas9 to a desired genetic locus, it is only necessary to recode the 17–20 base pair spacer located at the 5′-end of a single-guide RNA (sgRNA), a fusion of the crRNA and trRNA expressed as a single transcript ,5. To siAgrobacterium tumefaciens, aiming for stable integration of transgenes into the plant genome [A. tumefaciens are generally randomly inserted into the genome, are hemizygous in the regenerated generation (known as ‘T0’) and are often at low copy number. In many species, it has therefore been possible to segregate the transgene locus and the target locus in the progeny (or T1) generation of at least some transgenic events, resulting in transgene-free plants with mutations at the desired target. This has been demonstrated in many species, for example Arabidopsis thaliana (Arabidopsis) [Oryza sativa (rice) [Hordeum vulgare (barley) [Brassica oleracea [The typical workflow for obtaining plants with targeted mutations at one or more loci is to deliver a construct comprising a selectable marker gene; a Cas9 expression cassette and one or more sgRNA cassettes to plant tissues by an established DNA-delivery method. In many species, this is mediated by t genome . The trat genome –10. Howeidopsis) , Oryza sa (rice) , Hordeum(barley)  and Brasoleracea .BsaI, have been created to facilitate the simultaneous assembly of multiple sgRNAs to allow simultaneous induction of mutations at multiple genomic targets, sometimes referred to as multiplexed mutagenesis , it is highly desirable for all gene cassettes to be delivered together in a single multigene vector so that all elements integrate at a single genetic locus. Previously, the assembly of complex multigene constructs was considered a bottleneck in biotechnology. However, concurrent with the emergence of molecular tools for genome-editing, several new methods that enable the facile parallel (simultaneous) assembly of multiple DNA parts with minimal scars have emerged from the nascent field of synthetic biology ,15. The agenesis . A sgRNAZea mays (maize) and Arabidopsis, is to create plasmid backbones containing a selectable marker, Cas9 expression and sgRNA expression cassettes, with regulatory sequences, e.g. promoters and terminators, appropriate for the plant species of interest. The spacer is then inserted into the sgRNA cassette using a Type IIS enzyme to enable scarless cloning  [in N. benthamiana [BsaI. These gene expression cassettes can then be assembled into multigene constructs using a second Type IIS enzyme  toolkit  and Goldthamiana , Solanum(tomato)  and barl(tomato) , and thethamiana . The fleS enzyme . Čermák To avoid the necessity for multigene constructs and to increase the expression level of the sgRNA, viral delivery vectors have been used to transiently express sgRNAs in plants overexpressing Cas9 from a stably integrated transgene. Both Tobacco Rattle Virus (TRV)  and CabbMedicago trunculata, successfully recovering plants in which a 58 kb genomic fragment had been deleted [As described above, most efforts at multiplexed targeted mutagenesis have focused on tools for facile assembly of multiple sgRNA cassettes. However, polycistronic mRNAs with multiple sgRNAs have also been used to avoid the necessity for an individual promoter for each sgRNA. Xie and Yang  expresse deleted .Streptococcus pyogenies (SpCas9), which recognises the canonical NGG PAM. However, variants of SpCas9 with mutations in the PAM-recognition domain that enable recognition of NGA PAMs [Staphylococcus aureus [Francisella novicida (FnCpf1), Lachnospiraceae bacterium ND2006 (LbCpf1) and Acidaminococcus sp. BV3L6 (AsCpf1) were adopted for genome engineering in eukaryotes [The majority of studies have utilised the wild-type Cas9 from NGA PAMs  have beeNGA PAMs , and Cass aureus ,36. Recekaryotes . FnCpf1 karyotes . Xu et akaryotes  construckaryotes  flanked karyotes  reportedThe induced mutations mediated by CRISPR-associated proteins are the result of imperfect repair of DSBs, typically by the endogenous mechanism of non-homologous end-joining NHEJ; . Most reOne of the challenges of HDR in plants is the delivery of sufficient quantities of repair template concurrent with the creation of the DSB. Viral replicons based on Bean Yellow Dwarf Virus (BeYDV), Tomato Leaf Curl Virus (ToLCV) and Wheat Dwarf Virus (WDV) have been used to increase the number of copies of the repair template in many plant species, successfully increasing the frequency of targeted DNA insertion up to 10-fold ,42,46,47Cas9 has two nuclease domains, HNH and RuvC. Disruption of both domains results in deactivated Cas9 (dCas9), which has no nuclease activity but retains the ability to form a duplex with a sgRNA and to scan genomic DNA for PAM motifs allowing the sgRNA to pair with its cognate sequence . dCas9 cSeveral additional methods have been demonstrated in non-plant systems to co-recruit multiple activator domains to the same locus, thus increasing the level of transcriptional activation. A repeated peptide array, known as Suntag, was fused to dCas9 to recruit multiple VPS64 activators bound to single-chain variable fragments to the same locus ,50. In aEscherichia coli (in vitro transcription. RNP complexes have been delivered to plant tissues using particle bombardment (Nicotiana tabacum (tobacco), Lactuca sativa (lettuce) and rice. Mutations at the target were found in 46% of callus tissues regenerated from lettuce protoplasts. Kim et al. [LbCpf1 and AsCpf1 RNPs to induce targeted mutagenesis in protoplasts of soybean and tobacco  complex, avoids the introduction of DNA into the cell. RNP-mediated genome engineering was first shown in mammalian cells ,57 but hhia coli . Similarbardment  and alsobardment . Woo et m et al.  used LbC tobacco . SvitashThe CRISPR system has provided several reliable, flexible and robust methods for engineering plant genomes. The construction of molecular tools to enable their use in eukaryotes, including plans, has been facilitated by parallel assembly methods that allow multiple fragments of DNA to be assembled together in a single cloning reaction. Mutagenesis of the Cas9 nuclease and the adoption of new tools such as Cpf1 have removed the limitation of wild-type SpCas9 to targets associated with NGG PAMs, providing researchers with the opportunity to mutate or deliver protein cargos such as transcriptional activators to a much wider number of sites across plant genomes. Targeted insertion and the editing of endogenous gene to a desired sequence remains challenging, however, targeted mutagenesis has been shown to be efficient in many plant species , enablinMolecular tools adapted from bacterial CRISPR systems have been applied for the induction of targeted mutations in many plant species.The construction of molecular tools for genome engineering has been facilitated by techniques for parallel DNA assembly.Modifications of Cas9 and the exploitation of new CRISPR systems allow targeted engineering of an increasing number of genomic targets.The direct delivery of protein–RNA complexes to plant cells avoids the introduction of DNA into the genome.


Peripheral vascular catheterization (PVC) in pediatric patients is technically challenging. Ultrasound guidance has gained the most interest in perioperative and intensive care fields because it visualizes the exact location of small target vessels and is less invasive than other techniques. There have been a growing number of studies related to ultrasound guidance for PVC with or without difficult access in pediatric patients, and most findings have demonstrated its superiority to other techniques. There are various ultrasound guidance approaches, and a comprehensive understanding of the basics, operator experience, and selection of appropriate techniques is required for the successful utilization of this technique. This narrative review summarizes the literature regarding ultrasound-guided PVC principles, approaches, and pitfalls to improve its clinical performance in pediatric settings. Peripheral vascular catheterization (PVC) is an essential skill for perioperative, intensive, and emergency healthcare providers. In small children, operators often misjudge the exact location of vessels due to the presence of thick subcutaneous tissue and smaller vessel diameters, which in turn makes catheterization challenging. Currently, ultrasound guidance is an approach majorly used in such difficult cases.Numerous studies have demonstrated the utility of an ultrasound-guided approach in PVC in pediatric patients. Systematic reviews and meta-analyses have reported that ultrasound guidance improves pediatric PVC in terms of success rate, procedure time, number of attempts, and number of complications –4. RegarHowever, ultrasound-guided PVC in smaller children remains challenging for less-experienced operators. The first-attempt success rate of ultrasound-guided radial arterial catheterization in pediatric patients was significantly increased than the palpation technique when performed by operators who were familiar with ultrasound guidance ; therefoThis narrative review summarizes the accumulated knowledge and experiences to aid and improve ultrasound-guided PVC in pediatric patients. Notably, we proposed seven basic questions that arise while performing the procedure Table . We thenWe broadly searched for all types of articles in the PubMed and Medline bibliographic databases from 1970 to 2020 using the following term combination in the title/abstract: “ultrasound or ultrasonography or ultrasonographically or peripheral,” “artery or arterial or vein or venous or intravenous or vascular or intravascular or vessel,” and “catheterization or cannulation or insertion or placement or access or catheter or needle or line or cannula.” A total of 23,982 articles were identified, 270 of which were found to be duplicates.The first two authors independently screened the title/abstract of the identified articles, primarily related to vascular catheterization, with the potential to answer the seven questions. We limited the selection to 1057 articles. Thereafter, we excluded articles focusing on central venous catheter insertion, peripheral inserted central catheter insertion, midline catheter insertion, femoral arterial access, and axillary arterial access except for those referring to ultrasound approaches, techniques, pitfalls, or artifacts. A total of 384 full-text articles were assessed for eligibility, and 38 were finally included. Four additional articles were identified from the references.Peripheral arterial access sites include radial, brachial, dorsal pedis, ulnar, posterior tibial, and superficial temporal arteries . The radNumerous studies have confirmed the utility of ultrasound guidance at this site in pediatric and adult patients , 3. One To our knowledge, there are no strong recommendations of ultrasound guidance for ulnar, brachial, and superficial temporal artery catheterization. However, one previous retrospective study reported that ischemic and infectious complications of the ulnar artery were as low as those of the radial artery . AlthougThe posterior tibial and dorsal pedis arteries may be possible alternative access sites to the radial artery in pediatric patients without major complications , 12–14. The most common peripheral intravenous access site is the dorsum of the hands or feet . HoweverVeins at the cubital fossa are impalpable in most cases  and are One of the advantages of ultrasound-guided vascular catheterization is that the distinction between arteries and veins and the patency of the vessels can be confirmed by Doppler mode imaging achieved using the following techniques: (1) lightly squeezing the blood vessels with a probe under the color Doppler mode, (2) viewing the direction of blood flow under the color Doppler mode, and (3) distinguishing between arteries and veins using the wave pattern of the pulse wave Doppler mode , 21. FurThe position has been shown to alter arterial anatomical characteristics. In adult patients, the location and patency of the radial artery differed according to wrist position , 23, andThe determination of catheter size should be based on (1) the relationship between vessel and catheter diameter and (2) the catheter travel distance from skin to vessel. Larger arterial catheters may provide better tracings, easier blood sampling, and longer patency than smaller arterial catheters. However, comparing the use of 18G and 20G catheters in adult patients, the incidence of arterial occlusion after 24 h of cannulation increased linearly, as a larger vessel lumen area was occupied by the catheter 32]. In aIn the LAX-IP approach, the whole shaft of the needle and the entire course of the target vessel remain on the screen during the procedure for ultrasound guidance in “real time,” which is advantageous in terms of avoiding complications . HoweverIn the SAX-OOP approach, only the needle tip is visualized as a high-echoic point with acoustic shadow on ultrasound images, and the remaining shaft is off screen. Briefly, after puncturing the anterior wall at the maximum diameter and confirming the needle tip in the vessel and back flow in the catheter hub, the operator advances the needle slightly with a reduction in insertion angle and places the catheter without ultrasound guidance. The major advantage of the SAX-OOP approach, particularly in pediatric patients, is that the maximum vessel diameter can be constantly visualized until puncturing the anterior wall. However, due to the process being performed without ultrasound guidance, the SAX-OOP approach is considered inferior to the LAX-IP approach regarding “real time.” Additionally, accidental posterior-wall penetration during the procedure off screen is considered the main disadvantage of the SAX-OOP approach. For these reasons, the SAX-OOP approach is referred to as the “static approach” in contrast to “dynamic needle tip positioning” (DNTP), described below.DNTP was first described in 2012 and is an alternative derived from the “static” SAX-OOP approach . In DNTPThe approach is briefly described as follows. (1) The needle tip is visualized as a high-echoic point between the skin and the anterior vessel wall at the maximum diameter in the plane. 2) While holding the needle in position, the probe is advanced slightly forward along the travel course of the catheter until the high-echoic point disappears from the plane. (3) While holding the probe in position, the needle is advanced further until the high-echoic point reappears in the plane. Steps 1, 2, and 3 are repeated alternately until it is safe to thread off the catheter (at least until the whole needle tip is inserted into the vessel)  slightly rotate the probe toward the catheter, (2) move the catheter toward the center line of the probe, or 3) withdraw the catheter to near the skin and restart the procedure  Minimum catheter travel distance from skin to vessel should be calculated from the perpendicular distance corrected by the actual insertion angle. (B) Presumption of an insertion angle of 45° is the most common and convenient way to determine approximate catheter travel distance (Pythagorean theorem).Additional file 3. Representative ultrasound image of the vessel wall tenting. An example of when the needle does not puncture but presses the anterior wall of the vessel due to elasticity. Ultrasound-guided internal jugular venous catheterization of the long-axis in-plane approach in a pediatric patient is provided for improved visualization.Additional file 4. Representative ultrasound image of the vessel walls kissing by puncture pressure. A narrower vessel lumen  under puncture pressure before the puncturing of the anterior wall.Additional file 5. Representative ultrasound images of saline injection method. (A) Radial artery located at a depth of 1.3 mm from the skin surface (white arrow). (B) Saline injection increases the depth from 1.3 mm to 2.6 mm. Furthermore, it provides an anechoic area on the anterior arterial wall, which enhances the ultrasound signals and improves the visibility of the anterior arterial wall and needle tip. (★).


Maternal passage of immunoglobulin G (IgG) is an important passive mechanism for protecting the infant while the neonatal immune system is still immature and ineffective. IgG is the only antibody class capable of crossing the histological layers of the placenta by attaching to the neonatal Fc receptor expressed at the level of syncytiotrophoblasts, and it offers protection against neonatal infectious pathogens. In pregnant women with autoimmune or alloimmune disorders, or in those requiring certain types of biological therapy, transplacental passage of abnormal antibodies may cause fetal or neonatal harm. In this review, we will discuss the physiological mechanisms and benefits of transplacental transfer of maternal antibodies as well as pathological maternal situations where this system is hijacked, potentially leading to adverse neonatal outcomes. Immunological adaptative changes occurring during pregnancy allow maternal tolerance towards the fetus and placenta, which practically constitute semi-allografts as 50% of their antigens have a paternal provenience . This coThe placenta has an extremely important function throughout gestation. It is a histological barrier formed by three layers: (1) multinucleated syncytiotrophoblasts (STB) with a cytotrophoblast layer directly beneath, (2) stroma containing fibroblasts and Hofbauer cells and 3) endothelial cells of the fetal capillaries [ endothelIgG is a large protein with a tetrameric structure, containing two heavy and two light chains disposed in a Y-like shape. In its structure, there is an antigen binding site (Fab region) and a constant region, the crystallizable fragment (Fc), which binds to Fc receptors found on the surface of different cells in the immune system, most importantly on phagocytes. There are four types of IgG 1–4), and all of them are transferred across the placenta via syncytiotrophoblast cells that express receptors for the Fc domain, called neonatal Fc receptors (FcRn) , and all,6.Active transplacental transfer starts early in the course of pregnancy, at about 13 weeks of gestation, and fetal levels of IgG increase with advancing gestational age, being relatively low between 17 and 22 weeks at about 5–10% of maternal levels, then by 32 weeks reaching 50% of maternal levels, and finally exceeding maternal plasma IgG levels at birth [Besides gestational age as an important factor in the transfer of antibodies, the process is influenced by several aspects such as maternal immunocompetence, concomitant infections, specific antibody levels acquired postimmunization, placental integrity, class of IgG and type of antigen . These vNeonatal Fc receptor is a major histocompatibility complex class I (MHC)-related molecule that plays a central role in the regulation of IgG homeostasis and transport across the placental barrier . ExpressThe interaction between the Fc domain of IgG and FcRn is influenced by pH changes, presenting high affinity at acidic pH level < 6.5 and low affinity at physiologic pH . During In humans, FcRn are also expressed on both endothelial and bone marrow-derived cells, playing a role in prolonging the half-life of IgG and albumin ,19 by prThe transplacental transfer of maternal antibodies to the fetus begins during the first trimester of pregnancy. Very little maternal IgG reaches the embryo and could be found in the coelomic fluid and villous stroma as early as 6 weeks . ApproxiThe immunoglobulin rise in the fetal circulation is different between the four types of IgG. The fastest transfer was observed for IgG1 and the slowest for IgG2. At term, in the fetal circulation IgG1 level was seven times higher than IgG2 level. There are also differences between the two IgG types compared to maternal levels: fetal IgG1 level exceed the maternal concentration near term or after birth, while fetal IgG2 remain below maternal level ,30. ThesSerum maternal immunoglobulin levels correlate to the placental transfer and neonatal IgG levels. Higher maternal IgG levels, above 15 g/L, have a negative influence on transplacental IgG passage and correlate to lower neonatal IgG levels, although in normal conditions fetal IgG levels in the third trimester surpass the maternal concentration. Paradoxically, when maternal level is too high, as is the case in maternal hypergammaglobulinemia, FcRn are oversaturated and unbound IgG molecules are destroyed , leadingOn the other hand, in immunocompromised women secondary to HIV infection, reduced humoral immune response and reduced antibody production are linked with poor transfer of antibodies against specific pathogens such as group B streptoccocus, pertussis or Haemophilus influenzae ,34. In wWith an unclear mechanism of impairment, placental malaria has been associated with reduced IgG transplacental transfer . Pregnancy is associated with a particular background as the maternal immunological response is inhibited in order to tolerate the fetus and placenta. Some infectious diseases may be more severe during pregnancy, thus the most effective way to provide protection for the mother and the newborn is vaccination. Current guidelines recommend administration of inactivated vaccines, while live vaccines are contraindicated at least 4 weeks before conception and definitely during pregnancy. Vaccines routinely administered during pregnancy include inactivated influenza vaccine and Tdap, with some others such as those against pneumococcus, meningococcus, hepatitis A and hepatitis B being justified under specific circumstances . Influenza vaccine in pregnancy was introduced in 2004 as part of a routine immunization program. It was observed that pregnant women are at greater risk of developing severe complications following influenza infection and that immunization in pregnancy, irrespective of trimester , reducesA study on 112 mother–infant pairs highlights the strong correlation between maternal and neonatal antibody levels and the importance of passive immunity in newborns. As a consequence of low maternal immunity, only 30%, 42% and 82% of infants had protective antibody levels to pertussis, Haemophilus influenzae type b (Hib) and tetanus, respectively. Therefore, a high proportion of neonates are susceptible to those infections until active immunization is allowed . In thisIt is important to mention that higher doses of passively acquired antibodies may suppress the immune response after vaccination in early infancy . SeveralDue to general vaccination programs, most data showed high seropositivity for measles, mumps, rubella and varicella-zoster (VZV) in women of childbearing age. Maternal antibodies and transplacental passage protect newborns in the first few months of life against most vaccine-preventable diseases, and this protective level is maintained up to 6 months of age. In normal conditions, the rubella, measles or VZV antibody titer in the cord blood of neonates born at term exceeds maternal antibody levels. There is a high prevalence of anti-rubella and anti-VZV protective IgG titers, but there is a decreasing trend in anti-measles and anti-mumps immunity levels in pregnant women and neonates. Therefore, new preventive strategies have been proposed, such as measles-mumps-rubella (MMR) screening and vaccination of susceptible women of childbearing age or MMR vaccination of women who have no documentation of completed vaccination and no laboratory evidence of immunity ,48,49. LPassively acquired maternal antibodies with different antigen-specificities have been found to have different half-lives in infants. For example, although in normal pregnancy pertussis-specific IgG levels in cord blood achieve more than 100% of maternal levels, maternal pertussis-specific IgG has a half-life of 6 weeks in infants and decreases to undetectable levels as early as 4 months of life . In contOngoing efforts are being made to develop future vaccines for maternal immunization, the two most promising vaccines being respiratory syncytial virus vaccine, in phase III trial, and group B streptococcus vaccine to prevent late-onset neonatal infection, currently in the early stages of development. A vaccine against cytomegalovirus is also under development ,52,53,54New emerging viruses with potential harm for the fetus, such as the Zika and Ebola viruses, have also been under investigation for the development of future vaccines ,56. The greatest challenge of our time is the COVID-19 pandemic, and special attention has been drawn to pregnant women and their infants in terms of vertical transmission, severity of the disease and immunologic response. Although there is no clear evidence of vertical transmission due to lack of angiotensin-converting enzyme 2 (ACE2) receptors for SARS-CoV-2 virus in the placenta , the immIn a small cohort of six infants born to mothers with COVID-19 infection, IgG was present in all cases and the neonatal level was well correlated with maternal immunoglobulin levels. However, the study did not investigate whether the presence of antibodies in the neonates had a protective effect against infection as there was no long-term neonatal antibody level follow-up . One casTransplacental immunoglobulin transfer plays an important role in protecting the newborn in the first few months of life, when the immune system is immature and active immunization through vaccination is not yet recommended. Despite this clear benefit, there are situations in which transplacental transfer may be damaging to the fetus. Particular maternal conditions are further discussed.Fetal or neonatal goiter may be the result of transplacental passage of maternal antibodies in women with Graves’ disease and occurs in almost 20% of cases . AntibodDue to pregnancy-induced immunosuppression, TRAb levels detectable in the first trimester tend to decrease after 20 weeks of pregnancy, resulting in the amelioration of the thyrotoxicosis in women with Graves’ disease. TRAbs can cross the placenta from the first few weeks of pregnancy, and the rate increases with advancing gestation. Thus, it is recommended to measure maternal TRAb concentrations in the first trimester and at 24–28 weeks of gestation and to monitor the pregnancy more carefully if antibody levels surpass three times the normal values. Ultrasound evaluation along with maternal history and TRAb levels are important to establish the diagnosis. The fetal thyroid gland can be assessed by ultrasonography after 20 weeks of gestation, when fetal goiter appears as an anterior cervical echogenic mass . DependiAnti-Ro and anti-LA antibodies were initially described in association with systemic lupus erythematosus (SLE) and Sjögren’s syndrome, but they can also be found in relation to other autoimmune diseases such as mixed connective tissue disease or systemic sclerosis . Some paNeonatal lupus is a passively acquired autoimmune disorder as a result of transplacental passage of maternal anti-Ro/anti-La antibodies, and this condition can have cutaneous, hematologic or cardiac manifestations. The most threatening complication is fetal complete atrioventricular block, which occurs in 2% of nulliparous women with positive anti-Ro antibodies and increases to 20% in pregnancies with a previously affected fetus ,69. AntiPrenatal counseling is important because neonatal lupus is responsible for 80 to 95% of all cases of congenital complete heart block in the absence of structural defects diagnosed in utero or in the neonatal period ,76. CongThird-degree heart block can lead to heart failure and to hydrops. The risk of death amongst affected fetuses is about 15%, and 70–80% of the survivors require placement of a pacemaker within the first 10 years of life ,79.Due to increased mortality and morbidity, in pregnant women with known positive immune status, close monitoring should be carried out by a multidisciplinary team during pregnancy and immediately after birth . As a stRegarding the skin manifestations, the typical rash with annular lesions mainly located on the scalp or periorbital area with a raccoon-like appearance can be present at birth or may not develop until exposure to ultraviolet lamp. The rash is self-limiting, resolving in approximately six to eight months due to the half-life of IgG of about 21–25 days ,84.The risk of having a child with complete heart block increases with the titer of maternal antibodies and maternal history of previous affected pregnancies.Another autoimmune maternal condition that may affect the management of pregnancy and fetal outcome is the immune-mediated thrombocytopenia. Primary autoimmune thrombocytopenia (ITP) in pregnancy has an incidence that ranges between 1 in 1000 and 1 in 10,000 pregnancies ,86. Comp9/L for epidural anesthesia and >50 × 109/L for cesarean delivery [The majority of infants born to mothers with ITP may have normal or mild thrombocytopenia, but the full count may decrease sharply in two to five days after birth . The maidelivery .Prenatal or preconceptional screening for ITP is recommended, especially when the obstetric history shows an increased risk, for example a previously affected child. Myasthenia gravis (MG) is another autoimmune disease that may interfere with the development of a normal pregnancy and neonatal outcome. This neuromuscular disease is characterized by autoantibodies directed against the acetylcholine receptors (AChR) found at the neuromuscular junction of the skeletal muscles. Myasthenia gravis affects between 1 in 10,000 and 1 in 30,000 pregnant women, depending on the geographic area , and traThe severity and duration of maternal MG does not correlate with the risk of transient neonatal disease, but the history of a previous affected child can predict a risk of recurrence of about 75%.Autoimmune hemolytic anemia (AIHA), primarily caused by pregnancy, is characterized by erythrocyte autosensitization and development of antibodies against one’s own red blood cell antigens. Autoimmune hemolysis associated with pregnancy occurs very rarely, with an incidence estimated at 1 in 50,000 pregnancies . Autoimmune hemolytic anemia may be secondary to warm, cold, or a mix of warm and cold autoantibodies . Warm-acMaternal warm (IgG) autoantibodies can cross the placenta and determine variable forms of hemolytic disease in the fetus, ranging from mild disease to severe hemolysis. The risks to the infant were increased when erythrocyte autoantibodies were part of other active autoimmune conditions such as systemic lupus erythematosus. Evans syndrome, which is associated with autoimmune hemolytic anemia and immune thrombocytopenic purpura, carries high risks of fetal morbidity, including severe hemolysis and intracranial bleeding with neurological sequelae and death . Close fetal surveillance with Doppler examination of peak systolic velocity on the middle cerebral artery can predict cases at increased risk of fetal complications and dictate the appropriate management  Figure .Neonatal autoimmune blistering disease (AIBD) has a low incidence, and only a few case reports have been published so far. Neonates born to mothers who are known before conception to have autoimmune blistering diseases such as pemphigus vulgaris (PV), pemphigus foliaceus (PF), bullous pemphigoid (BP), linear Immunoglobulin A bullous dermatosis (LABD) or epidermolysis bullosa acquisita (EBA) may be affected due to transplacental passage of IgG autoantibodies. Usually these are transient conditions and improve spontaneously within a few weeks after birth, after maternal autoantibodies gradually decrease in the neonate’s blood. Autoimmune blistering disease might increase the risk of preterm birth and growth restriction, and there have also been reported cases of stillbirth and neonatal death related to pemphigus vulgaris. In the case of neonatal blistering disease, specific investigations should be undertaken in order to confirm the etiology. Confirmation of the autoantibodies involved is essential. Autoantibodies anti-desmoglein 1 and 3  are related to PV or PF (only anti-Dsg-3), anti-BP180 antibodies are related to bullous pemphigoid, and anti-type VII collagen antibodies are found in EBA. Cases of neonatal LABD were independent of maternal disease and appeared to have a more severe blistering form ,103.A rare autoimmune disease associated with pregnancy is pemphigoid gestationis (PG), previously known as herpes gestationis. This is an AIBD caused by the production of autoantibodies against adhesion molecules, mainly against bullous pemphigoid BP180 or collagen XVII . ClinicaAs a consequence of transplacental passage, neonatal PG can develop in 10% of cases. This is a mild condition that resolves spontaneously as antibody levels decrease. Another important implication is that BP180 antibodies can also attack chorionic cells with detachment of basement membranes and undeveloped hemidesmosomes, resulting in placental insufficiency associated with fetal growth restriction, preeclampsia or premature delivery ,107.There is little knowledge about the subject, but close fetal monitoring is recommended in the case of maternal symptoms due to risks for prematurity and fetal growth restriction .Around 50% of fetal antigens have a paternal origin, and maternal immunization to different fetal antigens inherited from the father, unrecognized as ″self″, determines an immune response with production of antibodies and placental transfer into fetal circulation leading to destruction of different fetal cells, mainly blood cells.Human red cells express hundreds of different blood group antigens, and some of them have been reported to determine a maternal alloimmune response. The Rh system is the most complex of the human blood group systems, consisting of about 45 antigens. Clinically, the most important antigen is D. This antigen is immunogenic and is well developed early in gestation; 12% of the Caucasian population is D-negative, and the maternal antibodies cause fetal hemolysis . Small vHDFN in the ABO system is restricted almost exclusively to the fetuses of group O mothers. ABO HDFN requiring any clinical intervention before birth is rare as A and B red cell antigens are not fully developed in the fetus. Also, A and B antigens are histo-blood antigens that can be found in other cells, for example in the placenta; therefore, antibodies transferred from the mother are held and destroyed at this level. In the context of generalized screening and prophylaxis against anti-D immunization, another antigen, part of the Kell blood group system, has attracted attention in the etiology of HDFN. The K-positive phenotype is present in about 9% of Europeans, and the anti-K antibody is considered the most common immune red cell antibody outside of the ABO and Rh systems . In immuAlloimmune thrombocytopenia is found in about 1 in 2000 pregnancies  and in mUnlike HDFN, in alloimmune thrombocytopenia, maternal sensitization to fetal platelet antigens often occurs in the first pregnancy, indicating that platelet antigens may be more immunogenic than red cell antigens. In alloimmune thrombocytopenia, compared to autoimmune maternal conditions, the severity of fetal platelet decrease is greater and consequently the risk of intracranial bleeding is higher, estimated at around 20% . Also, tUnfortunately, as currently there is no routine screening for HPA antibodies, most cases are diagnosed after birth or when severe fetal thrombocytopenia leads to intracranial bleeding that could be detected by ultrasound evaluation. Ultrasound findings in the case of intracranial hemorrhage are ventriculomegaly, clots within lateral ventricles, porencephaly, signs of severe anemia and hydrops  Figure . Due to Gestational alloimmune liver disease (GALD) is an alloimmune disorder and the main cause of neonatal acute liver failure. The disease is caused by maternal IgG directed against fetal hepatocytes, and although the exact antigen is not yet described, it is supposed to be expressed early in embryonic life on the hepatocytes . AnotherGALD has a poor prognosis associated with a high risk of mortality, close to 80%, and need for liver transplantation. A woman may have several unaffected pregnancies but once the condition occurs, the risk of recurrence in subsequent pregnancies is about 90%. Correct diagnosis and appropriate management initiated early in pregnancy might prevent recurrence in almost 90% of cases ,121.Different clinical implications in both the mother and the fetus are presented in Numerous autoimmune disorders have been identified so far, and almost 80% are encountered in women of childbearing age . The evoAnti-tumor necrosis factor alpha (anti-TNFα) antibodies are probably the most studied biological therapy used in pregnancy. Anti-TNFα antibodies such as infliximab and adalimumab are IgG1 antibodies that have a preferential transfer across the placenta starting at the end of the second trimester; therefore, neonatal drug levels may exceed maternal levels and may persist up to 12 months after birth . EtanercThe current data show that pregnancies exposed to anti-TNFα are not associated with an increased risk of fetal malformations, preterm delivery or pregnancy loss ,129,130.Initially used to compensate for the deficient immune response in immunocompromised patients, iv IgG is now a widely used therapy in autoimmune and systemic inflammatory diseases ,136. Iv Preventing harmful antibodies from reaching the fetal circulation, iv IgG is currently used in autoimmune thrombocytopenia, myasthenia gravis and systemic lupus erythematosus . Also, iThe transplacental passage of maternal IgG antibodies is of great importance to the fetus and newborn, offering suitable protection until maturation of the immune system and until active immunization through vaccination is allowed. On the other hand, in some conditions such as maternal autoimmune disorders or maternal immune responses against paternally inherited fetal antigens, the physiological passage of immunoglobulin can have a deleterious effect upon the fetus to various degrees of severity and with potential long-term implications.


Angiogenesis is a strictly controlled process defined as the formation of new blood vessels essential for certain physiologic and pathologic conditions where the latter includes tumor growth, development, and metastasis. Thus, inhibiting angiogenesis along with other anticancer strategies such as chemotherapy seems to be invaluable for reaching an optimal outcome in cancer patients. It has been shown that some natural plant-derived compounds are capable of preventing the formation of these new blood vessels in the tumor and also inhibit the proliferation and growth of the cancer cells. In this review, we intend to introduce plants with anti-angiogenic properties and discuss their related features. Angiogenesis plays an unquestionable role in some (certain) physiologic processes such as wound healing, diabetes, as well as pathologic conditions of tumor growth and metastasis.5 According to the significance of this cascade in cancers, researchers seek to find derivative compounds from herbal sources to inhibit angiogenesis. Due to fewer side-effects of herbal compounds in the treatment of angiogenesis-related disorders, investigations on plants in order to identify and discover such compounds could be a quite promising therapeutic approach. Use of herbs as treatment for cancer has a long history; so that herbs have been the primary source of traditional medications for treatment of various diseases. Although the real compounds derived from plants might not usually be utilized as medications, plants are still considered important sources for development of novel therapeutic factors for researchers. Herbal-rich food regimen not only provides the body with necessary vitamins and minerals, but also there are over 25 000 chemical compounds in different herbs; most of which hold biological effects and characteristics.7 The molecules in herbal compounds are able to bind the therapeutic agents to carrier molecules. These plant-derived tumor-targeting complexes shape the hope for producing natural drugs more effectively and highly toxic to tumors, but not normal tissues. After the identification of new proteins with important regulatory effects on the tumors’ cell cycle advancement, researchers have proven that the molecules derived from plants and other organisms could be among important inhibitors sources of synthesis or function of these key proteins. Therefore, these herbs have the potential to bring about developing novel anti-cancer agents and medications.9Man has long worked to fight the difficulties he encounters through his life and has tried various methods to control diseases. These efforts include investigating medicinal herbs which have long been used to cure diseases and still continue to keep their advantages despite the technological and scientific advances and the ever-growing use of chemical substances in modern medicine.13 Angiogenesis in tumors is intervened by targeting several molecules including nitric oxide (NO) which is a critical intermediate in angiogenesis that enhances endothelial cell survival, proliferation, and migration; so, it is basically considered a pro-angiogenic factor.15 On the other hand, some of the growth factors such as vascular endothelial growth factor (VEGF) are highly specific for endothelial cells while some others such as matrix metalloproteinases (MMPs), and basic fibroblast growth factor (bFGF) have a broader spectrum of target cells. Activating factors can be secreted by tumor cells, the surrounding tissues, fibroblasts, or by macrophages which enter the tumor microenvironment. Currently, the use of VEGF pathway inhibitors in angiogenesis is considered as an anti-cancer treatment strategy with clinical credit.23Angiogenesis is the formation of new capillaries from primary blood vessels and is involved in some pathologic processes such as tumor growth and metastasis. It is also play role in certain physiologic processes like organ growth and development as well as wound healing. Angiogenesis is a necessary procedure in natural physiology of the body and if the equilibrium between its inducers and inhibitors is disturbed, opportunity will be provided for some diseases to arise.26Plants contain several active chemical compounds simultaneously and unlike chemical drugs, they can have synergistic effects and therefore, influence different aspects of disease pathology at the same time. In another words, plant extracts rich in biologically active compounds can slow down the growth of cancer cells and induce apoptosis in them at the same time which leads to tumor eradication by hindering angiogenesis and therefore, metastasis. Interactions of the active ingredients in plants extracts with tumors can give this opportunity to the immune system to identify and respond to the tumor cell.28 Table 2 shows some of the discovered plants and their derivatives which inhibit VEGF.27 Studies have shown, fewer side-effects of herbal compounds could be imagined compared to the chemicals and anti-cancer agents. However, there are diverse unwanted effects regarding the application of crude plant extract in in vivo condition. The use of medicinal herbs should be standardized since their direct use could sometimes lead to severe poisoning, allergic reactions, bleeding, and cases of death. Herbal remedies may interfere with the absorption of certain necessary nutrients. Other types also increase or decrease the effect of a particular drug along with exhibiting serious side effects.28Nowadays, the importance of food of plant origins in preventing various diseases such as cancer, which depends on angiogenesis for growth, has been well proven. However, further studies are still required in order to study and discover more therapeutic plants with anti-angiogenic effects. Allium ascalonicum .35 In a study by Kwon et al in 2010 on human melanoma cells and mouse melanoma, it was shown that cinnamon extract down-regulated activator protein 1(AP1) and nuclear factor kappa B(NF-KB) levels and also increased apoptosis rate in various cancer cells such as lymphoma, cervical cancer, and colorectal cancer.Cinnamon  is a spi39 According to available information, the plant and its derivatives have antiseptic activity and play a role in regulating apoptosis.Oral administration of cinnamon extract in a melanoma model, exerted a significant anti-tumor effect and inhibited tumor growth.40 Therefore, oak can exert inhibitory effects on tumor growth by inhibiting factors involved in angiogenesis.Oak  is a shrP. harmala extract can induce apoptosis and inhibit tumor growth in vitro by affecting BCL-2 and P-Akt genes expression. Moreover, harmane has been shown to decrease NF-KB, MMP2, and MMP9 expression. These results show that HM acts as an anti-angiogenic factor in preventing cancer.43Peganum harmala  is a plaC. melo seeds (TICMS) inhibit endothelial cell migration and cell proliferation of human umbilical vein endothelial cells (HUVECs). TICMS affect the secretion of MMP2, MMP9 and VEGF from HUVEC and prevents their function. Therefore, it could be considered as an angiogenesis inhibitor.45Melon  is a natNigella sativa (black caraway)   is an an50Other studies have also shown that this plant prevents transcription of the angiogenesis factors of VEGF and HIF1α. In addition, it decreases the activity level of the enzymes MMP2 and MMP9.Marsdenia tenacissima  associates with suppressed tumor growth and decreased angiogenesis in A20 mouse lymphoma model.54The stem of acissima , also knCurcuma longa . Its anti-metastasis effects is partly mediated through decreasing expression of the enzymes MMP2,9 and COX-2 and also increasing the enzyme tissue inhibitor of metalloproteinases 1 (TIMP1). Studies have also demonstrated that this compound prevents the transcription of the angiogenesis factor VEGF.60 Wheatgrass inhibits the process of angiogenesis through accumulation of polyphenols via PI3K/AKT pathway. Thus, it could help to restrain angiogenesis and metastasis.61Wheatgrass  is a youTeucrium polium  expression and decreasing Bcl-2 expression (an anti-apoptotic factor).63 In 2019, Askari et al reported that the T. polium extract inhibited HUVEC cell growth in vitro and also VEGF secretion. These results show that T. polium could be a candidate for angiogenesis prevention.66The m polium  is a wil67Quercetin is a powerful flavonoid that has a wide range of benefits to human health including its ability to reduce inflammation, relieving pain, protection against cardiovascular diseases, preventing certain cancers, and boosting the immune system. Perhaps the most important of all could be its potent antioxidant activity. This compound, like other flavonoids, binds free radicals in the body and neutralizes them before they can cause any damages. Most importantly, it inhibits angiogenesis by interacting with VEGF, cyclooxygenase 2, and lipoxygenase 5. It could also induce cancer cell death through inhibiting Akt, mTOR, and HIF-1. A study on the other hand, shows that the antioxidant property of quercetin protects endothelial cells against high glucose levels which activates an autophagy response in them. However, the precise acting mechanism of quercetin has not been fully elucidated.68 This compound, by activating apoptosis, has an anti-proliferative effect on lung and breast cancer cells. Carvacrol significantly inhibits cell migration by blocking the phosphorylation of FAK and MMP-9 and MMP-2 and inhibited angiogenesis. In contrast, other studies have reported results of a VEGF-related angiogenesis induction by this compound. The reason behind such inconsistency could be related to the dose of carvacrol used as it reduces and inhibits MMP2 and 9 levels at high doses while increasing migration and stimulating angiogenesis at low doses.69Carvacrol is a phenol, a derivative of natural monoterpene with antioxidant and anticancer effects found in many plants such as wild bergamot, thyme, and tomato. Carvacrol has a potent anti-inflammatory effect exerted through inhibiting COX-2 enzyme and plays a role in reducing oxidative stress.70So far, numerous herbs that have been traditionally administered as anti-cancer medications in China are screened in various laboratory model settings to find whether they have anti-angiogenesis properties. 72Angiogenesis process is initiated by the activation of growth factors such as VEGF, Platelet-derived growth factor, bFGF, transforming growth factor-β (TGF-β), keratinocyte growth factor, hepatocyte growth factor, ephrin-B2, and angiopoietin. Hypoxia and the activated signaling pathway of HIF in tumor cells is an important stimulator for angiogenesis. HIF-1α and HIF-2α regulate the expression of pro-angiogenic genes including TIE-2, Ang1, Ang2, and VEGF. As already mentioned, VEGF is a target gene of HIF-1α which induced-expression by HIF-1α in endothelium leads to the activation of a VEGF-related autocrine signaling pathway. This cascade is involved in the survival and proliferation of endothelial cells. VEGF on the other hand, increases the permeability of blood vessels while modulating the secretion of extracellular matrix degrading enzymes. The latter in turn leads to the expansion of the vascular system. VEGF exerts its physiologic effects through binding homologues receptors of VEGFR1 and VEGEFR2 on endothelial cells to finally activate them. Upon activation, endothelial cells secret certain types of metalloproteinases that break down the basement membrane to facilitate endothelial cell’s migration. Once the extracellular matrix is degraded and being rearranged, tubulogenesis and therefore angiogenesis are triggered by angiopoietin TIE-2, a regulator of VEGF.The resistance of cancers to common treatments have always been a troublesome matter for specialists. Thus, researchers have focused a major part of their efforts on discovering and identifying new anti-cancer agents that could increase the sensitivity of cancer cells to drugs. Resistance of cancer cells to chemical medications has led to a reduction in their response to medications and as a result, failure of therapeutics. Therefore, investigating and development of more effective medications or those with fewer and less intense side-effects are of great necessity nowadays. So far, several chemical medications have been directly or indirectly derived from natural compounds found in extracts of plants. Some of the cytotoxic chemotherapy agents that are currently being administered have been designed to inhibit angiogenesis and leave the minimum toxicity in low doses. This strategy may provide a chance for patients with advanced cancers to have a longer life of a higher quality. This kind of treatment with low doses is termed metronomic dose. Metronomic model of traditional chemotherapy shows that it is also possible to administer herbal compounds reacting with the angiogenesis process to likely reinforce the positive effects of conventional chemotherapy. In other words, targeting the endothelium of vessels using non-toxic therapeutic agents with low and continuous doses can control the propagation of tumor without causing extra toxicity. The potential role of such treatments in order to extend patients’ survival and improve their quality of life needs further thorough investigation and research in clinical trials. Thus, clinical experts are also interested in identifying compounds that specifically fight different stages of angiogenesis when administered in low doses. These agents may somehow have lower toxicity in lower doses and they will most probably result in better therapeutic outcomes. The number of researches on herbs as treatments for various cancers is extensively growing because of their long-term therapeutic effects. Furthermore, researches on new herbs with anti-cancer effects look quite promising and will hopefully lead to the discovery of novel anti-cancer medications with herbal origin in the near future which would be a significant achievement in the field.Several plants have been reported to exhibit anti-angiogenic properties through various molecular pathways .Not applicable.None declared.We specially thank Dr Hamid Rza Mohammadimotlagh, Rezvan Asgari and Azadeh mahnam for their valuable discussions and help with the manuscript preparation.