Charinus and five Sarax species and found a wide range of diploid chromosome numbers (2n) in both genera. Representatives with lower 2n possessed generally a higher proportion of biarmed (metacentric and submetacentric) chromosomes. Our results indicate the important roles of chromosome fusions and pericentric inversions in the karyotype differentiation of Charinidae, similarly to that suggested previously for neoamblypygids. Our data, gathered from C-banding, fluorescent banding, and chromosomal mapping of ribosomal DNA and telomeric repeats, bring evidence for the action of these rearrangements and suggest the general trajectory towards 2n reduction from ancestral high chromosome counts. However, we cannot rule out the contribution of chromosome fissions. Whip spiders (Amblypygi) are spectacular arachnids characterized by powerful raptorial pedipalps and exceptionally elongated forelegs. Although cytogenetic data from amblypygids might be, given their phylogenetic position, important for the reconstruction of arachnid ka-ryotype evolution, cytogenetics of this order is still largely understudied. Here, we applied conventional and molecular cytogenetics to describe the karyotype patterns in Charinidae—the family placed almost at the base of amblypygid phylogeny, thereby providing insights into the ancestral amblypygid karyotype traits. We surveyed four in situ hybridization in four Charinus and five Sarax species. Both genera exhibit a wide range of diploid chromosome numbers . The 2n reduction was accompanied by an increase of proportion of biarmed elements. We further revealed a single NOR site , the presence of a (TTAGG)n telomeric motif localized mostly at the chromosome ends, and an absence of heteromorphic sex chromosomes. Our data collectively suggest a high pace of karyotype repatterning in amblypygids, with probably a high ancestral 2n and its subsequent gradual reduction by fusions, and the action of pericentric inversions, similarly to what has been proposed for neoamblypygids. The possible contribution of fissions to charinid karyotype repatterning, however, cannot be fully ruled out.Whip spiders (Amblypygi) represent an ancient order of tetrapulmonate arachnids with a low diversity. Their cytogenetic data are confined to only a few reports. Here, we analyzed the family Charinidae, a lineage almost at the base of the amblypygids, providing an insight into the ancestral traits and basic trajectories of amblypygid karyotype evolution. We performed Giemsa staining, selected banding techniques, and detected 18S ribosomal DNA and telomeric repeats by fluorescence  Paracharon, and the Euamblypygi, which includes four families  encompassing about 220 extant species placed in 17 genera .In the present work, we aim to elucidate karyotype differentiation in the family Charinidae, the earliest-diverging lineage among euamblypygids. Charinids consist of three genera  and the samples of C. pescotti are deposited in the collection of the Western Australian Museum, Perth .Twenty-one individuals belonging to four v/v) for 6, 10, and 20 min. A piece of fixed gonad was suspended in a drop of 60% acetic acid placed on a clean slide. The cell suspension was spread on a slide placed on a histological plate heated to 40 °C, using a tungsten needle. The chromosome preparations were subsequently stained in 5% Giemsa solution in Sörensen buffer (pH 6.8) for 30 min. To evaluate the chromosome morphology and to construct the karyotypes, at least five mitotic metaphases  or meiotic metaphases II  were analyzed. In S. ioanniticus, the quality of metaphase spreads only permitted the identification of the 2n and an approximate determination of chromosome morphology. We obtained just a limited number of chromosome plates due to the small size of the gonads and the low number of specimens collected. Therefore, selected techniques of chromosome banding and molecular cytogenetics could be applied only to some species. The sequential use of the slides for more techniques was met with varied success   and the pictures were were inverted for better contrast. This protocol modification provides a better resolution of the C-bands and, hence, allows visualization of the tiny blocks of constitutive heterochromatin. Although it has already been repeatedly used in some other arachnids , and the temperature of the histological plate was reduced to 35 °C for better preservation of the chromatin. The distribution of the constitutive heterochromatin was analyzed using the C-banding method following Sumner . Preparas e.g., ,29), we , we C. pC. neocaledonicus using the GC-specific fluorochrome chromomycin A3 (CMA3) and the AT-specific fluorochrome DAPI (both Sigma-Aldrich) following the method of Sola et al. [The composition of constitutive heterochromatin was determined in a et al.  and Mayra et al. .Silver staining of transcriptionally active NORs ,33 was pn was generated by non-template PCR according to Ijdo et al. [A telomeric probe corresponding to the insect telomeric motif (TTAGG)o et al. , followio et al. . The proDysdera erythrina (Dysderidae) by polymerase chain reaction (PCR) amplification using primers 18SF: 5′-CGAGCGCTTTTATTAGACCA-3′ and 18SR: 5′-GGTTCACCTACGGAAACCTT-3′  [The 18S rDNA probe was obtained from the spider epublic) . The proepublic) . In brieepublic) . The preepublic) ), and co3, green or blue for DAPI) and superimposed with the Cell^R software . Fluorescent banding was evaluated under immersion objective 100× using a Provis AX70 Olympus microscope with an appropriate fluorescence filter set. Images were photographed by a black and white DP30W CCD Olympus camera for each fluorescent dye using Olympus Acquisition Software. Karyotypes were arranged in Corel PHOTO-PAINT X4 software . Composed images were optimized and arranged using Adobe Photoshop CS6 .Standard preparations stained by Giemsa were inspected under a BX 50 microscope  and images were photographed under immersion objective 100× using a DP 71 CCD camera (Olympus). Pictures from FISH and C-banding were captured by an IX81 microscope (Olympus) equipped with an ORCA-AG CCD camera . The digital images from the FISH and fluorescent banding were pseudocolored  and its karyotype was composed of biarmed elements only , with a predominance of monoarmed elements centromeric region of biarmed chromosomes , the other one was AT-rich . CMA3-positive signals were also localized at one end of most chromosomes, while DAPI-positive regions were less pronounced. They were often placed at the opposite chromosome ends.C-banding was performed in two edonicus a and C. pescotti b. In botomosomes b. The meedonicus a. Additiedonicus b. While n was performed only in S. seychellarum. Since this species exhibits the lowest 2n in our sampling, it is the one with a possible high incidence of chromosome rearrangements, herein particularly fusions, which may produce interstitial telomeric sites (ITSs). The probe revealed signals at the ends of all chromosomes, and a single ITS on one chromosome (The FISH with the telomeric probe (TTAGG)romosome c.C. neocaledonicus   b.S. sarawakensis [The cytogenetic features of amblypygids are highly underexplored. Apart from an early report on the charinid wakensis , they arwakensis ,17,18. Wn [n in Sarax (2n = 22–74) and Charinus (2n = 42–76). The karyotypes with higher 2n were composed predominantly of mono-armed chromosomes, while the karyotypes with low 2n consisted exclusively of biarmed elements. These observations suggest the major role of chromosome fusions or fissions in the karyotype evolution of charinids. To infer the direction of this process and, hence, reconstruct the ancestral charinid karyotype, the obtained karyotype patterns were plotted onto the recent charinid phylogenetic hypothesis [Available cytogenetic reports on amblypygids point to a striking diversity in 2n ,16,17,18pothesis , as seenn (24–68) has been reported [n. We found very similar karyotypes in Charinus and Sarax with high chromosome counts . These species display a predominance of monoarmed chromosomes. It is much less probable that a series of independent chromosome fissions would convergently lead to such a similar chromosome count; hence, it is more likely that the process took the opposite trajectory, which has led to the reduction in high ancestral 2n. Unfortunately, we have no information about the degree of conserved synteny among the charinid karyotypes and, therefore, the real interspecific karyotype differences remain unknown. We may only suppose  that chromosomal fusions leading to the reduction in high ancestral 2n represent one of the main mechanisms of karyotype differentiation in Charinidae. This notion is corroborated by the comparably high 2n in two derived amblypygid lineages, specifically in Damon medius  [Heterophrynus longicornis, 2n = 66; Paraphrynus robustus, 2n = 64; Phrynus marginemaculatus, 2n = 68) [n of amblypygids may also be consistent with the recently proposed theory regarding the whole genome duplication in the common ancestor of scorpions and tetrapulmonate arachnids [n in ancestral tetrapulmonates is supported by high chromosome counts in most basal spiders, mesothelids [n in Charinidae and its subsequent convergent elevation within Sarax and Charinus by fissions. This alternative hypothesis is consistent with the basal phylogenetic position of C. dominicanus, a species with low 2n  as described in the derived amblypygid lineage Phrynidae, wherein the comparably wide range of 2reported . While tichidae)  and thre2n = 68) . The higrachnids ,40,41. Tothelids . Howeverh low 2n .C. neocaledonicus. This pair contains the two large adjacent segments of constitutive heterochromatin, which differ considerably by their sequence composition . Differential enrichment in AT vs. GC base pairs in these segments suggests that they belonged originally to two separate chromosomes, which fused during evolution. A similar situation was observed in animal taxa known for their high rate of chromosome fusions  of two smaller monoarmed chromosomes is likely, as (i) the AT- and GC-rich heterochromatin usually occupy the opposite ends of the chromosomes in this species, and (ii) the pair containing two tandemly arranged blocks is acrocentric. There are no data from other amblypygids concerning the distribution of AT- and GC-rich heterochromatin. Among other arachnids, diverse patterns of GC-rich heterochromatin have been recorded in spiders and scorpions, from a scattered distribution of many GC-rich bands throughout the chromosome complement to terminal bands localized on several chromosomes . In C. s e.g., ,45,46). ,46. C. nn cannot be fully explained by only a series of fusions. If this were the case, FN would be maintained roughly the same among the species and the number of biarmed elements would increase proportionally to the decrease in 2n [n. For example, the karyotypes of C. neocaledonicus and C. pescotti are composed of the same number of chromosomes, but these species display different proportions of monoarmed and biarmed chromosomes, and hence different FN . Our rDNA data (see below) strongly favor the action of inversions, although the involvement of other mechanisms leading to centromeric shift  cannot n = 5–152; [n = 5–186; [n = 10–109; [n = 7–143; [C. neocaledonicus suggest that this might also be the case in charinids. In turn, although not necessarily linked to speciation , = 5–186; ,52), har; ,49,50, 10–109; ,54), and= 7–143; ,56). Com= 7–143; —a condit= 7–143; ,20,21. T= 7–143;  and our n , chromon . However, the pre-sence of an X0 system in S. sarawakensis is unlikely, as we did not find heteromorphic sex chromosomes in other Sarax species. The lack of heteromorphic sex chromosomes in amblypygids, nonetheless, does not rule out the possibility that homomorphic sex chromosomes  might be present in them. Given that mostly only males (in seven out of nine species) provided chromosome plates, one cannot exclude female heterogamety with morphologically differentiated sex chromosomes in whip spiders. Heteromorphic sex chromosomes occur in two tetrapulmonate groups, spiders [S. ioanniticus females might potentially represent sex chromosomes. This hypothesis is amenable for experimental testing in further studies.We did not observe heteromorphic  sex chromosomes in any of the studied charinids, which is in line with the observations in other ka-ryotyped amblypygids ,18, excewakensis , where t spiders ,67,68,69 spiders . These o spiders , we suppCharinus species , the NOR occupies the terminal position on a chromosome pair, in a single surveyed Sarax species (S. seychellarum), the NOR is placed interstitially in the middle of one arm of the smallest metacentric chromosome pair. The most parsimonious explanation for the observed pattern is that a paracentric inversion shifted the NOR from the terminal location to the interstitial one in this species. Therefore, our data suggest that the major rDNA cluster might be a useful cytogenetic marker for the determination of mechanisms of karyotype repatterning in amblypygids.The major rDNA cluster 45S rDNA) is by far the most utilized molecular chromosomal marker across diverse eukaryotic organisms, and especially in non-model groups ,73,74. TS rDNA isn, which is ancestral for arthropods [Damon medius [n sequences, we also found one ITS in S. seychellarum. This ITS could have arisen by chromosome rearrangement , with the karyotype being composed of biarmed elements only. Therefore, the observed ITS might represent a hallmark of a previous chromosome fusion or translocation. The presence of ITS on only one of the homologues is puzzling but it might reflect the heterozygous constitution resulting from a different degree of sequence elimination/degeneration from the interstitial site. Besides chromosome rearrangement, the telomeric repeats might also be translocated to the interstitial region via transposition or during double-strand break repair processes [According to our data, DNA molecules of charinids are terminated by the so-called insect telomeric sequence (TTAGG)thropods . This mothropods ,79,89,91n medius . While tn medius ,93,94,95n medius . The pren medius . Besidest . S. . S. n, wrocesses ,99.n and chromosome morphology shaped mostly by fusions and inversions. While we suppose the general direction of karyotype change towards a reduction in 2n via fusions in amblypygids, and some of our findings support this view, larger sampling, including representatives of other amblypygid families, is needed to clarify this hypothesis.Our cytogenetic survey in the early-branching amblypygid family Charinidae provides important clues about the pace and trajectories of karyotype differentiation in amblypygids and in tetrapulmonates in general. The data corroborate the findings from the derived lineage Phrynidae in the way that amblypygids possess highly variable 2


Conventional magnetic resonance imaging (cMRI) is poorly sensitive to pathological changes related to multiple sclerosis (MS) in normal-appearing white matter (NAWM) and gray matter (GM), with the added difficulty of not being very reproducible. Quantitative MRI (qMRI), on the other hand, attempts to represent the physical properties of tissues, making it an ideal candidate for quantitative medical image analysis or radiomics. We therefore hypothesized that qMRI-based radiomic features have added diagnostic value in MS compared to cMRI. This study investigated the ability of cMRI (T1w) and qMRI features extracted from white matter (WM), NAWM, and GM to distinguish between MS patients (MSP) and healthy control subjects (HCS). We developed exploratory radiomic classification models on a dataset comprising 36 MSP and 36 HCS recruited in CHU Liege, Belgium, acquired with cMRI and qMRI. For each image type and region of interest, qMRI radiomic models for MS diagnosis were developed on a training subset and validated on a testing subset. Radiomic models based on cMRI were developed on the entire training dataset and externally validated on open-source datasets with 167 HCS and 10 MSP. Ranked by region of interest, the best diagnostic performance was achieved in the whole WM. Here the model based on magnetization transfer imaging (a type of qMRI) features yielded a median area under the receiver operating characteristic curve (AUC) of 1.00 in the testing sub-cohort. Ranked by image type, the best performance was achieved by the magnetization transfer models, with median AUCs of 0.79  in NAWM and 0.81 (0.71–0.90) in GM. The external validation of the T1w models yielded an AUC of 0.78 (0.47–1.00) in the whole WM, demonstrating a large 95% CI and a low sensitivity of 0.30 (0.10–0.70). This exploratory study indicates that qMRI radiomics could provide efficient diagnostic information using NAWM and GM analysis in MSP. T1w radiomics could be useful for a fast and automated check of conventional MRI for WM abnormalities once acquisition and reconstruction heterogeneities have been overcome. Further prospective validation is needed, involving more data for better interpretation and generalization of the results. Multiple sclerosis (MS) is an inflammatory disorder of the central nervous system, responsible for focal and diffuse damages, including both demyelination and neurodegeneration, and often leading to physical and mental disability . In 2016Early diagnosis in MS is challenging because the pathology mechanisms are not yet completely understood, and disease biomarker discovery is still ongoing. The McDonald criteria is currently used for diagnosis . It assi∗, respectively) or times , proton density (PD), magnetization transfer (MT) saturation, and a number of diffusion MRI (dMRI) metrics. Values in qMRI maps are linked to the physical properties of biological tissues, such as axonal myelination , iron accumulation (R2∗ and T2∗), and free water proportion (PD) (Quantitative MRI (qMRI) potentially overcomes these limitations by quantifying the physical micro-structural properties of brain tissues in standardized units. Commonly, some of the following parameters are estimated: longitudinal and effective transverse relaxation rates (R1 and R2ion (PD) . It has ion (PD) . Currention (PD) . It has ion (PD) . Furtherion (PD)  and earlion (PD) .The discovery of quantitative imaging biomarker is currently experiencing a large increase in research interest, and radiomics is rapidly emerging as a major tool in radiology. Radiomics is a high-throughput imaging data quantification approach aimed to calculate the quantitative descriptors of medical images to characterize the underlying biology and establish a correlation with clinical endpoints . RadiomiWithin the present study, we hypothesized that cMRI- and qMRI-based radiomic models have a diagnostic value in MS, while qMRI-based features have an advantage in the detection of diffuse damages. The objective of the study was to investigate the ability of radiomic features found in WM, NAWM, and GM, extracted from cMRI and qMRI maps, to distinguish between HCS and MSP. Radiomic classification models were developed and tested, and cMRI models were validated on external publicly available datasets.∗) of both MSP and HCS, dataset 2 (DS2) contains cMRI (T1W) of HCS, and dataset 3 (DS3) contains the cMRI of MSP (T1w and FLAIR) (see This study was performed on three datasets: dataset 1 (DS1) contains both cMRI (T1w and FLAIR) and four types of qMRI maps  see . DS2 andWith the addition of models combining features extracted from all four qMRI maps, a total of 18 models were trained on DS1 , of which three models  were validated on DSV. All feature selection and model training were performed in the respective training datasets. The testing and/or validation datasets were kept apart and were used only for evaluation purposes. The study design is detailed in in vivo imaging, University of Liège, Liege, Belgium) acquired within an MS cross-sectional study  retrospectively collected between 2013 and 2017 , and 36 HCS  challenge dataset. The MSSEG challenge dataset contains MRI data for 53 MSP, but only 15 participants from the training subset are publicly available . The datThere are some differences between DS1 and DSV, the main difference being the different image acquisition equipment and protocols see . Other dA summary of the datasets is presented in All the data processing and analysis hereafter were performed on a system containing 4 × 10 core 2.40 GHz Intel Xeon CPU and 64 GB RAM.1. In the absence of radiofrequency field sensitivity bias map acquisition, the radiofrequency field bias was corrected with a unified segmentation approach. The radiofrequency transmit field (B1) bias was corrected using B1 and B0 maps, which were acquired with 3D echo-planar imaging mapping protocols. The B1 data was processed with parameters which were identical to the standard default ones. The multiparameter input images included six MT-, eight PD-, and six T1-weighted images.The qMRI maps were generated in MATLAB 2017b  with the use of the hMRI toolbox, v0.2.0 , an exte3; hence, we decided to resample the scans within DS2 and DS3 to the same resolution. We used cubic spline interpolation as it performs well in terms of its Fourier properties, visual image quality, and interpolation errors . It was performed in MATLAB using hMRI for SPM12 with light regularization  and 60-mm cutoff for full-width at half-maximum of Gaussian smoothness of bias. The outputs were tissue probability maps for CSF, GM, and WM, with the voxel values between 0 (background) and 1 (corresponding brain tissue). In order to ensure the inclusion of only the relevant tissue class, binary masks for each tissue were obtained by thresholding the tissue probability maps at a high level of 0.9. For MSP, lesion masks were generated from the combination of T1w and FLAIR images with LST  when necessary. Multi-channel tissue segmentation was performed using multiple qMRI maps  with unified segmentation protocol in US-with-Lesion  unified segmentation protocol in MATLAB with SPM12, using T1w images.Following this step, tissue masks for CSF, GM, NAWM, and lesions within DS1 were estimated. Tissue segmentation in HCS was performed with a multi-channel unified segmentation protocol , using mwith LST 2 for SPMh-Lesion 3, addingAfter segmentation, total intracranial volume (TIV) was estimated for each patient as the morphological sum of the CSF, GM, NAWM, and lesion volumes (where applicable). This combined ROI was used for intensity normalization, as described below.As the magnetic field inside an MRI scanner is not ideally homogeneous and is affected by objects within it, a bias field signal is introduced, degrading image quality as a smooth, low-frequency signal that distorts segmentation results and feature values. To partially correct for this in T1w images, N4 bias field correction  was perfAs cMRI voxel intensities are expressed in arbitrary units, the Image Biomarker Standardization Initiative (IBSI) recommends using normalization for raw MR data . TherefoNbins50, in line with IBSI recommendations  was calculated for each feature.Feature analysis was performed in the whole DS1 to describe the data; its results were not included into model building. Statistical tests were performed to gauge diagnostic efficacy in such a small dataset. A univariate Mann–Whitney test was carried out using Bonferroni correction, and ∗ maps to arrive at a subset of N features each, attempting to adhere to published rules of thumb to estimate the optimal number  observations from the training subset. In each of the 100 iterations, a fixed number rS| > 0.85), we excluded a feature having the highest correlation on average with all the remaining features. The final selection was performed with recursive feature elimination  to investigate the value of each image type and ROI in the estimation of the MS status. For each image type, three binary classification models were trained using the same features from each image type and ROI: (i) random forest (RF), (ii) support vector machine (SVM) (Nfeatures ⋅ Var(X)), where Var(X) is the variance of the input feature X , and for LR, L2 penalty was used since this regularization does not lead to high values among the regression coefficients, with dual formulation, as recommended when the amount of observations exceeds the amount of features, and a liblinear solver, which is recommended for small datasets; inverse of regularization strength C = 1.0, which is optimal in terms of balance between accuracy and model complexity. Due to the small dataset sizes, DS1 was used again as an exploratory dataset.Separate binary classification models were trained on DS1 for different image types—T1w, PD, MT, R1, and R2ne (SVM) , and ; for each model, learning and curves were plotted. Since all the scores were estimated on the data subsets, containing equal numbers of HCS and MSP, the imbalanced data correction was not needed. The best model was selected based on these performance metrics for different ROIs and tissue types, giving the AUC score more weight and excluding models with median AUC scores below the threshold of 0.7, which is considered an underperforming classification model. In order to select the best model type , the number of highest AUC scores was used.The final models with the original coefficients were subsequently validated on DS2 and DS3. As the combined dataset containing DS2 and DS3 was highly unbalanced regarding the outcome, bootstrapping with balanced sampling was implemented. The models for qMRI were not validated externally due to the unavailability of similar datasets.To examine the models and methodology for overfitting, a permutation test was performed on DS1. The class labels in both training and testing subsets were randomized, maintaining the same distributions as in the original subsets. Without modifying the pipeline, feature selection was performed, models were trained and tested, and performance metrics were calculated to ascertain whether the pipeline detects patterns in randomly generated outcomes.p-values for comparison of age and gender distributions between HCS and MSP groups within development and validation data as well as between development and validation datasets can be found in Participants were drawn from DS2, aiming to match DS1 regarding age and magnetic field strength. Participants with MRI quality, which was not sufficient for robust automatic segmentation, were excluded after a visual check (ELa). Finally, 167 participants were selected from this dataset. Another 10 participants were selected from DS3, again trying to match the age and field strength distributions with those of DS1. An overview of the resulting feature sets is presented in ∗. In the entire feature set, there was only one feature (R1 first-order minimum in WM) that was highly correlated with the outcome, no feature was highly correlated with age, and 10 features out of 1,395 were highly correlated with ROI volume. A univariate analysis showed that 28% of the features  had an area under the receiver operating characteristic curve (ROC AUC) score > 0.75, most of which were obtained from the PD, MT, and R2∗ maps  were sampled from significantly different distributions in the HCS and MSP cohorts, mostly originating from WM in all image types but also from NAWM in MT and R2maps see .In the training subset of DS1, on average among all the image types and ROIs, 7% from the initial feature set were excluded by the low variance step, followed by 79% exclusion by the high correlation step. The number of features per set kept after each feature selection step is available in ∗ in NAWM and T1w and for PD in GM. According to the Mann–Whitney test, the highest number of features with significant differences in means in HCS and MSP is discovered in WM (15 features out of 15) when ranking by ROIs and on R1 (eight features out of nine) when ranking by image types. A list of the selected features with their Spearman correlations with age and the ROI volume, univariate ROC AUC scores, and Mann–Whitney test p-value is presented in No high correlations were discovered between the selected features, age, and ROI volume. For the selected features, the univariate AUC was below a threshold of 0.7 for PD, MT, and R2p = 0.01) AUC scores in all the cases, with the exception of MT and qMRIcomb in WM, R1 in NAWM, and PD in GM  in AUC scores obtained for WM in MT, R1, and qMRIcomb . The T1w model performed generally poorer than the MT and R1 models but better than the PD, R2∗, and qMRIcomb models.In NAWM, there were no significant differences in AUC scores obtained for highest scoring R2∗ and qMRIcomb. The highest overall performance was achieved with the MT-based model, which yielded a median accuracy of 0.88.In GM, there were no significant differences in AUC scores obtained for MT and R1 and for R2p ≤ 0.01) drop in AUC for all the models, except for PD and MT in NAWM and for T1w in GM. The full results obtained with the permutation test for different models and permutation test p-values can be correspondingly found in The permutation test results showed a significant  , which yIn this exploratory brain tissue MRI and qMRI radiomics study based on a unique dataset, we report on several hypothesis-generating findings for HCS vs. MSP classification. Previous studies on radiomics in MS have been focused on T2w cMRI data and aimed to distinguish between MS and neuromyelitis optica spectrum disorder  without Of the three machine learning models  tested, LR was the most stable with median accuracy, AUC, sensitivity, and specificity all exceeding a value of 0.7 while achieving the highest performance in terms of AUC. The fact that LR outperformed the other models could be due to the small number of observations, where the simplest models might perform best since they are less likely to overfit. The selected radiomics features were not correlated with age and volume , which indicates that radiomics could provide additional information to those simple variables.The best LR model performance concerning tissue type was achieved using features extracted from WM. This was expected since focal WM lesions (plaques) in the WM of MSPs affect the intensity distribution . In NAWM∗ weighting, as mentioned previously .This study demonstrates that brain cMRI and qMRI radiomic features have the potential to distinguish between MSP and HCS. In NAWM and GM analysis, having a potential in early automated diagnosis, stable results are achieved with qMRI-based data. This is a proof-of-concept clinical study demonstrating a strong signal in brain imaging, but further research is needed to develop and approve radiomic signatures for MS.Nevertheless, future large-scale studies should evaluate the reproducibility and generalizability of the proposed method and create an MS-specific radiomic signature. Because of fully automated pipeline and imaging data quantification, the proposed approach shows its potential in relevance to time-saving and reproducibility in MS diagnosis.https://github.com/CyclotronResearchCentre/brain-tissue-radiomics-on-clinical-and-quantitative-MRI-for-MS (str 1295-1296). The details on the packages, with the indication of versions and functions used, can be found in elommers@chuliege.be.The data analyzed in this study is subject to the following licenses/restrictions DS2 and DS3 are public datasets, the accession details can be found in The studies involving human participants were reviewed and approved by the Liege University Hospital-Faculty Ethics Committee, University Hospital Center of Liege, Liege, Belgium. The patients/participants provided their written informed consent to participate in this study.ELa, HW, AC, CP, ES, and PL contributed to study conceptualization and developed the methodology. PL and ES acquired the funding. ELo, PM, and CP performed data acquisition and curation. ELa, HW, and CP performed the analysis and wrote the original draft. HW, CP, PL, and ES performed the supervision. All authors contributed to manuscript revision and have read and approved the submitted version.PL reports, within and outside the submitted work, grants/sponsored research agreements from Radiomics SA, ptTheragnostic/DNAmito, and Health Innovation Ventures. He received an advisor/presenter fee and/or reimbursement of travel costs/consultancy fee and/or in kind manpower contribution from Radiomics SA, BHV, Merck, Varian, Elekta, ptTheragnostic, BMS, and Convert pharmaceuticals. PL has minority shares in the company Radiomics SA, Convert pharmaceuticals, Comunicare Solutions, and LivingMed Biotech. He is a co-inventor of two issued patents with royalties on radiomics (PCT/NL2014/050248 and PCT/NL2014/050728) licensed to Radiomics SA and one issued patent on mtDNA (PCT/EP2014/059089) licensed to ptTheragnostic/DNAmito, one non-issued patent on LSRT (PCT/P126537PC00) licensed to Varian Medical, three non-patented invention (softwares) licensed to ptTheragnostic/DNAmito, Radiomics SA and Health Innovation Ventures, and three non-issued, non-licensed patents on Deep & handcrafted Radiomics . He confirms that none of the above entities or funding was involved in the preparation of this paper. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Twenty to thirty percent of psoriasis (Pso) patients will develop psoriatic arthritis (PsA). Detection of Pso patients that are (at risk for) developing PsA is essential to prevent structural damage. We conducted a systematic search of five bibliographic databases, up to May 2020. We searched for studies assessing markers  associated with the development or presence of PsA in Pso patients. Study selection and quality assessment of the included studies was performed, followed by a qualitative best evidence synthesis to determine the level of evidence for a marker and its association with concomitant/developing PsA in Pso. Overall, 259 possible markers were identified in 119 studies that met the inclusion criteria. Laboratory markers related to inflammation and bone metabolism reached a strong level of evidence for the association (not prediction) of PsA in Pso. Only CXCL10 showed strong evidence for a positive predictive value for PsA in Pso. The importance of timely detecting PsA in a Pso population, and finding more (bio)markers contributing to early detection, remains high.The online version contains supplementary material available at 10.1186/s13075-021-02545-4. Psoriatic arthritis (PsA) is an immune-mediated inflammatory disease affecting joints and entheses and is strongly associated with psoriasis (Pso). Twenty to thirty percent of Pso patients will develop PsA, with an average lag time between Pso and PsA of 10 years . This laCurrent screening strategies mostly use questionnaires based on clinical characteristics to detect PsA , 7. BothTherefore, we conducted a systematic review to identify possible markers for the onset of PsA in a Pso population, with the purpose of providing a comprehensive summary of the available markers for PsA in Pso.The protocol was designed according to the Preferred Reporting Items for Systematic review and Meta-Analysis  and regiFive bibliographic databases  were searched for studies from January 1, 1990, up to April 29, 2020. Search terms compromised keywords involving study population, study design, and etiology (supplementary table n = 2), and none for genetic studies. To not miss potential relevant markers in these two categories, we also included genetic and laboratory studies with a cross-sectional design  as a “second best” option. While these might not be useful to identify predictors for development of PsA, they could provide information about possible markers for concomitant PsA.Studies were screened for eligibility based on title and abstract by two independent reviewers . Potentially relevant papers were assessed in full text . Any disagreement was resolved by consensus or by discussion with a third reviewer . Studies were excluded based on the following criteria: (1) < 10 patients per group , (2) age of patients < 18 years, (3) no statistical comparison between Pso and PsA, and (4) languages other than English, German, or Dutch. We primarily focused on studies with a longitudinal design, meaning that the marker was present before the presentation of PsA. A very low number of longitudinal studies was available for laboratory studies .Risk bias was assessed using the Newcastle Ottawa Scale for case-control and cohort studies . This to2 [2) compared to reference level . We then assessed the consistency of the results within and across studies. If within a study, a marker was represented in multiple non-hierarchical conceptually similar constructs, we considered the result consistent if ≥ 75% of the constructs pointed in the same direction. Otherwise, we considered the result for that marker “mixed.” For example, one study looked at fracture, any trauma, and trauma leading to medical care [For the best evidence synthesis (BES), we included markers that either showed a significant difference between Pso and PsA in at least one study or markers that showed no significant results in at least two studies . Markers were grouped into overarching categories  .Conflicting evidence was available for 13 of 51 clinical markers. These markers included several disease characteristics: younger age at Pso onset –25, longStrong evidence was available for nine of 137 investigated laboratory markers. CXCL10 (C-X-C motif ligand 10) was the only laboratory marker which showed a positive association with the development of PsA in Pso patients. It was also the only laboratory marker studied in a longitudinal design.Four markers showed a strong level of evidence for a positive association with the presence of PsA in Pso: a higher level of matrix metalloproteinase 3 (MMP3) , 52, 54,Five markers showed a strong level of evidence for no association with PsA in Pso: vitamin D –45, 130,Moderate evidence was available for 56 of 137 investigated laboratory markers. Fourteen of these 56 have been investigated in more than one study.Of those 14 markers, six showed a positive association with the presence of PsA in Pso: the presence of anti-citrullinated protein antibodies (ACPA) –40, a hiOnly one of the 14 markers which were investigated more than once showed moderate evidence of a negative association with the presence of PsA in Pso: a lower ratio of OPG to receptor activator of nuclear factor kappa-B ligand (RANKL) was associated with the presence of PsA in Pso , 56.There was moderate evidence for no association for seven laboratory markers: serum alkalic phosphate , 47, 48,Conflicting evidence was available for 14 of 137 laboratory markers: markers of bone metabolism (Dickkopf (DKK1) , 53; RANThere were no genetic markers which reached a strong level of evidence for a positive, negative, or no association with the presence of PsA.Moderate evidence was available for 30 of 71 investigated genetic markers. Twenty-two of those 31 have been investigated in more than one study.IL13 gene [Of these 22 markers, six showed a positive association with the presence of PsA in Pso: the presence of haplotype B*27-C*01 , 99, hapL13 gene , 117.IL12B gene [Moderate evidence of a negative association was available for three markers: the presence of haplotype B*57-C*06 , 99, the12B gene , 135.IL1RN [IL12B gene [IL23A [IL23R [TNFAIP3  [TNIP (TNFAIP3-interacting protein) [TSC1 (tuberous sclerosis 1) [There was moderate evidence for no association for 13 genetic markers: the presence of HLA-B*57 , 104, HLIL1RN , 135, thIL1RN , 115 and12B gene , 115, the [IL23A , 135, thA [IL23R , 115, thotein 3) , 135, thprotein) , 135, throsis 1) , 135, anrosis 1) , 122 androsis 1) , 122.IL13 gene [IL23R gene [Conflicting evidence was found for 17 of 71 genetic markers, of which 14 were investigated in more than one study. These were the presence of HLA-B*08 , 103, HLL13 gene , 135, an23R gene , 135.not associated with the development of PsA. Regarding laboratory markers, there was strong evidence for the predictive value of (a change in) CXCL10 serum titers [In this review, we summarized the available evidence for possible markers for the onset or presence of PsA in a Pso patient population in a systematic way. Thereby, we provide an update and addition to a recent narrative review regarding this subject by Scher et al. . When lom titers , 60. Them titers –55 and im titers –75, 130.In line with previous beliefs on possible clinical risk factors , 140, weNotably, we found conflicting evidence for the predictive value of obesity , 33, 34 When looking at BMI at one unspecified timepoint, this marker shows conflicting evidence for a relationship with the development of PsA. In three out of five high/fair quality studies, there was no association , 22, 27,not associated with a higher rate of PsA in Pso patients.The association of trauma and psoriatic arthritis was theorized to be due to a deep Koebner phenomenon . This phThe relationship between smoking and PsA development has been described previously as the “smoking paradox” . This en+ CD4+ and CD8+ T cells [With regard to laboratory markers, only CXCL10 was studied longitudinally. This cytokine was described in two good/fair quality studies; both found an association between CXCL10 and PsA. Pso patients who developed PsA had a higher CXCL10 serum level at baseline . It was  T cells . In the before the start of PsA in Pso. Therefore, it is unknown whether it can be used as a predictive marker. Also, a clear CRP cutoff value for the presence of PsA  is lacking.With regard to cross-sectional studies, and markers that may indicate the presence of PsA in Pso patients, we found strong evidence for a positive association with PsA in Pso for markers of inflammation and bone. CRP is a well-known, widely used inflammatory marker. We found strong evidence that the CRP level in PsA patients was higher than in patients with Pso only , 130. WeOther markers for which strong evidence of a positive association with the development of PsA in Pso exist were IL-6, MMP3, and OPG. IL-6 is widely regarded as a marker for systematic inflammation and an important contributor to the production of CRP by the liver. MMP3 and OPG are associated with bone metabolism; one of the hallmark signs of PsA is new bone formation . Also, uLaboratory markers for cardiovascular disease are studied extensively in psoriatic disease , 77, 130With respect to genetic markers, we focus here on the most important HLA-markers for Pso and PsA, and the IL-12 – IL-23 – IL-17 axis. The most important genetic marker for psoriasis is HLA-C*06, also known as PSOR1 . This maWe found a moderate level of evidence for the presence of concomitant PsA in Pso for HLA-B*27, known for its high prevalence (90%) in ankylosing spondylitis (AS) . In otheIL23 gene for which the presence differs significantly between PsA and Pso patients [IL17 gene was more common in PsA versus Pso patients [IL12 gene [IL12 (rs2082412) was lower in PsA versus Pso patients, while other SNPs in this gene showed no difference [When looking at the IL-17/IL-23 axis from a genetic viewpoint, there was moderate evidence that there are no SNPs in the patients , 147. Wepatients . With reL12 gene . We founfference , 115. WhThe strengths of this study include the extensiveness and systematic way of the search with respect to markers for PsA in patient cohorts with Pso, subsequentially providing a comprehensive overview of the available evidence. Also, the intertwining of clinical, laboratory, and genetic markers in a systematic way is unique. By conducting a best evidence synthesis, taking the study quality into account, we made a qualitative overview of the extensive data.presence of PsA in patients with Pso . The level of evidence was limited by a paucity of high or fair quality studies. Mostly, this was because of a lack of appropriate definition of patient and control groups, in addition to not adjusting for possible confounders.The limitations of this systematic review are mostly due to the limitations of the included studies. Since there were  no prospective/longitudinal studies looking at genetic and laboratory markers, we could only summarize the level of evidence with regard to the relationship between laboratory and genetic markers with the presence of PsA. Promising is CXCL10, which reached a strong level of evidence for predicting development of PsA in a Pso population [This comprehensive systematic review on clinical, laboratory, and genetic markers for PsA in patients with Pso revealed that a useful set of markers is not established yet. There were no clinical or genetic markers with strong evidence which could predict the development of PsA in Pso cohorts. There was strong evidence that laboratory markers related to bone metabolism and inflammation were associated with the pulation , 60. TheAdditional file 1: Supplementary table 1. Search strategy.Additional file 2: Supplementary table 2. Characteristics of included studies (n = 119).Additional file 3: Supplementary table 3. Statistical significance and effect sizes of clinical markers.Additional file 4: Supplementary table 4. Statistical significance and effect sizes of laboratory markers.Additional file 5: Supplementary table 5. Statistical significance and effect sizes of genetic markers.Additional file 6: Supplementary table 6. Quality assessment of cohort studies.Additional file 7: Supplementary table 7. Quality assessment of case control studies.


ARMC5, and the role of the adrenal paracrine regulation. Finally, the treatment of bilateral adrenal hyperplasia will be discussed, focusing on current data on unilateral adrenalectomy.Bilateral adrenal hyperplasia is a rare cause of Cushing’s syndrome. Micronodular adrenal hyperplasia, including the primary pigmented micronodular adrenal dysplasia (PPNAD) and the isolated micronodular adrenal hyperplasia (iMAD), can be distinguished from the primary bilateral macronodular adrenal hyperplasia (PBMAH) according to the size of the nodules. They both lead to overt or subclinical CS. In the latter case, PPNAD is usually diagnosed after a systematic screening in patients presenting with Carney complex, while for PBMAH, the diagnosis is often incidental on imaging. Identification of causal genes and genetic counseling also help in the diagnoses. This review discusses the last decades’ findings on genetic and molecular causes of bilateral adrenal hyperplasia, including the several mechanisms altering the PKA pathway, the recent discovery of  Cushing’s syndrome (CS) is characterized by an excess of cortisol production. In 80% of cases, it is due to an over-secretion of the adrenal corticotrophin hormone (ACTH) by a corticotroph pituitary adenoma, or more rarely, by a neuroendocrine tumor. The other 20% is due to a primary overproduction of cortisol by the adrenal glands, with the most frequent etiology being a benign cortisol-secreting adenoma. Other causes of adrenal CS include adrenocortical carcinoma and bilateral adrenal hyperplasia, that account for less than 10% of patients presenting with adrenal CS [ARMC5 (armadillo repeat containing 5) in PBMAH brought new insights into the pathogenesis of bilateral adrenal hyperplasia  and the other allele is inactivated at the somatic level . In addition, most of the genetic or molecular alterations described in these diseases lead to the activation of the protein kinase A (PKA) pathway. The cAMP pathway is a ubiquitous intracellular signaling pathway, regulating several cellular processes, such as proliferation, differentiation, and hormonal activity in endocrine tissues. In adrenals, ACTH binds to its seven-transmembrane domain receptor coupled to the G protein, the MC2R (melanocortin receptor 2), leading to the activation of the Gs subunit. The activated Gs protein stimulates the adenyl cyclase, which synthesizes cAMP. The cAMP binds to the regulatory subunits of the protein kinase A (PKA). The latter dissociates from the catalytic subunits, which then phosphorylate their targets, such as CREB (cAMP Response Binding Protein), a transcription factor involved in adrenal proliferation and steroidogenesis. The cAMP is degraded by phosphodiesterases [erplasia C. There is no consensus on the management of bilateral adrenal hyperplasia for several reasons: the disease is rare, patients may present with subclinical CS, and bilateral surgery exposes patients to definitive adrenal insufficiency. Bilateral adrenalectomy used to be the treatment of choice when the decision to treat CS patients was made. Since then, unilateral adrenalectomy has been proposed in both PBMAH and PPNAD, offering interesting results, especially in patients with PBMAH . This review states the current data on pathogenesis and treatment of bilateral adrenal hyperplasia. Bilateral micronodular adrenal hyperplasia is characterized by micronodules (<1 cm). Histologically, the adrenal cortex shows small pigmented nodules surrounding an atrophic cortex. Adrenal weight and size remain normal ,4,5. PPNA minority of patients develop PPNAD during the first three years of life, while most will develop the disease during the second and third decades . After pThe clinical presentation of the disease varies among patients, even in the same family. Patients may present with subclinical or overt CS, with sudden or insidious onset. Cyclic forms with sudden onset of intense hypercortisolism followed by spontaneous remission have also been described ,10. ArouBiologically, fluctuation in the urinary free cortisol (UFC) is frequently observed. A paradoxical elevation of cortisol after four days of a high-dose dexamethasone suppression test has been described in the disease , but itsA high-resolution computed tomography scan before and after contrast enhancement may detect micronodules that appear as hypodense spots scattered throughout the gland . OccasioTwo cases of adrenocortical cancer ,15, one The CNC was first described in 1985 by J. Aidan Carney at the Mayo Clinic . The endDiagnostic criteria were proposed in 2001 . ManifesPBMAH is characterized by the presence of several nodules > 1 cm, usually in both adrenal glands. It was described for the first time in 1964 . The disPBMAH is considered a rare disease, but its prevalence is probably underestimated. Most of the PBMAH have an incidental diagnosis by imaging performed for an unrelated reason. Prevalence of adrenal incidentaloma is estimated at 1% to 5% of abdominal imaging, and 2.7% to 10% of them are bilateral. Subclinical CS is observed in 35% to 40% of these bilateral incidentalomas, and a certain number of cases may correspond to PBMAH . Other pNR3C1 gene, encoding for the glucocorticoid receptor, has also been identified in 5% of a series of patients presenting with bilateral adrenal incidentalomas. These patients do not have features of CS despite an increased urinary free cortisol and an unsuppressed cortisol post-dexamethasone test, consistent with the resistance of cortisol due to the loss of function of the GR. In addition, their ACTH level is unsuppressed. However, they may develop hypertension due to an alteration of the 11β-hydroxysteroid dehydrogenase type 2 activity [Differential diagnosis includes bilateral adrenal hyperplasia observed in situations of chronic adrenal overstimulation by ACTH  . Mutatioactivity .In patients diagnosed with PBMAH, there is no specific recommendation for imaging follow-up. At diagnosis, the European Endocrine Society recommends an individual follow-up of each adrenal incidentaloma bigger than 4 cm or spontaneous density above 10 UH (these two features being often observed in PBMAH), with subsequential imaging at 6 months . RegardiMEN1 gene (11q13). It includes primary hyperparathyroidism (95%), pancreatic neuroendocrine tumors (50%), pituitary adenomas (40%), and thymic carcinoid tumors [MEN1 gene [MEN1: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disease linked to mutations in the d tumors . While ad tumors ,29,30, td tumors . The occd tumors ,31. The EN1 gene .fumarate hydratase (FH) gene (located on chromosome 1q43) are responsible for hereditary leiomyomatosis-kidney cancer syndrome (HLRCC). FH is an enzyme of the Krebs cycle that allows the conversion of fumarate to malate. Ten HLRCC patients presenting with PBMAH treated by adrenalectomy have been reported [FH mutations and the occurrence of PBMAH. Interestingly, a germline FH mutation was also characterized in a sporadic case of PBMAH [Fumarate Hydratase: Autosomal dominant mutations in the reported ,34. One reported , supportof PBMAH .APC gene is characterized by multiple colonic polyps and colon cancers at an early age. Patients may also present with pigmented retinal lesions, desmoid tumors, osteomas, thyroid nodules or cribriform thyroid cancers, and other malignancies [APC mutations and the occurrence of PBMAH [Familial polyposis coli: Familial polyposis coli or Gardner’s syndrome due to mutation in the gnancies . The devgnancies ,37,38. TH19, IGF2 (Insulin-like growth factor 2), and CDKN1C (Cyclin-Dependent Kinase Inhibitor 1C) genes. The phenotype includes overgrowth, neonatal hypoglycemia, congenital malformation, and predisposition to embryonic tumors [Beckwith–Wiedemann syndrome: Beckwith–Wiedemann syndrome is an imprinting disorder due to genetic or epigenetic alteration of the locus 11p15.5, including c tumors . If adrec tumors . Genetic studies have identified two independent loci at chromosomes 17p22–24 and 2p16 in patients with CNC. Currently, no candidate gene has been found at chromosome 2 . On chroPRKAR1A is a tumor-suppressor gene. The transmission of the disease is also autosomal dominant. The mutations are distributed over the 10 coding exons and their adjacent intronic sequences essential for splicing. They are mostly single-base substitutions and short deletions [eletions ,44. Largeletions .In 80% of cases, the mutations cause a premature stop codon and the degradation of the mutant mRNA by nonsense-mediated mRNA decay, leading the haploinsufficiency ,44,46. IMutations leading to the creation of a mutant protein are associated with a higher number of CNC manifestations.Mutations on the exons are more often associated with acromegaly, cardiac myxomas, lentigines, and schwannomas.Mutations on the intronic splice sites are associated with a milder phenotype.The c.491–492delTG hotspot mutation is more often associated with cardiac myxomas, lentigines, and thyroid tumors.The c.709(−7–2)del6 hotspot mutation and the c.1A > G mutation are associated with isolated PPNADs.Patients without the PRKAR1A mutation have fewer tumors that appear later in life.Some genotype–phenotype correlations have been established :MutationSeveral mouse models have been developed to study the consequence of PRKAR1A inactivation. The knockout of Prkar1a is lethal at the embryonic stage . HeterozCrosstalk between the PKA pathway and other signaling pathways involved in apoptosis and proliferation contributes to tumorigenesis observed with PRKAR1A deficiency . PRKAR1APRKACA: In a cohort of 35 patients with bilateral adrenal hyperplasia, germline amplification (duplication or triplication) of PRKACA has been described in 5 patients, including 3 patients with PPNAD and 2 patients with PBMAH, a mother and her son ,55. ThisMC2R: Mutations in the ACTH receptor (MC2R) have been described in two sporadic cases of PBMAH ,57. No oPhosphodiesterases: The presence of variants of the PDE11A (Phosphodiesterase 11A) gene appears to confer a genetic predisposition to PBMAH. In a series of 46 patients, the frequency of PDE11A variants was higher than in the control population . This obIn few children with iMAD, mutations in the PDE11A  and the GNAS: In children, the most common cause of Cushing’s syndrome of adrenal origin is the McCune Albright syndrome (MAS), related to post-zygotic mutations in the GNAS gene, encoding the Gαs subunit of the heterotrimeric G protein . The disEutopic receptors , such as the vasopressin V1 receptor, the luteinizing hormone/human chorionic gonadotropin (LH/HCG) receptor, the serotonin 5-HT4 receptor, and the leptin receptor.Ectopic receptors , such as the GIP receptor, the vasopressin V2 and V3 receptors, the serotonin 5-HT7 receptor, the glucagon receptor, the beta-adrenergic receptor, and the angiotensin II AT1-receptor.Abnormal cortisol secretion due to the activation of G-coupled protein receptors other than MC2R was one of the first pathogenic mechanisms demonstrated in PBMAH. In 1992, a food-dependent CS ,67 due tThe presence of these receptors can be clinically assessed by a combination of biological tests  Table 3Table 3. Abnormal expression or overexpression of these receptors has been confirmed by quantitative PCR  or transARMC5 inactivation may be responsible for the abnormal expression of the alpha-2 adrenergic receptor and the AVP-R1A receptor [The mechanism leading to this aberrant expression, which is likely to be an early event in PBMAH ,81, is ureceptor . At the receptor . In tranreceptor .ARMC5 gene (Armadillo repeat containing 5) had been identified by an integrated genomics approach as responsible for PBMAH [ARMC5, located at the chromosome 16p, as responsible for PBMAH in a series of 33 patients [ARMC5 mutations account for 25% of the PBMAH, except in Japan, where the prevalence may be higher [In 2013, mutations in the or PBMAH . LOH at patients . Severale higher ,87,88,89ARMC5 mutation leads to more severe disease with higher hypercortisolism, larger adrenal hyperplasia, and a higher number of nodules [ARMC5 variants have also been associated in African Americans with low renin hypertension, higher fasting glucose, and HbA1c [ARMC5 mutations undergo surgery more often [ARMC5 mutations in series including only operated patients. Interestingly, no food response has been observed in ARMC5-mutated patients, while a response to vasopressin or orthostatism can be observed [ nodules . Patient nodules , most lind HbA1c ,91. In and HbA1c . Due to re often , explainobserved ,92,93.ARMC5 mutations are responsible for nearly 80% of the familial forms [al forms ,92,93,94al forms ,92. In aal forms ,94. ARMC5-mutated patients have been described [ARMC5 mutations are responsible for meningioma [Meningiomas in escribed ,94,95,96ARMC5 inactivation followed the two-hits models observed for tumor-suppressor genes. The described mutations are distributed along the coding sequences. A multitude of mutations have been described since 2013, suggesting that most of them are unique for one patient and his family. There are no true hotspots, even though some mutations have been found by several teams [al teams . Deletioal teams ,98.ARMC5 was unknown when it was characterized as a causal gene of PBMAH in 2013. The ARMC5 protein is part of the Armadillo repeat containing gene family. Its structure contains two highly conserved domains involved in protein–protein interaction: the armadillo repeat domain and a broad complex Tramtrack bric-a-brac/Pox virus and zinc finger (BTB/POZ) domain. The protein is ubiquitously expressed [The function of xpressed . The firxpressed ,85,100. ARMC5 in vitro decreases the expression of genes involved in steroidogenesis and cortisol production [ARMC5 [Inactivation of oduction ,100. Intoduction , while aoduction . Therefooduction . Recent n [ARMC5 . ARMC5 deficiency decreases steroidogenesis. Interestingly, a decrease in the expression of Prkaca was observed in these mice [Knockout of Armc5 in mice has a high lethality rate at the embryonic stage ,103. Armese mice . Similarese mice . Howeverese mice , while aARMC5 is also involved in cell cycle regulation. ARMC5 interacts with Cullin 3 via its BTB/POZ domain, leading to the proteasomal degradation of ARMC5. Interestingly, ARMC5 overexpression alters the G1-S progression, and Cullin 3 blocks this effect. Mutations in the BTB domain of ARMC5 affect its degradation and its action on the cell cycle [ARMC5 in T-cell function has also been suggested by another knockout mice model study [ll cycle . Finallyel study .Paracrine and autocrine regulation of adrenal glands by peptides or neurotransmitters secreted by chromatin cells, nerve endings, or immune cells has been previously demonstrated ,107,108.ARMC5 in their appearance must be elucidated. Chromaffin cells in the medulla produce ACTH locally . In PBMAIn addition to ACTH, local production of serotonin by the perivascular mastocyte cells located in the subcapsular area of the adrenal cortex is observed. This local serotonin stimulates the production of aldosterone ,112. In In patients with overt CS, the indication to treat to normalize the cortisol level is consensual . HoweverDefinitive adrenal insufficiency also exposes patients to increased morbidity and mortality due to cardiovascular diseases and infections ,115,116.Finally, it is unclear if the results of studies on adrenal incidentaloma apply to patients with bilateral adrenal hyperplasia. Particularly, the onset of PPNAD is younger than the age of diagnosis of adrenal incidentaloma, and the balance between the long-term consequences of definitive adrenal insufficiency versus long-term consequences of subclinical CS must be determined. Therefore, unilateral adrenalectomy appears as an interesting option to treat patients with bilateral adrenal hyperplasia . Bilateral adrenalectomy used to be considered as the treatment of choice for patients with overt CS and adrenal hyperplasia . In pati131I- norcholesterol scintigraphy. Only 7 of 32 patients considered in initial remission needed contralateral adrenalectomy because of recurrence of the CS. Among the 25 patients that did not need completion of the surgery, follow-up was unavailable for 9 patients [Nevertheless, unilateral adrenalectomy has been proposed in patients with PPNAD to avoid definitive adrenal insufficiency. In a recent review of all published cases or series, the initial success rate was evaluated at 66% (32 of the 48 reported patients) . Most ofpatients ,122,123,patients . One patpatients . Follow-patients ,126. Finpatients ,128. Taken together, these results hence suggest that unilateral adrenalectomy could be an interesting option in selected patients presenting with PPNAD, especially in younger patients. However, interpretations of these data should be made with caution. In addition to the lack of biological and clinical data in several reports, one major limitation is the different criteria used to define remission. In some cases, alteration of the adrenal-pituitary axis tests, especially an abnormal circadian rhythm, was still observed . InteresUrinary free cortisol (UFC) level increases progressively with the evolution of adrenal hyperplasia. Unilateral adrenalectomy leads to a reduction of the adrenal mass and a reduction of the UFC at levels equivalent to those 10 to 20 years earlier.Regarding the late onset of the disease and the higher frequency of subclinical CS compared to PPNAD, unilateral adrenalectomy instead of bilateral adrenalectomy appears particularly interesting in patients with PBMAH. Unilateral adrenalectomy has also been proposed in PBMAH patients since the late 1990s. Currently, 23 case reports and small series totaling 117 patients have been reported . InitialUFC normalizes in almost every patient. A division of the UFC by nearly 8 has been described in patients one month after the unilateral adrenalectomy. In contrast, unilateral adrenalectomy leads to the division by a bit more than 2 of the adrenals. This observation may be explained in patients with high cortisol levels by the saturation of the corticosteroid-binding globulin that leads to the rapid increase of the ratio of free/bound cortisol. The division by 2 of the cortisol production will also lead to a bigger division of the UFC level . AdrenalRemoval of the largest adrenal is usually performed. Nordiodocholesterol scintigraphy shows in all cases a maximum uptake in the largest glands . Few stuIn most studies, improvement of cortisol-related comorbidities, including obesity, diabetes, and hypertension, is reported in patients treated by unilateral adrenalectomy ,133. In More recently, adrenal-sparing surgery  as performed in patients with some genetic predisposition to pheochromocytoma has been proposed . The intAll patients presenting with adrenal hyperplasia and subclinical CS must have a regular screening, symptomatic treatment of comorbidities, and strict control of cardiovascular risk factors.The presence of illegitimate receptors may offer the possibility to propose a specific medical therapy. Beta-blockers, especially propranolol, have been proposed in case of cortisol response to posture. Long-term control of hypercortisolism with such therapy has been described, but the side effects of the treatment are limiting factors ,136,137.Anti-cortisolic treatment can be temporarily used for patients presenting with overt CS waiting for surgery. Long-term treatment by steroidogenesis inhibitors, including ketoconazole, metyrapone, or mitotane, has been proposed in both PBMAH ,141 and ARMC5 mutations brought new insight into the pathogenesis of PBMAH. The role of ARMC5 may take another decade to be fully understood since its role appears to be complex, involving cell cycle, proteasomal degradation, and crosstalk with other pathways, such as the PKA pathway. In the coming years, whole-genome sequencing will likely bring new candidates to explain the nearly 30% and 75% unresolved cases of micronodular adrenal hyperplasia and PBMAH, respectively. Progress in understanding other mechanisms such as paracrine regulation in adrenal glands will also help to understand the evolution of this group of diseases.Significant advances in the understanding of bilateral adrenal hyperplasia pathogenesis have been made in the last three decades. The PKA pathway so far appears to be the main pathway dysregulated in bilateral adrenal hyperplasia, especially PPNAD. In agreement with its effect on steroidogenesis and cellular differentiation, its alterations lead to CS, and the slowly growing hyperplasia and nodularity. In the last decade, the discovery of PRK1R1A in patients with PPNAD and the analysis of ARMC5 in patients with PBMAH. Complementary analysis of phosphodiesterases genes and PRKACA should be discussed. Next-generation sequencing allows screening these genes in a unique array, including the research of amplification of PRKACA. Genetic counseling must be performed in first-degree relatives of PRKAR1A mutation carriers and should be discussed in first-degree relatives of ARMC5 mutation carriers.In all patients presenting bilateral adrenal hyperplasia, genetic screening should be offered, including at least the analysis of Bilateral adrenalectomy is an effective treatment, but clinicians must weigh the arising complications of adrenal insufficiency against the complications of hypercortisolism, especially in patients with subclinical CS. Unilateral adrenalectomy appears to be a compelling alternative in patients presenting with bilateral adrenal hyperplasia, particularly those with PBMAH. Prospective studies are needed to compare medical therapy and adrenalectomy in patients presenting subclinical CS. Both short- and long-term evaluations of the complications of hypercortisolism need to be carried out in these studies.


Burnout is prevalent among medical students. Throughout their training, medical students face many psychosocial stresses that, if not managed, might cause burnout syndrome. Burnout could negatively impact students’ life and their academic performance. This study aims to estimate the prevalence of burnout among medical students at the College of Medicine, University of Kerbala, and assess factors associated with it.P value of a level < 0.05 considered statistically significant.An analytic cross-sectional study. A sample of 424 students from all academic years from the College of Medicine, University of Kerbala, was approached. Data collection was conducted from June 11 to July 3, 2020, through a self-administered online questionnaire based on the Maslach Burnout Inventory Student Survey (MBI-SS). The MBI-SS included 15 questions with a 7-point rating ranging from 0 (never) to 6 . The tool measures three subscales: emotional exhaustion (5 questions), Cynicism (4 questions), and professional efficacy (6 questions). Statistical Package for the Social Sciences (SPSS) program version 24 was used for data analysis. Binary logistic regression was used to assess the association between burnout and students̓ variables, The prevalence of burnout syndrome among medical students was 38.2%. About 85.6% of students had high emotional exhaustion, 77.8% had high cynicism, and 32.5% exhibited low professional efficacy. Female gender, regular use of legal substances, and family history of mental diseases were associated with significantly high rates of burnout.Burnout is prevalent among medical students of the University of Kerbala with quite high levels of emotional exhaustion and cynicism and lower professional efficacy levels. Faculties of medicine need to consider burnout among their students and works to reduce unnecessary stresses by modifying and upgrading the educational and clinical environments. Burnout currently is a mental condition, that attains growing attention. It was firstly introduced by psychologists Herbert Freudenberger and Christina Maslach in the mid-1970s to describe the experience of being physically and emotionally drained as a result of extended stress. Kafry and Pines were the first to describe student burnout; they defined it as a syndrome characterized by a loss of interest in studying, a lack of motivation, and exhaustion. Student burnout refers to a feeling of exhaustion caused by study pressures, a cynical and detached behavior toward one's studies, and a sense of inadequacy as a student .The prevalence of student burnout varies significantly across different settings and geographical regions. Burnout is estimated to impact at least 50% of all medical students during their school, according to US research  . A systeThe increased years of learning, multiple negative life events, stress, feelings of having limited control over life, poor peer relationships and support, lower exercise levels, smoking cigarettes, gender, and increased alcohol consumption were all associated with burnout among medical students –12.Early detection of burnout is an effective strategy for preventing and/or reducing the burnout impacts, such as exhaustion, incredulity, and a sense of poor professional efficacy, and probably students’ academic performance. And medical colleges must assure that they have adequate support and interventions for students who may experience greater stress and emotional discomfort during their studies –15. No pAn analytical cross-sectional study was conducted at the College of Medicine, University of Kerbala, Kerbala, Iraq. The participants were medical students from all 6 stages.Kerbala Medical College (KMC) included 1016 medical students from 6 years of study.Due to the coronavirus COVID-2019 pandemic situation, most universities shifted to online classes around the world . So, then = Z2P (1-p)/d2 , n = sample size, Z = 1.96 which is the corresponding value for the 95% confidence interval, P = prevalence of burnout among medical students, d = the degree of precision was at 0.05 at 95% confidence interval, and anticipated prevalence of burnout among medical students = 50%According to the following formula for determination of sample size Supposing the non-response rate, 10% of the sample size was added, so, the minimum required sample size was 380+38 =418 students.A convenient sample of 424 students from the total number of 1016 medical students at the year 2019–2020  was approached in the study. Thus, the response rate was 42%.A self-administered online questionnaire using Google form was developed. Then it was distributed as a link to the student representatives of each stage who shared it with all (1016) other students through the telegram groups which were already formed for each stage academic year and requesting their voluntary participation. Email survey took about 3 weeks to complete. The email survey took about 5–8 min for one participant to be completed.Inclusion criteria include all of the undergraduate medical students including both gender  and 18 years or above, who are willing to participate in the study and to complete all items of the MBI-SS.The questionnaire consisted of 2 parts, the first part contains socio-demographic characteristics like gender, age, marital status, residency, academic stage, family income, smoking habit and type of smoking, exercise habit, daily sleep hours, regular use of legal substances , and family history of psychological disorders. Where to maintain maximum health, adults should sleep 7 or more hours every night regularly (according to the American Academy of Sleep Medicine and Sleep Research Society). And regular exercise can improve anxiousness, tension, and a general sense of well-being , 18.The second part of the questionnaire included Maslach Burnout Inventory -Student Survey (MBI-SS). The Arabic version was previously used and validated by other studies. It is a modified version of the Maslach Burnout Inventory-General Survey (MBI-GS), which had been adjusted for use on students and become the classically used tool to measure academic burnout syndrome , 20.The MBI-SS includes 15 questions that conform to three scales: Emotional Exhaustion (5 questions), Cynicism (4 questions), and Professional Efficacy (6 questions). The MBI-SS questions are scored by students on a 7-point scale, based on their indicated frequency, ranging from 0 to 6, where 0, never; 1, a few times a year; 2, monthly; 3, a few times a month; 4, weekly; 5, a few times a week; and 6, every day .a = 0.75) for the emotional exhaustion 5 questions, 0.8 (Cronbach’s a = 0.8) for the cynicism 4 questions, and 0.73 (Cronbach’s a = 0.73) for the professional efficacy 6 questions.The internal consistency for the MBI-SS total 15 questions was 0.76. The internal consistency for the subscales were 0.75 (Cronbach’s The authors used a similar classification of other studies for burnout diagnosis. Students were identified as exhibiting burnout (high level) if their scores were more than 14 for emotional exhaustion and cynicism (score >6) while the low for professional efficacy (score < 23). These results matched the 66th percentile of exhaustion and cynicism, as well as the 33rd percentile of efficacy. Low burnout level indicated emotional exhaustion (< 10) cynicism (< 2), and efficacy (scores > 27). A moderate level of burnout was identified according to the following scores: emotional exhaustion (10–14), cynicism (2–6), and professional efficacy (23–27) , 22.The study is a part of research that was approved by the Research Ethics Committee of the College of Medicine, University of Kerbala. The participation was entirely voluntary and anonymous, the participants were able to respond at their convenience, and the confidentiality of the answers was ensured.p value of less than 0.05 was considered statistically significant.Data analysis was done using the Statistical Package for Social Sciences (SPSS) version 24 computer software; categorical variables were presented as frequencies and percentages. Quantitative variables were expressed as mean and standard deviation (SD). Binary logistic regression had been used to evaluate the association between burnout and students̓ background variables. All predictor variables were tested in one block to assess their predictive ability. Adjusted odds ratios (AOR) and 95% confidence intervals (95% CI) were measured. A About 145 students (34.2%) were within the age group ≥22 years, 295 (69.6%) of them were females, 402 (94.8%) were singles, 313 (73.8%) were from Kerbala, and 392 (92.5%) were nonsmokers.Other characteristics are shown in Table Emotional exhaustion, cynicism, and professional efficacy categories had been used to classify burnout scales. As a result of this classification, 85.6% of students were classified as having high emotional exhaustion, 77.8% as having high cynicism, and 32.5% as exhibiting low professional efficacy as shown in Table According to the three-dimensional criterion of burnout, the burnout diagnosis was calculated as a combination of the 3 sub-domains ; 162 of the students (38.2%) were experiencing burnout, while 262 (61.80%) students had no burnout.P value < 0.05): female gender , regular use of legal substances , and family history of mental diseases (adjusted OR= 2.65 95% CI 1.251–5.624). Other variables did not show significant association with burnout syndrome as shown in Table Using binary logistic regression to find the association between socio-demographic variables and burnout syndrome, after adjusting for multiple variables in this analysis, burnout was associated with  of medical students have burnout. However, a variant range of burnout rates among medical students from different countries. The finding of the current study matched the previous study conducted by Shadid et al. in Saudi Arabia showed that 38.2% of the medical students exhibited a high burnout rate . While lThe high burnout level could reflect a stressful teaching environment in KMC and other medical colleges in Iraq, and perhaps this is a reality. However, other explanations for such differences in burnout rates might be related to variant instruments used to evaluate burnout among medical students. Further, some studies used the two-dimensional criteria, which were less delicate than those used in the current study . Furthermore, students’ psychological stress and burnout were considered to be increased by online learning. Thus, the curfew and the necessity accelerated shift to online teaching due to the COVID-19 pandemic could be a part of increasing burnout levels among medical students at the University of Kerbala , 27, 28.When each dimension of burnout was assessed separately, medical students from the University of Kerbala had high percentages of emotional exhaustion 85.6%) and cynicism (77.8%), but lower percentages in reduced professional efficacy (32.5%). Similar findings were described by other studies 5.6% and , which rHowever, these results differ from the results of Altannir et al., who indicated lower levels of emotional exhaustion, high cynicism, and high levels of reduced professional efficacy .The difference in these results between the current study and the previously mentioned study may be due to different instruments and cutoff points used to calculate burnout . The MBI Questionnaire that was used in Altannir et al.’s study was a 22-item instrument with the following scores for burnout diagnosis: emotional exhaustion ≥30, cynicism ≥12, and professional efficacy ≤33 , in addiBinary logistic regression analysis, reveals that three variables were associated with Burnout; gender , medications intake, history of mental disorders within the family, and the number of study days per week ≤ 2.The significantly higher percentages of burnout among female students are agreed with other studies done in Saudi Arabia, Pakistan, India , Ethiopia (Debre Berhan), and Croatia , 30–34, The regular use of legal substances among medical students was significantly among medical students was significantly related to high burnout rates in this study. Also, a significant relationship between the occurrence of burnout and drug intake was reported in a study done in Brazil among dental students .While other studies were done in Hong Kong and Srinagar, had reported no association between regular use of legal sunstances and burnout , 41. TheThe current study showed that the presence of mental diseases within the family can predict for burnout of medical students. While a study done in Nepal showed no association between family history of mental health problems and burnout among medical students . The stuThe limitations of this study include the non-probability sample of the students, due to the online approach. So probably, those who get interested in the subject had participated, and those who do not participate could have certain viewpoints differ than the participant. The study was planned to be on a random sample through direct contact, but the emergent COVID-19 pandemic changes the hall academic year and turned most of it to online learning. Similarly, the study plan changed to an online survey. However, this shift might give light to the shift to the electronic learning effect. Another limitation is that this study does not measure the impact of burnout on the academic performance of students.This study identifies that a considerable prevalence (more than one-third) of medical students of the University of Kerbala had burnout, with quite high rates of emotional exhaustion and cynicism, and a low level of professional efficacy. Burnout was significantly associated with female gender, regular use of legal substances, and family history of mental diseases. Faculty must focus on altering improving the academic and clinical conditions, context, and curricula to reduce unnecessary stresses and create more favorable teaching and clinical practice. As well as that professors need to understand the students’ needs, motivations, and experiences to keep students interested in education and to minimize their burnout rates.


Vitamin D supplementation is routinely introduced in infants, according to medical guidelines. However, vitamin D overdose can result in life-threatening hypercalcemia. We report the case of a 3-month-old infant who suffered from severe hypercalcemia. Upon detailed questioning of the parents, a vitamin D administration error has been identified. Indeed, the parents had followed the advice of their midwife. They substituted the prescribed medicinal vitamin D by a dietary supplement, different in concentration and dosing, without performing the dose conversion needed. In fact, many different medications and dietary supplements with vitamin D exist, offering various concentrations and units of measurement. This case highlights the pivotal role of therapeutic education. Broadly, there is a need for harmonization of the regulation and labeling of dietary supplements and medications containing vitamin D. Vitamin D is a fat-soluble hormone either synthetized endogenously or from external vitamin intakes. Vitamin D bears pleiotropic functions , 2, primIn August 2020, a 3-month-old infant was referred to the pediatric emergency department for severe anorexia, vomiting, and weight loss. After an uncomplicated pregnancy (36 weeks and 5 days of amenorrhea), he was born at term, weighing 2.3 kg. Since, he had been exclusively breastfed and had not presented any medical problem.At the time of admission, the infant weighted 4.5 kg. He had no fever, his heart rate was 109 beats per minute, his blood pressure was 107/85 mmHg, and his oxygen saturation was 99%. Blood pressure was later reassessed several times, and was found normal or at the upper limit of normal for the age. Clinical examination was unremarkable except a global hypotonia and the presence of moderate dark circles around the eyes. Natremia was 139 mmol/L, kalemia 4.4 mmol/L, alkaline reserve 21 mmol/L, hemoglobin 9.1 g/dL, leukocytes 11.11 G/L, platelets 471 G/L, and C-reactive protein (CRP) 13 mg/L with a negative procalcitonin. Blood gases analysis and liver function tests were within normal range. Natriuresis was <20 mmol/L for a kaliuresis of 33 mmol/L, suggestive of hypovolemia. Hemoculture, stool bacterial culture and virology, as well as lumbar puncture results were all negative. Lumbar puncture had been performed because the infant presented with global hypotonia and severe behavioral changes, in the absence of an obvious diagnosis. Immunoglobulin E (IgE) antibodies to cow milk were negative. Cranial ultrasonography and brain Magnetic Resonance Imaging (MRI) were normal. Five days after admission, an abdominal ultrasound confirmed 3 days later revealed some deposits in the bladder, which were deemed clinically not significant.Because of a suspicion of urinary infection, probabilistic intravenous antibiotic therapy was initiated, then discontinued 3 days later as no inflammatory syndrome nor bacteria in urinary analysis (including mycobacteria in a context of sterile leukocyturia) were found. Breastfeeding was supplemented with infant milk and intravenous hydration.Calcemia was assessed for the first time 12 days after admission to hospital and was at 3.08 mmol/L  with an albuminemia of 41 g/L . Parathormone (PTH) was below the limit of quantification , whereas calcidiol was above the upper limit of quantification  and calcitriol was 200 pg/mL . Phosphate was 1.8 mmol/L . Serum creatinine was 23 μmol/L  while urea was 2.3 mmol/L . Urinary calcium was 2.75 mmol/L with a creatininuria of 1 mmol/L.Electrocardiogram was normal and no arrhythmia was documented on the monitoring. The infant was kept hydrated and calcemia progressively decreased to reach 2.78 mmol/L in mid-September. Bisphosphonates were not deemed necessary. The child was finally discharged with a weight of 4.8 kg and with a close monitoring of his calcemia recommended. Throughout a follow-up consultation, 2 weeks later, calcemia was 2.6 mmol/L and the child weighed 5.2 kg. On last follow-up consultation 3 months after the onset of symptoms, he weighed 5.6 kg and was clinically asymptomatic. Renal ultrasound was suggestive of calcic deposits in the urinary tract.Upon detailed questioning of the parents, a chronic overdose of vitamin D was identified. In order to provide the recommended daily dose for breastfed newborn, the maternity medical staff initially had prescribed 4–5 drops per day of ZymaD®, a brand name containing cholecalciferol dosed at 10,000 International Unit (IU)/mL, one drop containing 300 IU of vitamin D . Once atper drop (and not per mL), and the manufacturer's recommendation consists of one drop every 10 days. However, as initially prescribed with ZymaD®, 4–5 drops per day were administered. This switch between two non-equivalent drugs, with different dosing, consequently exposed the infant to 40,000–50,000 IU per day, which represents 50-fold the Upper tolerable Level (UL) recommended. Such exposition conducted to a symptomatic severe vitamin D overdose, with a 15-day hospital stay and potential sequelae such as nephrocalcinosis and lithiasis.Thereupon, the parents bought on the Internet the DS Sunday Natural® brand of D3 10,000 IU (+ vitamin K2). This product is deemed to contain 10,000 IU This case highlights the pivotal role of health practitioners in limiting the risk of drug and dietary supplement misuse, even for so-called innocuous “vitamins.” Because of different regulatory specific domains, blurred frontiers between recommended use and potentially harmful errors may lead to serious health hazards. This situation may become more and more frequent, especially considering the current hype surrounding vitamin D.Vitamin D overdose is suspected when hypercalcemia coexists with calcidiol > 150 ng/mL  and low Vitamin D intoxications have been reported in patients consuming large doses of vitamin D-containing supplements, either voluntarily or as medication and dietary supplement errors. The latter pertain to the multiple brands, types of packaging and formulations of dietary supplements, resulting in prescription or administration errors , 16, 17.In this case, the lack of communication and/or comprehension between the parents and the midwife resulted in an error in drug administration. It took roots in substituting the medicinal vitamin D by a dietary supplement of different dosing. The risk is further increased by the plethora of alternate formulas available on the Internet, with various concentrations and expression of dosages. Up to 28 pharmaceutical brands of vitamin D are available in France, not to mention the DS and the various multivitamin complexes. The labeling of vitamin D products (drugs and dietary supplements) suffers from a lack of harmonization, misleading the consumer and exposing to hazardous titrations.Besides, products containing vitamin D are framed by two distinct regulations, regarding their qualification as drug or dietary supplement. Indeed, the sole notification to the competent authorities without any objection within 2 months allows the manufacturer to put a dietary supplement on the market. Manufacturers are free to mislead the consumer to benefit from the aura of the purported naturalness of their DS, maintaining confusion with equivalent drugs. However, vitamin D content of unlicensed DS is believed to vary widely from labeled claims, as DS are manufactured under less stringent quality standards . Yet, paThe error risk when dispensing or administering medications is further increased during the postpartum period, upon returning home. Mother and child both have their own prescriptions, sometimes including the same pharmacological classes or presentations  exposing to potential errors. Parents are receptive to marketing arguments insisting on the naturalness of the products intended for their child.inter alia ' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.