Cutaneous squamous cell carcinoma (SCC) is one of the most frequent malignancies in humans and academia as well as public authorities expect a further increase of its incidence in the next years. The major risk factor for the development of SCC of the general population is the repeated and unprotected exposure to ultraviolet (UV) radiation. Another important risk factor, in particular with regards to occupational settings, is the chronic exposure to polycyclic aromatic hydrocarbons (PAH) which are formed during incomplete combustion of organic material and thus can be found in coal tar, creosote, bitumen and related working materials. Importantly, both exposomal factors unleash their carcinogenic potential, at least to some extent, by activating the aryl hydrocarbon receptor (AHR). The AHR is a ligand-dependent transcription factor and key regulator in xenobiotic metabolism and immunity. The AHR is expressed in all cutaneous cell-types investigated so far and maintains skin integrity. We and others have reported that in response to a chronic exposure to environmental stressors, in particular UV radiation and PAHs, an activation of AHR and downstream signaling pathways critically contributes to the development of SCC. Here, we summarize the current knowledge about AHR’s role in skin carcinogenesis and focus on its impact on defense mechanisms, such as DNA repair, apoptosis and anti-tumor immune responses. In addition, we discuss the possible consequences of a simultaneous exposure to different AHR-stimulating environmental factors for the development of cutaneous SCC. Non-melanoma skin cancers, in particular basal cell carcinoma and SCC, are among the most frequent malignancies in humans –3. CutanIn this review article, we focus on the critical functions of AHR for DNA damage-dependent processes and immune responses which may contribute to the development of SCC in chronically UV- and/or PAH-exposed skin. Please note that while we are focusing on the mentioned aspects other functions of the AHR system might fall short, which may be also relevant for the process of skin cancer development.a]anthracene (DMBA) as tumor-initiator and phorbol ester-containing croton oil as tumor-promoting agent. Sequencing of the tumor DNA as well as further mechanistic studies provided evidence that PAHs initiate carcinogenesis by inducing mutations primarily in the Ha-Ras oncogene and that this process requires a metabolic conversion of the per se non-toxic chemicals to highly reactive metabolites, a process which is primarily carried out by AHR-regulated cytochrome P450 (CYP) monooxygenases  and 2-(1’H-Indole-3’-carbonyl)-thiazole-4-carboxylic acid methyl ester as well as tryptophan metabolites, such as kynurenic acid and xanthurenic acid, are considered as relevant endogenous agonists of AHR  molecules, the AHR-interacting protein, the co-chaperone p23 and the soluble tyrosine kinase c-Src  Figure–16. Typia]pyrene (B[a]P) to B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), a highly carcinogenic compound which forms bulky DNA adducts by binding to guanine at the N2 position  cell-related inflammatory responses in the skin , a family of cytosolic NADPH-dependent oxidoreductases . Severalthe skin , 52. Notpathways . Importapathways  and, morpathways . Taken t+ cytotoxic T cells and CD4+ type 1 T helper (Th1) cells which may prevent skin tumor development by secreting respective cytokines, such as interleukin (IL)-2 and interferon-γ (+ regulatory T cells (Tregs). The induction of keratinocyte apoptosis by cytotoxic T cells leads to the release of further mediators which may stimulate other immune cells to infiltrate the inflamed skin. In case the damage is not resolved, chronic inflammatory condition facilitates the growth and progression of skin tumors  radiation is part of the electromagnetic spectrum of sunlight and can be subdivided into UVA, UVB and UVC radiation , 69. TheSkin photocarcinogenesis is a multistep process, involving initiating and promoting events , 72. TheIn case NER fails, the remaining DNA photoproducts will cause DNA double-strand breaks (DSB), which have been denominated as lethal DNA lesions . In fact+ and CD25+, express FoxP3 and secrete IL-10. Although bearing lymph node-homing receptors, Tregs may switch to skin-homing receptors upon contact with epidermal Langerhans cells, migrate into the skin and suppress cancer cell-killing effector T cells (cis-urocanic acid in the stratum corneum (AHR and Immunosuppression), AHR has been identified to contribute to the suppression of immune responses in UVB-irradiated mice indole, which serve as high-affinity AHR ligands (in vivo (N-formylkynurenine (NFK), another tryptophan photoproduct formed in UVB-irradiated cells -9H-pyrido after UVB exposure . RB phosphorylation is necessary to activate E2F1, which controls the expression of CHK1 (p-dioxin (TCDD)-treated Chinese hamster ovary cells . An inhibition of both GGR and apoptotic clearance probably results in an accumulation of CPDs and thus may represent one mechanism through which active AHR maintains the UVB radiation-induced suppression of the immune system. Studies on a mouse model of contact hypersensitivity confirmed this hypothesis by demonstrating that a chemical or genetic inhibition of AHR attenuates the UV radiation-induced expansion of Tregs and associated immunosuppressive effects  (UV Radiation-Induced Immunosuppression) or airborne PAHs and PAH-rich PM (see AHR and PAH-Induced Immune Reactions) which stimulate the generation of Th17 cells and exacerbate autoimmune disorders. This discrepancy clearly points to a ligand-specific effect of the AHR system on fate and function of T lymphocytes. Interestingly, results from a study conducted by the Kerkvliet laboratory suggest that this ligand-specific effect is due to differences in the metabolic half-life of the respective AHR ligand and associated dose-dependent effects  , 143. Th effects . However effects –112, a p effects . Althoug effects , 146. Th effects , 148. Ne effects . In addi T cells . Hence,  T cells .Although experimental evidence is yet lacking, it is conceivable that at least in advanced stages of cutaneous SCC, the AHR may play a comparable role in modulating tumor immunity.ex vivo experiments (via NF-κB-mediated trans-repression, i.e. a competition for common transcriptional co-activators (Given that environmentally ubiquitous PAHs as well as UV radiation are capable of modulating AHR activity in keratinocytes and other epidermal cell populations, a simultaneous exposure to these factors may cause co-carcinogenic effects. However, as specified below, the data published so far on this topic produce a heterogenous and sometimes even contradictory picture. For instance, some early carcinogenesis studies reported an increased skin tumor formation upon UV irradiation of coal tar-treated mice , 153, wheriments . An enhaeriments . Intereseriments . In conteriments . As expeeriments . Thierryeriments , 172. Gieriments , 174. Ineriments , 176. Tutivators , 177. HoIn summary, the current data concerning the simultaneous exposure of the skin to UV radiation and PAHs are not coherent and illustrate the need for further studies to ensure a proper risk assessment, in particular for roofers, roadmen, and other occupational groups that are frequently exposed to high doses of both, PAHs and sunlight. Chronic exposure studies on rodents, for instance, using the same irradiation device for single and simultaneous exposure to UVA and UVB light in combination with a pre- and post-treatment with environmentally-relevant PAH mixtures may shed light on a potential interaction of both risk factors in skin carcinogenesis. Proper AHR signaling is indispensable for the development and physiology of the skin , 179. HoTH-S contributed to the conception and design of the review article. CV, KR, and TH-S wrote the manuscript. JK provided critical feedback during the preparation of the article and contributed to the revision of the manuscript. CV generated the figures. All authors contributed to the article and approved the submitted version.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.


Background: Decision-making regarding biochemical recurrence (BCR) in localized prostate cancer (PCa) patients after radical prostatectomy (RP) mainly relies on clinicopathological parameters with a low predictive accuracy. Currently, accumulating evidence suggests that immune-associated genes (IAGs) play irreplaceable roles in tumorigenesis, progression and metastasis. Considering the critical role of immune in PCa, we therefore attempted to identify the novel IAGs signature and validate its prognostic value that can better forecast the risk for BCR and guide clinical treatment.Methods: RNA-sequencing and corresponding clinicopathological data were downloaded from the Gene Expression Omnibus (GEO) database and the Cancer Genome Atlas (TCGA) database. Weighted gene co-expression network analysis (WGCNA) was utilized to screen out the candidate module closely related to BCR, and univariate and LASSO Cox regression analyses were performed to build the gene signature. Kaplan-Meier (KM) survival analysis, time-dependent receiver operating curve (ROC), independent prognostic analysis and nomogram were also applied to evaluate the prognostic value of the signature. Besides, Gene ontology analysis (GO), Kyoto encyclopedia of genes and genomes (KEGG) and gene set enrichment analysis (GSEA) were used to explore potential biological pathways.Results: A total of six IAGs  were eventually identified and used to establish a novel IAGs signature. The Kaplan-Meier analysis revealed that patients with low-risk scores had longer recurrence-free survival (RFS) than those with high-risk scores in both GSE70769 and TCGA cohorts. Further, our signature was also proven to be a valuable independent prognostic factor for BCR. We also constructed a nomogram based on the gene signature and related clinicopathologic features, which excellently predict 1-year, 3-year and 5-year prognosis of localized PCa patients after RP. Moreover, functional enrichment analysis demonstrated the vital biological processes, and stratified GSEA revealed that a crucial immune-related pathway  was notably enriched in the high-risk group.Conclusions: We successfully developed a novel robust IAGs signature that is powerful in BCR prediction in localized PCa patients after RP, and created a prognostic nomogram. In addition, the signature might help clinicians in selecting high-risk subpopulation, predicting survival status of patients and promoting more individualized therapies than traditional clinical factors. Worldwide, prostate cancer (PCa) ranks the second most commonly diagnosed malignancies in males As we known, the prognosis of PCa patients is closely related to prostate-specific antigen (PSA), Gleason score (GS) and clinical TNM stage https://www.ncbi.nlm.gov/geo/) database and the Cancer Genome Atlas . The results might offer a more powerful tool for clinicians to make individualized and precise medical decisions and have a better understanding of possible molecular mechanisms of BCR.However, there are few studies associating IAGs with BCR in localized PCa patients after RP. Considering the crucial role of the immune system in the prognosis of PCa, we determined to establish novel predictive IAGs signature to improve the risk stratification of biochemical recurrence in PCa patients via employing the high-throughput sequencing results and downloading clinical data from the Gene Expression Omnibus . Inclusion and exclusion criteria of eligible patients: The inclusion criteria were as follows: (I) biospecimens were concentrated from localized prostate cancer patients after radical prostatectomy; (II) clinicopathological characteristics, such as Gleason score (GS), clinical stage T (cT), prostate-specific antigen (PSA) or surgical margins (SM) were contained; (III) the outcomes  or BCR-free (BCR-F)) of samples were included; The exclusion criteria were as follows: (I) patients also following chemotherapy or radiotherapy in addition to the radical prostatectomy. In our study, GSE70769 was utilized as a training set, while the TCGA database was applied to validate. The whole RNA-seq data and microarray were log2 transformed and normalized with the manufacture-provided R packages. A collection of immune-associated genes (IAGs) were downloaded from the Immunology Database and Analysis Portal  In our study, 471 localized PCa patients following RP and full-scale clinical parameters from two independent cohorts were included. One was from the Gene Expression Omnibus (GEO), and the other was from the Cancer Genome Atlas (TCGA) cohort. The RNA-seq data of GSE70769 were produced with Illumina HumanHT-12 V4.0 Array, for 92 samples with full-scale clinical information. Besides, RNA-sequencing (RNA-seq) expression profile of 379 patients was downloaded from the TCGA dataset  in our study. Then, utilizing the topological overlap matrix (TOM)-based dissimilarity measure, with deep-Split of 2 and min-Module size (gene group) of 30 for the IAGs cluster dendrogram, average linkage hierarchical clustering was created. IAGs with similar expression modes were stratified into the same modules by a one-step network construction and module detection. Besides, two parameters were defined. One was gene significance (GS), which was used to quantify the connection between individual IAG and biochemical recurrence-free survival (RFS). The other was module eigengenes (MEs), which served as the first principal component-related module whose value could be regarded to represent the whole IAGs in the module. Under the two parameters, the excellent candidate module that had the highest absolute correlations with RFS was singled out for further analysis.In accordance with the microarray data from the training database, the weighted gene co-expression network analysis (WGCNA) was performed to establish a scale-free co-expression network IAGs deriving from the candidate module was submitted for the univariate Cox regression analysis, and those were significantly associated with biochemical recurrence-free survival (RFS) (p < 0.05) were screened for prognostic signature development. Subsequently, we performed the least absolute shrinkage and selection operator (LASSO) Cox regression analysis to single out the optimal prognostic IAGs Moreover, in light of the median risk score in the training set as the threshold, eligible PCa patients were divided into low- and high-risk groups. The Kaplan‐Meier survival curves were plotted to compare the prognostic difference in RFS between the two subgroups mentioned above. The time-dependent receiver operating characteristic curves (ROC) was generated by using the “SurvivalROC” package, and area under the curve (AUC) values were calculated to estimate the specificity and sensitivity of the risk score. To further determine the predictive ability of our model, the TCGA database was adopted as a validation cohort. The univariate and multivariate Cox proportional hazards regression analyses were performed to assess the BCR outcome predictive performance of our model and other related clinicopathological features.To offer a clinically quantitative tool to monitor and predict BCR outcomes in PCa patients, a novel nomogram model was established http://www.genome.jp/) connected genomic information with higher-order functional details. GO enrichment analysis and KEGG pathway analysis were carried out on the Database for Annotation, Visualization and Integrated Discovery (DAVID) online tool . Both FDR and P-value < 0.05 were regarded as statistically significant.Gene ontology analysis (GO) functioned as a standard method for noting genes and identifying biological attributes for high-throughput genome data, containing biological process (BP), cellular component (CC) and molecular function (MF). As a knowledge base for systematically analyzing gene functions, Kyoto encyclopedia of genes and genomes  was applied to identify the pathways, which were mainly enriched between high-risk and low-risk groups Then the downloaded gene set enrichment analysis  or Student's t-test for continuous variables. The univariate Cox proportional hazards regression model and LASSO regression analysis were used to calculate the hazard ratio and the regression coefficient. Survival curves were analyzed by the Kaplan‐Meier method utilizing the log-rank test. The Z-score method was applied to normalize risk scores in the training and validation cohort. The SPSS Statistics 26.0 and R software 3.6.3 were performed for all statistical analyses. It was considered significantly different that two-sided P values were less than 0.05.Figure 1). Sample clustering illustrated that the sample GSM1817916 was an outlier that were excluded from our study , and the other eligible samples along with different BCR, PSA, cT, GS, and SM rates were selected for IAGs expression clustering . With a power of β = 3 set as the optimal soft threshold, a total of six co-expressed modules were identified through a scale-free network establishment method . Among these co-expressed modules, one module (red) with the highest absolute correlation value with BCR was screened out (p = 0.002 and correlation coefficient = 0.34) . Next, the 58 IAGs from the red module was submitted for the univariate Cox regression analysis. Taking the cut-off value of P < 0.05, 24 IAGs were identified to be significantly linked to the RFS of localized PCa patients . Then, in order to generate the hub candidate IAGs, the LASSO Cox regression analysis was applied to get the optimal lambda value, which came from the minimum partial likelihood deviance . In the end, six critical prognostic IAGs comprising of SSTR1, NFATC3, NRP1, TUBB3, IL1R1 and GDF15 were selected, and their individual nonzero LASSO coefficients were shown in Figure . Furthermore, we established a six prognostic IAGs signature by employing the risk score model. The risk score of each sample was calculated according to the following formula:To identify the relationship between IAGs and localized PCa patients with different clinicopathologic features, the expression data profile of these 1811 IAGs was transformed into a gene co-expression network utilizing a WGCNA package in the training cohort . Utilizing the same regression coefficient (β) and algorithm, those findings were subsequently tested in the validation set, which presented the same result as expected, with a significantly longer RFS in the low-risk group (p < 0.0001) . The Cox's regression model was performed to determine whether the signature was an independent factor compared to other clinicopathological factors, such as GS, SM, PSA and age. After the multivariate Cox regression analysis, the results demonstrated that the IAGs signature was an independent prognostic parameter, which was significantly connected with RFS  . To estimate the prognostic power of the IAGs signature, the time-dependent ROC analyses were used, and the area under curve (AUC) values were calculated. We found that the average AUC value of the risk score for RFS was 0.778 at five years follow-up in the training set, which was significantly higher than those of GS (AUC = 0.733), SM (AUC = 0.640), PSA (AUC = 0.577) and cT (AUC = 0.577). Similarly, in the validation set, average AUC value of the risk score (AUC = 0.755) was also considerably higher than those associated with GS (AUC = 0.720) and cT (AUC = 0.609) . The outcomes mentioned above suggested that the IAGs signature served as a more powerful predictor for BCR than other clinical features in localized PCa patients.In accordance with the coefficient value of the six IAGs, the risk scores of all samples were calculated and ranked in the training set and the validation set. The risk scores of BCR-free (BCR-F) patients were obviously decreased compared with those of BCR ones in both cohorts. Based on the risk score, patients in the training set were divided into the high-risk and low-risk groups. Kaplan-Meier survival curves displayed that localized PCa patients in the high-risk group had a significantly poorer recurrence-free survival (RFS) compared to those in the low-risk one (p < 0.0001) in the training database .In addition, we normalized the risk score to Z-score for both TCGA and GSM1817916 cohorts. The results indicated that compared to BCR-free (BCR-F) patients, Z-score was significantly higher in BCR patients. What's more, with the extension of BCR time, Z-score also has a tendency to increase gradually . In the training set, the AUC value of 1-year, 3-year and 5-year RFS of the new nomogram were 0.801, 0.837 and 0.862, respectively, which presented the excellent prognostic power in predicting RFS . The calibration plots of our nomogram for survival prediction suggested an excellent conformity between the expected and the observed outcomes .To provide a clinically quantitative method to monitor and predict the prognosis of localized PCa patients undergoing BCR after RP, we constructed a novel prognostic nomogram that integrated the risk score, GS, SM, cT and PSA. A total point could be calculated by adding the scores of five variables in the nomogram, among which survival probabilities could be estimated easily. The results showed that the prognostic nomogram could better predict 1-, 3- and 5-year RFS of localized PCa patients .The GO enrichment and KEGG pathway analysis were performed to explore the potential functional and molecular mechanisms of IAGs in the candidate module (red module). GO enrichment analysis demonstrated that the candidate IAGs were mainly enriched in the biological processes (BP) connected with regulation of cell proliferation, response to wounding, cell surface receptor linked signal transduction, secretion by cell and antigen processing and presentation, etc. Regarding the cellular component (CC) analysis, we found that the candidate IAGs were notably associated with acellular region, extracellular space, integral to plasma membrane, intrinsic to plasma membrane and extracellular region part, etc. In terms of the molecular function (MF), the candidate IAGs were significantly involved in cytokine activity, cytokine binding, peptide binding, peptide receptor activity, peptide receptor activity and G-protein coupled, etc. Moreover, KEGG pathway analysis revealed that the candidate IAGs were mainly enriched in axon guidance, T cell receptor signaling pathway, cytokine-cytokine receptor interaction, natural killer cell mediated cytotoxicity and neuroactive ligand-receptor interaction, etc. (Table 2). Intriguingly, stratified GSEA demonstrated that an important immune-related pathway, namely T cell receptor signaling pathway, was notably enriched in the high-risk group .Additionally, we carried out the gene set enrichment analyses (GSEA) to identify the potential biological processes between high- and low-risk groups in the training cohort . However, the expression level of NRP1 was not significantly different between normal and prostate tissue (p = 0.5305). Moreover, to validate the protein expression of hub IAGs in prostate cancer, immunohistochemistry results from the Human Protein Atlas (HPA) database (http://www.proteinatlas.org/) were utilized, which showed the similar results as expected .Then the expression profiles of the six hub IAGs between tumor and normal tissue were presented in Prostate cancer is a common malignancy among elderly males in the world During the era of cancer immunotherapy, PCa is the first malignancy to demonstrate improved survival with a cancer-specific vaccine, proving that it is an immune responsive disease In our study, we singled out six immune-associated genes  to construct the BCR predictive model based on the GSE70769 dataset (training cohort) and validate it in the TCGA database . We performed WGCNA to identify the candidate gene module (red module) that was most significantly connected with BCR. Then, the univariate Cox regression analysis and LASSO regression analysis were utilized to select six hub genes from the red module. On the basis of six IAGs, we established the predictive signature and calculated the risk score of each patient according to the formula. Further, the enrolled patients were divided into high- and low-risk groups based on the median risk score value, and the Kaplan-Meier survival analysis demonstrated that patients with low risk scores had a significantly longer recurrence-free survival (RFS) than those with high risk scores in both TCGA and GSE70769 cohorts. Univariate and multivariate Cox regression analysis displayed that the risk score was an independent prognostic factor for BCR. What's more, we performed the time-dependent ROC analysis, which illustrated that our model exhibited the excellent predictive performance in both the training and validation datasets. The efforts above underscored that our six IAGs signature had the satisfactory sensitivity and specificity in predicting BCR of localized PCa patients after RP.In addition, to provide a quantitative tool for clinicians to predict BCR and improve risk stratification, a compound nomogram was established that integrated the risk score and four relative clinicopathological parameters . In the calibration plots of our novel prognostic nomogram, greatly satisfactory conformity was found between the predicted and observed outcomes. The results of time-dependent ROC analysis showed that the novel nomogram had the wonderful prognostic power in predicting RFS, with an AUC of 0.862 after 5-year follow-up. Therefore, our prognostic nomogram might better help clinicians monitor and predict the 1-, 3- and 5-year RFS of localized PCa patients following RP, and provide better individualized therapy than original clinical protocols.Some IAGs involved in our signature have been investigated to play critical roles in tumors, even in prostate cancer. For example, GDF15, namely a divergent TGF-beta superfamily cytokine, slowed the growth of PCa by irritating tumor immunity. And the overexpression of GDF15 was related to an increased number of CD8 T cells, an increased number of CD8+CD11c+ T cells and a reduced proportion of “exhausted” CD8+PD1+ T cells As the six IAGs signature demonstrated excellent ability in predicting BCR in PCa patients, the signaling pathways and biological processes also needed to explore in our study. The GO enrichment analysis and KEGG pathway analysis illustrated that IAGs in the candidate module were significantly enriched in T cell receptor signaling pathway, natural killer cell mediated cytotoxicity, regulation of cell proliferation, cell surface receptor linked signal transduction, extracellular region, extracellular space, cytokine activity, cytokine binding, peptide binding and peptide receptor activity, et al. Stratified GSEA indicated that in the high-risk group, biological processes were significantly enriched in T cell receptor signaling pathway, progesterone mediated oocyte maturation, blood vessel endothelial cell proliferation involved in sprouting angiogenesis, cell aggregation, mediator complex, anchored component of plasma membrane, protein deacetylase activity, glutamate receptor activity and so on. It was worth noting that one immune-related pathway, namely T cell receptor signaling pathway was enriched in both analyses. Therefore, we supposed that T cell receptor signaling pathway might make considerable contributions to tumorigenesis, progression and poor prognosis in localized PCa patients after RP, which further showed the high clinical value of our gene signature.Overall, our IAGs based signature was successfully established and carefully evaluated in the discovery and validation databases. The signature is of vital importance for clinical application, helping predict and monitor the progression and metastasis of prostate cancer. In spite of the merits, several limitations remained to be acknowledged. First, our study was the retrospective design, thus the reliability of our six IAGs signature is needed to verify in multi-center and prospective researches. Second, more and more experiments should be carried out to further investigate the potential biological progress and molecular mechanisms of these IAGs in prostate cancer progression.In conclusion, our study identified and established a novel robust IAGs  signature in predicting BCR for localized PCa patients following RP, which could help clinicians predict patients' recurrence-free survival (RFS) and improve the specific individualized management than original clinical parameters. Moreover, the signature could also serve as an independent prognostic factor, and nomogram based on it showed an excellent predictive efficacy of patients' survival status. However, further studies are expected to validate our prognostic model, and functional researches are required to better understand the molecular mechanisms in prostate cancer.Supplementary figures and tables.Click here for additional data file.


Amyotrophic lateral sclerosis (ALS) is increasingly recognised as a multi-system disorder, presenting with common and impactful non-motor symptoms, such as neuropsychiatric symtpoms, cognitive and behavioural changes, pain, disordered sleep, fatigue and problematic saliva.We aimed to systematically review 25 years of ALS clinical trials data to identify if non-motor features were evaluated, in addition to the traditional measures of motor functioning and survival, and where evaluated to describe the instruments used to assess. We hypothesised that assessment of non-motor symptoms has been largely neglected in trial design and not evaluated with ALS-suitable instruments.We reviewed clinical trials of investigative medicinal products in ALS, since the licensing of riluzole in 1994. Trial registry databases including WHO International Trials Registry, European Clinical Trials Register, clinicaltrials.gov, and PubMed were systematically searched for Phase II, III or IV trials registered, completed or published between 01/01/1994 and 16/09/2020. No language restrictions were applied.237 clinical trials, including over 29,222 participants, were investigated for their use of non-motor outcome measures. These trials evaluated neuropsychiatric symptoms , cognitive impairment , behavioural change , pain , sleep disturbances  and fatigue . Problematic saliva was assessed as part of composite ALS-FRS(R) scores in 184 trials (78%) but with no focus on this as an isolated symptom. 31 (13%) trials including 3585 participants did not include any assessment of non-motor symptoms.Non-motor symptoms such as neuropsychiatric, cognitive and behavioural changes, pain, disordered sleep, fatigue, and problematic saliva have not been consistently evaluated in trials for people with ALS. Where evaluated, non-symptoms were primarily assessed using instruments and impairment thresholds that are not adapted for people with ALS. Future trials should include non-motor symptom assessments to evaluate the additional potential therapeutic benefit of candidate drugs.CRD42020223648.The online version contains supplementary material available at 10.1007/s00415-021-10651-1. The focus of assessment and symptom management in amyotrophic lateral sclerosis (ALS) is traditionally on limb weakness, speech and swallowing difficulties, and respiratory failure. Despite this, a range of other symptoms are repeatedly reported as impactful and poorly evaluated in people with ALS (pwALS) including neuropsychiatric symptoms, cognitive and behavioural changes, pain, disordered sleep, fatigue, and problematic saliva .These symptoms are often collectively termed ‘non-motor’ or ‘extra-motor’ , 2 and rOur previous work has reported how neuropsychiatric, cognitive and behavioural assessments have been employed as outcome measures and exclusion criteria in ALS trials . This idThe focus on motor symptoms in clinical and research contexts is likely impacted by the limited availability of disease-modifying drugs for people with ALS. Riluzole is currently the only globally licensed disease-modifying therapy for ALS with limited efficacy, resulting in prolongation of survival by 2–3 months . There iTo deliver holistic disease management for people with ALS, it is necessary to expand our conceptualisation of ‘treatment’ beyond improved physical function and extended survival. Effective management, or ultimately slowed progression, of non-motor symptoms due to pharmacological intervention should be evaluated as part of any novel investigative medicinal products’ efficacy in clinical trials . InclusiThe method of assessment is also of particular relevance in trials of people with ALS. Due to progressive disability, overlap with somatic symptoms, disease-specific impairments and speech decline, traditional measures may not be as effective in detecting change in symptoms, directly reducing their suitability to evaluate people with ALS . This caWe aimed to systematically review historical clinical trials of interventional medicinal products (IMPs) in ALS, since the licensing of riluzole in 1994, to identify if non-motor features of ALS were evaluated as outcome measures. In addition, we aimed to review the assessment tools used, their characteristics and suitability for evaluating non-motor symptoms in people with ALS. We hypothesised that non-motor symptoms have been largely overlooked in trial design and that where evaluated, assessed with instruments that are not specifically designed to evaluate symptoms in this population.We completed a systematic, unbiased, search of trial registries including clinicaltrials.gov, World Health Organisation’s (WHO) International Clinical Trials Registry Platform (ICTRP), European Union Clinical Trials Register (EduraCT) and PubMed on 16/09/2020 for Clinical Trials of an Investigational Medicinal Product (CTIMP). Using the search terms “amyotrophic lateral sclerosis” or “motor neuron* disease”, we searched clinicaltrials.gov for interventional trials of investigative medicinal products. We searched European Union Clinical Trials Register (EudraCT) and WHO International Clinical Trials Registry Platform (ICTRP) for trials of “amyotrophic lateral sclerosis” with the filters “Phase II”, “Phase III” and “Phase IV” applied. Using the advanced search feature, we filtered PubMed with . We then applied the ‘Clinical Trial’ filter for Article Type, Human trials only and Publication Date within the criteria defined above.Phase II, III or IV trials assessing potential disease-modifying therapies in subjects with amyotrophic lateral sclerosis that were registered, completed or published between 01/01/1994 and 16/09/2020 were included. No language restrictions were applied. Extension trials, post hoc analysis papers, stem cell therapies, imaging studies, medical device studies, non-ALS subjects and trials focussed on symptom management were excluded.The following details of selected trials were extracted “Investigational Medicinal Product (IMP) Assessed”, “Number of Participants”, “Date of Commencement”, “Primary Outcome Measure(s)” and “Secondary Outcome Measure(s)”. We reviewed each assessment tool used as an exclusion criteria or outcome measure in the included trials to explore whether they evaluated non-motor symptoms; defined in this study as neuropsychiatric, cognitive impairment, behavioural changes, pain, disordered sleep, fatigue, and problematic saliva. Each assessment tool was categorised as ALS specific, symptom specific, both ALS and symptom specific or generic in content focus. We then reviewed each trial included in this review for their use of each assessment tool and subsequent evaluation of each non-motor symptom.Each assessment tool was reviewed and data extracted on the intended focus of assessment, administrator (clinician or self-report), if the scoring is affected by the presence of motor disability or speech impairment and the time to administer. We also explored the availability of disease-specific impairment thresholds where applicable, and the availability of non-English translations.The search identified 1507 records, ; 29 (48%) of these trials used ALS-specific quality of life measures, 29 (48%) generic and 3 a combination. Four trials used a combination of generic quality of life measures and neuropsychiatric assessments that were not ALS specific. No data on assessment tool used were available for one trial.Seven trials used neuropsychiatric measures which were not developed specifically for people with ALS; ESAS , HamiltTrials evaluating neuropsychiatric symptoms within quality of life measures utilised: Edmonton Symptom Scale (ESS ), ALS-SpCognition was evaluated in 16 trials (7%); within quality of life measures in 5 trials (Sickness Impact Profile/ALS-19 and ALSSQOL-R). Seven trials used the ECAS (Edinburgh Cognitive Assessment Screen ) and onePeople with ALS may lack insight into cognitive and behavioural changes , or down34 trials (14%) evaluated behavioural symptoms in participants. Five of these were within the context of quality of life measures: SIP and ALS-19. Nine were within assessment tools also evaluating cognition, eight trials using measures such as the ECAS and ALS-CBS which are specifically designed for ALS and one trial using the FBI , a non-Pain was evaluated in 55 trials (23%). Assessment was included in the context of quality of life measures  in 46 trials. Frequency of reporting changes in levels of pain are not reported separately when evaluated in quality of life measures.Cramp was specifically addressed in trials using questionnaires or visual analogue scales . Other o13 trials (5.5%) evaluated sleep, 9 of which utilised only quality of life measures. The quality of life measures did not provide a separate score for sleep-related symptoms as the scores were reported as an overall measure of quality of life. The remaining three trials used the symptom-specific Epworth Sleepiness Scale , visual 18 trials (7.6%) evaluated fatigue as an outcome measure. Eight of these trials measured fatigue within quality of life measures and as a result, no separate scores for each non-motor symptom were reported, only the aggregate score for quality of life. One trial utilised the ESAS, a generic tool to document change in patient-reported symptoms. Nine trials evaluated fatigue specifically, utilising visual analogue scales, presence/absence statements, the Krupp Fatigue Severity Scale .182 trials (77%) reported using the ALS-FRS or ALS-FRS-(R), as an outcome measure that evaluates saliva within the context of physical function. Ten of these trials also utilised additional saliva evaluations: the CNS-BFS , a visual analogue scale and the ALSSQOL-R . No disOf the 237 trials included in this study which evaluated non-motor symptoms, 49 versions or combinations assessment tools were used. In this study, we categorised assessment tools as ALS-specific , symptom specific (focussing only on the non-motor symptom under consideration), both (disease and symptoms specific), and generic . Six instruments used were ALS-specific , four were symptom specific, four were both disease and symptom specific, and seven were symptom-generic .Versions of the ALS-FRS-(R) most frequently utilised . A complete list of the assessment tools used, and the frequency that they are included as outcome measures, is available in Table 70 trials (30%) included quality of life assessments as outcome measures. These quality of life measures frequently contained questions on non-motor symptoms such as mood, pain and fatigue. Often these questionnaires include only a single item evaluating the presence of this non-motor symptom, with no additional information on its impact on the individual, severity or change over time , 43.In the trials included in this review, 20% of the times where non-motor symptoms were assessed, this occurred within a quality of life measure, rather than a scale specifically evaluating that symptom. As a result, often no score for the non-motor symptom is reported. Of the 70 trials that used a quality of life assessment as an outcome measure, only 21 included an additional tool to evaluate non-motor symptoms, which was not the ALS-FRS-(R).A range of quality of life (QoL) measures were used as outcome measures in the trials included in this review. ALS-specific measures; Sickness Impact Profile ALS, ALS Assessment Questionnaires, ALS-Specific Quality of Life enable us to evaluate how the candidate drug affects aspects of the individual’s life most likely to be affected by ALS. Disease- and symptom-specific measures are more likely to be sensitive and specific enough to detect changes, crucial in clinical trials.However, findings from drug trials using disease-specific measures are limited in their comparability across neurological conditions. In comparison, more general assessments of quality of life and physical functioning such as the Edmonton Symptom Assessment Scale, Short Form Health Surveys, EuroQol measures, Schedule for Individual QoL and McGill enable researchers to compare findings with existing health-related quality of life and disability data, but at the potential cost of evaluating disease-specific impairment.Neuropsychiatric outcome measure such as the NPI-Q are neither disease-specific nor symptom specific. Whilst useful to capture the potential presence of broad range of disorders, the utility of this measure to detect change over time is limited due to the dichotomous outcome of Yes/No to the presence of disorders. Symptom-specific measures such as the HADS, C-SSRS and HAM-D were used as outcome measures in other included trials, the suitability of these measures for people with ALS is uncertain, due to overlap with somatic symptoms and disease progression. This can be mediated through the use of revised disease-specific thresholds of impairment . The ADIThe ECAS and ALS-CBS are disease-specific measures of cognitive and behavioural symptoms in people with ALS. Designed and validated for use in this population, they focus on the aspects of cognition and behaviour which are most affected in this condition and are sensitive to detecting changes across repeated assessment . The MoCVerbal fluency is a measure of a specific aspect of cognition, often affected in people with ALS, however, using this assessment in isolation may be insufficient to detect the broad range of cognitive function that can be affected by ALS progression.As ALS exists on a disease spectrum with frontotemporal dementia (FTD) , measurePain was primarily evaluated within quality of life measures, both ALS-specific and generic measures. Symptom-specific assessments of pain and cramp prevalence and severity were limited to visual analogue scales and Cramp Questionnaires, which may not be sufficiently objective to detect the nuanced changes occurring within the progression of ALS and the potential impact of a candidate drug.Symptom-specific sleep measures used in the trials in this review are the Epworth Sleepiness Scale and Pittsburgh Sleep Quality Index are beneficial to understand participant perspective and acknowledge that sleep quality is greater than just time spent at rest. However, in ALS, disordered breathing and declining respiratory function can be a significant contributor to the multifactorial issue of sleep. In tools which are not ALS specific, the impact of respiratory symptoms may not be accounted for. In ALS, the Epworth Sleepiness Scale has the additional benefit of indicating the severity of respiratory symptoms.Contribution of other symptoms and evaluation using symptom-specific scales is also of consideration when evaluating fatigue. Using symptom-specific outcome measures such as the Krupp Fatigue Severity Scale, and the FSS, may not reflect the interwoven contributions to the conceptualisation and causes of physical and mental fatigue experienced in ALS.Whilst 184 (78%) trials evaluated saliva, in these trials saliva was assessed in the context of a single sub-domain score of the ALS-FRS(R) or with other bulbar symptoms, alongside swallowing and speech, in the CNS-BFS. Impact of the candidate drug, separate saliva score and change in saliva problems were not reported.ALS-FRS-(R) includes a single item on hypersalivation. CNS-BFS also considers problematic saliva, and the Norris Scale an item on behavioural change. As a result, despite apparent frequent measurement of saliva, as the measurement is within the ALS-FRS-(R), we know little about the potential impact of these candidate drugs on the saliva symptoms which can have a significant affect upon people with ALS .Of the 206 trials which included an assessment of any non-motor symptom, saliva  was the only non-motor symptom assessed in 102 (49%) of these trials. Ultimately, excluding the use of the ALS-FRS(R) as a physical function outcome measure, including the single item on salivation, saliva problems were under-evaluated. Only 80 (44%) of the 182 trials using the ALS-FRS-(R), evaluating saliva, assessed any additional non-motor symptom.As our conceptualisation of ALS broadens from a motor-only disorder to one of multi-system involvement, it is vital that clinical management guidelines and trial design continue to reflect this. Effective symptom management remains a major priority for ALS care, as stated in NICE 2016 care guidelines . ClinicaThis study considers the evaluation of non-motor symptoms as outcome measures in 237 trials proposed to include over 29,222 trial participants with ALS in the last 25 years, since the licensing of riluzole in 1994. The non-motor symptoms included in this review are neuropsychiatric symptoms, cognitive and behavioural changes, pain, disordered sleep, fatigue, and problematic saliva, all of which are prevalent in and impactful in ALS [Effective management and treatment of non-motor symptoms can have a significant impact on the lives of people with ALS and their caregivers, reducing disease burden and improving quality of life . Non-motUnder-evaluation of non-motor symptoms using disease specific measures is a potentially missed opportunity when considering the holistic impact of drug candidates on these troublesome symptoms. In our previous work, we found that neuropsychiatric and cognitive symptoms were consistently under-assessed in ALS trials . Whilst In other neurodegenerative conditions, such as Parkinson’s disease, where non-motor symptoms are common and impactful, disease-specific and symptom-specific scales such as Parkinson’s Disease Fatigue Scale , King’s However, measures designed to evaluate the non-motor features of ALS and other neurodegenerative diseases have not been frequently employed in clinical care, research or trial design . PotentiThis study indicates that non-motor symptoms have not been comprehensively or consistently evaluated within clinical trials of ALS. An improved understanding of the frequency that these symptoms occur, and their contribution to acquired disability, will enable us to provide a more holistic overview of an ALS diagnosis and potential impact of investigative medicinal products. The key strength of this study is that it provides a comprehensive evaluation of ALS trials completed, published or registered since 1994. In addition, we provide a detailed overview and critique of the assessment tools used to explore these non-motor symptoms in the included trials. However, a weakness of the current work is the focus on a limited number of non-motor symptoms in ALS, namely neuropsychiatric, cognitive and behavioural changes, pain, disordered sleep and fatigue and problematic saliva. However, the scope of non-motor symptoms can be extended in future studies to assessment of other symptoms including gastrointestinal issues, dysphagia and sexual dysfunction.We recommend that future clinical trials should include non-motor outcome measures. In addition, more research should focus on the association between these symptomatic outcomes and the potential benefit experienced by trial participants. Whilst it is appropriate that measurement of change in functional decline and improvement in survival remain as primary outcomes in confirmatory trials, we recommend future trials include disease-specific secondary outcome measures to establish the effect of investigative medicinal products on non-motor symptoms to enable a more complete profile of how a candidate drug may affect pwALS. Cognitive assessments are already gradually receiving greater prominence in trial design , reflectEvaluation of the key non-motor symptoms considered in this study was primarily using tools which may not be suitable for people with ALS, or generic assessments of physical function or quality of life where symptom-specific changes were not apparent. These non-motor symptoms should be evaluated with assessment tools which are ALS-specific or validated for use in people with ALS, including disease-specific impairment thresholds where possible.Future work should focus on evaluating the prevalence and impact of each of these non-motor symptoms in people with ALS. This research should also explore the comparison of different assessment tools for each non-motor symptom. Providing recommendations for assessment tools that are suitable to evaluate non-motor symptoms, or the availability of disease-specific impairment thresholds, will be a useful and relevant direction for future work.Supplementary file1 (XLSX 76 kb)Below is the link to the electronic supplementary material.


Obsessive-compulsive disorder (OCD) is among the most disabling neuropsychiatric conditions characterized by the presence of repetitive intrusive thoughts, impulses, or images (obsessions) and/or ritualized mental or physical acts (compulsions). Serotonergic medications, particularly Selective Serotonin Reuptake Inhibitors (SSRIs), are the first-line treatments for patients with OCD. Recently, dysregulation of glutamatergic system has been proposed to be involved in the etiology of OCD. We designed this systematic review and meta-analysis to evaluate clinical efficacy of glutamatergic medications in patients with OCD, according to the guidelines of Cochrane collaboration.We searched Medline, Scopus, and Cochrane library without applying any language filter. Two of the authors independently reviewed search results for irrelevant and duplicate studies and extracted data and assessed methodological quality of the studies. We transformed data into a common rubric and calculated a weighted treatment effect across studies using Review Manager.We found 476 references in 3 databases, and after exclusion of irrelevant and duplicate studies, 17 studies with total number of 759 patients with OCD were included. In the present review we found evidence for several drugs such as memantine, N-acetylcysteine (NAC), Minocycline, L-carnosine and riluzole. Glutamaterigic drug plus SSRIs were superior to SSRI+ Placebo with regard to Y-BOCS scale [standardized mean difference .Augmentation of glutamatergic medications with SSRIs are beneficial in obsessive-compulsive patients, no harmful significant differences in any safety outcome were found between the groups.The online version contains supplementary material available at 10.1186/s40360-021-00534-6. Obsessive-compulsive disorder (OCD) is among the most disabling neuropsychiatric conditions characterized by the presence of repetitive intrusive thoughts, impulses, or images (obsessions) and/or ritualized mental or physical acts (compulsions) . OCD affRecommended first-line pharmacotherapies for OCD are serotonergic antidepressants, such as selective serotonin reuptake inhibitors (SSRIs) and clomipramine , 6. HoweRecently, researchers have proposed that glutamatergic dysfunction, especially in the cortico-striato-thalamo-cortical (CSTC) circuitry may play a key role in the pathophysiology of OCD , 8. GlutThe most direct evidence suggesting altered glutamate homeostasis in OCD derived from cerebrospinal fluid (CSF) studies. These early studies demonstrated that glutamate is excessive in the CSF of a subset of untreated patients with OCD , 14. AddFurther to the aforementioned evidence on glutamatergic dysfunction in OCD, the potential benefits of some glutamate-modulating agents such as riluzole, memantine, N-acetylcysteine (NAC), D-cycloserine, and ketamine have been demonstrated in the treatment of OCD , 19. HowWe conducted this systematic review in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses for Protocols guidelines .In this systematic review and meta-analysis, controlled clinical trials (irrespective of blinding and randomization) investigating clinical efficacy of glutamatergic drugs  in patients with OCD  were included Appendix .The primary search process was conducted in Web of Science, PubMed, Scopus, ScienceDirect, Cochrane library and Google Scholar databases based on the search strategies described in the protocol Appendix  to gatheAfter the initial screening, the full texts were reviewed by two independent researchers to include eligible articles according to the inclusion criteria.Detailed data extraction was performed based on the pre-designed data extraction forms. Extracted information included the study design, name and address of the corresponding author, participants’ characteristics, interventions and outcomes. The methodological quality of the included studies was then evaluated according to the Cochrane risk of bias assessment tool . In caseWe developed an evidence synthesis of the findings of the included studies using systematic approaches such as textual descriptions, tabulation, and transforming data into a common rubric using Review Manager . Missing data were handled using sophisticated statistical analysis techniques.2(defined as I2 > 40% and/or chi-square statistic p < 0.1). A random-effects model was used in the case of statistical heterogeneity. Moreover, we applied a subgroup analysis in case of clinical heterogeneity.A meta-analysis was performed and the weighted average treatment effect was estimated. Heterogeneity across studies were evaluated by the chi-square statistic and calculation of IWe found 476 studies of interest in the initial electronic searches. We then excluded 111 duplicate citations using Endnote software and 204 articles due to obvious irrelevancy of their topics in primary screening . Details of the studies are described in Table Our primary outcome measure was the mean difference in Yale-Brown obsessive-compulsive score (Y-BOCS) ratings before and after pharmacological intervention in experimental and control groups. All except one of the included studies reported Y-BOCS final scores of each group. In the study which these data were not reported, we considered the proportion of treatment responders (as defined by a 35% decline in Y-BOCS scores) in the experimental group compared to the placebo group. A treatment response was significantly more likely in the glutamate-mediating-augmentation group than in the placebo-augmentation group .Four of seventeen studies in this review included memantine as an adjuvant therapy. In three of these studies reporting final Y-BOCS scores, the mean difference of Y-BOCS score between two groups with 95% confidence interval was − 5.68 (P < 0.001) .Riluzole, an anti-glutamatergic agent, is mainly known as a treatment of Amyotrophic Lateral Sclerosis (ALS) . A 10-weNAC is known as a regulator of glutamatergic system and can prevent pre-oxidant effect of glutamate. It has been proposed as a potential therapy for OCD since it can regulate the exchange of glutamate and prevent its pre-oxidant effects . Four stMany glutamate-modulating drugs have been reported to have clinical efficacy in the treatment of OCD. As previously mentioned, riluzole which is an anti-glutamate drug, can be effective in the treatment of refractory OCD. This therapeutic effect can be considered an evidence for the abnormal elevation of glutamate in the CNS of patients with OCD , 41.Based on the findings of this review, memantine has strong evidence supporting its clinical efficacy in the treatment of OCD which is in agreement with previous reviews . AdditioIn summary, there is supporting evidence for glutamate-modulating drugs in treating moderate to severe OCD as an alternative or adjunctive therapy. In the present review we found evidence for several drugs such as memantine, NAC, minocycline, L-carnosine and riluzole. Further research is needed to determine neuroanatomical, neurochemical and basic genetics for the new line of treatments in OCD. The efficacy, effectiveness and risks associated with these glutamate- modulating drugs for the treatment of moderate to severe OCD should be further investigated. In future investigations, it would also be interesting to identify and analyze possible moderator variables.Additional file 1.


Detection of diffraction-limited spots in single-molecule microscopy images is traditionally performed with mathematical operators designed for idealized spots. This process requires manual tuning of parameters that is time-consuming and not always reliable. We have developed deepBlink, a neural network-based method to detect and localize spots automatically. We demonstrate that deepBlink outperforms other state-of-the-art methods across six publicly available datasets containing synthetic and experimental data. While advances in smFISH-based spatial transcriptomics have enabled the quantification of thousands of mRNAs in single cells, these methodologies have focused on the development of transcript barcoding strategies for multiplexing and have relied on conventional threshold-based detection of single mRNA molecules  represent essential steps in various imaging applications including particle tracking in live cell imaging data and quantification of mRNAs in single-molecule fluorescent olecules ,2. Accurolecules  are baseDeep Learning is a machine learning method that uses artificial neural networks to automatically learn features and patterns from raw data. Even though deep learning requires labeled data, once a model is trained, it can be used out of the box without further modifications or parameter adjustments. Recent advances in deep learning have consolidated the convolutional neural network (CNN) as the state-of-the-art for computer vision applications ,5. PreviHere we present deepBlink, a command-line interface to automatically detect and localize diffraction-limited spots. deepBlink exploits neural networks to achieve sub-pixel localization of spots in microscopy images. By benchmarking deepBlink against state-of-the-art methods in spot detection across multiple datasets with different signal-to-noise ratios (SNRs), we show that deepBlink can detect spots more accurately and with higher precision.http://smal.ws/wp/software/synthetic-data-generator/) described in Smal et al.  was defined as the mean Euclidean distance of all true positive coordinates at a cutoff value of 3 px. True positive coordinates are predicted coordinates with an assignment to a ground truth coordinate.https://youtu.be/vlXMg4k79LQ).Here we present deepBlink, a novel neural network-based tool to detect and localize diffraction-limited spots. deepBlink can batch process images with different background brightness levels and spot qualities without relying on manual adjustments of thresholds. deepBlink’s interface provides the ability to detect and localize spots on images using a pre-trained model Figure , step 4.The current default network architecture is a fully convolutional neural network that exploits the U-Net architecture  to encodx, and the y position of a single spot. The performance of the network critically depends on the cell-size  as too small sizes can lead to a class imbalance. Given a fixed number of spots in an image, decreasing the cell-size results in fewer grid-cells containing a spot. This will create an imbalance for the classification part of the loss, favoring background (grid-cells without spots). Images with higher spot densities perform better even at smaller cell-sizes  for the localization  is shown in Equation The basic building blocks of our architecture consist of three convolutional layers followed by a squeeze and excitation layer . We emplTo determine the importance of each component in our network, we performed ablation experiments where we removed individual elements to measure the decrease in performance . This shTo compare the performance of deepBlink to previously published methods, we used a new metric we termed the ‘F1 integral score’ , we used two experimental, manually labeled microscopy datasets . Since TrackMate, however, requires manual adjustment of parameters on an image-by-image basis, the gain in execution speed is offset by the time required to manually set these parameters. DetNet is marginally faster than deepBlink, but as previously mentioned, has markedly worse performance.To determine the effect of spot densities and SNRs on detection performance, we used the official categories from the ISBI particle tracking challenge and divided the Microtubule, Receptor, and Vesicle datasets accordingly (see Methods). In general, both spot density and SNR do not affect the performance of deepBlink except for very low SNRs where no model is able to perform well .https://github.com/BBQuercus/deepBlink/tree/master/examples).deepBlink detects and localizes spots on 2D data, but importantly, one can easily use coordinate outputs for multi-dimensional data analysis. This allows deepBlink’s application in multi-channel colocalization, particle tracking across time, and three-dimensional spot detection. To this end, deepBlink is applied on every channel/frame/slice of a multi-dimensional image separately. Multi-dimensional detection is then achieved by linking identified particles. Example workflows are shown on GitHub  or through the command-line interface. We benchmarked deepBlink on six publicly available datasets that contain both synthetic images as well as smFISH and live cell (SunTag) imaging data, each with a variety of spot sizes, shapes, densities, and signal-to-noise ratios. We show that deepBlink outperforms all benchmark methods, both in detection and localization across these datasets. We expect that our improved detection efficiency and localization precision will also enhance current particle tracking analyses and make image processing more reliable and reproducible.https://figshare.com/projects/deepBlink/88118) together with all trained models used for benchmarking. Further data will be made available from the corresponding authors upon request.All datasets with their training, validation, and test splits are available on Figshare (https://github.com/BBQuercus/deepBlink. The KNIME node is available on KNIME Hub (https://hub.knime.com/bbquercus/spaces/Public/latest/deepBlink).deepBlink’s command-line interface is available on PyPI. Updated versions of the software can also be found on GitHub gkab546_Supplemental_FileClick here for additional data file.


Comparative structural/molecular biology by single-molecule analyses combined with single-cell dissection, mass spectroscopy, and biochemical reconstitution have been powerful tools for elucidating the mechanisms underlying genome DNA folding. All genomes in the three domains of life undergo stepwise folding from DNA to 30–40 nm fibers. Major protein players are histone (Eukarya and Archaea), Alba (Archaea), and HU (Bacteria) for fundamental structural units of the genome. In Euryarchaeota, a major archaeal phylum, either histone or HTa  were found to wrap DNA. This finding divides archaea into two groups: those that use DNA-wrapping as the fundamental step in genome folding and those that do not. Archaeal transcription factor-like protein TrmBL2 has been suggested to be involved in genome folding and repression of horizontally acquired genes, similar to bacterial H-NS protein. Evolutionarily divergent SMC proteins contribute to the establishment of higher-order structures. Recent results are presented, including the use of Hi-C technology to reveal that archaeal SMC proteins are involved in higher-order genome folding, and the use of single-molecule tracking to reveal the detailed functions of bacterial and eukaryotic SMC proteins. Here, we highlight the similarities and differences in the DNA-folding mechanisms in the three domains of life. Genomic DNA must be properly folded inside the cell to simultaneously achieve compaction and relaxation (for transcription and replication) at the same time in all living organisms. Chromosome architectures in the three domains—Eukarya, Bacteria, and Archaea—are different in their protein components, but similar at the nanometer scale at both fundamental and higher-order structure levels .2 tetramer and two H2A/H2B dimers [In eukaryotes, the most fundamental structural unit of chromosomes is the nucleosome. In a nucleosome, approximately 146–147 bp of double-stranded DNA is wrapped around a histone octamer, which is composed of a core H3/H4)/H42 tetrB dimers . The staB dimers ,9.Escherichia coli) encode factor for inversion stimulation (Fis), integration host factor protein (IHF), host factor for phage Qbeta RNA replication (Hfq), histone-like nucleoid structuring protein (H-NS) and its paralog suppressor of T4 Td mutant phenotype A (StpA), and DNA-binding protein from starved cells (Dps) [E. coli and related strains, and Fis is conserved among Enterobacteriaceae [In bacteria, genomic DNA is folded into nucleoids through the aid of nucleoid-associated proteins (NAPs). Heat-unstable nucleoid protein (HU) is a major NAP that is widely distributed among bacteria; at least one HU subunit is present in most bacterial genomes . Other Nls (Dps) . Among teriaceae . These seriaceae .E. coli nucleoids form a hierarchical structure consisting of 10, 30, and 80 nm fibers and 300 nm loops [Structural studies have shown that nm loops . RNase dnm loops . The chlnm loops . Thus, sE. coli protein increases towards the stationary phase, thereby contributing to nucleoid compaction [S. aureus [The structure of bacterial nucleoids changes dynamically during growth phase progression. The expression of Dps of mpaction ,15. The . aureus .Salmonella enterica serovar Typhimurium [Streptococcus pneumoniae [Porphyromonas gingivalis [Helicobacter pylori [NAPs are associated with bacterial pathogenicity. For example, NAPs, including HU, H-NS, Fis, and IHF, affect the expression of virulence genes in himurium . HU is ieumoniae  and is ingivalis . It alsor pylori .Archaea is a domain of life, along with Eukarya and Bacteria . AlthougThermococcales) that binds to the entire genome without sequence specificity implies a link between higher-order chromosome architecture and gene expression [Archaea have a eukaryotic-like basal transcription apparatus, but their gene expression is mainly controlled by bacterial-like transcription factors ,26. Growpression ,28, and pression . Archaea can be divided into two major groups based on the proteins they use to build their chromosomes. The majority of species in the phylum Euryarchaeota encode proteins homologous to eukaryotic histones, while those in Crenarchaeota use other DNA-binding proteins such as Alba and Cren7 . AlthougIn this review, we provide an overview of what we have learned to date about the chromosome structure across the three domains, focusing on the similarities and differences in the principle of their chromosome organization. First, we outline strategies to study chromosome structures. We then try to illustrate the principle of chromosome organization in the three domains.To understand the hierarchical structure of chromosomes and their functions, it is essential to combine different levels of analysis. The structure of chromosomes or nucleoids from the fundamental unit to higher-order structures was uncovered by combining various approaches . These aMicrococcal nuclease (MNase) is both an endonuclease and an exonuclease and is widely used in the analysis of chromatin structure in eukaryotic cells . When itIn contrast to eukaryotic chromosomes, digestion of bacterial nucleoids does not result in the protection of DNA of a specific size, neither does it form a ladder-like pattern of protected DNA sizes  Figure b. This mIn Archaea, MNase digestion has shown that chromosomal DNA of Euryarchaeota is wrapped around DNA-binding proteins (histone or HTa) ,36,37 Fc, whereaT. kodakarensis [Thermococcus gammatorelans cells confirmed the outstanding intracellular abundance of TrmBL2 compared to other normal transcription factors [Mass spectrometry allows for the identification of proteins in a sample. By combining chromosome isolation, protein separation by SDS-PAGE, and mass spectrometry, chromosome-associated proteins can be identified along with their mass ratios. The advantage of this method is that it enables the discovery of novel chromosomal proteins without relying on homology to other known proteins. For example, chromatin purification, followed by SDS-PAGE and MS identified a novel, abundant chromosomal protein, TrmBL2, in karensis . In fact factors . Recentl factors . In euka factors . Thus, MAs a top-down approach, an on-substrate cell lysis method was developed. In this method, cells attached to a substrate (such as a thin cover glass) are treated with a mild detergent to achieve cell lysis. The combination of this on-substrate lysis with atomic force microscopy (AFM) provides a powerful tool for visualizing and analyzing three-dimensional chromosome structures that are exposed or spread out from the cells. Using this method, hierarchical chromosome structures characteristic of eukaryotic cells , bacteriIn vitro chromatin reconstitution is a powerful bottom-up approach. In this method, naked DNA is first mixed with chromosomal protein(s)  at a relatively high salt concentration, and then the salt concentration is gradually reduced to physiological levels by dialysis . The resReconstitution of eukaryotic chromosome structures beyond the level of eukaryotic 30 nm fiber has not been successful to date. This may be due to the fact of their complexity and the need for a real cellular environment. Recently, a more sophisticated in vitro approach has been proposed. It involves assembling chromosomes using deproteinized genomic DNA and purified DNA organizing elements (such as proteins), followed by encapsulation into cell-sized containers using microfluidics . The appPlasmodium species, a causative agent of human malaria, have also been shown by Hi-C analysis [Chromosome conformation capture (3C) technology and its derivatives, such as Hi-C, have enabled the detection of three-dimensional chromosome organization by analyzing the interactions between chromosomal regions inside the cell . In thesanalysis . Single-analysis , althouganalysis ,53. The Hi-C techniques have revealed that, similar to eukaryotic chromosomes, bacterial nucleoids are also segmented into highly self-interacting regions called chromosomal interaction domains (CIDs), and CIDs are further organized into macrodomains . Hi-C haThe three domains clearly diverged in how they organize their chromosome structures. However, there are proteins that are commonly used in more than one domain such as histone, HU, and SMC proteins. In this section, we focus on the roles of individual chromosomal proteins in understanding the principles of chromosome folding.Methanothermus fervidus, a methanogenic archaeon [Thermoplasmata, encode proteins homologous to eukaryotic histones. Species in other phyla, such as Nanoarchaeota, Thaumarchaeota, and Lokiarchaeota, also encode histones [T. gammatolerans suggests that archaeal histones undergo acetylation [Histones are the most fundamental building blocks of eukaryotic chromosomes. In 1990, the first archaeal histone was discovered in archaeon . Later, histones . Archaeahistones . A recentylation . Howevertylation ,59. Studtylation   suggested the presence of structures other than the histone tetramer ” [Since the first proposal of an alternative chromatin structure model specific to archaea , a numbe (AHCP)” . In thisThe structure of the hypernucleosome may dynamically change depending on the state of the cell and the environment, thereby regulating gene expression in response to changes in the environment ,62. AnotInterestingly, although a majority of eukaryotic nucleosomes contain regular histone octamers, several types of non-canonical nucleosomes exist. For example, histone H3 is replaced with the centromere-specific histone CENP-A in eukaryotic centromeres . MoreoveAlba is a 10-kDa DNA/RNA-binding protein found widely in archaea including Euryarchaeota, Crenarchaeota, and newly proposed phyla such as Nanoarchaeota, Korarchaeota, Thaumarchaeota, and Lokiarchaeota . Alba unA comparison of chromosome structures of different archaeal lineages using a combination of in-vitro reconstitution and AFM revealed that Alba-mediated chromosome structures may differ depending on the type of other chromosomal proteins expressed in the cell . For exaAlba superfamily proteins are also found in eukaryotes and have been suggested to be involved in RNA metabolism . It remaE. coli cells has shown that its nucleoid is composed of 30–80 nm structures, and RNase A degraded the size of the 30–80 nm structures into 10 nm fibers, indicating that RNA is involved in the formation of the 30–80 nm higher-order structure, but not in the 10 nm fiber [E. coli strains that lack a single NAP gene indicates that there is no single NAP that is responsible for the formation of the 10 nm fiber. However, it is clear that proteins (NAPs or topoisomerase) are involved in the formation of the 10 nm fiber, because naked DNA appears only after proteinase K treatment of the nucleoid [HU is a major bacterial NAP that bends DNA. AFM analysis of lysed nm fiber . AFM ananucleoid . HU protein is associated with the survival of some bacterial species and regulates a variety of cellular processes, such as growth and SOS response . Single-Thermoplasma genomic DNA and protects about 40 bp of DNA at a minimum [Thermoplasma wraps DNA, forming particles of approximately 6 nm [In the case of archaea, species in Thermoplasmatales are unique in that, although they belong to Euryarchaeota, they lack histones. Instead, they encode HTa, a protein homologous to the bacterial HU . Early s minimum . Althoug minimum , it remaely 6 nm ,37  is fundamental to archaeal and bacterial evolution ,72. Varinarchaea ,72.H-NS is an NAP that functions as a global repressor of transcription in bacteria. It preferentially binds to AT-rich sequences and can bridge two DNA strands together . H-NS caT. kodakarensis competes with archaeal histones for DNA binding [T. kodakarensis [Recently, a model was proposed that the archaeal chromosomal protein TrmBL2 plays a role similar to that of H-NS in suppressing horizontally transferred genes . TrmBL2  binding . Therefokarensis ,77. We pAs discussed above, proteins that serve as transcriptional repressors and genome folding factors also play an important role in suppressing horizontally transferred genes. Are there any such silencer proteins in eukaryotes? The horizontal transfer of genes from bacteria to eukaryotes is known, and several mechanisms have been proposed for how the expression of these genes is initially regulated, but it is not assumed that transcriptional repressors are involved . In factSMC family proteins are essential for higher-order chromosome folding in the three domains of life. In general, SMC proteins are composed of ATPase domains, coiled-coil regions, and a hinge domain a. SMC prE. coli and Smc-ScpAB in Bacillus subtitles are involved in the formation of higher-order nucleoid structures [B. subtilis nucleoids revealed that SMC operates by different patterns of motion compared to other DNA-condensing NAPs such as gyrase and HBsu (an HU family protein) [E. coli MukBEF entraps two distinct DNA strands when bound to the unloader MatP [Bacterial species encode proteins homologous to eukaryotic SMCs. Bacterial SMCs have a domain structure similar to that of eukaryotic condensin and cohesin a, and thructures   ,86. Elecder MatP .Sulfolobus species lack condensin. Recently, coalescin (ClsN), a novel Sulfolobus-encoded SMC protein, was shown to be involved in the organization of chromosomes into a two-domain compartment structure resembling the eukaryotic A/B compartments [The condensin complex is highly conserved across all three domains of life and plays an important role in higher-order chromosome folding  Figure c. Althouartments . ClsN isartments .Haloverax volcanii, an extreme halophilic euryarchaeon, and T. kodakarensis, contains multiple large chromatin loops and self-interacting domains similar to bacteria and eukaryotes. Unlike Sulfolobus species, these euryarchaeal chromosomes are not organized in a way that separates transcriptionally active and inactive compartments [Hi-C analysis of euryarchaeal chromosomes showed slightly different structures. The chromosome of artments . It remaSulfolobus. Investigating this association between fundamental and higher-order chromosome structures in Sulfolobus and comparing it to other lineages of life may provide clues as to how different genome folding mechanisms have evolved.Structurally diverse SMC proteins were found to play similar roles in the higher-order genome folding in the three domains. Interestingly, the absence eukaryotic condensin-like protein and the use of a lineage-specific SMC  coincides with the absence of DNA wrapping at a fundamental level of genome folding in Many studies on chromosome structure have been obtained from studies on eukaryotes and bacteria, and new insights are coming from archaeal chromosome studies. Further studies may contribute to the understanding of the general principles of genome folding across the three domains. Comparative structural/molecular biology using single molecule/cell techniques has been successful and revealed hierarchic structures in all three domains of life. Histone, Alba, and HU are the main proteins that build-up the initial steps for this hierarchy in Eukarya, Archaea, and Bacteria, respectively. At this level, bacterial H-NS and archaeal TrmBL2 are possibly domain-specific global regulatory proteins that also function as suppressors of horizontally acquired genes. SMC proteins, such as condensin (Eukarya), MukBEF (Bacteria), and ClsN (Archaea), are involved in the later stages of the construction of higher-order chromosomal/nucleoid structures. At present, little is known about how intermediate levels of hierarchical genome folding are achieved in these three domains. Further interesting information on genome-folding mechanisms will be obtained from single-molecule and single-cell analyses.


In absence of complete reference genomes, this requires the reconstruction of metagenome assembled genomes (MAGs) from sequencing reads. We present the nf-core/mag pipeline for metagenome assembly, binning and taxonomic classification. It can optionally combine short and long reads to increase assembly continuity and utilize sample-wise group-information for co-assembly and genome binning. The pipeline is easy to install-all dependencies are provided within containers-portable and reproducible. It is written in Nextflow and developed as part of the nf-core initiative for best-practice pipeline development. All codes are hosted on GitHub under the nf-core organization  Shotgun metagenomic approaches enable genomic analyses of all microbes within, for example, environmental or host-associated microbiome communities. Since most bacteria cannot be cultured, isolated and individually sequenced, reference databases for microbial genomes are often incomplete. Thus, one of the main tasks in metagenomic data analysis is to reconstruct the individual genomes directly from the given mixture of metagenomic reads.A typical reference-independent metagenomic workflow consists of preprocessing raw reads, assembly and binning to generate so-called metagenome assembled genomes (MAGs), as well as taxonomic and functional annotation of MAGs. Assemblies based on short reads typically suffer from being highly fragmented. In contrast, long reads can be used to generate continuous assemblies but suffer from high error rates. Hybrid assembly approaches combine the advantages of both short and long reads and thus, can produce continuous and accurate assemblies at reasonable cost . AnotherSeveral pipelines have been developed for the assembly and binning of metagenomes . HoweverHere we introduce nf-core/mag, a Nextflow pipeline for hybrid assembly of metagenomes, binning and taxonomic classification of MAGs. The nf-core/mag pipeline is ideal for standardized, large-scale and high-throughput analysis. It is also versatile and can use mixed sequencing data (hybrid assembly) or single sequencing technology data, and allows the use of group information to perform co-assembly and the computation of co-abundances used for genome binning. The pipeline is part of the nf-core collection of community curated best-practice pipelines .https://nf-co.re/mag/results). Moreover, since the nf-core framework uses DSL2, commonly used processes can be shared across pipelines via nf-core/modules (https://github.com/nf-core/modules). This allows the efficient integration of new analysis tools into the pipeline in the future.nf-core/mag is written in Nextflow, making use of the new DSL2 syntax (see Supplementary Data: Section S2). DSL2 enables a modularized pipeline structure, where each individual process, containing ideally only one tool, is provided as a ‘module’. Additionally, sub-workflows can be integrated. The pipeline strongly benefits from the nf-core framework, which enforces a set of strict best-practice guidelines to ensure high-quality pipeline development and maintenance . For exaAlthough the nf-core framework facilitates reproducibility at several layers, it is further crucial to ensure that the individual tools that are part of the pipeline can be run in a deterministic and reproducible manner. For this purpose, nf-core/mag offers dedicated reproducibility settings, for example, to set a random seed parameter, to fix and report multi-threading parameters as well as to generate and/or save required databases, whose public versions do not always remain accessible (see Supplementary Data: Section S3).https://doi.org/10.5281/zenodo.5155395.To show exemplary results generated with the nf-core/mag pipeline, we simulated metagenomic time series data with CAMISIM . For thiAn overview of the nf-core/mag pipeline is shown in Figure https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) for quality control (QC) on the raw and preprocessed reads. For long Nanopore reads, porechop (https://github.com/rrwick/Porechop) is used for adapter trimming, NanoLyse (https://github.com/rrwick/Filtlong) to remove phage lambda control contamination, and Filtlong (https://github.com/rrwick/Filtlong).The pipeline starts with preprocessing the raw reads. For short Illumina reads, fastp  is used Filtlong  for qualThe preprocessed reads are then de novo assembled using MEGAHIT  or SPAdeFinally, MAGs are taxonomically annotated using GTDB-TK  or CAT/BPreprocessed short reads are classified using Kraken2  or CentrTable We ran nf-core/mag v2.1.0 on the metagenomic time series data simulated with CAMISIM. Figure https://nf-co.re/mag) and community support via a dedicated Slack channel (https://nfcore.slack.com/channels/mag). One important advantage over existing pipelines is that it can utilize sample-wise group information to perform co-assembly and/or to compute co-abundances used for the genome binning step. This can be particularly useful for the analysis of enrichment cultures, of longitudinal datasets—as often generated in clinical studies—or for studies with interest also in lower abundant genomes. Users are enabled to choose the approach most suitable to their specific research question and experimental setup, or even to compare different settings.We implemented nf-core/mag, an easy to install, reproducible and portable pipeline for hybrid assembly, binning and taxonomic classification of metagenomes. It facilitates the analysis of large metagenomic datasets, while allowing the generation of results that can be reproduced by other scientists. It provides comprehensive usage documentation (The pipeline was already successfully applied in microbial studies ,31 and, https://github.com/nf-core/mag and released under the MIT license. The with CAMISIM simulated metagenomic data used to generate the exemplary results is available at https://doi.org/10.5281/zenodo.5155395.nf-core/mag code is hosted on GitHub under the nf-core organization lqac007_Supplemental_FileClick here for additional data file.


The present study aims to assess the success of controlled ovarian stimulation in intrauterine insemination cycles stimulated by recombinant-FSH and Clomiphene citrate for either mono- or bi-follicular development.We assessed 870 infertile patients treated with controlled ovarian stimulation in intrauterine insemination cycles at a university-based infertility clinic between January 2012 and December 2017. We compared the cycles stimulated by clomiphene citrate and recombinant-FSH in two set-ups; mono- and bi-follicular development. The main outcome measure was the clinical pregnancy rate per cycle.p=0.940). Regarding the entire cohort, clinical pregnancy was significantly higher in cases of bi-follicular development when compared to mono-follicular development .The demographic and cycle parameters were similar between the groups, except for endometrial thickness on the day of hCG administration, which was higher in the recombinant-FSH group than the clomiphene citrate group. The overall clinical pregnancy rates in clomiphene citrate and recombinant-FSH groups were 9.8% and 10.3%, respectively (Clomiphene citrate and recombinant-FSH have similar success rates in terms of clinical pregnancy, in either mono-follicular development or bi-follicular development. Clomiphene citrate and recombinant-FSH cycles resulted in comparable rates of bi-follicular development, which significantly increases clinical pregnancy rate. Clomiphene citrate and recombinant-FSH have similar success rates in terms of clinical pregnancy, in either mono-follicular development or bi-follicular development. Controlled ovarian stimulation (COS) combined with intrauterine insemination (IUI) is a common fertility treatment, approved as a first-line treatment option for unexplained infertility, mild male factor infertility and minimal-mild endometriosis . HoweverThe superiority of FSH regarding pregnancy rates seems to be due to increased multifollicular growth. However, there is still a gap in the literature regarding the success of FSH and CC when an equal number of follicles develop. This study aimed to compare the pregnancy rates of COS-IUI cycles utilizing FSH and CC in the case of both mono-follicular and bi-follicular development.In this retrospective cohort study, we assessed infertile patients treated with COS-IUI at a university-based infertility clinic between January 2012 and December 2017. The Institutional Review Board of Ankara University approved the study . The inclusion criteria were females, aged <38 years, failure to conceive for ≥12 months of unprotected sexual intercourse, with at least one patent fallopian tube confirmed by hysterosalpingography (HSG) or laparoscopy, basal FSH levels <12 IU/l. We have also determined the causes of infertility, as female factor, mild male factor and unexplained infertility. Semen samples compatible with the criteria from the World Health Organization (WHO) were determined as normal . We statA baseline ultrasound exam was performed on day 2 or 3 before starting a cycle. Clomiphene citrate  was administered at a 100 mg daily dose for five days, starting from cycle days 3-5. Recombinant FSH  was administered according to body mass index (BMI). Starting rFSH dose was 75 IU/day for women with BMI <25 kg/m2 and 100 IU/day for women with BMI ≥25 kg/m2. rFSH injections were commenced on cycle day 2-4. The first monitoring scan was performed on the 6th day of gonadotrophin stimulation, and repeated after 2 or 3 days, depending on the follicular growth. HCG  10,000 IU was administered when the leading follicle reached ≥18 mm in both groups. A single IUI was performed 36-40 h after hCG injection.Semen samples were taken by a masturbation and collected in sterile containers. Semen samples used for insemination were processed within one hour after ejaculation, using a density gradient centrifugation followed by washing with culture medium. The women had bed rest for 15-20 min after the IUI.th gestational weeks. The primary outcome measurement was the clinical pregnancy rate (CPR).The first study group consisted of patients stimulated by CC and the second study group consisted of patients stimulated by rFSH. The distribution of the type of stimulations with CC and rFSH were not different within years, and approximately 25% and 75% for CC and rFSH, respectively. The success rates of the groups were compared under two separate conditions in cases of mono-follicular development or case of bi-follicular development. Bi-follicular development was defined as two dominant follicles reaching a minimum of 17 mm in diameter. Biochemical pregnancy was defined by positive serum β-hCG levels 2 weeks after IUI. Clinical pregnancy was defined as the presence of a heartbeat at 7-8p-value of <0.05 was considered statistically significant.We analyzed the data using the SPSS Version 21.0 . The samples were tested with the Kolmogorov-Smirnov test to determine the normality of distributions. Continuous variables were compared with the Student’s t-test  and the Mann-Whitney U test (for skewed data). Categorical variables were compared with the Chi-square test. We used the multivariable logistic regression analysis with a model-building strategy to determine independent predictors of clinical pregnancy in IUI cycles. A p=0.003 and p=0.009, respectively). As expected, endometrial thickness on the day of hCG was significantly lower in the CC group than in the rFSH group, due to the anti-estrogenic effect of CC . The etiologies of infertility, basal antral follicle count (AFC) and TMSC were significantly different between the CC and rFSH groups. We did not find any impact of these differences on pregnancy outcomes, although AFC and TMSC were higher in the CC group than in the rFSH group.A total of 1081 womens’ cycles were assessed for eligibility. The cycles stimulated with other than CC or rFSH were excluded, as well as the cycles in which three or more follicles developed . Only fip=0.512). Regarding the entire cohort, the CPR was significantly higher in the case of bi-follicular development when compared to mono-follicular development . There were no significant differences regarding to the number of intermediate follicles between the mono- and the bi-follicular growth groups, although we found higher intermediate follicles in the cycles that had bi-follicular growth . Multiple pregnancy rates were similar between mono- and bi-follicular developments . We only had twin pregnancies in our study, there were no triplets or other multiple pregnancies. We also recorded the number of accompanying intermediate follicles (14-16 mm), and found significant tendencies in favor of the rFSH group. The overall clinical pregnancy rates in the CC and rFSH groups were 11.8% and 12.0%, respectively , IMC (inseminated motile count), type of stimulation, endometrial thickness on the hCG day, number of intermediate follicles and bi-follicular development were analyzed on the logistic regression analysis. Backward stepwise logistic regression was performed and then, bi-follicular development and endometrial thickness on the day of hCG significantly increased the odds of clinical pregnancy . HoweverIn the present study, we identified that the CPR was similar between IUI cycles stimulated by CC or rFSH, in cases of mono- and bi-follicular development, separately. We also determined that bi-follicular development and increment in endometrial thickness increased the likelihood of clinical pregnancy in COS-IUI.p<0.05) (The impact of multi-follicular development on the pregnancy rates in COS-IUI cycles has been investigated for many years and contradictive results have been achieved. p=0.009). Furthermore, we performed logistic regression analyses, which showed bi-follicular development as one of the two significant predictors of success in COS-IUI cycles (OR: ~ 2.5).In a recent study by Female age is one of the most important predictors in COS-IUI cycles, and it has been speculated that female age negatively affected the reproductive outcomes. p=0.02; RR:1.8). The authors explained this incompatibility with the lower dose of gonadotrophins in their study. On the contrary, Several studies have compared CC and FSH in IUI cycles, and most suggested superiority of FSH to CC in terms of CPR . In a muIn general, it has been thought that multi-follicular growth was significantly higher in FSH cycles than in CC cycles. In our previous study, we observed increased multi-follicular growth in the FSH group without a significant impact on CPR (The cost-effectiveness analysis showed controversial results about using gonadotrophins or CC in intrauterine insemination cycles. We showed non-significant higher multiple gestation rates in the bi-follicular development group as similar to the literature. Despite the retrospective nature of the analyses and heterogeneity between the groups in terms of age, antral follicle count, and TMSC, that we recognize as limitations of our study due to unequal groups compromising the rFSH cohort, the systematic exploration of individual parameters, large sample size in each group, and the multivariate approach add credence to our observations. We focused on evaluating the CPR among the study population so we did not observe the LBR. It may be another limitation of our study. Endometrial thickness was significantly higher in the rFSH group than in the CC group. However, this was not reflected on the CPR outcome, possibly due to higher age, lower antral follicle count, and lower TMSC values in the rFSH group.This clinical study found that the development of two preovulatory follicles increased the clinical pregnancy rate compared to mono-follicular development in intrauterine insemination cycles. However, gonadotropin did not have a significantly higher pregnancy rate than clomiphene citrate in both mono- and bi-follicular growth. We identified the importance of two preovulatory follicles development on the clinical pregnancy rate, without increasing the multiple pregnancies.In the present study; bi-follicular development has a significant impact on IUI cycles, and CC and rFSH cycles result in comparable rates of bi-follicular development. In any indication, CC and FSH have similar success rates in terms of CPR, in either mono-follicular development or bi-follicular development due to the compromised rFSH cohort having higher age and longer duration of infertility.