Plasmodium falciparum kelch 13 propeller domain associated with partial artemisinin resistance, which we determined by using Sanger sequencing samples from patients enrolled in therapeutic efficacy studies from 9 sub-Saharan countries during 2014–2018. Of the 2,865 samples successfully sequenced before treatment (day of enrollment) and on the day of treatment failure, 29 (1.0%) samples contained 11 unique nonsynonymous mutations and 83 (2.9%) samples contained 27 unique synonymous mutations. Two samples from Kenya contained the S522C mutation, which has been associated with delayed parasite clearance; however, no samples contained validated or candidate artemisinin-resistance mutations.The spread of drug resistance to antimalarial treatments poses a serious public health risk globally. To combat this risk, molecular surveillance of drug resistance is imperative. We report the prevalence of mutations in the  Plasmodium falciparum parasites, as recommended by the World Health Organization (WHO)  (with focus on the propeller domain region), a molecular marker associated with delayed clearance of parasitemia after therapy with artemisinin monotherapy or an ACT  every 2 years to evaluate antimalarial treatments currently used in a particular region , 2017–141 (Benin), 2018–035 (DRC), 2016–046 (Guinea), 6696.0 (Kenya), 6029.0 , 2016–012a , 2015–073a (Tanzania), and 2016–200 (Zambia). Dried blood spots were collected from TESs conducted in 9 countries in Africa  according to the manufacturer’s instructions. We amplified the propeller domain region from codon positions 389–649 by PCR and Sanger sequenced according to methods previously described . We trimmed and quality filtered forward and reverse sequence reads for each sample  from the 3′ and 5′ ends to remove low-quality bases. We aligned trimmed sequences to the Pfk13 National Center for Biotechnology Information gene reference no. PF3D7_1343700 (https://www.ncbi.nlm.nih.gov/gene/814205) and assessed for SNPs. We only considered SNPs if they had a Phred quality score of >30 and were present in both forward and reverse strands. Mixed infections were detected by using the heterozygous caller plug-in tool in Geneious with a threshold of >30%. A second analyst confirmed all SNP and heterozygous calls by manual technical review. We submitted all Pfk13 sequences with SNPs reported in this study to GenBank (accession nos. MN072940–3042). We used R software version 4.0.1  to generate a map showing the distribution of mutations in the 9 countries  from the 9 countries  contained mutations . Of the Of the 669 day of failure samples, 562 were successfully sequenced; 107 (16.0%) samples failed to amplify, produced poor-quality sequences, or both . A totalPfk13 genetic markers in 9 countries in Africa with endemic malaria. Although clinical resistance to ACTs has yet to be confirmed in Africa  to the presence of P. falciparum histidine-rich protein 2 and 3 deletions (which might affect rapid diagnostic test performance). Generating phenotypic  and genotypic data on the same sample provides an opportunity to identify novel mutations associated with resistance and enables detection of known mutations in samples with well-characterized efficacy outcomes. Because the PARMA network encourages standardization of laboratory methods and data reporting, such explorations might detect trends over time in a single country or produce insightful observations by using data from multiple countries. With the increased use of next-generation sequencing, the PARMA network has embarked on applying these principles of data generation, capacity building, networking, and standardization to this emerging technology (The results described in this article represent the collaborative output of the PARMA network, which originated in 2015 with the objectives of assisting countries in Africa in testing malaria samples from TESs for genetic markers associated with antimalarial resistance and supporting training and capacity building of collaborators in Africa (Plasmodium falciparum kelch13 mutations, 9 countries in Africa, 2014–2018.Additional information on Plasmodium falciparum kelch 13 mutations, 9 countries in Africa, 2014–2018.Reanalysis of sequence data and results in a study of


Epidemiological investigations show that mosaic loss of chromosome Y (LOY) in leukocytes is associated with earlier mortality and morbidity from many diseases in men. LOY is the most common acquired mutation and is associated with aberrant clonal expansion of cells, yet it remains unclear whether this mosaicism exerts a direct physiological effect. We studied DNA and RNA from leukocytes in sorted- and single-cells in vivo and in vitro. DNA analyses of sorted cells showed that men diagnosed with Alzheimer’s disease was primarily affected with LOY in NK cells whereas prostate cancer patients more frequently displayed LOY in CD4 + T cells and granulocytes. Moreover, bulk and single-cell RNA sequencing in leukocytes allowed scoring of LOY from mRNA data and confirmed considerable variation in the rate of LOY across individuals and cell types. LOY-associated transcriptional effect (LATE) was observed in ~ 500 autosomal genes showing dysregulation in leukocytes with LOY. The fraction of LATE genes within specific cell types was substantially larger than the fraction of LATE genes shared between different subsets of leukocytes, suggesting that LOY might have pleiotropic effects. LATE genes are involved in immune functions but also encode proteins with roles in other diverse biological processes. Our findings highlight a surprisingly broad role for chromosome Y, challenging the view of it as a “genetic wasteland”, and support the hypothesis that altered immune function in leukocytes could be a mechanism linking LOY to increased risk for disease.The online version contains supplementary material available at 10.1007/s00018-021-03822-w. For over 50 years it has been noted that chromosome Y is frequently lost in the leukocytes of aging men , 2 repreRecent large-scale population studies have highlighted a heritable component to LOY and begun identifying individual genetic determinants. The largest to date, studying 205,011 men in UK Biobank, identified 156 genetic loci associated with LOY, which were preferentially found near genes involved in cell-cycle regulation, cancer susceptibility, somatic drivers of tumor growth, and cancer therapy targets . The emeTCL1A was dysregulated in CD19 + B-lymphocytes missing the Y chromosome, providing a proof-of-concept that LOY may not be functionally neutral in these cells  uWe used R (version 3.5.2) and the edgeR (version 3.24.3) library to compare the expression between LCLs with the Y chromosome and LCLs with LOY in two steps. As all the clones with LOY were derived from the same individual , we designed the analysis to remove inter- and intra-individual differential expression effects. As step one, to identify the intra-individual differences, we targeted the differential expression genes among the normal LCLs by comparing each non-LOY individual’s clones with all other non-LOY clones. The union of all differentially expressed genes resulted out of each comparison was gathered into List1 . This gave us a set of genes that are differentially expressed between subjects but not due to LOY status. As step two, we targeted the differentially expressed genes regarding LOY status in two steps: first by comparing the non-LOY versus LOY clones derived from the same individual; second by comparing all non-LOY versus LOY clones and the overlap of the results of steps one and two was gathered into List2. The final list of LATE genes was generated by comparing List2 and List1 and extracting the differentially expressed genes that exist in List2 but not in List1, to remove the intra-individual differentially expressed genes from the LATE genes.6 cells/ml.Blood was collected using BD Vacutainer CPT tubes (BD Biosciences). PBMCs were isolated using density gradient centrifugation and resuspended in 0.04% BSA 1X PBS solution. Concentrations were measured using an EVE cell counter  and cells were diluted to 10The collected PBMCs were loaded on a 10X Genomics Chromium Single Cell 3′ Chip v2 for sequence library generation using protocol CG00052 (10X Genomics) using a 3′-end protocol. The single cell libraries were sequenced on Illumina HiSeq2500 and NovaSeq instruments The single cell library preparation and sequencing were performed at the Science for Life technology platform SNP&SEQ, Uppsala University, Sweden.Sequence reads were mapped to the hg19 version of the human genome using CellRanger v2 (10X Genomics) and recommended settings. The produced raw reads matrices were analyzed using R v3.4) and the R library Seurat (v2.2) . For eac and the RPS4Y1, ZFY, USP9Y, DDX3Y, KDM5D, and EIF1AY). Next we identified the degree of co-expression between these six MSY genes and autosomal genes using the default settings in the SEEK database (http://seek.princeton.edu/). The applied algorithm identifies genes showing similar expression patterns across human samples in a large number of databases containing gene expression data from numerous sources [We investigated if changes in expression as an effect of LOY would be larger in autosomal genes that are normally co-expressed with MSY genes, compared with genes that are not co-expressed with MSY genes. We first identified six MSY genes that showed expression in normal leukocytes  in the single cell data set  using the R library bioMart (v2.38). The list was filtered for protein coding genes and annotated as either positioned in the male-specific part of the Y chromosome (MSY) or in the pseudo-autosomal regions (PAR). The GO categories for each gene were inspected manually and genes were assigned into four different categories: “https://www.ncbi.nlm.nih.gov/grc/human/data (assembly GRCh37).Over-representation of known biological connections between genes was calculated using the STRING web tool . To evalTo test the hypothesis that LATE genes might be preferentially located near LOY-associated variants highlighted by GWAS  we perfoThe 489 autosomal LATE genes were compared to the background of 11,884 tested genes. Gene symbols were mapped to Entrez Gene IDs using the org.Hs.eg.db Bioconductor package  resultinhttp://www.rproject.org/), using two-sided tests and α-levels at 0.05 and default parameters, unless otherwise specified. When appropriate, correction of p values for multiple testing was applied. To define the LATE genes, we applied three stringency levels. The first was based on standard p value at α-level 0.05 and the second was a more stringent p value correcting for multiple testing (FDR < 0.1) using Benjamini and Hochberg (BH) correction. Furthermore, an additional criterion was applied to identify only the highly expressed LATE genes, i.e., using threshold of at least an average of 100 reads per gene in RNAseq data.Statistical analyses such as logistic regression, linear regression, Fisher exact test, Kolmogorov–Smirnov test, and Wilcoxon rank sum test were performed using R version 3.3.1 .We observed a high concordance in LOY estimates generated from pairwise samples studied in vivo with scRNAseq, RNAseq, and DNA based technologies . Furthermore, we studied in vitro changes of gene expression using RNAseq in 13 lymphoblastoid cell lines (LCLs) with or without LOY . Concordant levels of LOY were observed in LCLs studied with SNP-array as well as with a new method for LOY analysis  using drLOY Associated Transcriptional Effects (LATE), we tested for differential gene expression between the LOY and non-LOY cellular populations  and 2.5 lower  abundance of transcripts, respectively. A considerable number of genes were independently identified as showing LATE in different cell types and by both technologies (Supplementary Table 2). Co-expression analysis showed that autosomal genes that are normally co-expressed with MSY genes displayed a higher level of differential expression in single cells with LOY compared with control genes .Outside of the Y chromosome, we found evidence for 489 autosomal LATE genes and 10 on the non-PAR X chromosome across the two expression datasets and all types of leukocytes, from in vivo studied samples . The level and direction of differential expression of LATE genes detected independently by both technologies are illustrated in Fig. LY6E gene located on chromosome 8 and downregulation of the PAR gene CD99.In the monocytes and NK cells that were studied by two RNA sequencing methods, the identified LATE genes showed an overall concordance in the direction of change in gene expression between technologies  and in the scRNAseq dataset . For instance, up to 15% of normally expressed genes showed LATE in NK cells, but only about 2% of LATE genes were shared between NK cells and granulocytes or monocytes . This result emphasizes that transcriptomic alterations associated with LOY vary between different types of immune cells, likely because each cell type normally express a set of genes that is only partially overlapping with the gene expression of other cell types.Furthermore, we observed that the fraction of LATE genes within specific cell types was substantially larger than the fraction of LATE genes shared between different subsets of cells, in both the RNAseq dataset (ANOVA: p < 1 × 10–16) biological connections amongst each other than expected by chance for a matched randomly selected set of genes. Furthermore, LATE genes were not randomly distributed throughout the genome, with clusters of genes evident . This was most pronounced in the 11q13.1 region, where we identified 17 LATE genes (13 over-expressed in LOY) in an 8 Mb genomic window (Supplementary Table 1). To explore the potential biological relevance of the identified genes, we next performed pathway enrichment analyses, controlling for the background set of all genes expressed in our tested cell types. This resulted in the identification of a large amount of significantly over-represented (FDR < 0.05) GO categories and pathways, the majority of which focused on aspects of RNA processing, immune function and the viral life cycle. Specifically, 191 pathways and GO categories was identified using the DAVID functional annotation tools and 104 was identified using the Panther classification system (Supplementary Table 3). A summary of our literature review for detected LATE genes is presented in Supplementary Table 4.The 489 autosomal LATE genes had twice as many (p = 0.010), with 41 LATE genes mapping within 300 kb of a genome-wide significant SNP association in 25 genomic regions. In several instances, the proximal LATE gene was the same as that prioritized through gene mapping approaches in the GWAS study. For example, LATE gene cyclin-dependent kinase inhibitor 1C (CDKN1C) was linked via expression changes to a nearby LOY-associated variant (rs60808706). Here, the LOY risk increasing allele increases expression of CDKN1C and the expression of CDKN1C is increased in cells with LOY. As with our previously reported example of TCL1A [CDKN1C—genetically increased expression of the gene promotes LOY and LOY subsequently further dysregulates gene expression in a cell-specific manner, possibly leading to a negative spiral of increased clonal mosaicism via perturbed cell-cycle processes.We next tested whether LATE genes were preferentially located near genetic variants associated with LOY . Gene seN = 121) or prostate cancer (N = 107) vs. healthy controls (N = 156). Overall, there was a general trend for LOY to be increased in patients vs. controls, and this rate varied considerably between cell types . The fraction of Alzheimer’s disease cases with high levels of LOY in NK cells was about four times larger compared with controls and supports previous observations that LOY is associated with incident Alzheimer’s disease [p = 0.033 and p = 0.031, respectively). Although not proving direct evidence of causality, the observation that prostate cancer and Alzheimer’s disease patients were primarily affected with LOY in different types of immune cells would support a disease-specific link. This suggests that associations between LOY and disease cannot solely be attributed to a confounding age-dependent process of genomic instability, which would be expected to equally affect different types of leukocytes. Our results are thus compatible with both hypotheses presented in the introduction regarding aetiology of LOY and disease; accumulation of LOY in leukocytes as a barometer of genomic instability in somatic cells, as well as increasing risk for different types of disease when specific types of immune cells are affected with LOY.Finally, we investigated the distribution of LOY in six types of sorted leukocytes—CD19 + B lymphocytes, CD4 + T lymphocytes, CD8 + T-cytotoxic lymphocytes, Natural Killer (NK) cells, granulocytes, and monocytes—among men diagnosed with Alzheimer’s disease , suggests that LOY might have pleiotropic effects. Pleiotropy is defined as an effect of one feature at the genetic level to multiple characteristics at the phenotypic level . ConsideLAG3 .Second, our study highlights altered immune cell function that could conceivably link LOY in immune cells directly to disease mechanism(s). The combined number of LATE genes derived from analyses of various cells is surprisingly large, which suggests that a loss of ~ 2% of the male haploid genome (via LOY) could have an impact on cellular homeostasis. Enrichment analyses showed that LATE genes are important for a range of physiological functions (Supplementary Table 3) including many normal functions of the immune system. One of the LATE genes that showed the strongest downregulation was the autosomal gene activity , 51. TheLY6E and CD99 showed LATE by both RNAseq and scRNAseq in NK cells and monocytes , interleukin-1β (IL1B), and interleukin 1 receptor antagonist (IL1Ra), preventing them from binding to other receptors [IL1R2 in LOY cells could reduce the inhibiting effect of this receptor.Furthermore, the genes tes Fig. . The prounctions , 53. A lgnancies  and it hgnancies . Among aeceptors . Hence, GABRR1, PRLR, and LEP. GABRR1 encodes a receptor for Gamma Aminobutyric Acid (GABA), the major inhibitory neurotransmitter in the brain. A recent study found these receptors were functionally expressed in human and mouse hematopoietic stem cells and megakaryocyte progenitors [GABRR1 in these cells led to increased rates of megakaryocyte and platelet differentiation. In contrast, we observed 1.26-fold (p = 7.9 × 10–5) increased expression of the receptor in NK cells lacking the Y chromosome, extending the biological role of this receptor in immune cell function. Sex hormone receptors have also been described to have functional effects on hematopoietic stem/progenitor cells [PRLR), which we see over-expressed in monocytes with LOY (Supplementary Table 2). We also observed over-expression of the hunger inhibiting hormone Leptin (LEP) in granulocytes with LOY, consistent with studies describing its lesser known roles in regulating innate immune response [Additionally, several of the LATE genes we identified had diverse and well-described roles in other biological processes, notably genitors . Overexpor cells  includinresponse .As mentioned in the introduction, LOY is the most common post-zygotic mutation from analyses of bulk DNA from peripheral blood. The current study adds new information on this as all 29 men  studied by scRNAseq carried leukocytes without chromosome Y. Thus, LOY is more common than so far appreciated and the future analyses of single- or sorted-cells from various tissues will be instrumental for establishment of true frequency of this mutation as well as for identification of cell subtypes most often associated with disease. Lastly, we describe that LOY is associated with dysregulation of ~ 500 autosomal genes, for example, involved in immune functions but also with roles in other biological processes. The present study provide insights into the molecular role of the Y chromosome and bring forth clues on how its mosaic loss in leukocytes could be associated with increased risk for disease in affected men, which ought to stimulate further research.Supplementary file1 (PDF 2535 KB)Supplementary file2 (XLSX 141 KB)Supplementary file3 (XLSX 469 KB)Supplementary file4 (XLSX 81 KB)Supplementary file5 (XLSX 76 KB)Below is the link to the electronic supplementary material.


Women with uterine adenomyosis seeking assisted reproduction have been associated with compromised endometrial receptivity to embryo implantation. To understand the mechanisms involved in this process, we aimed to compare endometrial transcriptome profiles during the window of implantation (WOI) between women with and without adenomyosis.We obtained endometrial biopsies LH-timed to the WOI from women with sonographic features of adenomyosis (n=10) and controls (n=10). Isolated RNA samples were subjected to RNA sequencing (RNA-seq) by the Illumina NovaSeq 6000 platform and endometrial receptivity classification with a molecular tool for menstrual cycle phase dating . The program language R and Bioconductor packages were applied to analyse RNA-seq data in the setting of the result of accurate endometrial dating. To suggest robust candidate pathways, the identified differentially expressed genes (DEGs) associated with the adenomyosis group in the receptive phase were further integrated with 151, 173 and 42 extracted genes from published studies that were related to endometrial receptivity in healthy uterus, endometriosis and adenomyosis, respectively. Enrichment analyses were performed using Cytoscape ClueGO and CluePedia apps.Out of 20 endometrial samples, 2 were dated to the early receptive phase, 13 to the receptive phase and 5 to the late receptive phase. Comparison of the transcriptomics data from all 20 samples provided 909 DEGs  in the adenomyosis group but only 4 enriched pathways . The analysis of 13 samples only dated to the receptive phase provided suggestive 382 DEGs  in the adenomyosis group, leading to 33 enriched pathways . These included pathways were already associated with endometrial biology, such as “Expression of interferon (IFN)-induced genes” and “Response to IFN-alpha”. Data integration revealed pathways indicating a unique effect of adenomyosis on endometrial molecular organization  and its interference with endometrial receptivity establishment .Accurate endometrial dating and RNA-seq analysis resulted in the identification of altered response to IFN signalling as the most promising candidate of impaired uterine receptivity in adenomyosis.The online version contains supplementary material available at 10.1186/s12958-021-00871-5. Adenomyosis has been associated with lower pregnancy rate in infertility treatments.Molecular knowledge of endometrial receptivity in women with adenomyosis is scarce and limited to studies with selected candidate genes and one genome-wide study performed using microarrays.Therefore, we performed the first transcriptome sequencing of endometrial samples LH-timed to the expected WOI (LH+7 – LH+9) between women with (n = 10) and without (n = 10) sonographic features of adenomyosis.Transcriptomics data comparison of 10 adenomyosis cases and 10 normal controls provided 909 DEGs , but functional enrichment analysis identified 4 pathways  that were not directly associated with endometrial biology.Retrieved endometrial biopsies were applied for the external molecular tool beREADY®  to verify their receptivity status on the basis of the gene expression signature associated with endometrial receptivity. Out of 20 samples, 2 were classified as early receptive, 13 as receptive and 5 as late receptive.Two early- and 5 late-receptive samples were excluded from the RNA-seq dataset to prevent the impact of early- and late-secretory phases of the menstrual cycle on transcriptomics analysis associated with endometrial receptivity. The RNA-seq dataset of the remaining 8 adenomyosis cases and 5 control receptive endometrial samples was reanalysed, and 382 DEGs  were identified, resulting in 33 enriched pathways  that have already been associated with endometrial biology.The 382 identified DEGs were further integrated with the most extensive set of genes from the literature associated with endometrial receptivity in the healthy uterus, endometriosis  and adenomyosis to provide candidate pathways characterizing the role of adenomyosis on endometrial molecular organization.Integrative enrichment analysis provided candidate pathways that may indicate a unique effect of adenomyosis on endometrial molecular organization  and its interference with endometrial receptivity establishment .Identification of robust endometrial pathways and associated genes could lead to the development of molecular tools for endometrial receptivity examination that would be specific for women with adenomyosis.Accurate endometrial receptivity examination in infertile adenomyosis patients could better verify whether endometrial-associated factors are a source of recurrent implantation failures.Adenomyosis is a common acquired uterine anomaly characterized by the presence of endometrial glands and stroma within the myometrium. Advances in imaging techniques in the last decade have enabled the diagnosis of adenomyosis  in a larSeveral functional and molecular aberrations could be responsible for altered endometrial receptivity to embryo implantation and lower fecundity in women with adenomyosis. It has been suggested that the disruption of the junctional zone architecture by adenomyosis could lead to altered contractility and interrupt endometrial receptivity , 8. OtheLIF, SOCS3, IL10, IL6, JUNB and FOS genes were validated. Since downregulated expression levels of selected genes in adenomyosis compared to the control group showed no statistical significance, we assumed that comprehensive endometrial transcriptomics profiling would be an appropriate next step to identify adenomyosis-specific loci [In our previous study , we gathfic loci .To date, there is only one transcriptomics study  profilinThe first aim of this study was to perform RNA-seq of endometrial samples dated to the WOI between women with and without sonographic features of adenomyosis to identify DEGs. The second aim was to perform enrichment analysis of identified DEGs alone and together with endometrial receptivity genes from the literature to provide robust candidate pathways related to altered molecular background of endometrial receptivity in adenomyosis.We designed a prospective observational study including women scheduled for medically assisted reproduction at the Department of Reproductive Medicine and Gynaecological Endocrinology, University Medical Centre Maribor, Slovenia between 2018 and 2020.The inclusion criteria were as follows: age ≤ 42 years, regular menstrual cycle 24 – 36 days in length, no current hormonal treatment, controlled ovarian stimulation (COS), ovulation triggering or vaginal progesterone for luteal support at least two months prior to endometrial biopsy. The exclusion criteria were anovulatory menstrual cycles, polycystic ovary syndrome (PCOS), previous surgical treatment of endometriosis or uterine surgical procedures, sonographic evidence of fibroids, endometrial polyps, hydrosalpinges, and evidence of ovarian or deep infiltrating endometriosis . In all women, comprehensive 2-D and 3-D ultrasound using high-range equipment was performed with a 10 MHz transvaginal transducer . Diagnostic criteria for adenomyosis were based on previously published criteria . The diaDemographic and clinical characteristics of participants, including age, body mass index (BMI), endometrial thickness at the time of endometrial biopsy and the number of previous ART cycles, are presented as the median (range) and were compared between study groups using the nonparametric Mann–Whitney U-test in SPSS 25.0 software . Statistical significance was set at p value < 0.05.Endometrial biopsy sampling was conducted in a natural menstrual cycle, and women were scheduled for cycle monitoring by urinary luteinizing hormone (LH) tests . Women were scheduled for endometrial sampling conducted by the Pipelle endometrial suction curette  in the expected WOI on the day between LH+7 to LH+9 after a participant’s LH surge determination (day LH+0). Retrieved endometrial samples were immediately placed in RNAlater solution , stored overnight at +4 °C and then transferred to –80 °C until RNA isolation was performed.2O.Total RNA was isolated using the miRNeasy Mini Kit  according to the manufacturer’s instructions. Each whole-tissue endometrial sample was first disrupted with a Bullet Blender Storm Pro homogenizer  using 1 mm zirconium oxide beads in 700 µL of QIAzol Lysis Reagent from the miRNeasy Mini Kit. After 5 min of incubation at room temperature, 140 µL of chloroform was added to the homogenate, and the solution was shaken vigorously. The sample was then centrifuged at 12 000 rfc for 15 min at 4 °C. The upper aqueous phase (approximately 300 µL) was transferred to a new Eppendorf tube, and 1.5 volumes of ethanol were added. The samples were then pipetted to RNA binding miRNeasy Mini spin columns and washed using RWT Buffer and RPE Buffer solutions of the miRNeasy Mini Kit. Total RNA was eluted in 50 µL of RNase-free HThe quantity and purity of each RNA sample were assessed with Synergy 2 spectrophotometric measurements . RNA integrity number (RIN) was estimated on the 2100 Bioanalyser system  using the RNA Nano 6000 Assay Kit . After passing those quality controls, each RNA sample was used for cDNA library construction and subsequent RNA-seq and for accurate endometrial dating of retrieved biopsies.https://beready.ccht.ee/). Endometrial dating was performed according to the established protocol using targeted allele counting by sequencing (TAC-seq) methodology [One part of each RNA sample was shipped on dry ice to the Competence Centre on Health Technologies, CCHT, Tartu, Estonia, where endometrial receptivity testing was performed using the beREADY® test  . Briefly, a total amount of 2 µg of RNA per sample was used for cDNA sequencing library preparation. Ribosomal RNA (rRNA) was removed using the Epicentre Ribo-zeroTM rRNA Removal Kit , and the remaining RNA was used for library generation by the NEBNext® UltraTM Directional RNA Library Prep Kit for Illumina® . First, rRNA-depleted RNA samples were fragmented followed by first- and second-strand cDNA synthesis. The sequencing adaptors were ligated, and library fragments were purified to obtain cDNA fragments 150~200 bp in length. Polymerase chain reaction (PCR) amplification of size-selected, adaptor-ligated cDNA was performed using universal PCR primers and index primers. Index-coded samples were clustered by Illumina TruSeq PE Cluster Kit v3-cBot-Hs. Libraries were sequenced on an Illumina NovaSeq 6000 platform, which generated 150 bp paired-end reads.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) to obtain a quality profile of the reads.Raw sequence reads were trimmed by Novogene in-house Perlscript to remove raw reads with adapter contamination and reads containing poly-N and low-quality reads. The RNA-seq data presented in this study are deposited in the Gene Expression Omnibus (GEO) database with accession number GSE185392. Provided raw fastq files were first evaluated with FastQC v.0.11.9 software (http://www.bioconductor.org/) were used for high-throughput sequence data analysis. Raw paired-end reads were aligned to the UCSC Homo sapiens hg19 reference genome using the Rsubread v.2.2.4 R package [The statistical environment R v.4.0.2  and contributed packages from the R software repository Bioconductor  that were identified by transcriptomics data comparison of endometrial samples between adenomyosis cases and controls were subjected to functional enrichment analyses using ClueGO v.2.5.8  and ClueWhen analysing identified sets of DEGs associated with the present adenomyosis groups, up- and downregulated genes were separately uploaded as two clusters in the ClueGO app, which gave a unique colour marker to each gene set. When performing enrichment analyses of integrated gene lists associated with different gynaecological conditions, each gene list was uploaded as a cluster in the ClueGO app to distinguish study groups according to colour markers of the cluster.Each enrichment analysis was applied by representative Gene Ontology Biological Process (GO_BP), Reactome Pathways and Reactome Reactions ontologies. Only enriched pathways (Reactome pathways/reactions and GO_BP terms) with corrected p values < 0.05 according to the Bonferroni step down test were considered. The identified pathways were sorted into groups based on their common biological role and associated genes (kappa score) and further projected into functionally organized networks. The size of nodes in the generated networks was correlated with the obtained p value. The pathway with the highest significant value was considered to be the leading term of a group and was therefore highlighted in the network by a large name label and a statistical summary. The CluePedia app was further applied to visualize shared initial genes within or between functional network groups. The proportion of visible genes mapped to each pathway was also determined. When more than 60% of mapped genes originated from one of the clusters, a pathway was shown in the network with the predefined colour of this cluster.An overview of the study is outlined in Fig. The demographic and clinical characteristics of the study cohorts are summarized in Table Total RNA was isolated from 20 endometrial samples, 10 from the adenomyosis group and 10 from the control group. The A260/A280 ratios and RIN values of all RNA samples were above 2.0 and >8.5, respectively, and were further used for endometrial dating and RNA-seq.The results of endometrial receptivity testing performed on each endometrial RNA sample are provided in Table The timing of endometrial biopsy and measured receptivity status are provided for each sample, followed by a summary of mapped RNA-seq reads and library size after filtering for low gene expression. Abbreviations “A” refer to adenomyosis and “K” to control samples. Timing of biopsy refers to the day after luteinizing hormone (LH) peak determination (LH+0) by urinary LH test.RNA-seq of endometrial samples utilizing the Illumina NovaSeq 6000 platform generated between 53,026,608 and 75,561,205 reads per sample, with an average of 64,266,576 reads. Quality analysis of raw RNA-seq reads by FastQC revealed that each fastq file contained reads 150 base pairs (bp) in length with a mean per base sequence quality score (Phred score) of 36 and thus each file was considered for downstream bioinformatics analysis. Table Differential expression analyses were conducted using two RNA-seq datasets constructed of samples according to the results of endometrial receptivity testing. The first RNA-seq dataset was composed of all 20 samples: 13 receptive, 2 early- and 5 late-receptive samples. The second RNA-seq dataset was composed of 13 receptive samples only, while 2 early- and 5 late-receptive samples were omitted to exclude the influence of early- and late-secretory phases of the menstrual cycle on endometrial transcriptomic analysis associated with endometrial receptivity.https://www.genenames.org/), different locus types were identified, including 829 protein-coding genes (mRNAs), 27 long noncoding RNAs (lncRNAs), 5 microRNAs (miRNAs), 5 small nucleolar RNAs, 28 pseudogenes, 1 complex locus constituent and 14 loci that were not mapped in the HGNC database. Among 909 DEGs, 487 genes  were upregulated, and 422 genes  were downregulated. However, the fold change (FC) of expression levels between study groups was nonsignificant after the application of multiple comparison correction.Transciptomics data comparison of 10 adenomyosis and 10 control samples resulted in 909 DEGs (p<0.05) associated with the adenomyosis group  associated with the adenomyosis group  and “Icosanoid receptor activity” . Sorted pathways with associated genes in networks are presented in Fig. Functional enrichment analysis of 909 DEGs associated with the adenomyosis group that were obtained from transcriptomics comparison of all 20 endometrial samples provided only 4 enriched GO terms sorted within 2 functionally organized network groups: “Intracellular lipid transport” , “Response to interferon-alpha” , “ISG15-protein conjugation”  and “Homophilic cell adhesion via plasma membrane adhesion molecules” . Upregulated genes were mapped in the specific network group “Cysteine metabolic process” . Nonspecific network groups characterized by equal proportions of mapped up- and downregulated genes were “Diseases associated with O-glycosylation of proteins”  and “Retina homeostasis” . The 33 identified enriched pathways are summarized in Additional file Functional enrichment analysis of 382 DEGs associated with the adenomyosis group that were obtained by transcriptomics analysis of endometrial samples in the receptive phase resulted in 33 enriched pathways, including 20 GO_BP terms, 6 Reactome pathways and 7 Reactome reactions. They were sorted into 7 network groups to remove redundancy, which is visualized in Fig. Only a set of 382 DEGs associated with the adenomyosis group that were identified by transcriptomics data comparison of adenomyosis case and control samples dated to the receptive phase were used for integrative enrichment analyses with endometrial receptivity genes from the literature.Integration of lists with 382, 151 and 173 genes associated with adenomyosis, healthy uterus and endometriosis, respectively, provided 40 enriched pathways sorted in 11 network groups, which are presented in Fig. Integration of lists with 424 (382 DEGs of the present sequencing experiment and 42 genes from the literature), 151 and 173 genes associated with adenomyosis, healthy uterus and endometriosis, respectively, provided 57 enriched pathways sorted in 18 network groups, which are presented in Fig. Altered endometrial molecular mechanisms obstructing successful embryo implantation in women with adenomyosis are poorly understood. The focus of the present transcriptomics analysis was to apply two novel molecular approaches to identify gene expression differences in LH-timed endometrial samples between women with and without adenomyosis: genome-wide profiling using RNA-seq and accurate classification of endometrial receptivity as assessed by the molecular tool beREADY®, measured from the same biopsy. Lists of DEGs associated with the adenomyosis group that were identified by analysing RNA-seq datasets in the setting of the endometrial dating results were applied for enrichment pathway analyses to predict their role in the context of endometrial molecular organization. In addition, a set of 382 DEGs obtained after transcriptomics data comparison of confirmed receptive samples was used for further bioinformatics analysis. They were integrated with the most extensive set of genes from the literature associated with endometrial receptivity in healthy uterus, endometriosis  and adenomyosis to predict mechanisms in which adenomyosis mediates an effect on endometrial receptivity.Recently, Devesa-Peiro et al.  comparedEnrichment analysis using 382 DEGs also provided a higher number of pathways tightly sorted in connected network groups compared to analysis of 909 DEGs, which were also more meaningful to relate with endometrial molecular biology (Fig. The 382 Identified DEGs were also applied for the integration approach to repeat our previous enrichment pathway analysis  orientedA limitation of our study is the relatively small sample size, which prevents definitive conclusions regarding the impact of adenomyosis on the endometrial transcriptome . The resIn this study, we focused on the molecular background of infertility-related adenomyosis based on our research and the available literature. We applied accurate endometrial receptivity classification of retrieved endometrial samples LH-timed to the expected WOI to avoid menstrual cycle bias in downstream transcriptomics analysis. The 382 DEGs identified in the adenomyosis group using the RNA-seq dataset of only confirmed receptive endometrial samples resulted in 33 enriched pathways further projected in the network from which “Expression of IFN-induced genes”, “Response to interferon-alpha” and “ISG15-protein conjugation” were highlighted as connected processes. Additional integration of 382 DEGs with candidate genes associated with endometrial receptivity in healthy uterus, endometriosis and adenomyosis based on a literature review revealed that cytokine signalling impairments in endometrial pathologies could interfere with mechanisms of endometrial receptivity. According to our results, an altered response to IFN signalling is suggested as a candidate mechanism of impaired uterine receptivity in adenomyosis that needs to be further studied in a larger sample size.Additional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.Additional file 6.Additional file 7.


Background: Delayed cerebral ischemia and cerebral vasospasm remain the leading causes of poor outcome in survivors of aneurysmal subarachnoid hemorrhage. Refractory cerebral vasospasms can be treated with endovascular vasodilator therapy, which can either be performed in conscious sedation or general anesthesia. The aim of this study is to compare the effect of the anesthesia modality on long-term clinical outcomes in patients undergoing endovascular vasodilator therapy due to cerebral vasospasm and hypoperfusion.Methods: Modified Rankin Scale (mRS) scores were retrospectively analyzed at time of discharge from the hospital and six months after aneurysmal subarachnoid hemorrhage. Additionally, National Institutes of Health Stroke Scale (NIHSS) was assessed 24 hours before, immediately before, immediately after, and 24 hours after endovascular vasodilator therapy, and at discharge and six months. Interventional parameters such as duration of intervention, choice and dosage of vasodilator and number of arteries treated were also recorded.Results: A total of 98 patients were included in this analysis and separated into patients who had interventions in conscious sedation, general anesthesia and a mix of both. Neither mRS at discharge nor at six months showed a significant difference for functionally independent outcomes (mRS 0-2) between groups. NIHSS before endovascular vasodilator therapy was significantly higher in patients receiving interventions in general anesthesia but did not differ anymore between groups six months after the initial bleed.Conclusion: This study did not observe a difference in outcome whether patients underwent endovascular vasodilator therapy in general anesthesia or conscious sedation for refractory cerebral vasospasms. Hence, the choice should be made for each patient individually. Commonly used rescue treatments for symptomatic CVS include induced hypertension, and in refractory CVS angioplasty or intra-arterial application of vasodilators, e.g. nimodipine or papaverine.4 Both have been shown in case series to improve neurological outcome in said patients.Cerebral vasospasms (CVS) and delayed cerebral ischemia still remain among the leading causes of morbidity and mortality in survivors of aneurysmal subarachnoid hemorrhage (aSAH). Up to 40% of aSAH patients experience symptomatic CVS, resulting in disability in up to 50% thereof. While initially in mostly retrospective studies, data showed conscious sedation (CS) to be superior,9 a recent meta-analysis showed no significant difference in outcomes for CS and general anesthesia (GA)17 if only randomized controlled trials were considered.18 To the best of our knowledge, no studies comparing CS and GA in endovascular treatments for refractory CVS after aSAH have been performed.In recent years, several studies investigated the best method of anesthesia for endovascular treatment in acute ischemic stroke.The aim of this study is to compare six-month outcomes for choice of sedation in patients treated with endovascular vasodilators for CVS after aSAH.This is a single-center retrospective case-control study analyzing clinical outcomes in patients with symptomatic CVS after aSAH treated with endovascular vasodilators at the University Hospital Bern, Bern, Switzerland.The University Hospital Bern conducts a prospective database for patients treated with aSAH. This database was retrospectively searched for patients hospitalized between September 2011 and October 2019. Only patients aged >18 and <85 years were included. Inclusion criteria were: 1) aSAH of all severities (World Federation of Neurosurgeons (WFNS) score I–V), 2) secured aneurysm either by endovascular or surgical treatment, 3) refractory CVS treated by intra-arterial admission of either nimodipine and/or papaverine. Exclusion criteria were: 1) incomplete data, 2) loss of follow up, 3) continuous intra-arterial nimodipine treatment, 4) re-rupture of aneurysm during the hospital stay.Patients were divided into three treatment groups: patients who underwent treatment with endovascular vasodilators in CS only (“CS”), in GA only (“GA”), or patients who received intra-arterial treatments in CS and GA (“both”). Choice of anesthesia modality was made by the treating physician on an individual basis. However, according to institutional guidelines, GA was preferred in patients with impaired consciousness (GCS ≤ 8) or insufficient swallowing.TM Critical Care, General Electric Company, GE Healthcare, United States of America). Vital signs are automatically recorded and the bedside team additionally enters clinical scores and administered drugs into the system.All data was acquired from patient records and the institutional electronic Patient Data Management System (Centricity1), directly before (t2), directly after (last) treatment (t3), 24 hours after (last) treatment (t4) and consecutively at discharge from the hospital (t5) and after six months (t6). Further parameters consisted of interventional parameters such as duration of intervention, choice of vasodilator (nimodipine or papaverine), number of treated arteries and vasodilator dosage.The primary endpoint of this study was functional outcome at six months, analyzed by the modified Rankin Scale (mRS). Secondary outcome parameters included mRS at discharge and National Institutes of Health Stroke Scale (NIHSS) assessed 24 hours before the (first) intra-arterial vasodilator treatment , Fisher, Hunt & Hess and WFNS scores were obtained from institutional patient records.The statistical analysis was performed using SPSS Statistics 21.0 . The Shapiro-Wilk normality test was used to test for normal distribution.Univariate Analysis of Variance (ANOVA) test was used to compare “CS”, “GA” and “both” groups for differences in age as well as BNI, Fisher, Hunt & Hess and WFNS scores. Differences in sex and aneurysm treatment were explored with Pearson Chi Squared analysis.For mRS at discharge and six months, outcomes were divided in functionally independent (mRS 0-2) and functionally dependent (mRS 3-6). A Chi Squared test was used to test for significant group differences between CS, GA and both. An additional subgroup analysis was performed using a Chi Squared test with “CS” and “GA” groups divided into single versus multiple interventions, resulting in five groups .1 – t6).For the analysis of NIHSS, a 3 × 6 analysis of variance (ANOVA) for repeated measures with post hoc Bonferroni correction for multiple comparisons was conducted. The factors were (i) treatment  and (ii) time  in brackets and in figures as mean with +1 SD as error bars. A p-value of p < 0.05 was considered statistically significant.This study was carried out in accordance with the recommendations of the local ethics committee . All subjects gave written general consent in accordance with the Declaration of Helsinki. The protocol was approved by the local ethics committee .In total, 109 patients with refractory CVS treated by intra-arterial admission of either nimodipine and/or papaverine between September 2011 and October 2019 at the University Hospital Bern, Bern, Switzerland were included. Of those, 11 patients had to be excluded. Reasons for exclusion were incomplete data (n = 2), re-rupture of aneurysm during the hospital stay (n = 2), continuous intra-arterial nimodipine treatment (n = 1) and loss of follow-up at six months (n = 6). The final study population consisted of 98 patients, 23 patients in the “CS” group, 53 patients in the “GA” group and 22 patients in the “both” group. In the “CS” group, 16 patients received a single intervention (“single CS”) and seven patients received up to five interventions (“multiple CS”). In the GA group, 26 patients received one intervention (“single GA”) and 27 patients received 2-10 treatments (“multiple GA”). As per definition, all patients in the “both” group received more than one and up to 10 interventions.mRS at six months is displayed in1 and t5, meaning between 24 hours before (first) intervention and discharge from the hospital. At the six-month follow up appointment, “CS”, “GA” and “both” did not differ significantly regarding NIHSS.The NIHSS time course analysis is presented inInterventional parameters are displayed inThis retrospective study found no significant differences in functionally independent outcomes (mRS 0-2) six months after aSAH in patients who were treated with intra-arterial vasodilators in CS, GA or a combination of both. While NIHSS was significantly higher in patients undergoing endovascular therapy in GA compared to patients of the “CS” or “both” group in the time window 24 hours before intervention up to discharge from the hospital, this difference was no longer found at six months. Most articles describe both CS and GA to be generally safe for treatment of unruptured aneurysms or aSAH with no clear recommendation for either one.To the best of our knowledge, the effect of anesthesia modality (CS versus GA) on functional outcome has not yet been studied for intra-arterial admission of either nimodipine and/or papaverine in patients with refractory CVS after aSAH. Albeit, there have been several research papers published regarding anesthesia in aneurysm treatment.et al. (2010) showed in their analysis of the “North American SOLITAIRE Stent-Retriever Acute Stroke” (NASA) registry, that patients treated in GA experienced poorer neurologic outcome at 90 days and higher mortality rates than patients treated in CS.8 Berkhemeret al. (2016) reported similar results in a post-hoc analysis of a prospective trial.15 Correspondingly, a recent analysis of the “Endovascular Therapy Following Imaging Evaluation for Ischemic Stroke 3” (DEFUSE 3) trial by Powerset al. (2019) showed higher rates of functional independence (mRS 0-2) and a lower NIHSS score at 24 hours for patients treated in CS. At discharge they did not find a statistically significant difference in NIHSS scores anymore.11Additionally, multiple studies have had similar research questions in relation to the choice of anesthesia during endovascular therapy of acute ischemic stroke. Abou-Cheblet al. (2017) reported no difference in outcome at three months or NIHSS after 24 hours in their Anesthesia During Stroke (AnStroke) Trial.16 Schönenbergeret al. (2016) and Simonsen et al. (2018) report, that GA produced better 3-month outcomes, with the former even finding this result to be significant.19 Finally, a recent meta-analysis determined no significant difference between GA and CS if only randomized controlled trials were considered.17In contrast, most recent randomized controlled trials found a non-inferiority of GA when compared to CS. Hendénet al. (2016), Hendénet al. (2017), Simonsenet al. (2018) and Kimet al. (2019), we also found no significant difference in functional independency (mRS 0-2) at discharge or six months.Our results are in line with these recent randomized controlled trials published for endovascular treatment in acute ischemic stroke. Similar to Schönenberger8 Disadvantages of CS may be procedural discomfort for patients, more difficult interventions because of patients’ movements, emergency conversion to GA or increased risk for aspiration.8 Many of the conceived disadvantages of GA have also been analyzed in prospective studies concerning ischemic stroke treatment. For example, Berkhemeret al. (2016) as well as Hendénet al. (2017) found no treatment delay in the GA group.16 Schönenbergeret al. (2016) reported no difference in feasibility, safety and intra-interventional complication between the groups.18 They did however discover more postprocedural complications after GA such as delayed extubation and pneumonia. Different authors mention the possibility of blood pressure drops and decreased cerebral blood flow as possible additional complications of GA, which could potentially worsen outcomes because of an increase of the ischemic area.15 Others argue that GA on its own has a neuroprotective effect by lowering the neuronal oxygen need.25 Overall, the similar functional outcome in our study as well as in the above cited studies in ischemic stroke suggest that these factors are of minor relevance.Similar considerations regarding choice of anesthesia hold true in intra-arterial vasodilator therapy after aSAH and treatment for acute ischemic stroke alike. Disadvantages of GA may be a delay in treatment, hemodynamic changes and complications associated with intubation such as an increased risk for pneumonia.The major limitations of this study are its retrospective design and the single-center approach. A potential bias could lie in the choice of sedation for treatment. Even before intra-arterial vasodilator treatment, patients who would go on to receive treatments in GA showed significantly higher NIHSS scores when compared to the “CS” and “both” groups. This indicates that patients in clinically and neurologically worse conditions were more likely to be treated in GA. However, this bias was not reflected in our results, as we did not find a significant difference in mRS scores between the anesthesia groups at discharge or at six months and also no significant difference in NIHSS scores at six months. The “GA” group were therefore in an initially worse state but still managed to reach similar outcomes in the long-term clinical course. This suggests to an even greater degree, that GA will not negatively affect long term outcome in these patients.Furthermore, some subgroups consisted of a small number of patients. The results of this study will have to be replicated by a larger prospective trial in the future.Our preliminary results indicate that choice of anesthesia method does not negatively affect six-month outcome in aSAH patients who undergo intra-arterial vasodilator treatment for CVS. Treating physicians should therefore decide between CS and GA individually based on patient characteristics and circumstances.https://doi.org/10.5061/dryad.g4f4qrfq5.26Dryad: Functional Outcome after intraarterial vasodilator therapy in CS vs GA,Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).Data are available under the terms of the I thank the authors for the changes they made in the revised manuscript and for the answers to our comments.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Neurocritical careWe confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This is a retrospective study. How were the NIHSS extracted from the patients files? Is NIHSS performed routinely? If the patient was already intubated, how was NIHSS evaluated? While aneurysma location as well as Fisher score seem to be similar, WFNS/ H&H seem to differ between CS + GA (e.g. 52% vs 28% WFNS 1). Is there a statistical difference? If so, how do you explain the lack of association thereof with the outcome? Patients with high WFNS + Fisher scores commonly remain intubated in case of unsuccessful sedation-reduction trial. How many patients were already under GA before intra-arterial vasodilatation. Is there difference in delay between non-intubated patients to CS or GA intra-arterial vasodilatation? When mRS at 6 months is compared between CS and GA only (excluding both) irrespective of if they received a singular or multiple CS/GA statistical significance is reached. How do you explain this finding?Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Neurocritical careWe confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above. https://www.ninds.nih.gov/sites/default/files/NIH_Stroke_Scale_Booklet.pdf). Most notably, intubated patients get a default score of 1 for verbally assessed categories such as orientation if they are unable to speak or write. Additionally, aphasia is assessed through writing. Dysarthria is not assessable in the intubated patient.In our institution, NIHSS is routinely assessed in stroke patients at least once a day as well as before and after interventions. In intubated patients, the NIHSS is determined after depth of sedation is decreased as much as clinically possible. The NIHSS assessment is performed according to the recommendation of the National Institute of Neurological Disorders and Stroke . The p-values for WFNS, Hunt&Hess, BNI and Fisher scores were added to Table 1. Only the WFNS and Hunt&Hess scores differed significantly between the groups. However, as it has been shown in two earlier publications cited below, WFNS 5 scores at admission are often over-estimated due to accompanying factors e.g. early seizures. In addition, if factors such as intubation, ventilation, sedation, muscle relaxation or insufficient pain stimuli inhibit said motor response, the WFNS grading can be over-estimated while the outcome is not necessarily impacted. Similar effects can alter Hunt&Hess gradings. In contrast the radiologically determined scores (BNI and Fisher) are not influenced by the above factors, which can at least partially explain that these two sores are not significantly different while the clinical ones are.  Twenty-two patients were already ventilated before the first intra-arterial vasodilator treatment . Unfortunately, due to the retrospective study design, the additional delay of intra-arterial vasodilatation therapy due to intubation was not assessable within the obtained documentation. Considering only mRS at 6 months and reducing the analysis to patients who were only treated with one modality (CS or GA), the outcome differed significantly between the two groups (Fisher exact p = 0.044). In our opinion this analysis has to be interpreted with caution as the number of patients undergoing intra-arterial vasodilator treatment in CS who showed an unfavorable outcome is very small (n = 5). Furthermore, if the above mentioned patients who remained intubated after the initial bleeding are excluded from this analysis, the result is no longer statistically significant (p = 1.000). This indicates that the difference in outcome of this additional analysis is probably mainly due to the initial disease severity and not due to the anesthesia modality chosen for intra-arterial vasodilator treatment. This is an interesting preliminary study on the anesthesia modality for endovascular rescue therapies in patients with aneurysmal subarachnoid hemorrhage (aSAH). This single-center retrospective study included 98 aSAH patients (admitted in an 8-year period) selected for endovascular vasospasm therapy. Circa half of these patients were treated in general anesthesia, 25% were treated in conscious sedation and 25% had multiple interventions and both anesthesia modalities were applied. No differences in outcome (6-months mRS) were observed between these groups. Carefully interpreted and fully aware of the limitations associated with the study design and data collection, this study may indicate towards safety and non-inferiority of both general anesthesia and conscious sedation for endovascular interventions after aSAH. These findings are in keeping with the pertinent literature on anesthesiologic management of endovascular interventions for stroke. I thank the authors for sharing their first experience with conscious sedation as an alternative to general anesthesia for endovascular vasospasm therapy. I encourage investigating the clinical algorithm that triggers the choice for either method. 50% of patients treated in general anesthesia had admission WFNS grades 4 or 5 (i.e. poor grade aSAH) compared to 30% of patients selected for conscious sedation. A fact that may very well explain the presumed tendency towards better outcomes of the latter group. The rate of delayed infarction should have been reported. Given that safety of conscious sedation seems to apply to endovascular vasospasm therapy, future studies are warranted. They should follow a prospective and randomized design. Potential benefits of conscious sedation  might be of high clinical relevance and could improve outcome of patients who develop clinical vasospasm.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?No source data requiredIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:NeurosurgeryI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The following text was added to the methods section of the manuscript: “Choice of anesthesia modality was made by the treating physician on an individual basis. However, according to institutional guidelines, GA was preferred in patients with impaired consciousness (GCS <=8) or insufficient swallowing.”


Pseudomonas aeruginosa isolates that have been recovered from different infected human sites. Convergent changes in gene expression patterns were found in different groups of clinical isolates. The majority of repeatedly observed expression patterns could be attributed to a defective lasR gene, which encodes the major quorum-sensing regulator LasR. Strikingly, the gene expression pattern of the lasR-defective strains appeared to reflect a transcriptional response that evolves in a direction consistent with growth within a biofilm. In a process of genetic assimilation, lasR-deficient P. aeruginosa isolates appear to constitutively express a biofilm-adapted transcriptional profile and no longer require a respective environmental trigger. Our results demonstrate that profiling the functional consequences of pathoadaptive mutations in clinical isolates reveals long-term evolutionary pathways and may explain the success of lasR mutants in the opportunistic pathogen P. aeruginosa in a clinical context.The overall success of a pathogenic microbe depends on its ability to efficiently adapt to challenging conditions in the human host. Long-term evolution experiments track and predict adaptive trajectories and have contributed significantly to our understanding of the driving forces of bacterial adaptation. In this study, we conducted a cross-sectional study instead of long-term longitudinal evolution experiments. We analyzed the transcriptional profiles as well as genomic sequence variations of a large number of clinical  RNA sequencing data offer the potential for large-scale comparative studies, which aim at deciphering coordinated regulation of genes within an organism. Thus, new insights into complex bacterial adaptation strategies, for example during an infection process, can be achievedPseudomonas aeruginosa is considered one of the most important pathogens involved in nosocomial infections2. The bacterium causes high morbidity and mortality, especially in patients with weakened immune systems and burn victims3. In cystic fibrosis (CF) patients, lifelong chronic P. aeruginosa infections lead to an over-activation of the immune system and to extensive tissue damage in the patients’ lungs6. In the course of these long-term infections, P. aeruginosa not only adapts by orchestrating the transcription of complex regulatory gene networks, but the bacteria also develop a number of pathoadaptive mutations that promote survival in the harsh environment of the human host10. The adapted phenotypes have fitness advantages over the originally infecting wild-type strain, e.g., by escaping immune recognition, saving energy by using public goods or developing resistance to antimicrobial agents12. Understanding the evolutionary trajectories that P. aeruginosa follows during long-term infections is an important task in order to optimize treatment and predict the clinical course of the infection15.The Gram-negative, opportunistic pathogen P. aeruginosa isolates previously recorded under both planktonic and biofilm growth conditions17. We found that complex gene expression patterns evolved independently in different clinical P. aeruginosa isolates and that a majority of the observed expression patterns could be attributed to a defective lasR gene, encoding the major quorum sensing (QS) regulator LasR19. Strikingly, whereas a great number of genes were differentially regulated in the lasR-proficient isolates upon shifting from planktonic to biofilm growth conditions, the lasR-defective isolates exhibited a transcriptional signature under planktonic culture conditions that already resembled a biofilm transcriptional profile. Our results demonstrate that the analysis of transcriptomic and genomic data from a large variety of clinical isolates provides new insights into how adaptive mutations drive gene expression programs and regulate phenotypic outcomes responsible for bacterial adaptation to altered and challenging host niche habitats.In this study, we analyzed the genomes and transcriptomes of a large number of clinical P. aeruginosa isolates following growth in lysogeny broth (LB) media until the early stationary phase (OD600 of 2)20. Here, we re-analyzed the data and recorded the distribution of individual gene expression values across all 414 clinical isolates. As exemplified in Fig. We have previously transcriptionally profiled a collection of 414 clinical We identified overall 136 genes with a bi-modal gene expression distribution by the use of an algorithm that distinguishes between uni- and at least bi-modality expressed genes  or pre-mature stop codons (STOP), there were also non-silent mutations that led to changes in the amino acid sequence of LasR. As depicted in Supplementary Fig. 22, indicating that they might result in the functional inactivation of LasR23.As many of the genes of these functional categories are under the control of the major QS regulator LasR, we evaluated whether mutations of the lasR gene and underscores the dominant role of LasR in shaping the transcriptional profile in our collection of clinical isolates. We did not identify large groups of clinical isolates that exhibited a characteristic expression pattern of genes belonging to the two other major clusters  that influences a comparably large fraction of the bi-modally expressed genes. Nevertheless, future work should concentrate on the identification of additional genes, which influence the expression of smaller groups of bi-modal genes.There was a clear correlation between the expression status of bi-modal genes and the allele status of the transcriptional regulator LasR  and 21 clinical isolates with lasR inactivating (InDels/stop) mutant alleles . Both, planktonic and biofilm transcriptomes, were available for those 49 clinical isolates. We then calculated the differential gene expression between the lasRWT and lasR* group. In total, 722 genes were differentially regulated . Of those, 412 genes were up- and 310 genes were downregulated in lasR* isolates compared to lasRWT isolates , the protease LasA (lasA), the elastase LasB (lasB) or rhamnolipids (rhlAB). Accordingly, we identified genes belonging to the KEGG24 pathways of “phenazine biosynthesis”, “quorum sensing”, and “biofilm formation” as being significantly enriched  in the group of genes that were differentially expressed between the clinical lasRWT and lasR* isolates. We next phenotypically characterized a random selection of lasRWT (n = 10) and lasR* isolates (n = 8). In agreement with the finding that LasR governs the production of a range of virulence factors, we found a significantly reduced virulence of the lasR* isolates in a Galleria mellonella infection model compared to the isolates with functional LasR  as compared to a clean lasR deletion mutant (PA14 ∆lasR26) under the same growth conditions as applied for the clinical isolates  in at least 10 lasR* isolates and 74 genes were downregulated in all 21 lasR* isolates as compared to the group of lasRWT isolates , and which were also significantly downregulated in PA14 ∆lasR as compared to its PA14 wild-type.The complementary analysis of 27. However, we also found that lasR mutations in the clinical isolates led to an increased expression of certain genes when compared to lasRWT isolates  are associated with the degradation of complex carbon sources, such as aromatic compounds or lipids. Another 21 genes are involved in various mechanisms of iron acquisition (such as hasRDE and pvdGEFN), and the kdpABC operon is associated with sensing low extracellular potassium levels. In addition, 12 genes were upregulated in the lasR* isolates that encoded for cup fimbriae or the biosynthesis of the pel exopolysaccharide. Moreover, the spermidine transport system encoding potABCD exhibited increased expression levels in lasR* isolates compared to lasRWT isolates. Furthermore, acquisition of extracellular phosphate either via the phn transport system, the (extracellular) degradation of phosphatidylcholine (glpQ and glpD), or the type 2 secretion system  seems to be pronounced in the lasR* isolates. Interestingly, also multiple resistance genes including the multi-drug efflux pump mexCD-oprJ were significantly upregulated in lasR* isolates.It is well established that LasR is a transcriptional activator that governs a large QS regulontes Fig. . Table 2lasR* isolates  and lasRWT (n = 28) isolates under planktonic growth conditions, we also recorded their transcriptomes under biofilm growth conditions14. While the planktonic gene expression profiles of the two groups of clinical isolates were clearly separated in a multi-dimensional scaling plot (MDS), their transcriptomes did not cluster separately when the isolates were grown under biofilm growth conditions  under biofilm growth conditions (as compared to 722 genes under planktonic conditions). We also recorded the transcriptional profiles of PA14 WT and PA14 ∆lasR under biofilm growth conditions  were expressed at even higher levels in the lasR* isolates under biofilm growth conditions , 15 isolates with an inactivating mutation (InDel/Stop) exclusively in lasR (lasR*), and 7 isolates with InDel/stop mutations in rhlR and an InDel/stop and/or non-synonymous mutations in lasR (lasR**/rhlR*). For all of the isolates both, planktonic and biofilm transcriptomes, were available. As expected, the isolates harboring a rhlR inactivating mutation exhibited a significantly reduced expression of rhlI, the autoinducer synthase, which is under the direct control of RhlR , whereas elastase production under biofilm conditions (static growth for 48 h) was at a similar level in lasR* but not in the lasR**/rhlR* strains as compared to lasR wild-type isolates  among the lasRWT, lasR* and lasR**/rhlR* isolates under planktonic and biofilm growth conditions. The data confirm that RhlR becomes activated under biofilm growth conditions independent of LasR to induce expression of the LasR-regulon genes.Our results thus demonstrate that RhlR acts downstream of LasR to impact the expression of many genes and that RhlR becomes activated under biofilm growth conditions independent of LasR to induce expression of genes that belong to the LasR core regulon under planktonic conditions. Supplementary Fig. aprIAFEDX) was dependent on lasR even under biofilm conditions and the additional inactivation of rhlR did not reduce gene expression further  biosynthetic gene cluster (pqsABCDE) seemed to be independent of both lasR and rhlR under biofilm growth conditions  genes, which have been identified to be upregulated in the lasR-deficient isolates, among the lasRWT, lasR* and lasR**/rhlR* isolates confirm that RhlR acts downstream of LasR to induce the expression of genes in the absence of lasR under planktonic conditions. However, this lasR-mediated inhibition of gene expression is lost under biofilm conditions.We also analyzed the genes that were expressed at higher levels in the absence of ons Fig. . Again, ons Fig.  seemed tons Fig.  these gerhlR acts downstream of lasR also in the PA14 type strain to induce the expression of genes in the absence of lasR under biofilm growth conditions. We, therefore, generated clean ∆rhlR and ∆lasR/∆rhlR PA14 deletion mutants using a CRISPR/Cas9-based approach and recorded their transcriptional profiles under planktonic and biofilm growth conditions. Again, we found that the expression of the lasR core regulon genes was dependent on the presence of lasR under planktonic, but not under biofilm conditions, while the lack of rhlR reduced the expression of the lasR regulon genes under both, planktonic and biofilm conditions  that were expressed at a higher level in the clinical lasR* vs. lasRWT isolates. Expression of those genes was demonstrated to be less consistent across the clinical isolates  conditions. Although growth e.g. in a synthetic sputum medium might have reflected the conditions within the human host more accurately, the non-selective cultivation conditions accommodated the growth of all clinical isolates. Despite the fact that the transcriptional profiles were recorded under rich medium growth conditions, the recorded transcriptional patterns are nevertheless the result of genomic variations that have evolved during the infection process in the human host.In this study, we took advantage of the extensive transcriptome as well as whole-genome sequencing data on a large collection (>400) of clinical P. aeruginosa strains. Our analysis identified mutations within lasR as major contributors to evolved gene expression patterns and therefore as main drivers of adaption towards the habitat of the human host. Indeed, inactivating mutations within lasR are well-known pathoadaptive mutations, frequently found in P. aeruginosa isolates from the respiratory tract of CF patients33. LasR-deficient isolates have been associated with advanced disease and major changes in the expression of QS-regulated genes34. However, especially the apparent reduced ability to produce virulence factors raises the question of the fitness advantage of such an evolutionary path36.The extensive data allowed us to cluster the clinical isolates according to a high vs. low expression status of a large subset of expressed genes. Although we did not account for absolute differences in gene expression levels, we were able to group isolates according to the complex high/low gene expression profiles that developed repeatedly in different lasR deletion mutants in P. aeruginosa type strains has previously uncovered the LasR regulon37. This regulon comprises many virulence-associated genes, which are positively regulated by LasR activity. In this study, we identified genes that are commonly found to be differentially regulated in lasR-deficient (lasR*) versus lasR-proficient (lasRWT) clinical isolates. This core regulon included many genes, which were also found to be LasR-dependent in the PA14 type strain. However, we also found additional pathoadaptive traits in the lasR* isolates. Recording of transcriptional profiles of clinical lasR* isolates informed on important co-selected or possibly compensatory adaptations that might explain the success of the lasR mutations in the clinical context. Most remarkably, we found that the gene expression profile of the lasR* isolates did not differ to the same extent to those of lasRWT clinical isolates when cultured under biofilm growth conditions. Indeed, it seems that the lasR* isolates exhibit a transcriptional profile typical for growth in a biofilm, already under planktonic culture conditions. The lasR* isolates were found to regulate 1.52-fold less genes compared to lasRWT isolates . It seems that the switch to the biofilm mode of growth, which is expected to be triggered by the environmental conditions in the human host, might have become fixed in the lasR* population46.In a process of genetic assimilationlasR regulon do not depend on the presence of lasR, but on the presence of rhlR under biofilm growth conditions. An exception was the alkaline protease gene cluster (apr) and the PQS synthesis operon, whose expression remained dependent on the presence of a functional lasR even under biofilm growth conditions. It has been described previously that LasR becomes dispensable under conditions where RhlR is activated by different means, in order to drive genes belonging to the LasR regulon. A decoupling of the QS networks has been described under diverse environmental conditions such as low phosphate30, late stationary phase28 and now biofilm growth conditions. Interestingly, the phosphate-starvation regulator PhoB was previously shown to interact either directly or indirectly via RhlR and PqsR on the expression of the otherwise LasR-dependent pyocyanin synthesis gene clusters47. However, our results indicate that the phosphate starvation response regulator PhoB does not play a role in the activation of lasR/rhlR dependent genes under biofilm growth conditions, despite the fact that many genes that are activated in the clinical lasR mutants are involved in the acquisition of phosphate.The results of our study also showed that the genes of the In conclusion, we demonstrate in this study that the analysis of large sample sizes of clinical isolates provides an interesting alternative to in vitro long-term experimental evolution experiments. Our data illustrate the power of profiling large sample sizes of clinical isolates to discover novel evolutionary trajectories in pathogen populations on their way of adaptation to the conditions encountered during an infection process within the human host. An integrated analysis of large-scale genomic and transcriptomic data together with clinical metadata, including detailed knowledge about the isolate-specific history (e.g. duration of infection and infection site), would further extend our knowledge and contribute to a more comprehensive understanding of adaptive evolution during infection. Profiling the consequences of pathoadaptive mutations promises to identify predictable pathways to successful long-term persistence within a host and thus highlights opportunities for developing novel strategies to combat therapy-refractory chronic infections.E. coli or 50 µg/ml for P. aeruginosa), or streptomycin  to retain plasmids.Bacteria were grown in standard rich medium culture conditions (LB) with constant shaking (180 rpm) at 37 °C unless otherwise stated. All strains and plasmids used in this study are listed in Table P. aeruginosa. For this endeavor, we resorted to the pS448·CsR plasmid developed for the accelerated genome engineering in the closely related bacterium species P. putida48. This vector controls the constitutive expression of a target-specific small guide RNA (sgRNA) and the inducible production of the Cas9 endonuclease upon addition of 3-methylbenzoate (3-mBz). In Pseudomonas spp., it has been previously shown that using synthetic, linear DNA (either ssDNA or dsDNA) as a mutagenic template for genomic manipulations is futile unless an efficient DNA recombinase is also expressed50. Thus, we constructed a second vector expressing the gene encoding for the Ssr recombinase from P. putida DOT‐T1E51. The ssr coding sequence was excised from pSEVA658-ssr as an AvrII/BamHI restriction fragment and cloned anew into the same restriction sites of the isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible vector pSEVA624, resulting in plasmid pSH624-ssr.We developed a targeted CRISPR/Cas9-recombineering system in order to generate clean deletion mutants in 48. Briefly, 100 pmol of each primer pair constituting the gene-specific spacer sequence were mixed and phosphorylated for 30 min at 37 °C in a 50-μl mixture with the T4 Polynucleotide Kinase . Next, the primers were denatured for 5 min at 95 °C and annealed to each other by allowing the mixture to slowly cool down to room temperature for at least 2 h. Both complementary oligonucleotides were designed to generate the protruding ends of the Eco31I restriction site (BsaI isoschizomer) when hybridizing  of 0.05, and bacteria were cultivated at 37 °C and constant shaking (180 rpm) until they reached an OD600 of 2.02–2.28 (early stationary phase). For the harvest, 1 ml of bacterial suspension was mixed with an equal volume of RNAprotect (Qiagen), incubated for 10 min, and centrifuged at 8000 rpm for 10 min.We re-analyzed the transcriptional profiles of previously characterized clinical n = 77) of our strain collection was chosen to be analyzed under biofilm growth conditions14. Biofilms were inoculated at a starting OD600 of 0.002 in LB and grown statically in half-area 96-well µClear microtiter plates (Greiner Bio-One) at 37 °C in a humid atmosphere. After 48 h, mature biofilms (10 wells per replicate) were resuspended and harvested in an equal volume of RNAlater (Qiagen). Three independent biological replicates were used for each strain and condition.A randomly selected sub-group  following an initial QIAshredder step according to the manufacturer’s instructions. DNA was removed by DNase I (Ambion) treatment, and the RNase inhibitor RNAsin (Promega) was added to protect the eluted RNA.54, total RNA was fragmented (150–350 bp) in Fast AP-Buffer (Thermo Scientific), DNA was digested by TURBO™ DNase (Invitrogen), and RNA fragments were phosphorylated by FastAP alkaline phosphatase (Thermo Scientific). Custom-made barcodes were ligated to the RNA using the T4 ligase (New England Biolabs) and fragments were subsequently purified and concentrated using the RNA Clean & Concentrator™-25 Kit (Zymo Research) following the manufacturer’s instructions. Ribosomal RNA was removed by using the RiboZero Bacteria kit (Illumina). cDNA libraries were synthesized using the SMARTScribe Reverse Transcriptase (Takara) followed by a PCR enrichment using the AccuPrime HiFi Taq polymerase (Invitrogen). Enzymatic reactions were carried out in the presence of SUPERase·In™ RNase Inhibitor (Invitrogen); RNACleanXP beads (Agencourt) were used for all RNA purification steps. Quality checks were performed before, during, and after cDNA library preparation with the RNA Nano Kit and an Agilent Bioanalyzer 2100 (Agilent Technologies). Libraries were sequenced on an Illumina HiSeq  or an Illumina NovaSeq 6000 .For cDNA library preparationR statistical environment (version 3.6.3)55 Reads were mapped against the UCBPP-PA14 reference genome (NCBI Reference sequence: NC_008463.1) using stampy (version 1.0.23)56. Normalization was performed using the command calcNormFactors of the R-Package edgeR (version 3.28.1)57 and normalized read counts were extracted using the cpm command of the same package. Genes with less than 1 count per million (cpm) in at least 21 isolates were excluded from the analysis. p-value correction outside of the edgeR-based differential gene expression analyses was calculated by p.adjust of the stats R-package (version 3.6.1) with the adjustment method ‘fdr’.Data analysis was performed in the P. aeruginosa isolates using the command modetest of the R-package multimode (version 1.4)58 in default settings. Multimodality was assigned when the calculated and corrected p-value was <0.1. Isolates were sorted into either high or low expressing group by determining the last minimum before the last clear maximum of the distribution of expression values 59 package in R with the “pau” pathways to map the differentially regulated genes. Enrichment was performed via the p.hyper function of the stats package with a corrected p-value of 0.05 as a cut-off for a significant enrichment.KEGGlasR* isolates against all lasRWT isolates (n = 28). The number of isolates with log2FC ≥ 1 or log2FC ≤ −1 for each gene was counted and summed over the number of tested lasR* isolates (n = 21). Finally, the lasR core regulon was determined as the intersection of genes that showed a log2FC above or below the respective threshold in at least 90% of the isolates (n = 19), and genes that are significantly regulated in the clean PA14 ∆lasR compared to PA14 WT.The regulon robustness, as well as the core regulon analysis, were performed by calculating a differential gene expression of each individual edgeR package in R, taking all analyzed genes into account for the calculation of the dissimilarity matrix. The data was visualized using the ggplot2 R-package with the command stat_ellipse for the 95% confidence interval ellipses.MDS plots were calculated based on the normalized read counts created in the differential gene expression analysis described above and the PlotMDS function in the R; values described as “nrpg”) and a value of 1 was added to the all nrpg values prior to log10 transformation in order to prevent the creation of infinite values.Comparisons of gene cluster expression were generally performed by combining the complete transcriptional profiles of isolates of interest into a DGEList object as described above to subsequently perform the described normalization and low coverage gene exclusion. Expression values of the genes of interest were then extracted as library size-normalized counts per million (cpm command in lasR (PA14_45960) and rhlR (PA14_19120), previously published whole-genome sequencing data of 414 clinical isolates17 were screened for sequence variations in the respective genes. Mapping was accomplished using stampy (version 1.0.23)56 and variant calling was performed using SAMtools (version 0.1.19)60 with UCBPP-PA14 (NCBI Reference sequence: NC_008463.1) or PAO1 (NCBI Assembly: GCA_000006765.1 ASM676v1) as a reference. The strain background of the clinical isolates was assessed based on the phylogenetic analysis documented in Khaledi et al.16. PA7-like isolates were excluded in this study.In order to identify mutations in the sequences of lasR wild-type allele status , strains harboring a non-functional lasR  and isolates with inactivating mutations in both lasR and rhlR  was collected in previous studies17. In vivo virulence was determined 48 h post infection as % killing of Galleria mellonella larvae. For infection, bacterial strains were grown overnight and serially diluted in PBS. 20 µl of the bacterial suspension was injected into the last left proleg of the larvae with an MOI (multiplicity of infection) of 100 cfu . Relative proteolytic activity was assessed by measuring clearing zones of spotted bacteria on cation-adjusted Müller-Hinton (MH) broth containing 10% (v/v) milk after 24 h of incubation. Overnight cultures were adjusted to an OD600 of 0.025 and 5 μl of the bacterial suspensions were spotted on top of the agar plates. An Elastin Congo Red (ECR) assay was applied to determine the elastolytic activity of secreted proteases. Bacteria were grown for 24 h in planktonic cultures (shaking at 180 rpm), or for 48 h in biofilms (static incubation) as described above. After harvest, 100 µl of bacteria-free culture broth was incubated with the substrate  for 3 h at 37 °C and 900 rpm. Elastase secretion was determined by measuring the absorbance of the supernatant at OD = 495 nm.Phenotypic data for randomly selected clinical isolates representing a stats package in R (v 3.6.1) was applied to test for normal distribution of the compared groups. If no normal distribution was observed  in one of the groups, the wilcox.test command with default settings from the same package was employed to test for statistical significance.The shapiro.test function of the stats package in R with the lower.tail option set to “FALSE” to test for enrichment.Overrepresentation analysis was calculated using the phyper command of the Statistical analyses for phenotypic data were performed in GraphPad Prism (v 8.3.0) by using Kruskal-Wallis (comparison of three groups) and Mann–Whitney tests (pair-wise comparison).Further information on research design is available in the Supplementary InformationSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Reporting Summary


FBN1 variant. A 67-year-old female with a history of lens dislocation and retinal detachment in the left eye was admitted for the evaluation of resting dyspnea several months prior. Transesophageal and transthoracic echocardiography revealed severe aortic valve regurgitation and a large left coronary sinus of Valsalva aneurysm in the proband. Sanger sequencing identified a heterozygous p.Gly1127Cys variant in the FBN1 gene; previously, a mutation at this amino acid position was described as pathogenic . A 3D printed personalized external aortic root model based on a multidetector computed tomography scan was constructed to illustrate the location of the ostium of the left main coronary artery on the aneurysm of the left coronary artery cusp. Aortic root replacement with the Bentall procedure matched the exact shape of the 3D printed model. Creation of a 3D printed patient-specific model could be useful in facilitating the development of next-generation medical devices and resolving the risks of postoperative complications and aortic root disease.The major cause of death in Marfan syndrome (MFS) is cardiovascular complications, particularly progressive dilatation of the proximal aorta, rendering these patients at risk of aortic dissection or fatal rupture. We report a 3D printed personalized external aortic root model for MFS with an isolated sinus of Valsalva aneurysm caused by a novel pathogenic  FBN1), which is the primary disease-associated gene of MFS [FBN1 variants, with no differences according to sex, ethnicity, or geographical location [FBN1 mutations encoding a major component of the extracellular matrix microfibril, namely, fibrillin-1 [FBN1, and the disease phenotype depends on the type of mutation involved and its effect on the fibrillin-1 protein [Marfan syndrome  is an autosomal dominant systemic disorder that affects connective tissue, primarily in the skeletal , ocular (ectopia lentis), and cardiovascular (aortic aneurysm and dissection) systems. MFS is caused by the fibrillin1 gene  printed personalized external aortic root model to understand abnormal aortic structure in a patient with MFS.In this report, we described a Korean patient in which a novel p.Gly1127Cys variant in A 67-year-old female was admitted to the Department of Cardiology, Daejeon St. Mary’s Hospital  for the evaluation of resting dyspnea several months prior. She grew up in an orphanage when she was young and had no information about her parents. During childbirth, she developed a lens dislocation in her left eye. Lens removal surgery was performed but caused retinal detachment that led to blindness in the left eye after several ophthalmic surgeries. She had glaucoma in the right eye. She was 158 cm tall, but she had a slender build and arachnodactyly and a positive thumb sign. Transesophageal echocardiography (TEE) and transthoracic echocardiography (TTE) were performed to evaluate dyspnea and revealed severe aortic valve regurgitation (AR) and a large left coronary sinus of Valsalva aneurysm in the proband A–C. The The proband was going to undergo aortic root replacement (ARR) with the Bentall procedure due to the isolated left coronary sinus of Valsalva aneurysm and severe AR. To help understand the anatomic structure before the Bentall procedure, a 3D printed personalized external aortic root model based on the MDCT scan was constructed to illustrate the location of the ostium of the left main coronary artery on the aneurysm of the left coronary artery cusp. The patient’s CT datasets were imported into a medical 3D image-based engineering software  and the 3D model was printed on a Stratasys Connex3 Objet500 3D printer  using the form2 clear resin (FLGPCL02). At first, we considered valve sparing ARR for this case because aortic leaflets were relatively preserved even though annular dilatation was combined (26 mm). However, 3D printed model revealed that thinning of the cusp due to asymmetrically severely enlarged left coronary cusp can cause stress fenestration toward the commissures and turbulent blood flow inside the aorta A–D. ProbFBN1 gene were amplified by polymerase chain reaction using the primers described previously [FBN1 sequence with RefSeq ID NM_000138.4 was used as a reference for cDNA nucleotide numbering. Mutations predicted to cause strong and moderate alterations in gene functions were manually reviewed by laboratory geneticists based on ACMG-AMP standards and guidelines [FBN1 gene  substitutions are more frequently associated with a high prevalence of ectopia lentis such as partial or complete displacement of the eye lens [FBN1 mutations in exons 24–32 define a high-risk group for cardiovascular manifestations at all ages [FBN1 variants such as nonsense and out-of-frame variants that presumably cause nonsense-mediated mRNA decay have more severe aortic phenotypes than those with DN-type mutations such as missense and in-frame variants that are expected to exert loss-of-function effects [FBN1 gene as others that have caused different substitutions of the Gly1127 residue. The proband had complete displacement of the left eye lens during childbirth, and subsequent retinal detachment and total blindness developed. She had glaucoma in the right eye. She suffered from all kinds of ocular complications of MFS. The pathogenic criterion for this novel variant is weighted as moderate 5 (PM5) based on ACMG-AMP standards and guidelines [FBN1 gene produces a mild form of autosomal dominantly inherited weakness of elastic tissue, which predisposes patients to ascending aortic aneurysm and dissection later in life. Additionally, in our case, the proband had an isolated left coronary sinus of Valsalva aneurysm without dissection in late life. Recently, Hernándiz A and colleagues [FBN1 variants and of males versus females. In addition, the observation of a “Carter effect” in the cardiovascular phenotype provides further evidence of the existence of inherited modifiers and supports the high heritability of cardiovascular clinical features [Mutated fibrillin-1 results in dysregulation of TGF-β signaling as well as structural weakness of connective tissue, both of which contribute to complicated pathogenesis in MFS . It playedictive . The corthe gene ,15,16. Feye lens . FBN1 mu effects . Deleteridelines . A novelidelines . Franckelleagues  reportedfeatures .The major cause of death in MFS is cardiovascular complications, particularly progressive dilatation of the proximal aorta, rendering these patients at risk of aortic dissection or fatal rupture . AlthougFBN1 p.Gly1127Cys variant. Creation of a 3D printed patient-specific model could be useful in facilitating the development of next-generation medical devices and resolving the risks of postoperative complications and aortic root disease. Genetic analysis of the FBN1 gene is essential to discover its genetic origin in MFS patients with isolated left coronary sinus sinuses of Valsalva aneurysm.In conclusion, we reported a 3D printed personalized external aortic root model in MFS with an isolated left coronary sinus of Valsalva aneurysm caused by a novel pathogenic