Role of ROS and ROS generating enzymes in the human ovary

Reactive oxygen species (ROS) have long been regarded as destructive molecules that have harmful effects. However, research data emerging over the last decade have demonstrated that ROS can positively influence a range of cellular events in a manner similar to that seen for traditional second messenger molecules. Hydrogen peroxide (H2O2) appears to be the main ROS with such signalling properties, and this molecule has been shown to affect a variety of cellular functions. Its synthesis by the NADPH oxidase (NOX) family of enzymes and how these enzymes are regulated in the human ovary are poorly investigated topics. In the ovary, ROS are involved in fundamental reproductive processes, as implicated by previous studies. However, ROS can also cause oxidative stress, which is associated with impaired oocyte quality and a negative outcome of assisted reproductive techniques. Oocytes grow and mature in ovarian follicles, formed by granulosa cells (GCs) and theca cells (TCs). NOX4, a member of the ROS-producing NOX family, was identified in cultured human in-vitro fertilization (IVF)-derived GCs as well as in corresponding GCs and TCs of growing preantral and antral follicles in human ovarian sections. It was further detected in GC- and TC-derived luteal cells of the corpus luteum. IVF-derived GCs resemble ovulatory GCs and/or the corpus luteum, but do not proliferate. Therefore, as a model for the growing follicle, the granulosa-like tumour cell line KGN was further studied. These cells also express NOX4. Accumulation of ROS in the medium of cultured GCs and KGN could be detected and was inhibited by the specific NOX4 blocker GKT137831. This blocker reduced specifically the production of H2O2 by around 45 % in GCs and KGN. This indicates a major contribution of NOX4 activity to the generation of H2O2. This is a rather long-lived and the only membrane-permeable ROS. H2O2 may diffuse to neighbouring cells, and studies implicated aquaporins in the uptake of extracellular H2O2 into GCs. The gonadotropins follicle stimulating hormone (FSH) and human chorionic gonadotropin (hCG) play a key role in reproduction. They induce the maturation of ovarian follicles, and the ovulation process. FSH and hCG, however, did not alter expression of NOX4, but elevated mRNA expression of antioxidant enzymes including catalase. This indicates a role of these hormones in ovarian ROS homeostasis. H2O2 and FSH also increased MAPK-phosphorylation, suggesting convergence of the signalling pathways. Furthermore, H2O2, when added to GCs, elevated several cytokines. This implicates H2O2 in inflammatory events, which are possibly involved in ovulation and/or regression of the corpus luteum. Inhibition of H2O2 production by GKT137831 did not affect cell viability of GCs but lowered expression of CYP11A1, a crucial enzyme for steroid synthesis. This suggests involvement in the maintenance of the steroidogenic phenotype of GCs. In proliferating KGN cells, GKT137831 reduced cell growth. This may implicate a role of H2O2 in cell proliferation and possibly in follicular growth. Taken together, the results imply important roles of H2O2 in the regulation of GCs. As ROS are potentially harmful molecules, the full elucidation of the two sides of the ROS-signalling system in the human ovary is important to guide future therapeutic strategies.Reaktive Sauerstoffspezies (englisch reactive oxygen species, ROS) wurden lange Zeit als potentiell schädliche Moleküle betrachtet, die nachteilige Effekte haben. Forschungsdaten des letzten Jahrzehnts zeigen jedoch, dass ROS eine Reihe von zellulären Ereignissen positiv beeinflussen können, ähnlich wie traditionelle Second Messenger Moleküle. Wasserstoffperoxid (H2O2) scheint das wichtigste ROS mit einer solchen Signaleigenschaft zu sein. Es wurde beobachtet, dass dieses Molekül eine Vielfalt von zellulären Funktionen beeinflusst. Seine Synthese durch die Enzymfamilie NADPH Oxidasen (NOX) und dessen Regulierung im menschlichen Ovar sind kaum erforschte Themen. ROS sind im Ovar in fundamentalen Reproduktionsprozessen involviert, was frühere Studien implizieren. Allerdings können ROS auch oxidativen Stress verursachen, welcher mit schlechter Oozytenqualität und negativem Ergebnis bei assistierten Reproduktionstechniken verbunden ist. Oozyten wachsen und reifen in ovariellen Follikeln, welche aus Granulosazellen (GZs) und Thekazellen (TZs) gebildet werden. NOX4 ist ein Mitglied der ROS-produzierenden NOX Familie und wurde sowohl in kultivierten menschlichen GZs, die aus In-Vitro-Fertilisation (IVF) stammen, als auch in den korrespondierenden GZs und TZs von wachsenden Preantral- und Antralfollikeln in menschlichen Ovarschnitten identifiziert. Des Weiteren wurde es in Lutealzellen des Gelbkörpers, welche aus den GZs und TZs hervorgehen, festgestellt. IVF-abstammende GZs ähneln den ovulatorischen GZs und/oder dem Gelbkörper, aber proliferieren nicht. Deshalb wurde zusätzlich die Granulosa-ähnliche Tumorzelllinie KGN als weiteres Model für den wachsenden Follikel untersucht. Diese Zellen exprimieren auch NOX4. Die Akkumulation von ROS im Medium durch kultivierte GZs und KGN konnte ermittelt werden und wurde von einem spezifischen NOX4 Blocker GKT137831 gehemmt. Dieser Blocker reduzierte spezifisch die H2O2-Produktion um 45 % in GZs und KGN, was auf einen erheblichen Beitrag der NOX4-Aktivität an der H2O2-Produktion hinweist. Dies ist vielmehr ein langlebiges und das einzig membran-permeable ROS. H2O2 könnte zu Nachbarzellen diffundieren und Studien weisen auf eine Aufnahme von extrazellulärem H2O2 in GZs hin, an welcher Aquaporine beteiligt sind. Die Gonadotropine, follikelstimulierendes Hormon (FSH) und humanes Choriongonadotropin (hCG), spielen eine Schlüsselrolle in der Reproduktion. Sie induzieren die Reifung von ovariellen Follikeln und den Eisprung. FSH und hCG veränderten jedoch nicht die Expression von NOX4, aber erhöhten die Expression von antioxidativen Enzymen, einschließlich der Katalase. Dies weist auf einen Einfluss dieser Hormone auf die ovarielle ROS-Homöostase hin. H2O2 und FSH erhöhten auch die MAPK-Phosphorylierung, was auf eine Konvergenz der Signalwege hinweist. Zusätzlich erhöhte H2O2 nach Zugabe zu den GZs einige Zytokine. Dies bringt H2O2 mit inflammatorischen Ereignissen in Verbindung, welche möglicherweise beim Eisprung und/oder dem Abbau des Gelbkörpers beteiligt sind. Die Hemmung der H2O2-Produktion durch GKT137831 zeigte keinen Einfluss auf die Zellviabilität, aber senkte die Expression von CYP11A1, ein zentrales Enzym der Steroidsynthese. Dies deutet auf eine Beteiligung des Erhalts des steroidogenen Phänotyps der GZs hin. In proliferierenden KGN Zellen reduzierte GKT137831 das Zellwachstum. Dies könnte eine Rolle von H2O2 in der Zellproliferation und möglicherweise im Follikelwachstum implizieren. Die Ergebnisse dieser Studie deuten zusammenfassend auf eine wichtige Signalrolle von H2O2 in der Regulierung der GZs hin. Da ROS potenziell schädliche Moleküle sind, ist die vollständige Aufklärung der gegensätzlichen Seiten des ROS-signalisierenden Systems im menschlichen Ovar wichtig, um zukünftige therapeutische Ansätze zu entwickeln

Role of ROS and ROS generating enzymes in the human ovary

Reactive oxygen species (ROS) have long been regarded as destructive molecules that have harmful effects. However, research data emerging over the last decade have demonstrated that ROS can positively influence a range of cellular events in a manner similar to that seen for traditional second messenger molecules. Hydrogen peroxide (H2O2) appears to be the main ROS with such signalling properties, and this molecule has been shown to affect a variety of cellular functions. Its synthesis by the NADPH oxidase (NOX) family of enzymes and how these enzymes are regulated in the human ovary are poorly investigated topics. In the ovary, ROS are involved in fundamental reproductive processes, as implicated by previous studies. However, ROS can also cause oxidative stress, which is associated with impaired oocyte quality and a negative outcome of assisted reproductive techniques. Oocytes grow and mature in ovarian follicles, formed by granulosa cells (GCs) and theca cells (TCs). NOX4, a member of the ROS-producing NOX family, was identified in cultured human in-vitro fertilization (IVF)-derived GCs as well as in corresponding GCs and TCs of growing preantral and antral follicles in human ovarian sections. It was further detected in GC- and TC-derived luteal cells of the corpus luteum. IVF-derived GCs resemble ovulatory GCs and/or the corpus luteum, but do not proliferate. Therefore, as a model for the growing follicle, the granulosa-like tumour cell line KGN was further studied. These cells also express NOX4. Accumulation of ROS in the medium of cultured GCs and KGN could be detected and was inhibited by the specific NOX4 blocker GKT137831. This blocker reduced specifically the production of H2O2 by around 45 % in GCs and KGN. This indicates a major contribution of NOX4 activity to the generation of H2O2. This is a rather long-lived and the only membrane-permeable ROS. H2O2 may diffuse to neighbouring cells, and studies implicated aquaporins in the uptake of extracellular H2O2 into GCs. The gonadotropins follicle stimulating hormone (FSH) and human chorionic gonadotropin (hCG) play a key role in reproduction. They induce the maturation of ovarian follicles, and the ovulation process. FSH and hCG, however, did not alter expression of NOX4, but elevated mRNA expression of antioxidant enzymes including catalase. This indicates a role of these hormones in ovarian ROS homeostasis. H2O2 and FSH also increased MAPK-phosphorylation, suggesting convergence of the signalling pathways. Furthermore, H2O2, when added to GCs, elevated several cytokines. This implicates H2O2 in inflammatory events, which are possibly involved in ovulation and/or regression of the corpus luteum. Inhibition of H2O2 production by GKT137831 did not affect cell viability of GCs but lowered expression of CYP11A1, a crucial enzyme for steroid synthesis. This suggests involvement in the maintenance of the steroidogenic phenotype of GCs. In proliferating KGN cells, GKT137831 reduced cell growth. This may implicate a role of H2O2 in cell proliferation and possibly in follicular growth. Taken together, the results imply important roles of H2O2 in the regulation of GCs. As ROS are potentially harmful molecules, the full elucidation of the two sides of the ROS-signalling system in the human ovary is important to guide future therapeutic strategies.Reaktive Sauerstoffspezies (englisch reactive oxygen species, ROS) wurden lange Zeit als potentiell schädliche Moleküle betrachtet, die nachteilige Effekte haben. Forschungsdaten des letzten Jahrzehnts zeigen jedoch, dass ROS eine Reihe von zellulären Ereignissen positiv beeinflussen können, ähnlich wie traditionelle Second Messenger Moleküle. Wasserstoffperoxid (H2O2) scheint das wichtigste ROS mit einer solchen Signaleigenschaft zu sein. Es wurde beobachtet, dass dieses Molekül eine Vielfalt von zellulären Funktionen beeinflusst. Seine Synthese durch die Enzymfamilie NADPH Oxidasen (NOX) und dessen Regulierung im menschlichen Ovar sind kaum erforschte Themen. ROS sind im Ovar in fundamentalen Reproduktionsprozessen involviert, was frühere Studien implizieren. Allerdings können ROS auch oxidativen Stress verursachen, welcher mit schlechter Oozytenqualität und negativem Ergebnis bei assistierten Reproduktionstechniken verbunden ist. Oozyten wachsen und reifen in ovariellen Follikeln, welche aus Granulosazellen (GZs) und Thekazellen (TZs) gebildet werden. NOX4 ist ein Mitglied der ROS-produzierenden NOX Familie und wurde sowohl in kultivierten menschlichen GZs, die aus In-Vitro-Fertilisation (IVF) stammen, als auch in den korrespondierenden GZs und TZs von wachsenden Preantral- und Antralfollikeln in menschlichen Ovarschnitten identifiziert. Des Weiteren wurde es in Lutealzellen des Gelbkörpers, welche aus den GZs und TZs hervorgehen, festgestellt. IVF-abstammende GZs ähneln den ovulatorischen GZs und/oder dem Gelbkörper, aber proliferieren nicht. Deshalb wurde zusätzlich die Granulosa-ähnliche Tumorzelllinie KGN als weiteres Model für den wachsenden Follikel untersucht. Diese Zellen exprimieren auch NOX4. Die Akkumulation von ROS im Medium durch kultivierte GZs und KGN konnte ermittelt werden und wurde von einem spezifischen NOX4 Blocker GKT137831 gehemmt. Dieser Blocker reduzierte spezifisch die H2O2-Produktion um 45 % in GZs und KGN, was auf einen erheblichen Beitrag der NOX4-Aktivität an der H2O2-Produktion hinweist. Dies ist vielmehr ein langlebiges und das einzig membran-permeable ROS. H2O2 könnte zu Nachbarzellen diffundieren und Studien weisen auf eine Aufnahme von extrazellulärem H2O2 in GZs hin, an welcher Aquaporine beteiligt sind. Die Gonadotropine, follikelstimulierendes Hormon (FSH) und humanes Choriongonadotropin (hCG), spielen eine Schlüsselrolle in der Reproduktion. Sie induzieren die Reifung von ovariellen Follikeln und den Eisprung. FSH und hCG veränderten jedoch nicht die Expression von NOX4, aber erhöhten die Expression von antioxidativen Enzymen, einschließlich der Katalase. Dies weist auf einen Einfluss dieser Hormone auf die ovarielle ROS-Homöostase hin. H2O2 und FSH erhöhten auch die MAPK-Phosphorylierung, was auf eine Konvergenz der Signalwege hinweist. Zusätzlich erhöhte H2O2 nach Zugabe zu den GZs einige Zytokine. Dies bringt H2O2 mit inflammatorischen Ereignissen in Verbindung, welche möglicherweise beim Eisprung und/oder dem Abbau des Gelbkörpers beteiligt sind. Die Hemmung der H2O2-Produktion durch GKT137831 zeigte keinen Einfluss auf die Zellviabilität, aber senkte die Expression von CYP11A1, ein zentrales Enzym der Steroidsynthese. Dies deutet auf eine Beteiligung des Erhalts des steroidogenen Phänotyps der GZs hin. In proliferierenden KGN Zellen reduzierte GKT137831 das Zellwachstum. Dies könnte eine Rolle von H2O2 in der Zellproliferation und möglicherweise im Follikelwachstum implizieren. Die Ergebnisse dieser Studie deuten zusammenfassend auf eine wichtige Signalrolle von H2O2 in der Regulierung der GZs hin. Da ROS potenziell schädliche Moleküle sind, ist die vollständige Aufklärung der gegensätzlichen Seiten des ROS-signalisierenden Systems im menschlichen Ovar wichtig, um zukünftige therapeutische Ansätze zu entwickeln

A framework for human microbiome research

A variety of microbial communities and their genes (the microbiome) exist throughout the human body, with fundamental roles in human health and disease. The National Institutes of Health (NIH)-funded Human Microbiome Project Consortium has established a population-scale framework to develop metagenomic protocols, resulting in a broad range of quality-controlled resources and data including standardized methods for creating, processing and interpreting distinct types of high-throughput metagenomic data available to the scientific community. Here we present resources from a population of 242 healthy adults sampled at 15 or 18 body sites up to three times, which have generated 5,177 microbial taxonomic profiles from 16S ribosomal RNA genes and over 3.5 terabases of metagenomic sequence so far. In parallel, approximately 800 reference strains isolated from the human body have been sequenced. Collectively, these data represent the largest resource describing the abundance and variety of the human microbiome, while providing a framework for current and future studies

Structure, function and diversity of the healthy human microbiome

Author Posting. © The Authors, 2012. This article is posted here by permission of Nature Publishing Group. The definitive version was published in Nature 486 (2012): 207-214, doi:10.1038/nature11234.Studies of the human microbiome have revealed that even healthy individuals differ remarkably in the microbes that occupy habitats such as the gut, skin and vagina. Much of this diversity remains unexplained, although diet, environment, host genetics and early microbial exposure have all been implicated. Accordingly, to characterize the ecology of human-associated microbial communities, the Human Microbiome Project has analysed the largest cohort and set of distinct, clinically relevant body habitats so far. We found the diversity and abundance of each habitat’s signature microbes to vary widely even among healthy subjects, with strong niche specialization both within and among individuals. The project encountered an estimated 81–99% of the genera, enzyme families and community configurations occupied by the healthy Western microbiome. Metagenomic carriage of metabolic pathways was stable among individuals despite variation in community structure, and ethnic/racial background proved to be one of the strongest associations of both pathways and microbes with clinical metadata. These results thus delineate the range of structural and functional configurations normal in the microbial communities of a healthy population, enabling future characterization of the epidemiology, ecology and translational applications of the human microbiome.This research was supported in part by National Institutes of Health grants U54HG004969 to B.W.B.; U54HG003273 to R.A.G.; U54HG004973 to R.A.G., S.K.H. and J.F.P.; U54HG003067 to E.S.Lander; U54AI084844 to K.E.N.; N01AI30071 to R.L.Strausberg; U54HG004968 to G.M.W.; U01HG004866 to O.R.W.; U54HG003079 to R.K.W.; R01HG005969 to C.H.; R01HG004872 to R.K.; R01HG004885 to M.P.; R01HG005975 to P.D.S.; R01HG004908 to Y.Y.; R01HG004900 to M.K.Cho and P. Sankar; R01HG005171 to D.E.H.; R01HG004853 to A.L.M.; R01HG004856 to R.R.; R01HG004877 to R.R.S. and R.F.; R01HG005172 to P. Spicer.; R01HG004857 to M.P.; R01HG004906 to T.M.S.; R21HG005811 to E.A.V.; M.J.B. was supported by UH2AR057506; G.A.B. was supported by UH2AI083263 and UH3AI083263 (G.A.B., C. N. Cornelissen, L. K. Eaves and J. F. Strauss); S.M.H. was supported by UH3DK083993 (V. B. Young, E. B. Chang, F. Meyer, T. M. S., M. L. Sogin, J. M. Tiedje); K.P.R. was supported by UH2DK083990 (J. V.); J.A.S. and H.H.K. were supported by UH2AR057504 and UH3AR057504 (J.A.S.); DP2OD001500 to K.M.A.; N01HG62088 to the Coriell Institute for Medical Research; U01DE016937 to F.E.D.; S.K.H. was supported by RC1DE0202098 and R01DE021574 (S.K.H. and H. Li); J.I. was supported by R21CA139193 (J.I. and D. S. Michaud); K.P.L. was supported by P30DE020751 (D. J. Smith); Army Research Office grant W911NF-11-1-0473 to C.H.; National Science Foundation grants NSF DBI-1053486 to C.H. and NSF IIS-0812111 to M.P.; The Office of Science of the US Department of Energy under Contract No. DE-AC02-05CH11231 for P.S. C.; LANL Laboratory-Directed Research and Development grant 20100034DR and the US Defense Threat Reduction Agency grants B104153I and B084531I to P.S.C.; Research Foundation - Flanders (FWO) grant to K.F. and J.Raes; R.K. is an HHMI Early Career Scientist; Gordon&BettyMoore Foundation funding and institutional funding fromthe J. David Gladstone Institutes to K.S.P.; A.M.S. was supported by fellowships provided by the Rackham Graduate School and the NIH Molecular Mechanisms in Microbial Pathogenesis Training Grant T32AI007528; a Crohn’s and Colitis Foundation of Canada Grant in Aid of Research to E.A.V.; 2010 IBM Faculty Award to K.C.W.; analysis of the HMPdata was performed using National Energy Research Scientific Computing resources, the BluBioU Computational Resource at Rice University

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity.

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Novel multiple sclerosis susceptibility loci implicated in epigenetic regulation.

We conducted a genome-wide association study (GWAS) on multiple sclerosis (MS) susceptibility in German cohorts with 4888 cases and 10,395 controls. In addition to associations within the major histocompatibility complex (MHC) region, 15 non-MHC loci reached genome-wide significance. Four of these loci are novel MS susceptibility loci. They map to the genes L3MBTL3, MAZ, ERG, and SHMT1. The lead variant at SHMT1 was replicated in an independent Sardinian cohort. Products of the genes L3MBTL3, MAZ, and ERG play important roles in immune cell regulation. SHMT1 encodes a serine hydroxymethyltransferase catalyzing the transfer of a carbon unit to the folate cycle. This reaction is required for regulation of methylation homeostasis, which is important for establishment and maintenance of epigenetic signatures. Our GWAS approach in a defined population with limited genetic substructure detected associations not found in larger, more heterogeneous cohorts, thus providing new clues regarding MS pathogenesis

Low-Frequency and Rare-Coding Variation Contributes to Multiple Sclerosis Risk

Multiple sclerosis is a complex neurological disease, with 3c20% of risk heritability attributable to common genetic variants, including >230 identified by genome-wide association studies. Multiple strands of evidence suggest that much of the remaining heritability is also due to additive effects of common variants rather than epistasis between these variants or mutations exclusive to individual families. Here, we show in 68,379 cases and controls that up to 5% of this heritability is explained by low-frequency variation in gene coding sequence. We identify four novel genes driving MS risk independently of common-variant signals, highlighting key pathogenic roles for regulatory T cell homeostasis and regulation, IFN\u3b3 biology, and NF\u3baB signaling. As low-frequency variants do not show substantial linkage disequilibrium with other variants, and as coding variants are more interpretable and experimentally tractable than non-coding variation, our discoveries constitute a rich resource for dissecting the pathobiology of MS. In a large multi-cohort study, unexplained heritability for multiple sclerosis is detected in low-frequency coding variants that are missed by GWAS analyses, further underscoring the role of immune genes in MS pathology

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

SummaryBackground Azithromycin has been proposed as a treatment for COVID-19 on the basis of its immunomodulatoryactions. We aimed to evaluate the safety and efficacy of azithromycin in patients admitted to hospital with COVID-19.Methods In this randomised, controlled, open-label, adaptive platform trial (Randomised Evaluation of COVID-19Therapy [RECOVERY]), several possible treatments were compared with usual care in patients admitted to hospitalwith COVID-19 in the UK. The trial is underway at 176 hospitals in the UK. Eligible and consenting patients wererandomly allocated to either usual standard of care alone or usual standard of care plus azithromycin 500 mg once perday by mouth or intravenously for 10 days or until discharge (or allocation to one of the other RECOVERY treatmentgroups). Patients were assigned via web-based simple (unstratified) randomisation with allocation concealment andwere twice as likely to be randomly assigned to usual care than to any of the active treatment groups. Participants andlocal study staff were not masked to the allocated treatment, but all others involved in the trial were masked to theoutcome data during the trial. The primary outcome was 28-day all-cause mortality, assessed in the intention-to-treatpopulation. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936.Findings Between April 7 and Nov 27, 2020, of 16 442 patients enrolled in the RECOVERY trial, 9433 (57%) wereeligible and 7763 were included in the assessment of azithromycin. The mean age of these study participants was65·3 years (SD 15·7) and approximately a third were women (2944 [38%] of 7763). 2582 patients were randomlyallocated to receive azithromycin and 5181 patients were randomly allocated to usual care alone. Overall,561 (22%) patients allocated to azithromycin and 1162 (22%) patients allocated to usual care died within 28 days(rate ratio 0·97, 95% CI 0·87–1·07; p=0·50). No significant difference was seen in duration of hospital stay (median10 days [IQR 5 to >28] vs 11 days [5 to >28]) or the proportion of patients discharged from hospital alive within 28 days(rate ratio 1·04, 95% CI 0·98–1·10; p=0·19). Among those not on invasive mechanical ventilation at baseline, nosignificant difference was seen in the proportion meeting the composite endpoint of invasive mechanical ventilationor death (risk ratio 0·95, 95% CI 0·87–1·03; p=0·24).Interpretation In patients admitted to hospital with COVID-19, azithromycin did not improve survival or otherprespecified clinical outcomes. Azithromycin use in patients admitted to hospital with COVID-19 should be restrictedto patients in whom there is a clear antimicrobial indication