Jean-Paul Cristol

Alain R Thierry

Anne-Sophie Bargnoux

Marion Morena-Carrere

Bernard Canaud

What is the role of the neutrophil extracellular traps in the cardiovascular disease burden associated with hemodialysis bioincompatibility?

Keywords: neutrophil extracellular traps, ROS, NADPH oxidase, myeloperoxidase, circulating DNA, bioincompatibility, hemodialysis, CKD

Despite significant progress in dialysis modalities, intermittent renal replacement therapy remains an "unphysiological" treatment that imperfectly corrects uremic disorders and may lead to low-grade chronic inflammation, neutrophil activation, and oxidative stress due to repetitive blood/membrane interactions contributing to the "remaining uremic syndrome" and cardiovascular disease burden of hemodialysis patients. Understanding dialysis bioincompatibility pathways still remains a clinical and biochemical challenge. Indeed, surrogate biomarkers of inflammation including C-reactive protein could not discriminate between all components involved in these complex pathways. A few examples may serve to illustrate the case. Cytokine release during dialysis sessions may be underestimated due to their removal using high-flux dialysis or hemodiafiltration modalities. Complement activation is recognized as a key event of bioincompatibility. However, it appears as an early and transient event with anaphylatoxin level normalization at the end of the dialysis session. Complement activation is generally assumed to trigger leukocyte stimulation leading to proinflammatory mediators' secretion and oxidative burst. In addition to being part of the innate immune response involved in eliminating physically and enzymatically microbes, the formation of Neutrophil Extracellular Traps (NETs), known as NETosis, has been recently identified as a major harmful component in a wide range of pathologies associated with inflammatory processes. NETs result from the neutrophil degranulation induced by reactive oxygen species overproduction via NADPH oxidase and consist of modified chromatin decorated with serine proteases, elastase, bactericidal proteins, and myeloperoxidase (MPO) that produces hypochlorite anion. Currently, NETosis remains poorly investigated as a sensitive and integrated marker of bioincompatibility in dialysis. Only scarce data could be found in the literature. Oxidative burst and NADPH oxidase activation are well-known events in the bioincompatibility phenomenon. NET byproducts such as elastase, MPO, and circulating DNA have been reported to be increased in dialysis patients more specifically during dialysis sessions, and were identified as predictors of poor outcomes. As NETs and MPO could be taken up by endothelium, NETs could be considered as a vascular memory of intermittent bioincompatibility phenomenon. In this working hypothesis article, we summarized the puzzle pieces showing the involvement of NET formation during hemodialysis and postulated that NETosis may act as a disease modifier and may contribute to the comorbid burden associated with dialysis bioincompatibility.

Introduction

The development of maintenance hemodialysis (HD) for end-stage kidney disease patients is a successful therapeutic story supporting the lives of almost 3 million patients worldwide [START_REF] Himmelfarb | The current and future landscape of dialysis[END_REF]. Despite improvement in dialysis methods, intermittent renal replacement therapy remains an unphysiological treatment that only partially clears uremic toxins, imperfectly corrects fluid imbalance, and cyclically induces osmotic and electrolytic shifts contributing to dialysisinduced systemic stress (2, 3). In addition, repetitive blood contact with artificial material and dialysis fluid is recognized as a cause of low-grade chronic inflammation, neutrophil activation, and oxidative stress [START_REF] Morena | Why hemodialysis patients are in a prooxidant state? What could be done to correct the pro/antioxidant imbalance [in process citation[END_REF]. Neutrophil oxidative burst can result in the extrusion of neutrophil extracellular traps (NETs), which consist of extracellular chromatin containing various granular proteins, proteolytic enzymes, and reactive oxygen species (ROS)-producing proteins. Hemoincompatibility of the extracorporeal circuit contributes to endothelium dysfunction, increases thrombogenicity, and ultimately cardiovascular disease burden [START_REF] Assa | Hemodialysisinduced regional left ventricular systolic dysfunction and inflammation: a cross-sectional study[END_REF][START_REF] Mcintyre | Recurrent circulatory stress: the dark side of dialysis[END_REF].

In recent years, the management of HD patients has evolved in two directions. First, improvements in dialysis techniques, such as the use of ultrapure dialysate, online hemodiafiltration (HDF), and surfaces with reduced complement activation, have significantly enhanced biocompatibility. Conversely, HD patient demographics have shifted from younger individuals with minimal comorbidities to older, frail patients with preexisting complex comorbidities. The intricate interplay between neutrophil activation, platelets, and endothelial cells plays a crucial role in HD-induced endothelial dysfunction, potentially exacerbating vascular impairment and organ damage in this vulnerable population.

In this working hypothesis article, we provide a concise overview of the contribution of HD bioincompatibility to dialysis-induced systemic morbidity. We postulate that dialysis triggers an innate immune response through the formation of NETs. Given that uncontrolled NET formation or NETosis has previously been identified in various inflammatory systemic diseases, NETs could be a pivotal piece in the bioincompatibility puzzle, potentially contributing to the development of vascular diseases and poor outcomes in HD-treated patients.

Imbalance and maladaptive response to NET formation contribute to poor outcomes of major systemic inflammatory diseases in the general population 2.1. NET as a first-line defense against microorganisms

Neutrophils are regarded as the first line of innate immune defense. A part of neutrophil function aims at producing NETs, which were discovered only two decades ago [START_REF] Brinkmann | Neutrophil extracellular traps in the second decade[END_REF] and were first described as a special form of programmed cell death different from necrosis and apoptosis [START_REF] Takei | Rapid killing of human neutrophils by the potent activator phorbol 12-myristate 13-acetate (PMA) accompanied by changes different from typical apoptosis or necrosis[END_REF]. In response to stimuli, NETs extracellularly release extensive fibrous structures mainly composed of chromatin containing granulomatous and cytosolic proteins assembled on a scaffold of released chromatin. The classical NETosis is strictly dependent on NADPH oxidase activation [START_REF] Fuchs | Novel cell death program leads to neutrophil extracellular traps[END_REF]. ROS overproduction induces neutrophil degranulation leading to the release of the protein complex "azurosome," which includes serine proteases, such as neutrophilic elastase (NE), cathepsin G, azurocidin, and S100 family proteins, such as calprotectin [START_REF] Sprenkeler | S100A8/A9 is a marker for the release of neutrophil extracellular traps and induces neutrophil activation[END_REF] and myeloperoxidase (MPO) (Figure 1). Serine proteases as well as MPO could break down lamin and histones contributing to the chromatin decondensation and destruction of the nuclear envelope. At the final stage of NETosis, pores are formed in the plasma membrane and chromatin is released into the environment with NET formation. Proteins released from the granules are strongly bound to the decondensed chromatin [START_REF] Fuchs | Novel cell death program leads to neutrophil extracellular traps[END_REF]. These structures physically entrap microbes and exploit the strong enzymatic power of anchored enzymes and coagulant function to suppress their dissemination. In addition, NET byproducts (nuclear and mitochondrial DNA fragments associated with nucleosomes, histones, granule proteins, etc.) trigger an inflammatory response. Thus, NETs appear to have a "double-edged sword" function. On the one hand, NETs participate in an early efficient response to neutralize microorganisms. On the other hand, NET byproducts in exaggerated or uncontrolled amounts may be harmful to the host by being toxic to endothelial cells and parenchymal tissue [START_REF] Thierry | SARS-CoV2 may evade innate immune response, causing uncontrolled neutrophil extracellular traps formation and multi-organ failure[END_REF] and by leading to immunothrombosis [START_REF] Thierry | Neutrophil extracellular traps and by-products play a Key role in COVID-19: pathogenesis, risk factors, and therapy[END_REF].

NET-associated toxicity

NETs are macromolecular complexes, and their byproducts in excess may potentially lead to toxic effects. As they are the major NET constituents, nucleic acids appear as the most toxic ones mainly due to their high degradation in the blood resulting in the accumulation of circulating DNA (cirDNA) in the form of mononucleosomes and circulating mitochondrial (mt) DNA. The presence of cirDNA was first described in 1977, and chronic vascular toxicity was hypothesized [START_REF] Leon | Free DNA in the serum of cancer patients and the effect of therapy[END_REF]. Both types of nucleic acids appear to be the primary source of damage-associated molecular patterns (DAMPs), also known as alarmins. These endogenous molecules are recognized by pattern recognition receptors (PRRs), and their activation leads to the synthesis of proinflammatory cytokines, inducing inflammation [START_REF] Thierry | Neutrophil extracellular traps and by-products play a Key role in COVID-19: pathogenesis, risk factors, and therapy[END_REF]. Additionally, short DNA fragments from mononucleosomes are recognized by DNA sensors such as Tolllike receptor 9 (TLR9), which is more specific for microbial DNA, and stimulator of interferon genes (STING), capable of recognizing a wide range of DNA. TLR9 and STING are located in endosomes and the cytosol, respectively. This recognition triggers the secretion of type I interferons and proinflammatory cytokines, thereby regulating host defense mechanisms. Histones from mononucleosomes were reported to be toxic by triggering inflammation as well. Because of its bacterial antecedent, mtDNA can serve as a DAMP, in particular, mtDNA can bind to the TLR9 receptor of leukocytes via hypomethylated CpG motifs and trigger an inflammatory reaction [START_REF] West | Mitochondrial DNA in innate immune responses and inflammatory pathology[END_REF]. As exacerbated inflammation has been associated with a failure in innate immune tolerance to self-DNA, in particular in inflammatory disorders, it is hypothesized that cirDNA, mostly of mitochondrial origin, may be a mediator of transfusion-related acute lung injury [START_REF] Lee | Blood transfusion products contain mitochondrial DNA damage-associated molecular patterns: a potential effector of transfusion-related acute lung injury[END_REF]. It was shown that mtDNA DAMP levels may predict the development of acute respiratory distress syndrome after multiple transfusions [START_REF] Simmons | Potential contribution of mitochondrial DNA damage associated molecular patterns in transfusion products to the development of acute respiratory distress syndrome after multiple transfusions[END_REF].

In addition to DNA and histones, granule proteins such as MPO and NE also contribute to the triggering of an inflammatory process. In the presence of hydrogen peroxide and halides, MPO catalyzes the production of hypochlorous acid, the latter could generate long-lived oxidants, such as chloramines, enhancing long-lasting oxidative stress [START_REF] Morena | Oxidative stress in hemodialysis patients: is NADPH oxidase complex the culprit?[END_REF]. Multiple lines of evidence demonstrate that MPO is associated with oxidative stress and inflammation [START_REF] Ndrepepa | Myeloperoxidase -a bridge linking inflammation and oxidative stress with cardiovascular disease[END_REF][START_REF] Maiocchi | Myeloperoxidase: a versatile mediator of endothelial dysfunction and therapeutic target during cardiovascular disease[END_REF]. MPO activation is involved in endothelial dysfunction, reduced NO availability, and impaired vasoreactivity leading to cardiovascular disease [START_REF] Ndrepepa | Myeloperoxidase -a bridge linking inflammation and oxidative stress with cardiovascular disease[END_REF]. During inflammatory cardiovascular disease, MPO could be released by activated leukocytes and transported to the endothelium resulting in sequestration and accumulation within the vascular endothelium. Thus, the synergistic action of NADPH oxidase and MPO activation could be a pivotal event in NETinduced endothelial dysfunction and vascular damage [START_REF] Bonaventura | Neutrophil extracellular traps and cardiovascular diseases: an update[END_REF]. In addition, MPO-damaged biomolecules can act as chemo-attractants and modulate gene expression enhancing inflammation and tissue damage [START_REF] Maiocchi | Myeloperoxidase: a versatile mediator of endothelial dysfunction and therapeutic target during cardiovascular disease[END_REF][START_REF] Hawkins | Role of myeloperoxidase and oxidant formation in the extracellular environment in inflammation-induced tissue damage[END_REF]. NE is known as one of the most destructive enzymes in the body and overwhelming NE release together with simultaneously produced ROS can cause local tissue injury and neutrophil-mediated endothelial injury1 [START_REF] Henriksen | Human neutrophil elastase: mediator and therapeutic target in atherosclerosis[END_REF].

NETs as components of inflammatory diseases

Unbalanced NET formation and maladaptive response were observed in infectious and non-infectious diseases [START_REF] Thierry | SARS-CoV2 may evade innate immune response, causing uncontrolled neutrophil extracellular traps formation and multi-organ failure[END_REF][START_REF] Thierry | Neutrophil extracellular traps and by-products play a Key role in COVID-19: pathogenesis, risk factors, and therapy[END_REF][START_REF] Pastor | Association of neutrophil extracellular traps with the production of circulating DNA in patients with colorectal cancer[END_REF]. They have been observed in inflammatory diseases, such as pre-eclampsia, small-vessel vasculitis, and systemic lupus erythematosus (SLE). The deleterious role of NETs has been particularly studied in the context of autoimmune diseases, especially lupus. NETs expose many autoantigens in the extracellular space, in particular double-strand DNA or MPO. Numerous studies have demonstrated the existence of the deregulation of NETs during lupus. Increased circulating levels of NETs and evidence for NET in SLE patients' tissue are associated with the number of immune complexes typically containing nucleic acids associated with various proteins, such as the chromatin-associated protein HMGB1, the antimicrobial peptide LL39, ribonucleoproteins, and others [START_REF] Lamphier | Novel small molecule inhibitors of TLR7 and TLR9: mechanism of action and efficacy in vivo[END_REF]. Of note, Elkon (25) found in patients with SLE an impaired ability to degrade NET. The unbalanced NET formation was also shown in acute respiratory distress syndrome, acute lung injury, diabetes, and sepsis and was suggested in sickle cell disease, cystic fibrosis, periodontitis, autoimmune vasculitis, rheumatoid arthritis, severe obesity, pre-eclampsia, or kidney diseases [START_REF] Thierry | Neutrophil extracellular traps and by-products play a Key role in COVID-19: pathogenesis, risk factors, and therapy[END_REF]. NET deleterious effects were observed in infectious diseases as well. Microbe-induced respiratory illnesses such as lung endothelial injury were associated with high NET formation. Among the numerous studies reporting it, NETs were detected in the bronchoalveolar lavage fluid of children with severe respiratory syncytial virus infection of the lower respiratory tract [START_REF] Caudrillier | Platelets induce neutrophil extracellular traps in transfusion-related acute lung injury[END_REF]. In addition, pathologies of chikungunya virus, simian immunodeficiency virus, influenza, parvovirus, rhinovirus, and influenza pneumonia involve neutrophils and NETs [START_REF] Thierry | Neutrophil extracellular traps and by-products play a Key role in COVID-19: pathogenesis, risk factors, and therapy[END_REF]. Among infectious respiratory diseases, NETs have been involved in SARS-COV-2 infection [START_REF] Guéant | Elastase and exacerbation of neutrophil innate immunity are involved in multi-visceral manifestations of COVID-19[END_REF][START_REF] Pisareva | Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosomeassociated circulating DNA[END_REF]. Recently, it has been shown that NETosis was a key factor for acute respiratory distress [START_REF] Thierry | Neutrophil extracellular traps and by-products play a Key role in COVID-19: pathogenesis, risk factors, and therapy[END_REF][START_REF] Guéant | Elastase and exacerbation of neutrophil innate immunity are involved in multi-visceral manifestations of COVID-19[END_REF]. Interestingly, NETosis persists in the post-acute COVID phase, suggesting a feedback loop to NET formation due to the combined stimulation (i) from the NETs byproducts, in particular, the cirDNA fragments acting as DAMPs and neutrophil activation; (ii) from the subsequent platelets activation; and (iii) the generation of auto-antibodies [START_REF] Pisareva | Persistence of neutrophil extracellular traps and anticardiolipin auto-antibodies in post-acute phase COVID-19 patients[END_REF]. In this context, the COVID post-acute phase NETosis could enhance the uremia-and HD-induced low-grade inflammation and could account in part for the higher risk of death in patients on maintenance HD [START_REF] Li | Mortality and risk factors for COVID-19 in hemodialysis patients: a systematic review and meta-analysis[END_REF][START_REF] Bruchfeld | The COVID-19 pandemic: consequences for nephrology[END_REF].

In brief, the unbalanced formation of NETs contributes to vascular disease progression in various diseases associated with chronic inflammatory processes [START_REF] Thierry | Neutrophil extracellular traps and by-products play a Key role in COVID-19: pathogenesis, risk factors, and therapy[END_REF][START_REF] Vorobjeva | NETosis: molecular mechanisms, role in physiology and pathology[END_REF]. NETs result from neutrophil degranulation induced by ROS overproduction via NADPH oxidase, consisting of a mixture of modified chromatin, cirDNA, and bactericidal proteins (Figure 1). Despite its role in the vascular disease pathway, NETosis remains poorly investigated in HD. As NET components could be taken up and could lead to damage in the endothelium, these components might be considered as a marker of vascular memory of intermittent bioincompatibility phenomenon.

NET formation and link with bioincompatibility of the extracorporeal circuit: highlighting the prominent role of the hemodialyzer acting as bioincubator

Bioincompatibility phenomenon

Bioincompatibility (or more specifically, hemobioincompatibility) of the extracorporeal circuit is recognized as a cause of dialysis-related low-grade inflammation contributing to morbidity and mortality through systemic effects [START_REF] Zhang | Prognostic role of C-reactive protein and interleukin-6 in dialysis patients: a systematic review and meta-analysis[END_REF]. HD allows the removal by diffusion and convection of low molecular weight uremic toxins and excess water by the use of a semipermeable polymeric membrane in an extracorporeal system [START_REF] Canaud | Hidden risks associated with conventional short intermittent hemodialysis: a call for action to mitigate cardiovascular risk and morbidity[END_REF][START_REF] Bonomini | Biocompatibility of surface-modified membranes for chronic hemodialysis therapy[END_REF]. However, intermittent HD remains an imperfect treatment that only partially corrects uremic abnormalities and could be harmful to patients. The "unphysiological" nature of intermittent HD and HD-induced systematic stress have long been recognized as a leading cause of dialysis intolerance [START_REF] Kjellstrand | The "unphysiology" of dialysis: a major cause of dialysis side effects?[END_REF]. Bioincompatibility complex syndrome results from the interaction of blood with multiple components of the extracorporeal circuit. In brief, as soon as blood leaves the vascular component and enters the extracorporeal circuit, it encounters multiple stimuli that contribute to the activation of a cascade of serum proteins and blood cells. Dialysate itself is a potential biological stressor. Indeed, the chemical composition including acetate or citrate could result in metabolic stress [START_REF] Gubensek | Influence of citrate concentration on the activation of blood cells in an in vitro dialysis setup[END_REF]. On the other hand, dialysis fluid contaminants, bacterial translocation, and bacterial-derived products (lipopolysaccharide and muramyl dipeptide) could activate leukocytes (2). Blood-dialyzer interaction that occurs in the warm aquatic environment of the dialyzer creates the perfect conditions for a bioincubator to nurture biocell reactions. Other exogenous stressors including venipuncture, blood-air interface, mechanical cell trauma (i.e., pump), nature of blood tubing, and contact with foreign material of the extracorporeal circuit contribute to acute phase reactions [START_REF] Bowry | Blood-incompatibility in haemodialysis: alleviating inflammation and effects of coagulation[END_REF]. Artificial membrane packed in the hemodialyzer represents the centerpiece of HD, and the material of the dialysis membrane bathed by dialysis fluid and its components are the main determinants of hemobioincompatibility. HD strategy has developed membrane technology in order to better clear uremic toxins and improve biocompatibility. Indeed, the use of synthetic membranes [made of polysulfone, polyethersulfone, poly(methyl methacrylate), polyester polymer alloy, polyacrylonitrile, polycarbonate, and polyamide] has allowed an improvement of patient outcomes compared to the first HD cuprophan membranes based on cellulose derived from cotton. New strategies have been considered in the development of HD membranes by incorporating bioactive compounds, such as vitamin E [START_REF] Bargnoux | Vitamin E-coated polysulfone membrane improved red blood cell antioxidant status in hemodialysis patients[END_REF], lipoic acid, or heparin [START_REF] Morena | Biocompatibility of heparin-grafted hemodialysis membranes: impact on monocyte chemoattractant protein-1 circulating level and oxidative status[END_REF]. Recently, a new generation of dialyzer membranes emerged, comprising a blend of polysulfone and polyvinylpyrrolidone (PVP), with small amounts of α-tocopherol added to stabilize the blood-side surface of the membrane. The robust hydrophilic layer on the membrane surface enhances the hemocompatibility of polysulfone membranes through improved antifouling ability, which yields less protein adsorption and lower coagulation activation [START_REF] Ehlerding | Performance and hemocompatibility of a novel polysulfone dialyzer: a randomized controlled trial[END_REF].

Dialysis-related inflammation, enhanced role of dialysis fluid contamination

Observational studies have shown that low-grade inflammation, as assessed by highly sensitive C-reactive protein (CRP), was highly predictive of cardiovascular disease (CVD) in the general population [START_REF] Ridker | Clinical application of C-reactive protein for cardiovascular disease detection and prevention[END_REF][START_REF] Carriere | Biomarkers of inflammation and malnutrition associated with early death in healthy elderly people[END_REF], advanced chronic kidney disease patients [START_REF] Menon | C-reactive protein and albumin as predictors of all-cause and cardiovascular mortality in chronic kidney disease[END_REF] and dialysisdependent patients [START_REF] Terrier | Association between novel indices of malnutrition-inflammation complex syndrome and cardiovascular disease in hemodialysis patients[END_REF]. The CRP increase results from an imbalance between pro-and antiinflammatory cytokines involving several factors. The first hit is the decrease in glomerular filtration rate (GFR), which is associated with an increase in pro-inflammatory cytokines [START_REF] Shlipak | Elevations of inflammatory and procoagulant biomarkers in elderly persons with renal insufficiency[END_REF]. Further hits contribute to the low-grade inflammation being part of the uremic complex syndrome, such as toxin accumulation [START_REF] Wojtaszek | Uremic toxins, oxidative stress, atherosclerosis in chronic kidney disease, and kidney transplantation[END_REF], vascular access type [START_REF] Colì | Role of the hemodialysis vascular access type in inflammation status and monocyte activation[END_REF], intercurrent or subclinical infection including periodontal disease [START_REF] Miyata | Periodontal disease in patients receiving dialysis[END_REF], cytokine genetic background, or dialysis-induced mainly mediated through bioincompatibility reactions. As a result, the net prominence of proinflammatory cytokines contributes significantly to the poor outcome of end-stage kidney disease patients [START_REF] Zhang | Prognostic role of C-reactive protein and interleukin-6 in dialysis patients: a systematic review and meta-analysis[END_REF][START_REF] Badiou | Fine-tuning of the prediction of mortality in hemodialysis patients by use of cytokine proteomic determination[END_REF].

Although high-sensitive CRP and interleukin levels have been extensively reported as biomarkers of uremic-and dialysis-induced inflammatory response, they have serious limitations when exploring bioincompatibility pathways. With the improvement of dialysis modalities (i.e., high-flux synthetic polymer with better biocompatible profile) and generalization of ultrapure dialysis fluid, CRP levels have significantly decreased in dialysis patients. As an example, the median CRP locally reported by our group reached 7.5 mg/L at the beginning of the century [START_REF] Terrier | Association between novel indices of malnutrition-inflammation complex syndrome and cardiovascular disease in hemodialysis patients[END_REF], further decreased at 5.7 mg/L in the FRENCHIE randomized clinical trial study [START_REF] Morena | Treatment tolerance and patient-reported outcomes favor online hemodiafiltration compared to high-flux hemodialysis in the elderly[END_REF] down to 4 mg/L in the Fragil HD study (unpublished data). However, low-grade inflammation persists in a geriatric population from the South of France with a median CRP value of 2 mg/L (42). In addition, cytokine dosages are more difficult to accurately assess in a clinical setting. Indeed, dialysis technique, high-flux membranes used in HD or HDF alone have no significant impact on predialysis plasma cytokine levels [START_REF] Morena | Treatment tolerance and patient-reported outcomes favor online hemodiafiltration compared to high-flux hemodialysis in the elderly[END_REF], indicating that membrane type or fluxes are not sufficient per se, but highlighting the necessity of using ultrapure dialysis fluid to achieve such reduction. Provided these conditions are fulfilled, predialysis levels of proinflammatory cytokines and CRP might remain stable for 2 years, which could rule out any cumulative effect of intermittent stimulation [START_REF] Morena | Treatment tolerance and patient-reported outcomes favor online hemodiafiltration compared to high-flux hemodialysis in the elderly[END_REF]. Finally, plasma cytokine levels in dialysis patients reflect a delicate balance between cytokine production from the patient side and cytokine removal from the dialysis side. As a consequence, cytokine level changes observed during dialysis sessions could not be considered sensitive biomarkers of bioincompatibility.

Biological pathways of dialyzer bioincompatibility

In order to explore more precisely the bioincompatibility phenomena and pathways, we have to expand the scope of our canonical markers to understand which ones are the components of bioincompatibility reactions. Although several dialysis-related factors such as exposure to endotoxin from dialysis fluid, silent vascular access infection, and dialysate chemical composition could be involved in bioincompatibility reactions, blood interaction with the dialyzer membrane is usually assumed to be the main component. Contact phase activation, complement pathway, leukocyte activation, and thrombogenicity are wellidentified pathways of bioincompatibility. Platelet binding to the membrane is enhanced by protein adsorption such as collagen, fibronectin, and fibrinogen, which could activate in turn glycoprotein IIb/IIIa receptors. Activation of platelets may then in turn initiate the clotting cascade leading to the thrombus formation [START_REF] Frank | Role of contact system activation in hemodialyzer-induced thrombogenicity[END_REF][START_REF] Skinner | Hemodialysis-related complement and contact pathway activation and cardiovascular risk: a narrative review[END_REF]. Complement activation is also well recognized as a key event of bioincompatibility, in particular with cellulosic-based membranes. However, complement activation is an early and transient event that tends to turn down across dialysis sessions leading to the normalization of anaphylatoxin levels at the end of the dialysis [START_REF] Skinner | Hemodialysis-related complement and contact pathway activation and cardiovascular risk: a narrative review[END_REF][START_REF] Stepniewska | The activation of complement system in different types of renal replacement therapy[END_REF]. This transient complement activation has been shown to trigger leukocytes and other immunocompetent cells leading to proinflammatory mediator secretion with a subsequent oxidative burst and decrease in antioxidant defense and oxidative stress [START_REF] Vida | Does inflammation affect outcomes in dialysis patients?[END_REF].

NADPH oxidase and MPO activation as main triggers of dialyzer bioincompatibility

Once activated, immunocompetent cells are able to produce large amounts of cytokines and ROS which are clearly linked by amplification loops (55, 56). Among the functional consequences of neutrophil activation, the enhancement of ROS production results in the translocation and the assembly of cytosolic components of the NADPH oxidase enzyme [START_REF] Ward | Polymorphonuclear leukocyte oxidative burst is enhanced in patients with chronic renal insufficiency[END_REF]. The activated NADPH oxidase complex catalyzes the reduction in molecular oxygen O2 into superoxide anion O2-, which then rapidly dismutates to form hydrogen peroxide, a reaction catalyzed by superoxide dismutase. Thereafter, hypochlorous acid and chloramines could be produced by MPO [START_REF] Morena | Oxidative stress in hemodialysis patients: is NADPH oxidase complex the culprit?[END_REF]. In addition, O2-could react with large amounts of NO° produced by the inducible NO synthase, leading to a deleterious peroxynitrite (ONOO-). Such reactions triggered by the NADPH oxidase are beneficial for bacterial killing in healthy people but represent an additional deleterious source of ROS production in chronic kidney disease patients.

NADPH oxidase activation and cytokine secretion are closely linked. As such, proinflammatory cytokines induce the activation of NADPH oxidase. ROS overproduction may stimulate proinflammatory transcriptional factors such as NFκB, which in turn may increase the synthesis of cytokines and may further activate the NADPH complex. MPO activity is increased in chronic kidney disease patients and further enhanced in HD according to the type of membrane and contamination of dialysis fluid. Recently, it has been shown that, despite using biocompatible membranes including polysulfone, vitamin E-bonded type, polyester type, or PMMA, MPO was significantly increased after dialysis and was associated with polynuclear cell apoptosis [START_REF] Fukushi | Enhanced neutrophil apoptosis accompanying myeloperoxidase release during hemodialysis[END_REF]. In addition to its direct effect on the endothelium, MPO activation leads to protein carbamylation [START_REF] Lim | The role of nonenzymatic post-translational protein modifications in uremic vascular calcification[END_REF][START_REF] Koeth | Protein carbamylation predicts mortality in ESRD[END_REF][START_REF] Gillery | Usefulness of non-enzymatic post-translational modification derived products (PTMDPs) as biomarkers of chronic diseases[END_REF]. These non-enzymatic post-translational-derived product modifications alter the physiologic functions of proteins and could promote new deleterious properties. Carbamylation affects circulating proteins, such as hemoglobin, albumin, and low-and high-density lipoproteins. Carbamylation modification has also been evidenced in tissue proteins, such as collagen, elastin, and mitochondrial proteins [START_REF] Gorisse | Carbamylated proteins in renal disease: aggravating factors or just biomarkers?[END_REF]. Such protein modifications enhance vascular disease progression and the occurrence of metabolic complications, such as erythropoietin resistance, hemostatic dysfunctions, immune response disorders, insulin resistance, vascular damage, and ultimately poor outcome [START_REF] Gorisse | Carbamylated proteins in renal disease: aggravating factors or just biomarkers?[END_REF]. Interestingly, endothelial and vascular smooth muscle cells do not express the MPO enzyme. However, after leukocyte activation, MPO could be released and bind to endothelial cells and the extracellular matrix of vascular cells [START_REF] Kubala | The potentiation of myeloperoxidase activity by the glycosaminoglycan-dependent binding of myeloperoxidase to proteins of the extracellular matrix[END_REF]. Since after exposure to urea, ROS overproduction is sustained in endothelial cells for up to 48 h, one can postulate that colocalization and coactivation of NADPH oxidase and MPO have a remaining, longlasting vascular effect after dialysis session during the interdialytic period [START_REF] Apolito | Urea memory: transient cell exposure to urea causes persistent mitochondrial ROS production and endothelial dysfunction[END_REF]. MPO has been identified as a predictive biomarker of poor outcomes in patients with chest pain reflecting coronary ischemic insult and in HD patients [START_REF] Brennan | Prognostic value of myeloperoxidase in patients with chest pain[END_REF][START_REF] Kalantar-Zadeh | Serum myeloperoxidase and mortality in maintenance hemodialysis patients[END_REF].

NETosis as a unifying process of bioincompatibility: putting the pieces of the puzzle together

NETosis has been evidenced during HD mainly by exploring cirDNA and its involvement in bioincompatibility phenomenon has been suggested [START_REF] Korabecna | NETosis provides the link between activation of neutrophils on hemodialysis membrane and comorbidities in dialyzed patients[END_REF]. However, there is no "gold standard" for detecting NETs in blood or in tissue and NETosis may be easily evidenced by the detection of surrogate biomarkers, such as cirDNA, MPO, or elastase. Several pieces of the NETosis puzzle have been identified in HD (Figure 2). ROS overproduction and NADPH oxidase stimulation acting as starting points of NETosis have been extensively reported in this setting. In addition, MPO release, one of the main protein components of NETs, has been evidenced during HD sessions. Furthermore, the serine protease elastase, another component of NETs, which is significantly increased in end-stage kidney disease patients, is further enhanced after 1 h of HD [START_REF] Li | The effects of citrate dialysate in hemodialysis on polymorphonuclear elastase interaction with tissue factor and its inhibitor[END_REF]. Exacerbated elastase activity in uremic patients prior to dialysis sessions has been reported by Khatib-Massalha and colleagues (69) and elastase concentration increases progressively during dialysis sessions [START_REF] Stefoni | Inflammatory response of a new synthetic dialyzer membrane. A randomised cross-over comparison between polysulfone and helixone[END_REF]. By contrast to the early and transient activation of complement, elastase activity is enhanced after 1 h of dialysis and then sustained till the end of the session [START_REF] Ehlerding | Performance and hemocompatibility of a novel polysulfone dialyzer: a randomized controlled trial[END_REF]. Using three different synthetic polymer membranes, the new polysulfone-based FX CorAL 600 (Fresenius Medical Care, Bad Homburg, Germany), the polyarylethersulfone-based Polyflux 170H (Baxter Healthcare Corporation, Deerfield, IL), and the cellulose triacetate-based SureFlux 17UX (Nipro Medical Europe, Mechelen, Belgium), it has been shown that activation of elastase was dependent on the membrane nature and maybe on other components such as sterilization process [START_REF] Ehlerding | Performance and hemocompatibility of a novel polysulfone dialyzer: a randomized controlled trial[END_REF]. Furthermore, as recently shown, the surface modification of the new polysulfone membrane, the so-called hydrophilic layer, resulting from a PVP enrichment and a stabilization by alphatocopherol of the polysulfone polymer, decreases significantly complement activation and elastase release. It is noteworthy that early complement activation is significantly reduced but not entirely eliminated with the use of new membranes and features. As complement activation could trigger NETosis, it is important to consider this residual complement activation when planning for future complementary actions [START_REF] Ehlerding | Performance and hemocompatibility of a novel polysulfone dialyzer: a randomized controlled trial[END_REF].

NETosis has been recently evidenced during a dialysis session using a multimarker approach, through a simultaneous increase in cirDNA and a release of MPO and calprotectin [START_REF] Bieber | Neutrophil activation and neutrophil extracellular trap formation in Dialysis patients[END_REF]. Moreover, it has been shown that endothelial dysfunction, assessed by the kinetic of adhesion molecules, was also related to NET occurrence [START_REF] Bieber | Neutrophil activation and neutrophil extracellular trap formation in Dialysis patients[END_REF]. In vitro, uremic serum obtained from HD was shown to increase NET formation, MPO, and nucleosome release from polymorphonuclears and induce apoptosis of endothelial cells [START_REF] Lee | Uremic serum damages endothelium by provoking excessive neutrophil extracellular trap formation[END_REF]. Also, neutrophils isolated from HD patients tend to express an increased basal level of ROS production and cirDNA release, suggesting a uremia-induced NET formation [START_REF] Kim | Increased neutrophil extracellular trap formation in uremia is associated with chronic inflammation and prevalent coronary artery disease[END_REF]. However, after PMA or lipopolysaccharide stimulation, oxidative burst and NETosis are significantly decreased in agreement with the decreased capacity of HD neutrophils to kill extracellular bacteria and an increased susceptibility to infections in these patients [START_REF] Talal | Neutrophil degranulation and severely impaired extracellular trap formation at the basis of susceptibility to infections of hemodialysis patients[END_REF]. Levels of cirDNA were positively correlated with elastase concentrations, inflammatory markers, and oxidative stress as assessed by the lipid peroxidation index [START_REF] Coimbra | Cellfree DNA as a marker for the outcome of end-stage renal disease patients on haemodialysis[END_REF]. In this context, recent outcome-based studies have shown that cirDNAs were the best predictors of all-cause and cardiovascular mortality in end-stage kidney disease patients at 1 year, in both unadjusted and adjusted models for basic confounding factors [START_REF] Coimbra | Cellfree DNA as a marker for the outcome of end-stage renal disease patients on haemodialysis[END_REF][START_REF] Goligorsky | NET formation in Dialysis: a valuable, albeit mysterious and enticing predictor of mortality[END_REF]. Interestingly, it has been shown in previous studies that cirDNA increase was triggered by HD sessions [START_REF] Opatrna | Cell-free plasma DNA during hemodialysis[END_REF]. Furthermore, in a multivariate model exploring the role of cirDNA kinetic (pre-and post-dialysis) on mortality, it has been reported that only post-dialysis cirDNA levels were predictors of poor outcome [START_REF] Einbinder | Elevated circulating cell-free DNA in hemodialysis-treated patients is associated with increased mortality[END_REF]. It is therefore tempting to postulate that cirDNA released during dialysis sessions reflects such NET formation which then contributes to patient outcome worsening.

The direct link of cirDNA with NETs as well as considering cirDNA as a NET marker may be based on the recent study demonstrating that NETs lead to the production of circulating mononucleosomes, which were shown as the main components of cirDNA-associated structures. The detection of cirDNA in blood is typically associated with the protective effect of mononucleosomes, which determines its fragmentation [START_REF] Thierry | Circulating DNA fragmentomics and cancer screening[END_REF]. The concomitant observation that (i) mononucleosomes clearly accumulate in cirDNA extracts after incubation of very high molecular weight DNA in serum or plasma extracts of cirDNA from mice or human subjects, in combination with (ii) the observation that activated neutrophils in culture produce NETs and high molecular weight DNA, which are further degraded mainly into mononucleosomes, demonstrates that, in a physiological setting, NETs can produce cirDNA as a byproduct, independent of apoptosis. These observations directly and unequivocally provide the mechanistic bridge linking NETs and cirDNA, advocating the use of cirDNA fragments or mononucleosome amounts as NET markers.

NETosis as a significant component of dialysis-induced cardiovascular disease burden: an attractive working hypothesis for improving biocompatibility

Hemodialysis patients are exposed to periodic "systemic circulatory stress" resulting from the treatment schedule, leading to a multiorgan injury superimposed on preexisting comorbidities [START_REF] Canaud | Hidden risks associated with conventional short intermittent hemodialysis: a call for action to mitigate cardiovascular risk and morbidity[END_REF]3,[START_REF] Canaud | Multitargeted interventions to reduce dialysis-induced systemic stress[END_REF]. Intermittent HD (e.g., conventional thrice-weekly 4-h treatment schedule) generates cyclical fluctuations in volume (hyper to hypovolemia), blood pressure changes, osmotic shifts and swings in solutes, and electrolyte levels [START_REF] Kjellstrand | The "unphysiology" of dialysis: a major cause of dialysis side effects?[END_REF]. Dialysis-induced systemic stress resulting from the "unphysiology" of intermittent dialysis is dramatically worsened by bioincompatibility phenomena that conspire with the limited efficiency of the overall renal replacement therapy. In addition, conventional high-flux HD has limited effect on the removal of medium-and high-molecular-weight uremic compounds as well as on protein-bound uremic toxins, suggesting a "residual uremic syndrome" contributing to patient morbidity (3). All these factors contribute to patients' end-organ damage, including vasculature, cardiac, gut, liver, and cognitive dysfunction, and negatively impact patients' perception and quality of life (Figure 3).

The endothelial system has been identified as a critical target pathway in dialysis-induced multiorgan morbidity. NETosis emerges as a key player in the interplay between endothelial cells, platelets, and neutrophils. It has been demonstrated that adhering cells onto the dialyzer membrane at the end of a dialysis session primarily activate neutrophils, with the presence of NETs detectable via immunofluorescence [START_REF] Lakbakbi | Tissue factor expressed by adherent cells contributes to hemodialysis-membrane thrombogenicity[END_REF]. Interestingly, NETs are associated with thrombin generation in a tissue factor (TF)-dependent manner. Furthermore, the presence of TF mRNA in adhering neutrophils has been confirmed [START_REF] Lakbakbi | Tissue factor expressed by adherent cells contributes to hemodialysis-membrane thrombogenicity[END_REF]. Recent research has shed light on the interaction between NETs and platelets [START_REF] Bonaventura | Neutrophil extracellular traps and cardiovascular diseases: an update[END_REF], as NETs can express tissue factor, further triggering thrombin generation and platelet activation, ultimately increasing the thrombogenic potential of NETs. Conversely, activated platelets could stimulate neutrophils to form NETs [START_REF] Maugeri | Activated platelets present high mobility group box 1 to neutrophils, inducing autophagy and promoting the extrusion of neutrophil extracellular traps[END_REF]. The NET network could impede blood flow and promote intravascular occlusion of blood vessels independently of plasmatic coagulation [START_REF] Burmeister | Impact of neutrophil extracellular traps on fluid properties, blood flow and complement activation[END_REF]. Therefore, NET activation could significantly favor microthrombus formation and endothelial dysfunction.

Additionally, both NETs and extracellular vesicles (EVs) are simultaneously released upon activation. However, it remains unclear whether NETs and EVs act as antagonistic, synergistic, or independent mediators [START_REF] Pfister | Neutrophil extracellular traps and neutrophil-derived extracellular vesicles: common players in neutrophil effector functions[END_REF]. Nevertheless, EV-NET complexes can also form and seem to synergistically enhance the procoagulative and prothrombotic capacity of NETs by delivering tissue factor [START_REF] Leal | Tumorderived exosomes induce the formation of neutrophil extracellular traps: implications for the establishment of Cancer-associated thrombosis[END_REF]. EV contains lipids, informative molecules, and coding or noncoding mRNA. Microvesicles could thus induce phenotypic changes in endothelial cells [START_REF] Pomatto | Noncoding RNAs carried by extracellular vesicles in endocrine diseases[END_REF][START_REF] Tetta | Extracellular vesicles as an emerging mechanism of cell-to-cell communication[END_REF]. It has been reported that procoagulant microvesicles are increased in HD patients. Therefore, NETs and microvesicles may conspire to induce a procoagulant milieu in HD patients [START_REF] Martin | Exercise during hemodialysis does not affect the phenotype or prothrombotic nature of microparticles but alters their proinflammatory function[END_REF][START_REF] Burton | Elevated levels of procoagulant plasma microvesicles in dialysis patients[END_REF].

NET formation occurring during a single HD session might be related to acute-phase inflammation. However, the repetition of dialysis sessions almost every other day might result in chronic low-grade inflammation, making it challenging to assess the contribution of NETosis to this biological systemic stress. Interestingly, NETosis, such as elastase or cirDNA release, appears as a late event in the bioincompatibility phenomenon, rising after the first hour of dialysis and persisting until the end of the session, raising questions about sustained NETs during the interdialytic period [START_REF] Ehlerding | Performance and hemocompatibility of a novel polysulfone dialyzer: a randomized controlled trial[END_REF][START_REF] Einbinder | Elevated circulating cell-free DNA in hemodialysis-treated patients is associated with increased mortality[END_REF].

During the interdialytic period, several factors affecting the kinetics and circulating levels of NETs must be considered. First, an increase in uremic toxins could activate polymorphonuclears, resulting in NET formation. Indeed, it has been shown that uremic toxins induce DNA-histone complexes in neutrophils in vitro [START_REF] Jeong | Significance of the DNAhistone complex level as a predictor of major adverse cardiovascular events in hemodialysis patients: the effect of uremic toxin on DNA-histone complex formation[END_REF]. Additionally, cirDNA has been proposed as a potential biomarker of renal damage [START_REF] Celec | Cell-free DNA: the role in pathophysiology and as a biomarker in kidney diseases[END_REF]. MPO and elastase levels have been reported to be elevated in chronic kidney diseases prior to dialysis [START_REF] Khatib-Massalha | Free circulating active elastase contributes to chronic inflammation in patients on hemodialysis[END_REF][START_REF] Kisic | Role of myeloperoxidase in patients with chronic kidney disease[END_REF][START_REF] Janus | Myeloperoxidase is independently associated with incident heart failure in patients with coronary artery disease and kidney disease[END_REF] but they are enhanced in dialysis patients compared with end-stage renal failure patients [START_REF] Li | The effects of citrate dialysate in hemodialysis on polymorphonuclear elastase interaction with tissue factor and its inhibitor[END_REF]. On the other hand, the disappearance rate of NETs after a dialysis session is unknown. CirDNA is rapidly degraded in the blood due to the numerous plasma DNases [START_REF] Korabecna | NETosis provides the link between activation of neutrophils on hemodialysis membrane and comorbidities in dialyzed patients[END_REF][START_REF] Goligorsky | NET formation in Dialysis: a valuable, albeit mysterious and enticing predictor of mortality[END_REF]. However, DNase expression or activity could be decreased in chronic pathologies, increasing the half-life of cirDNA. In contrast to cirDNA, MPO could be bound and sequestered in the vascular wall [START_REF] Ndrepepa | Myeloperoxidase -a bridge linking inflammation and oxidative stress with cardiovascular disease[END_REF]. In association with the prolonged urea-induced NADPH oxidase stimulation [START_REF] Apolito | Urea memory: transient cell exposure to urea causes persistent mitochondrial ROS production and endothelial dysfunction[END_REF] activity, the binding of MPO to endothelial cells (63) could produce hypochlorous acid and chloramines after the end of the dialysis session, potentially exerting a long-lasting effect on endothelial function. The interdialytic kinetics of NET components warrant further investigation, but it is tempting to speculate about a "vascular memory" of acute bioincompatibility phenomenon linked to the potential persistence of NETs, as observed after an acute phase of viral insult [START_REF] Thierry | Neutrophil extracellular traps and by-products play a Key role in COVID-19: pathogenesis, risk factors, and therapy[END_REF][START_REF] Pisareva | Persistence of neutrophil extracellular traps and anticardiolipin auto-antibodies in post-acute phase COVID-19 patients[END_REF]. Several recent studies tend to support this concept. Indeed, in a recent prospective study, NETosis was associated with endothelial dysfunction and positively correlated with a reduced flow-mediated dilation of the brachial artery despite adjustment for cardiovascular risk factors and uremic toxin concentrations [START_REF] Kim | The relationship between autophagy, increased neutrophil extracellular traps formation and endothelial dysfunction in chronic kidney disease[END_REF]. It has been also shown that NETosis was able to induce platelet trapping and promote microvascular occlusions [START_REF] Liaw | DAMP and DIC: the role of extracellular DNA and DNA-binding proteins in the pathogenesis of DIC[END_REF][START_REF] Pertiwi | Neutrophil extracellular traps participate in all different types of thrombotic and haemorrhagic complications of coronary atherosclerosis[END_REF] leading to multiorgan failure. NET was also detected as essential for the development of profound venous thrombosis [START_REF] Fuchs | Neutrophil extracellular trap (NET) impact on deep vein thrombosis[END_REF]. The prothrombotic effect of NETs, mainly initiated by platelet activation, is further enhanced by DNA-bound elastase that degrades plasminogen, reduces plasmin formation, and decreases fibrinolysis [START_REF] Cruz | DNA-bound elastase of neutrophil extracellular traps degrades plasminogen, reduces plasmin formation, and decreases fibrinolysis: proof of concept in septic shock plasma[END_REF]. Here, again these prothrombotic actions could be sustained during the interdialytic period.

In addition to this prothrombotic effect, NETosis might be involved in CV diseases [START_REF] Bonaventura | Neutrophil extracellular traps and cardiovascular diseases: an update[END_REF], a leading cause of mortality in dialysis patients. Recent intradialytic imaging studies have evidenced a reduction in myocardial perfusion and contractility [START_REF] Mcintyre | Haemodialysis-induced myocardial stunning in chronic kidney disease -a new aspect of cardiovascular disease[END_REF]. Dialysis-induced cardiac disorders, known as cardiac stunning, are due to transient ischemia injury resulting in myocardial death and fibrosis. HD session per se has been shown to be a significant hemodynamic stressor able to precipitate such recurrent ischemic insults (100). Furthermore, NETs have been identified as key contributing components in ischemia-reperfusion injury as shown in the myocardial no-reflow phenomenon (101). NET formation participates in the extension of myocardial infarction size as confirmed by large amounts of NETs in coronary thrombi (102). Surrogate markers of NET burden as cirDNA and citrullinated histones were increased in the infarct site (103). Levels of cirDNA and infarct size were found to be strong and independent predictors of in-hospital major adverse CV events (104). Such a positive association between high levels of cirDNA, nucleosomes, MPO complexes, and ST-elevation myocardial infarction with poor patient outcomes has been already described (105). In HD patients, high levels of cirDNA have been associated with major cardiovascular events [START_REF] Jeong | Significance of the DNAhistone complex level as a predictor of major adverse cardiovascular events in hemodialysis patients: the effect of uremic toxin on DNA-histone complex formation[END_REF]. By contrast, a progressive decrease in these biomarkers was observed after a successful revascularization procedure (106). Finally, markers of NETs are associated with adverse clinical outcomes in stable coronary artery disease patients (107).

It has been evidenced that cerebral vascular disease of HD patients could translate into brain alteration, cognitive impairment, and morbi-mortality (108). Intradialytic Doppler ultrasonography and cerebral magnetic resonance imaging have recently established a link between the deleterious effects of HD and cognitive function as mediated by changes in cerebral arterial flow velocity (109, 110). In a prospective observational cohort study of 97 adults receiving HD, the degree of decline in flow velocity in the middle cerebral arteries correlated with the decline in cognitive function (110). Moreover, repetitive HD-induced brain ischemia has been implicated in white matter damage and leukoaraiosis development (111). Recently, it has been also shown that surrogate NET biomarkers, such as cirDNA, MPO, and calprotectin, were increased in stroke patients (112) while NETs exacerbate ischemic brain injury (113). The NET formation is also accompanied by reduced neovascularization and increased blood-brain barrier damage (114).

The gut and liver are also targets of dialysis-induced hemodynamic stress. Dialysis-induced acute circulatory stress contributes to the development of gastrointestinal dysfunction and "gut stunning" [START_REF] Mcintyre | Recurrent circulatory stress: the dark side of dialysis[END_REF]. As shown, gut ischemia leads to bacterial endotoxin translocation and the release of gut-derived uremic toxins from the gut to the bloodstream (115). Gut-derived uremic toxin and endotoxin levels are contributing to maintaining low-grade inflammation in HD patients (116,117). Circulating endotoxin levels are increased in dialysis patients and correlate with intradialytic instability, systemic inflammation, cTnT levels, ultrafiltration rate, dialysis-induced myocardial stunning, and risk of subsequent mortality (115). NETs are increased in the inflamed intestinal mucosa, feces, or blood, and NET abundance positively correlates with the degree of inflammatory intestinal disease (118,119). In an experimental intestinal ischemia-reperfusion injury model, NETs were involved in organ damage and intestine inflammation (120). Interestingly, NETs were able to damage the cytoskeleton of human enterocyte-like cells and destroy the intestinal epithelium facilitating bacterial translocation (121).

In brief, all these facts strongly suggest that dialysis-induced systemic stress and NETosis process that act synergistically during dialysis may have a long-lasting systemic effect contributing to end-organ damage and morbidity of dialysis patients (Figure 3).

Conclusion and perspectives

Bioincompatibility in HD is driven by complex molecular and cellular pathways that intricately act in a cascade of phases. To outline this process schematically, the initial event involves protein adsorption, platelet activation, and complement activation, occurring within the first 15 min of a dialysis session. This transient activation sets the stage for prolonged leukocyte activation, which persists until the end of the dialysis session. Leukocyte activation leads to a cytokine storm, accompanied by an overproduction of ROS due to NADPH oxidase activation. Subsequently, oxidative stress amplifies the production of NETs, resulting in the release of serine proteases, neutrophil elastase, MPO, calprotectin, decondensed chromatin, and cirDNA. NETosis emerges as a late event in the bioincompatibility phenomenon, and the interplay between NETs, platelets, and coagulation may persist during the interdialytic period, suggesting a "vascular memory" of dialysisinduced bioincompatibility. From this perspective, NETs can be considered as markers of the vascular memory of dialysis-induced bioincompatibility, contributing to the systemic circulatory stress observed in dialysis patients. The ongoing activity of NETs during the interdialytic period may, in turn, impact systemic circulation, contributing to end-organ damage and dialysis-induced systemic morbidity. Further studies are needed to investigate the kinetics of NETosis during both intradialytic and interdialytic periods, assessing NET involvement in target organs affected by systemic stress. In this context, it will also be interesting to explore the relative contributions of more efficient renal replacement therapies, such as intensive dialysis treatment schedules (e.g., nocturnal or daily treatments), versus more biocompatible hemodialysis, to the NET imbalance phenomenon. As DNase and anti-coagulant treatments have been proposed for NET-related diseases, these therapeutic options warrant further investigation [START_REF] Thierry | Neutrophil extracellular traps and by-products play a Key role in COVID-19: pathogenesis, risk factors, and therapy[END_REF].

https://clinicaltrials.gov/ct2/show/NCT04338126

Funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. The authors declare that this study received funding from Fresenius Medical Care. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication. The funding was received to pay the article processing charge.

Conflict of interest

BC was employed by the MTX Consulting Int. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.


A Bergougnoux1

L Gaspari2

M Soleirol4

N Servant1

S Soskin5

S Rossignol5

K Wagner-Mahler6

J Bertherat7

C Sultan2

N Kalfa8

F Paris
email: f-paris@chu-montpellier.fr

Virilization at puberty in adolescent girls may reveal a 46,XY disorder of sexual development

Keywords: 46, XY DSD, pubertal virilization, 46, XY sex reversal --

Although hyperandrogenism is a frequent cause of consultation in adolescent girls, more severe forms with virilization must lead to suspicion of an adrenal or ovarian tumor. However, they may also reveal a 46,XY disorder of sexual development (DSD). Here, we describe four adolescent girls referred for pubertal virilization and in whom we diagnosed a 46,XY DSD. We performed gene mutation screening by Sanger sequencing (all patients) and by next-generation sequencing (NGS) in patient #4. We identified new heterozygous NR5A1 gene variants in patients #1 and #2 and a homozygous SRD5A2 gene deletion in patient #3. Patient #4 received a diagnosis of complete androgen insensitivity in childhood; however, due the unusual pubertal virilization, we completed the gene analysis by NGS that revealed two heterozygous HSD17B3 variants. This work underlines the importance of considering the hypothesis of 46,XY DSD in adolescent girls with unexplained virilization at puberty.

Introduction

Mild/moderate hyperandrogenism is frequent in adolescent girls (moderate acne) and is often associated with irregular menstrual cycles. It is most often transient and related to the hypothalamic-pituitary-ovary axis immaturity during this period and will decrease/disappear in the first years following menarche [START_REF] Rosenfield | Puberty in the female and its disorders[END_REF]. In some girls, clinical signs of hyperandrogenism may persist after 2 years post menarche and worsen (hirsutism) [START_REF] Catteau-Jonard | Hyperandrogenism in adolescent girls[END_REF][START_REF] Sultan | Management of Adolescent hyperandrogenism: still a challenge?[END_REF]. These persistent and severe forms of hyperandrogenism warrant investigations to rule out an adrenal or ovarian disorder, particularly polycystic ovary syndrome and nonclassical congenital adrenal hyperplasia [START_REF] Catteau-Jonard | Hyperandrogenism in adolescent girls[END_REF][START_REF] Sultan | Management of Adolescent hyperandrogenism: still a challenge?[END_REF]. In few patients, the clinical expression of hyperandrogenism is more severe with rapidly worsening hirsutism and other signs of virilization, such as clitoromegaly, voice deepening and muscle mass increase. This must suggest an adrenal or ovarian tumor [START_REF] Catteau-Jonard | Hyperandrogenism in adolescent girls[END_REF][START_REF] Sultan | Management of Adolescent hyperandrogenism: still a challenge?[END_REF]. In all patients, the workup should include at least the quantification of testosterone and gonadotropins in plasma. Moreover, quantification of plasma 17-hydroxyprogesterone, and possibly of other androgens (e.g. delta-4 androstenedione (A4) and dehydroepiandrosterone sulfate (DHEA-S)) and abdominopelvic ultrasonography are useful to identify the hyperandrogenism cause. Independently of the presence or not of Müllerian structures, karyotyping must also be performed to exclude a 46,XY disorder of sex development (DSD) resulting in sex reversal. Indeed, in the absence of any particular warning sign (e.g. inguinal gonads or neonatal clitoral enlargement), 46,XY individuals with severe undervirilization (i.e. with normal female external genitalia) are unsurprisingly identified and reared as girls from birth. In these patients, 46,XY DSD will only be diagnosed at puberty because of amenorrhea and/or virilization. Among the various causes of 46,XY DSD, 5α-reductase type 2 (encoded by SRD5A2) [START_REF] Maimoun | Primary amenorrhea in four adolescents revealed 5α-reductase deficiency confirmed by molecular analysis[END_REF][START_REF] Maimoun | Phenotypical, biological, and molecular heterogeneity of 5{alpha}-reductase deficiency: an extensive international experience of 55 patients[END_REF][START_REF] Fénichel | Molecular diagnosis of 5α-reductase deficiency in 4 elite young female athletes through hormonal screening for hyperandrogenism[END_REF][START_REF] Mendonca | Reprint of "Steroid 5α-reductase 2 deficiency[END_REF][START_REF] Bonnet | Changes in the clinical management of 5α-reductase type 2 and 17β-hydroxysteroid dehydrogenase type 3 deficiencies in France[END_REF] or 17β-hydroxysteroid dehydrogenase type III (encoded by HSD17B3) deficiency [START_REF] George | The clinical and molecular heterogeneity of 17βHSD-3 enzyme deficiency[END_REF][START_REF] Hiort | Pubertal development in17Beta-Hydroxysteroid dehydrogenase Type 3 deficiency[END_REF][START_REF] Mendonca | 46,XY disorder of sex development (DSD) due to 17β-hydroxysteroid dehydrogenase type 3 deficiency[END_REF] and alteration of steroidogenic factor 1 (SF-1; encoded by NR5A1) function [START_REF] Hasegawa | Testicular dysgenesis without adrenal insufficiency in a 46,XY patient with a heterozygous inactive mutation of steroidogenic factor-1[END_REF][START_REF] Lourenço | Mutations in NR5A1 associated with ovarian insufficiency[END_REF][START_REF] Tajima | A novel heterozygous mutation of steroidogenic factor-1 (SF-1/Ad4BP) gene (NR5A1) in a 46, XY disorders of sex development (DSD) patient without adrenal failure[END_REF][START_REF] Cools | Pubertal androgenization and gonadal histology in two 46,XY adolescents with NR5A1 mutations and predominantly female phenotype at birth[END_REF][START_REF] Tantawy | Testosterone production during puberty in two 46,XY patients with disorders of sex development and novel NR5A1 (SF-1) mutations[END_REF][START_REF] Sıklar | A novel heterozygous mutation in steroidogenic factor-1 in pubertal virilization of a 46,XY female adolescent[END_REF][START_REF] Domenice | Wide spectrum of NR5A1-related phenotypes in 46,XY and 46,XX individuals. Birth Defects Research[END_REF][START_REF] Colson | SF1 and spleen development: new heterozygous mutation, literature review and consequences for NR5A1-mutated patient's management[END_REF][START_REF] Adachi | Spontaneous virilization around puberty in NR5A1-related 46,XY sex reversal: additional case and a literature review[END_REF] have been associated with virilization at puberty. Through the description of four cases, we wanted to demonstrate that virilization in adolescent girls (i) may reveal a 46,XY DSD and (ii) requires a precise genetic evaluation.

Patients and methods

Patients

The expertise of the DevGen (genital development) team of Montpellier University Hospital, France, includes the medical (pediatric/adult endocrinology services) and surgical management of patients with DSD and also the molecular study of these disorders. In the past 20 years, we analyzed more than 1500 DNA samples from patients with 46,XY DSD followed by our service and by other French teams. In addition, we followed more than 900 patients with complex 46,XY DSD in our clinical practice in the past 20 years.

The present study focused on the clinical, biochemical, and molecular characterization of four patients with pubertal virilization and 46,XY DSD diagnosed at our center between 2002 and 2022. The only other case of authentic pubertal virilization in an adolescent girl, referred to our pediatric endocrinology department during this period, was an adolescent 46, XX girl with adrenocortical cancer. The study was approved by Montpellier University Hospital Ethics Committee. Written informed consent was obtained from all four patients.

Pubertal development was assessed using the pubic hair (P) and breast (B) development stages 1-5 of the Tanner scale [START_REF] Tanner | Growth at Adolescence: with a General Consideration of the Effects of Hereditary and Environmental Factors upon Growth and Maturation from Birth to Maturity[END_REF]. Hirsutism was quantified using the modified Ferriman-Gallwey (FG) scoring system that includes nine androgen-sensitive body areas [START_REF] Ferriman | Clinical assessment of body hair growth in women[END_REF]. All hormone reference values are reported in Table 1 [START_REF] Adeli | The Canadian laboratory initiative on pediatric reference intervals: a CALIPER white paper[END_REF][START_REF] Kulle | A novel ultrapressure liquid chromatography tandem mass spectrometry method for the simultaneous determination of androstenedione, testosterone, and dihydrotestosterone in pediatric blood samples: age-and sex-specific reference data[END_REF].

Patient #1 was a 17-year-old girl referred to the pediatric endocrinology service because of primary amenorrhea. At the first consultation, the pubertal status was B1P5 (Tanner scale) and a clear clinical hyperandrogenism was noted: hirsutism (FG score = 18), acne, and clitoromegaly. The plasma concentrations of testerosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were 10.4 nmol/L, 58.3 IU/l, and 16.4 IU/L, respectively. The karyotype was 46,XY. MRI showed the presence of a Müllerian duct remnant of 27 mm × 15 mm not visible on ultrasonography. Surgery was performed to remove the gonads due to testicular dysgenesis highlighted by the hormonal workup. The anatomopathological study confirmed partial testicular dysgenesis and showed also the presence of a right gonadal seminoma. The clinical and biological hyperandrogenism regressed after gonadectomy.

Patient #2 was referred to the pediatric endocrinology service at the age of 14 years due to clinical hyperandrogenism. At clinical examination, height was 176 cm, weight was 56 kg, without breast development, the pubertal status was B1P5 (Tanner scale), but with important hirsutism (FG score = 20). The gynecological examination found clitoris enlargement (60 mm). The plasma concentrations of testerosterone, FSH, and LH were 21.9 nmol/L, 27.3 IU/L, and 9.2 IU/L, respectively. Anti-Müllerian hormone (AMH) and inhibin B concentrations in blood were 0.49 ng/mL and 5 pg/mL, respectively. The karyotype was 46,XY. MRI showed the presence of a prepubertal uterus (33 mm in height) and two pelvic gonads.

Patient #3 reported virilization at the age of 12 years with hirsutism (FG score = 15), gradual voice deepening, and development of the genital tubercule that bothered the patient who started gender reassignment at the age of 17 years. This patient of Iraqi origin came to France at the age of 18 years and was referred to the endocrine department. The clinical examination showed absence of breast development, the pubertal status was B1P5 (Tanner scale), hirsutism (FG score = 20), clitoromegaly (35 mm), and a palpable left inguinal gonad (the right gonad might have been removed during childhood following a trauma, without any tissue or genetic analysis). The plasma concentrations of testerosterone, dihydrotestosterone (DHT), A4, and DHEA-S were 19.8 nmol/L, 0.3 nmol/L, 7.9 nmol/L, and 9.4 μmol/L, respectively. They were associated with elevated FSH and LH concentrations: 33.1 IU/L and 10.3 IU/L. AMH and inhibin B concentrations were 4.5 ng/mL and 16 pg/mL, respectively. The karyotype was 46,XY. The parents were first cousins. Ultrasonography and pelvic MRI did not find any Müllerian duct remnant. Patient #4 was followed by the pediatric endocrinology service unit of the DevGen team of Montpellier for complete androgen insensitivity syndrome (CAIS) diagnosed at the age of 10 years in the context of bilateral inguinal hernia associated with palpable inguinal gonads. At that time, the pubertal status was B1P1 (Tanner scale). Ultrasonography confirmed the presence of inguinal gonads but did not find the uterus. The karyotype was 46,XY. The plasma concentrations of testerosterone, FSH, LH, AMH, and inhibin B were <0.87 nmol/L, 1.7 IU/L, 1.3 IU/L, 516 ng/mL, and 277 pg/mL, respectively. Sanger sequencing of the androgen receptor (AR) gene identified the c.2395C>G, p.(Gln799Glu) variant that was previously reported in patients with partial and mild androgen insensitivity (McGill University database). The mother was a heterozygous carrier of the same mutation. At the age of 14 years, the patient complained of hirsutism and acne, as well as of feeling lumps in the inguinal area. At clinical examination, the height was 169 cm, weight 49.5 kg, and pubertal status B3P5 (Tanner scale). Hirsutism was marked on the face and inner thighs (FG score = 15), associated with acne on the face and back, and deepened quality of voice. Gynecological examination found clitoromegaly (50 mm). The plasma concentrations of testerosterone, FSH, LH, A4, AMH, and inhibin B were 20.8 nmol/L, 7.8 IU/L, 35.2 IU/L, 40.2 nmol/L, 78.55 ng/mL, and 379.9 pg/mL, respectively.

The patients' phenotypes are summarized in Table 2.

Methods

Hormone concentrations were measured by independent laboratories.

Genomic DNA was extracted from peripheral blood leucocytes using the QIAamp DNA blood mini-kit (Qiagen) following the manufacturer's instructions. As previously reported, exons 1-5 of the SRD5A2 gene (25) and exons 2-7 of the NR5A1 gene (26) were amplified by PCR, and direct sequencing was performed with the BigDye terminator version 1.1 kit (Applied Biosystems) and an ABI Prism 3130 genetic analyzer (Applied Biosystems).

For patient #4, the unusual observation of pubertal virilization in the context of CAIS led to complete the genetic analysis by next-generation sequencing (NGS) of genes implicated in DSD. Mutations in these genes have been associated with sexual determination/differentiation alterations, hypogonadotropic hypogonadism, and steroidogenesis alteration. The implication of these genes has been validated in human diseases or is suspected because of the results of knockout and/or knockdown experiments showing repercussions on sex determination or differentiation in animal models. Massive Parallel Sequencing libraries were prepared following the manufacturer's instructions. Briefly, DNA libraries were prepared using SeqCap EZ probes, the KAPA HyperPrep and HyperCap Target Enrichment Kits (Roche Diagnostics®). Libraries were sequenced in independent runs using a MiniSeq High Output Reagent Cartridge and Flowcell on a MiniSeq (Illumina, San Diego, CA, USA). Samples reached high quality control levels. Target coverage at 50× was 99.0%. Reads were aligned using Local Run Manager (Illumina) or the MobiDL workflow developed by the MoBiDiC team (https://github.com/ mobidic/MobiDL). This workflow is dedicated to NGS data obtained using SeqCap libraries and focuses on gene panels/exomes. It uses the Genome Analysis ToolKit (GATK) 4 HaplotypeCaller and Google DeepVariant for variant calling. Copy number variants were detected using the MobiCNV tool (https://github.com/mobidic/ MobiCNV). The Captain Achab workflow and the MoBiDiC prioritizing algorithm [START_REF] Yauy | MoBiDiC prioritization algorithm, a free, accessible, and efficient pipeline for single-nucleotide variant annotation and prioritization for next-generation sequencing routine molecular diagnosis[END_REF] were used for single-nucleotide variant filtering and prioritization. The pathogenicity of rare variants was predicted by the MoBidetails Online DNA Variant Interpretation tool that exploits multiple sources (https://mobidetails.iurc.montp.inserm.fr/MD/).

The suspected disease-causing HSD17B3 variants were confirmed by Sanger sequencing with the following primers: exon 3 forward 5′-TAACACAAGCCCTCC CTGTC-3′; reverse 5′-GAGCAGGCTTGGTTGGAG-3′; exon 4 forward 5′-GGGCATTTGGATCCCTG-3′; reverse 5′-CATCAGTGTCAGGTTATTTCACTG-3′.

Results

Sequencing allowed identification of two new heterozygous NR5A1 gene variants, c.1069C>T, p.(Gln357Ter) in patient #1 and c.38G>C, p.(Cys13Ser) in patient #2; a new homozygous deletion of one base pair in SRD5A2, c.453del, p.(Leu152TyrfsTer8) in patient #3; and two compound heterozygous variants of the HSD17B3 gene, c.277+4A>T, p.(?) (inherited from the father) and c.278-1G>C, p.(?) (inherited from the mother) in patient #4. Both variants are recurrent variants in patients with 17β-hydroxysteroid dehydrogenase type III deficiency [START_REF] Boehmer | 17Beta-hydroxysteroid dehydrogenase-3 deficiency: diagnosis, phenotypic variability, population genetics, and worldwide distribution of ancient and de novo mutations[END_REF]. They were confirmed (c.277+4A>T) or predicted (c.278-1G>C) to disrupt canonical splicing sites, leading to aberrant splicing and to the occurrence of premature stop codons inside exon 4.

All variants detected in the four patients and their interpretation according to the American College of Medical Genetics and Genomics (ACMG) guidelines and standards are reported in Table 3.

Discussion

Here, we described four cases of adolescent girls with virilization in whom the clinical, laboratory, and sequencing analyses led to the diagnosis of 46,XY DSD caused by NR5A1 (patients #1 and #2), SRD5A2 (patient #3), and HSD17B3 variants (patient #4; initial diagnosis of CAIS).

NR5A1 alterations are associated with a large spectrum of clinical phenotypes, from isolated infertility in male patients to 46,XY sex reversal, without adrenal failure in most patients [START_REF] Ferraz-De-Souza | Steroidogenic factor-1 (SF-1, NR5A1) and human disease[END_REF][START_REF] Philibert | Phenotypic variation of SF1 gene mutations[END_REF]. In the literature, there are only nine reports on pubertal virilization revealing 46,XY DSD in patients who were identified as females at birth [START_REF] Hasegawa | Testicular dysgenesis without adrenal insufficiency in a 46,XY patient with a heterozygous inactive mutation of steroidogenic factor-1[END_REF][START_REF] Lourenço | Mutations in NR5A1 associated with ovarian insufficiency[END_REF][START_REF] Tajima | A novel heterozygous mutation of steroidogenic factor-1 (SF-1/Ad4BP) gene (NR5A1) in a 46, XY disorders of sex development (DSD) patient without adrenal failure[END_REF][START_REF] Cools | Pubertal androgenization and gonadal histology in two 46,XY adolescents with NR5A1 mutations and predominantly female phenotype at birth[END_REF][START_REF] Tantawy | Testosterone production during puberty in two 46,XY patients with disorders of sex development and novel NR5A1 (SF-1) mutations[END_REF][START_REF] Sıklar | A novel heterozygous mutation in steroidogenic factor-1 in pubertal virilization of a 46,XY female adolescent[END_REF][START_REF] Domenice | Wide spectrum of NR5A1-related phenotypes in 46,XY and 46,XX individuals. Birth Defects Research[END_REF][START_REF] Colson | SF1 and spleen development: new heterozygous mutation, literature review and consequences for NR5A1-mutated patient's management[END_REF][START_REF] Adachi | Spontaneous virilization around puberty in NR5A1-related 46,XY sex reversal: additional case and a literature review[END_REF]. We and others previously reported that pubertal virilization [START_REF] Colson | SF1 and spleen development: new heterozygous mutation, literature review and consequences for NR5A1-mutated patient's management[END_REF] and also isolated primary amenorrhea [START_REF] Philibert | Steroidogenic factor-1 (SF-1) gene mutation as a frequent cause of primary amenorrhea in 46,XY female adolescents with low testosterone concentration[END_REF][START_REF] Kulkarni | Comprehensive molecular analysis identifies eight novel variants in XY females with disorders of sex development[END_REF] should orient towards a diagnosis of 46,XY sex reversal related to NR5A1 alterations (MIM #612965).

Different hypotheses have been proposed to explain the paradox of androgen production failure during the fetal male sexual differentiation period and recovery of testicular androgen production at puberty. Specifically, fetal Leydig cell (FLC) steroidogenesis could be more SF-1-dependent than adult Leydig cell (ALC) steroidogenesis [START_REF] Shaughnessy | The foetal Leydig cell--differentiation, function and regulation[END_REF]. It is currently acknowledged that FLC and ALC are different cell populations [START_REF] Shaughnessy | The foetal Leydig cell--differentiation, function and regulation[END_REF][START_REF] Griswold | Fetal Leydig cell origin and development[END_REF]. In 2015, Karpova et al. demonstrated in male mice, that SF-1 is needed for the proper development of FLC and ALC, but with distinct functions: cell differentiation in FLC and progenitor cell formation and/or survival in ALC [START_REF] Karpova | Steroidogenic factor 1 differentially regulates fetal and adult leydig cell development in male mice[END_REF]. However, Shima et al. reported that most ALC derive from dedifferentiated FLC and that NR5A1 is essential for the initial FLC differentiation and also for ALC redifferentiation at puberty [START_REF] Shima | Fetal Leydig cells persist as an androgen-independent subpopulation in the postnatal testis[END_REF]. Besides SF-1 essential role in FLC and ALC, ALC development involves many other transcription factors, growth factors, luteinizing hormone and androgens [START_REF] Ye | Insights into the development of the adult leydig cell lineage from stem Leydig cells[END_REF]. Therefore, it could be hypothesized that some of these factors may partly compensate for the SF-1 defect, particularly at puberty, when luteinizing hormone signaling is critical for ALC differentiation and precursor proliferation. Moreover, in the adult testis, liver receptor homolog-1 (LRH-1), also known as nuclear receptor subfamily 5, group A, member 2 (NR5A2; a totipotency pioneer factor), could replace SF-1 to regulate steroidogenesis. Indeed, it is abundantly expressed in ALC, but not in FLC [START_REF] Yazawa | Liver receptor homolog-1 regulates the transcription of steroidogenic enzymes and induces the differentiation of mesenchymal stem cells into steroidogenic cells[END_REF]. In addition, FLC cannot convert A4 to testosterone. This is done by Sertoli cells, the only cells that express 17βhydroxysteroid dehydrogenase type III in fetal testes. Therefore, impaired Sertoli cell function in patients with pathogenic NR5A1 variants may be implicated in the more severe testosterone defect observed in fetal life compared with adult life [START_REF] Shaughnessy | The foetal Leydig cell--differentiation, function and regulation[END_REF]. Indeed, the increase of testosterone production at puberty is preserved because of the shift in 17β-hydroxysteroid dehydrogenase type 3 production from fetal Sertoli cells to ALC [START_REF] Shaughnessy | The foetal Leydig cell--differentiation, function and regulation[END_REF].

Patient #3 had a 5α-reductase type 2 deficiency (MIM #264600). Our group and others have described the large clinical phenotype variability of patients with 5α-reductase enzyme deficiency [START_REF] Maimoun | Primary amenorrhea in four adolescents revealed 5α-reductase deficiency confirmed by molecular analysis[END_REF][START_REF] Maimoun | Phenotypical, biological, and molecular heterogeneity of 5{alpha}-reductase deficiency: an extensive international experience of 55 patients[END_REF][START_REF] Okeigwe | 5-alpha reductase deficiency: a 40-year retrospective review[END_REF]. Nevertheless, we clearly demonstrated that severe undervirilization is the most frequent clinical sign, as indicated by the finding that >72% of patients are identified as females at birth [START_REF] Maimoun | Phenotypical, biological, and molecular heterogeneity of 5{alpha}-reductase deficiency: an extensive international experience of 55 patients[END_REF]. We [START_REF] Maimoun | Primary amenorrhea in four adolescents revealed 5α-reductase deficiency confirmed by molecular analysis[END_REF][START_REF] Maimoun | Phenotypical, biological, and molecular heterogeneity of 5{alpha}-reductase deficiency: an extensive international experience of 55 patients[END_REF][START_REF] Fénichel | Molecular diagnosis of 5α-reductase deficiency in 4 elite young female athletes through hormonal screening for hyperandrogenism[END_REF] and others [START_REF] Mendonca | Reprint of "Steroid 5α-reductase 2 deficiency[END_REF] showed that patients with 46,XY DSD and sex reversal present almost systematically some degree of pubertal virilization, associated with disease-causing compound heterozygous or homozygous SRD5A2 variants. In these patients, virilization is explained by the rise in serum testosterone at puberty, which may be metabolized to dihydrotestosterone by the type 1 isoenzyme that is expressed in skin from puberty. Our previous study found that 9.1% of patients from our cohort (total n = 55) requested female-to-male reassignment (5), as done by patient #3. On the basis of the better knowledge on the consequences of 5α-reductase type 2 deficiency in 46,XY patients and the increased success rates of assisted reproduction treatments in individuals with less severe undervirilization, currently, we favor male sex rearing from birth in this condition [START_REF] Mendonca | Reprint of "Steroid 5α-reductase 2 deficiency[END_REF].

Patient #4 presented 17β-hydroxysteroid dehydrogenase type 3 deficiency (MIM #264300), a well-known cause of 46,XY DSD with sex reversal and virilization at puberty [START_REF] Mendonca | 46,XY disorder of sex development (DSD) due to 17β-hydroxysteroid dehydrogenase type 3 deficiency[END_REF]. At puberty, the isoenzyme 17βhydroxysteroid dehydrogenase type 5 can convert Δ4 to testosterone [START_REF] Hiort | Pubertal development in17Beta-Hydroxysteroid dehydrogenase Type 3 deficiency[END_REF]. However, our initial diagnosis (at the age of 10 years) was CAIS related to the presence of the AR variant c.2395C>G, p.(Gln799Glu), inherited from the mother. This variant is located in the ligand binding domain of AR, and has a frequency of 1.3 × 10 -3 in the general population (gnomAD V3). It has been detected in patients with less severe phenotype, ranging from mild androgen insensitivity syndrome with infertility [START_REF] Wang | Azoospermia associated with a mutation in the ligand-binding domain of an androgen receptor displaying normal ligand binding, but defective trans-activation[END_REF][START_REF] Hiort | Significance of mutations in the androgen receptor gene in males with idiopathic infertility[END_REF] to partial androgen insensitivity syndrome [START_REF] Bevan | Functional analysis of six androgen receptor mutations identified in patients with partial androgen insensitivity syndrome[END_REF][START_REF] Kalfa | Minor hypospadias: the "tip of the iceberg" of the partial androgen insensitivity syndrome[END_REF]. To our knowledge, only one study reported the association in the same patient with CAIS of this variant and another AR gene variant [START_REF] Petroli | Functional impact of novel androgen receptor mutations on the clinical manifestation of androgen insensitivity syndrome[END_REF]. In vitro studies reported impaired AR transactivation without binding [START_REF] Wang | Azoospermia associated with a mutation in the ligand-binding domain of an androgen receptor displaying normal ligand binding, but defective trans-activation[END_REF][START_REF] Hiort | Significance of mutations in the androgen receptor gene in males with idiopathic infertility[END_REF][START_REF] Bevan | Functional analysis of six androgen receptor mutations identified in patients with partial androgen insensitivity syndrome[END_REF] or N-and C-terminal domain interaction alteration [START_REF] Jääskeläinen | Human androgen receptor gene ligand-binding-domain mutations leading to disrupted interaction between the N-and C-terminal domains[END_REF]. This variant remains of uncertain significance according to the ACMG classification (PM1, PP3, PP5, BS1). However, in our patient, it might have contributed to the fetal undervirilization and less severe virilization at puberty. Moreover, this patient demonstrates that any clinical sign or disease progression discordant with a previous molecular diagnosis should lead to the reevaluation of the etiological diagnosis and to more in-depth genetic analyses.

In conclusion, this work demonstrates that it is important to consider the hypothesis of a 46,XY DSD in adolescent girls with unexplained pubertal virilization. This clinical presentation warrant prompt investigations to exclude the presence of an adrenal or ovarian tumor. Karyotyping should be performed, independently of the presence/absence of Müllerian structures. Genetic analyses, including most genes previously described in 46,XY DSD, are required to establish the precise molecular diagnosis, to optimize patient management, and to propose genetic counseling.

Funding

This work has not received any funding.

Declaration of interest

The authors declare that there is not conflict of interest that could be perceived as prejudicing the impartiality of the study reported.