TSC1orTSC2genes were discovered to be responsible for mTOR overactivation, which is the underlying mechanism of pathogenesis. TSC is a highly heterogenous clinical entity with variable presentations and severity of disease. The brain, heart, skin, eyes, kidneys, and lungs are commonly involved in this syndrome, with neurologic symptoms comprising a significant source of morbidity and mortality. In 2012, the diagnostic criteria for TSC were revised by the International Tuberous Sclerosis Complex Consensus panel, and genetic testing was incorporated into the guidelines. Early detection of cardiac rhabdomyomas or TSC-associated skin lesions can suggest the diagnosis and underlie the importance of clinical vigilance. Animal studies have demonstrated the benefit of using mTOR inhibitors for various symptoms of TSC, and they have been successfully translated into clinical trials with significant improvement in symptom burden. Subependymal giant cell astrocytomas, renal angiomyolipomas, and epilepsy are the three FDA-approved indications in relation to TSC for the use of everolimus, which is a first generation mTOR inhibitor. Rapamycin has been FDA approved for lymphangioleiomyomatosis. Other TSC symptoms that could potentially benefit from this class of medication are currently under investigation. TSC constitutes a unique combination of protean physical symptoms and neurobehavioral abnormalities. TSC associated neuropsychiatric disorders (TAND), including intellectual disability, mood disorders, and autism spectrum disorder, represent significant challenges but remain underdiagnosed and undertreated. The TAND checklist is a useful tool for routine use in the clinical evaluation of TSC patients. A multidisciplinary treatment plan, based on the specific problems and needs of individuals, is the key to management of this genetic condition. Ongoing research studies have been providing promising leads for developing novel mechanistic strategies to address the pathophysiology of TSC. Tuberous sclerosis complex (TSC) is an autosomal dominant, multisystem disorder that is characterized by cellular and tissue dysplasia in several organs. With the advent of genetic and molecular techniques, mutations in the TSC1orTSC2genes. TSC was first identified by German pathologist Friedrich Daniel von Recklinghausen, in 1862, in a baby with cardiac myotomas and sclerotic brain lesions who died shortly after birth [1].Tuberous sclerosis complex (TSC) is a rare, multisystem, autosomal dominant syndrome characterized by tumorigenesis and is associated with neurologic and behavioral abnormalities. The pathogenesis is driven by hyperactivation in the mTOR pathway due to de novo or inherited mutations in theIt was better defined in 1880 by French neurologist Désiré-Magloire Bourneville as “tuberous sclerosis of the cerebral convolutions”; hence, the disease was named “Bourneville’s disease” after him. His patient reportedly had seizures, intellectual disability, and renal angiomyolipomas (AMLs) in the form of “hard masses, one the size of a walnut” [2]. In 1908, another German neurologist, Heinrich Vogt, established the Vogt triad for TSC, comprising intellectual disability, intractable seizures, and facial angiofibromas [3]. The first use of the term tuberous sclerosis complex was by Sylvan Moolten in 1942 [4]. In 1972, Spanish-American neurologist Manuel Rodriguez Gomez established the first diagnostic criteria for TSC, and he has since been viewed as the father of TSC in the USA [5]. TSC1orTSC2genes and guide genetic counseling. As a matter of fact, improvements in the realm of genetics opened a new era for TSC in the 1990s, when theTSC1andTSC2genes were identified, which led to the exploration of the molecular pathways involved [6].Even though there are no pathognomonic signs for TSC, various clinical stigmata are commonly seen as part of the syndrome, which raise suspicion for the diagnosis. Common manifestations include cortical tubers, subependymal nodules, subependymal giant cell astrocytomas , seizures, cardiac rhabdomyomas, renal AMLs, retinal hamartomas, pulmonary lymphangioleiomyomatosis (LAM), facial angiofibromas , ash-leaf spots, shagreen patches, intellectual disability, and autism spectrum disorder . Once the diagnosis is suspected, genetic testing can be performed to look for mutations in theTSC1andTSC2genes encode the proteins hamartin and tuberin, respectively. These proteins compose the TSC complex, which acts as a brake on the mTOR signaling cascade . In agreement with this, mTOR inhibitors, such as rapamycin and its analogs , are commonly used in a clinical setting for various symptoms of this condition [9]. The current management of TSC is mostly symptomatic, with pharmacologic, surgical, or behavioral intervention options. Due to the vast phenotypic heterogeneity encountered, not all therapeutic approaches benefit the entirety of symptoms or patients to the same extent; hence, a personalized treatment strategy is critical [10]. The incidence of TSC has been estimated as occurring in 1/6000–10,000 newborns annually, and therefore, it is categorized as a rare disease. It affects approximately 2 million people globally [11]. The prevalence in Europe is approximately 11,500–25,000 [12]. There is no sex or ethnicity predilection [13]. However, differences in sex predominance have been observed in numerous symptoms . In 2012, a conference was held in Washington, DC, USA, by the International Tuberous Sclerosis Complex Consensus group to revise the diagnostic criteria for TSC, providing a comprehensive list consisting of major and minor criteria for use by physicians in a clinical setting  (Table). TSC1orTSC2genes, which were discovered through the use ofDrosophilamodels in the 1990s [16].TSC1is found on chromosome 9 (9q34) andTSC2is found on chromosome 16 (16p13.3), encoding the proteins hamartin and tuberin, respectively . TSC is also genetically linked to autosomal dominant polycystic kidney disease, as thePKD1andTSC2genes are closely located (48 base pairs apart) on chromosome 16. When both genes are affected due to a contiguous gene deletion, it may lead to a clinical picture called PKD-TSC (MIM #600273) with severe renal symptoms [17].TSC is caused by mutations in either of thede novoor inherited mutations.De novomutations constitute two-thirds of all TSC diagnoses. The remaining one-third is inherited in an autosomal dominant fashion [18]. The genetic pattern of TSC can be described by Knudson’s 2-hit hypothesis, where the acquisition of a somatic mutation in a previously functional allele ofTSC1orTSC2, in addition to the existing germline mutation in the other allele, leads to the disease state [19]. MostTSC2cases are sporadic and have more severe manifestations, while the ratio ofTSC1toTSC2mutations in familial cases is close to one [20]. TSC shows almost complete penetrance with wide phenotypic variability. This means that any individual carrying a TSC mutation would be afflicted with the disease, but to varying degrees. The large number of possible mutations in the TSC genes also contributes to the heterogeneity within the patient population [21]. TSC can arise due toTSC1andTSC2genes differ from each other in thatTSC1mutations are mostly nonsense or frameshift mutations, leading to protein truncation, whereas missense mutations, large deletions, or rearrangements are seen more withTSC2[22]. Additionally,TSC1mutations have been identified in ~10–20% of patients clinically diagnosed with TSC, whileTSC2mutations have been identified in ~70–90% . Several thousand small mutations have been shown to cause TSC, which can be found in the online Leiden Open Variation Database http://chromium.lovd.nl/LOVD2/TSC/home.php. However, genetic testing may be negative in 10–25% of inherited cases due to reasons such as somatic mosaicism, or mutations in intron or promoter regions [25]. TheThe protein products of the TSC genes, hamartin and tuber, work together within the same intracellular pathway, which explains why a mutation in either gene can give rise to the same disease. The downstream target of these proteins is the mammalian target of rapamycin complex 1 (mTORC1), which is a protein serine/threonine kinase complex involved in many important anabolic and catabolic processes, such as translation, cellular growth, proliferation, stress response, and autophagy . mTORC1 is a protein complex that contains mTOR, a rapamycin-associated protein of TOR (raptor), and mLST8 [28] . The major driver of the cellular hyperplasia and tissue dysplasia seen in TSC is the overactivation of the mTORC1 signaling pathway. Hamartin and tuberin bind to a third protein, TBC1D7, to form the TSC protein complex as part of this cascade [29]. The heterotrimeric TSC complex acts as a GTPase-activating protein for RAS homologue enriched in brain (Rheb), which is the functional mediator between the TSC complex and mTORC1 [30]. Under normal circumstances, the TSC complex functions to keep it in an inactive GDP-bound state, thus rendering Rheb unable to stimulate mTORC1.TSC1orTSC2, the brake on Rheb by the TSC complex is released as the complex can no longer be formed [9]. Therefore, mTORC1 is constitutively activated by Rheb, regardless of the upstream signals. The mechanism by which Rheb regulates mTORC1 still needs exploration. Rheb-induced mTOR activation results in the stimulation of S6 kinase and inhibition of 4EBP1, the eukaryotic translation initiation factor 4E-binding protein 1, leading to unrestricted protein synthesis and proliferation [31].When second hit mutations affect eitherThe mTOR pathway can be pharmacologically manipulated by rapamycin (Sirolimus), which binds to FKPB12 and causes the mTORC1 complex component raptor to dissociate, rendering mTORC1 unable to stimulate the downstream targets of anabolism . Rapamycin was discovered in the 1970s on Easter Island and was originally used as an antifungal compound [34]. Later, it drew further attention due to its immunosuppressant and antiproliferative properties, causing it to become an important agent in oncology. In the early 2000s, rapamycin was demonstrated to be effective for symptom control in TSC mouse models by several labs, and the bench-to-bedside studies that followed . The activity of mTORC1 is also influenced by the upstream components of several intersecting pathways. mTORC1 is negatively regulated by PRAS40, the proline-rich Akt substrate of 40 kDa and DEPTOR, the DEP domain containing mTOR-interacting protein, which works as a component of the GATOR complex. [37]. In addition to growth-inducing states in the presence of sufficient energy and nutrients, mTORC1 is also turned on by tyrosine kinase growth factor receptors, such as insulin, insulin-like growth factor 1, brain-derived neurotrophic factor, and epidermal growth factor receptor [38]. This results in the activation of the Ras/ERK and PI3K/Akt signaling pathways, which converge on the TSC complex and cause its activation favoring a progrowth state [39]. mTORC1 has been demonstrated to regulate both anabolic and catabolic processes. When activated, it stimulates protein synthesis, nucleotide synthesis, gluconeogenesis, lipogenesis, and ATP production. When it is turned off, it inhibits cell growth through several mechanisms including autophagy . mTORC2 complex is less well understood, especially in terms of its role in the pathogenesis of TSC [42]. It is involved in the regulation of cytoskeletal dynamics. Additionally, it is not sensitive to the effects of acute rapamycin treatment because the core component, RICTOR, does not bind FKBP12 [43]. Further studies could help to characterize its functions and explore the possibility of interactions with the mTORC1 pathway. Both mTOR and Rheb are ubiquitous in the body, causing multisystem involvement in the presence of TSC mutations [43]. Thanks to the successful implementation of genetic and molecular discoveries into clinical settings, TSC serves as model for many other diseases involving similar pathways or cellular processes [44].The clinical manifestations of TSC are protean in terms of severity and the range of tissues it can involve. Despite the potential of TSC to involve any organ system in the body, some organs are more affected than others. Neurologic manifestations, including seizures and cognitive impairment, are the primary source of patient and caretaker burden, followed by renal abnormalities . Although there is no single symptom specific to TSC, a constellation of findings on physical examination and imaging raises the suspicion for diagnosis [47]. The revision of the diagnostic criteria for TSC by the International Tuberous Sclerosis Complex Consensus group incorporated genetic testing into the clinical framework of TSC [14]. Even before genetic testing is undertaken, the presence of a first-degree relative with TSC puts the patient at 50% risk for having the disease [48]. The 2012 diagnostic criteria list includes major and minor features, determining a TSC diagnosis to be categorized as definite or possible (Table). A definite diagnosis can be made when 2 major or 1 major and 2 minor criteria are fulfilled. Patients with 1 major or 2 and more minor criteria meet the criteria for having a possible TSC diagnosis. A thorough clinical evaluation should be followed by genetic testing for confirmation of the disease and prognostication [45].The neurologic issues of TSC comprise the most important cause of impairment in the majority of patients, owing to their prevalence and the severity of symptoms [46]. The array of manifestations include epilepsy, cortical tubers, subependymal nodules and giant cell astrocytomas, intellectual disability, autism spectrum disorder, and behavioral problems . The foremost neurologic symptom in TSC is epilepsy, afflicting 90% of patients [51].The onset of epilepsy is variable, but most patients present before the age of 1 year [52]. All seizure types can be seen with TSC. The most common seizure type in early life is infantile spasms, which affects nearly 40% of patients with TSC-associated epilepsy [53]. Although earlier studies supported the hypothesis of seizures originating from cortical tubers, the exact origin and mechanism of epileptogenesis are still debated [54]. Several studies have demonstrated a lack of correlation between tuber burden and epilepsy severity [55]. Up to 38–50% of seizure patients are refractory to the point of necessitating surgical intervention [56]. The age of onset and severity of seizures are most predictive of long-term cognitive and behavioral outcomes . Cortical tubers (80–90%) are one type of brain malformation that present as part of TSC and give the disease its name. They are thought to be caused by a failure of cellular differentiation and neuronal migration during neurodevelopment [59]. Radial migration lines occur due to a similar process and can be observed with the tubers [45]. The cortical tubers contain giant dysplastic neurons and astrocytes, and they tend to stay stable in size [60]. Microtubers may also be found in normal appearing white matter [61]. Subependymal nodules (SENs) are formations that arise mostly along the lateral and third ventricle walls, and are seen in >80% of patients. Approximately 5–15% of these growths transform into subependymal giant cell astrocytomas (SEGAs) [47]. SEGAs are composed of ganglion-like giant cells expressing both neuronal and astrocytic markers [62]. SEN/SEGAs may remain asymptomatic [51]. However, if they are located at the foramen of Monro, they can potentially cause obstructive hydrocephalus and increased intracranial pressure. Both SENs and SEGAs can be detected prenatally or at birth, and it is rare for SEGAs to grow after the age of 20 [14]. Most of these lesions tend to progressively calcify [63]. Renal abnormalities can also lead to significant morbidity and mortality, as they are commonly encountered in TSC patients, and can lead to complications if left untreated [64]. The most common renal lesion is angiomyolipoma, a hamartoma composed of blood vessels, smooth muscle, and adipose tissue [65]. They are often bilateral and multiple. [66]. Although benign in nature, these lesions have a tendency to bleed, and therefore should be watched closely for timely intervention. In severe cases, renal AMLs may lead to renal failure [67]. The second most frequent lesion in the kidney is single or multiple simple cysts. They are seen in 45% of patients and can result in hypertension or kidney failure [68]. The combination of AMLs and cysts in the same patient is highly suspicious for a TSC diagnosis [69]. In early-onset severe cases, they may constitute stigmata of PKD-TSC syndrome . The dermatologic lesions seen with TSC are of paramount value, as their presence heralds the diagnosis in a considerable number of cases [70]. Many of the major features listed in the 2012 diagnostic criteria for TSC included cutaneous manifestations. Among these, hypomelanotic macules or ash-leaf spots, which were named after the European Mountain Ash Tree, can be seen in up to 90% of patients [71]. They are detected at birth or during early infancy. They may be difficult to visualize especially in fair-skinned individuals or small babies; therefore, the use of ultraviolet light (Wood’s lamp) can be helpful [51]. Another important dermatologic finding is facial angiofibromas or adenoma sebaceum. These lesions are comprised of connective and adipose tissue and are found in 75% patients over 9 years of age . They appear in the central face and increase in number with age, which is a common source of concern [73]. TSC patients can also present with fibrous plaques in the forehead (20%), shagreen patches in the lumbosacral region (20%), and ungual or gingival fibromas (20%) [74].Intracardiac rhabdomyomas are seen in nearly 50% of patients [75]. They are one of the earliest TSC lesions to emerge as the cardiac rhabdomyomas can be detected on prenatal ultrasound as early as 22 weeks of gestation [11]. On average, the lesions tend to cluster as a few lesions, grow to a size of 3–25 mm, and are mostly found in the ventricles of the heart along the septum. They tend to regress within 3 years of life [76]. Although rare, some rhabdomyomas may lead to arrhythmia, valvular defects, or cardiac failure, so prenatal and postnatal surveillance are critical until regression [77].Pulmonary involvement is much less common than the previously mentioned manifestations of TSC. The most frequent pulmonary lesion is LAM, which is almost exclusively seen in adult females [78]. This suggests that the pathogenesis may be driven by estrogen, which has also been evidenced by animal models of LAM [79]. Infiltration of the lung tissue by smooth muscle cells is characteristic of LAM, leading to cystic lung changes and potential complications of pneumothorax, pleural effusion and hemoptysis [52]. Multifocal micronodular pneumocyte hyperplasia (MMPH) is another type of pulmonary lesion associated with TSC [80].The eye is another organ that can be commonly affected in TSC, as they arise from ectoderm like the central nervous system. Retinal astrocytic hamartomas are seen in 35–50% of patients and are typically benign unless they compress the optic disc [81]. Additionally, areas of hypopigmentation around the retina called retinal achromic patch can be observed in 39% of TSC patients [82].TSC can involve the gastrointestinal system , thyroid, pituitary gland, pancreas, and gonads  [83]. However, the number of cases reported in the literature is limited.The group of cognitive and behavioral problems associated with TSC causes a great burden, both for TSC patients and their caretakers. Most neuropsychiatric symptoms of TSC present a challenge for treatment and ironically do not receive as much attention as the physical stigmata of the disease [84]. The gap between burden and treatment exhibited for the tuberous sclerosis associated neuropsychiatric disorders (TAND) was found to be similar to those seen in the approach to HIV, where the major focus is also on the physical symptoms [85]. The umbrella term TAND was coined through the inspiration by HAND, which defines the HIV-associated neurocognitive disorders [86].Among the neuropsychiatric manifestations of TSC, ASD has a special place, both in terms of clinical approach and research focus. Approximately 26–50% of TSC patients fulfill the criteria for autism spectrum disorder (ASD) [87]. ASD in TSC has many overlaps with idiopathic ASD, which is another factor that makes TSC an important genetic model to study this condition [88]. It has been observed that TSC patients with ASD tend to have a more severe epilepsy phenotype, in line with several studies demonstrating that poorly controlled seizures contribute to worse cognitive outcomes [89]. Overall in TSC, there is a wide range of severity for intellectual disability and patients are affected by the neurologic comorbidities at varying degrees [90]. The exact nature of the relationship between autism, intellectual disability, and epilepsy needs further investigation.TAND is a broad category of symptoms encompassing multiple dimensions of cognitive, psychological, and social issues encountered within the context of TSC. The 2012 International Tuberous Sclerosis Complex Consensus group established the guidelines for evaluation of neuropsychiatric manifestations of TSC, by providing the practitioners with a TAND checklist. This proved to be a practical clinical tool for practitioners to address TAND, which are often missed and therefore undertreated. Properly addressing TAND can dramatically improve the quality of life of TSC patients and their families [91].The multiple levels covered in the TAND checklist included the following: behavioral level , psychiatric level , intellectual level , academic level  and psychosocial level  [84]. The different levels provide a common ground between patients and physicians to have a conversation and come up with a personalized management plan. This is of great importance, as up to 90% of TSC patients are observed to have TAND features during at least one period of their life [92].One should bear in mind that the TAND checklist was not designed to be used as a rating scale, but rather a tool to make an individual action plan based on the TAND profile [84]. Neuropsychiatric reevaluations are recommended by the 2012 TSC guidelines, as the profile of the individual may change over time. Sudden changes in behavior should prompt evaluation for underlying medical causes, such as new brain lesions or seizures [93].As a consequence of the multisystem involvement in TSC, each symptom demands evaluation and management within the relevant clinical context. mTOR inhibitors have been groundbreaking in the TSC world due to their ability to target the molecular defect in the disorder. However, animal models and clinical studies have shown that not all TSC-related symptoms benefit from mTOR inhibitors to the same extent [94]. Research is still ongoing for the optimization of mTOR inhibition for each symptom and what other pharmacologic and nonpharmacologic approaches could be employed for tackling the challenges in TSC. In particular, the timing of treatment may be crucial for the neurocognitive symptoms, and treatment during early critical windows may be necessary [95]. Regardless of the choice of intervention, genetic counseling should be included in the discussion with families.Once a diagnosis of TSC is reached, a baseline MRI is recommended to look for the presence of any cortical malformations as tubers, SENs, or SEGAs [68]. Surgery and mTOR inhibitors are the current treatment options for asymptomatic SEGAs; however, surgical intervention is recommended in acute cases [91]. After the success of the EXIST-1 clinical trial, the FDA approved the use of everolimus for individuals with tuberous sclerosis who present with SEGAs that are not amenable to surgery [96].As mentioned previously, epilepsy remains one of the primary sources of morbidity for TSC patients. Early intervention is key for both better control of seizures and improved neurocognitive outcomes. For infantile spasms, vigabatrin is the best choice of medication, and ACTH is an alternative in cases with insufficient response [97]. Vigabatrin is an irreversible inhibitor of GABA transaminase. It helps to increase the GABA concentration and potentially reset the imbalance between GABA and glutamate neurotransmitters, which is a proposed mechanism of epileptogenesis [98]. Patients should be counseled about the possible side effects of retinal toxicity and evaluated for vision changes. Most antiseizure medications can be used for epilepsy in TSC. Alternative or complementary therapeutic options include surgery, ketogenic low glycemic index diet, and vagal nerve stimulation. Despite the presence of these modalities, refractory epilepsy is still a big concern, with seizures persisting in more than 60% of patients [53]. The EXIST-3 trial demonstrated the benefit of everolimus in patients with treatment-resistant focal seizures [99]. Neuropsychiatric conditions associated with TSC should be managed with a multidisciplinary team, focusing on the individual’s level of psychosocial and neurocognitive functioning. Despite contributing greatly to the burden of care, TAND has received little clinical attention, with less than 20% of cognitive and behavioral symptoms being treated [84]. During clinical visits, the TAND checklist is a useful diagnostic tool to determine which neuropsychiatric symptoms require special attention. An individualized educational plan should be established for school-aged patients [14].Animal studies have shown correlations between seizure frequency and the extent of social deficits, which are improved by the early treatment of epilepsy [100]. Several clinical trials have aimed to investigate the relationship between mTOR inhibition and neurocognition. In a 6-month clinical trial of an mTOR inhibitor (everolimus) in children and adolescents with TSC (6–21 years of age), no improvement was detected in the active drug group when compared to those taking a placebo [101]. Another trial in Europe with a similar age group also failed to show an effect on cognitive abilities or neuropsychological functioning [102]. While there are several possible explanations for these results, one important aspect may be the age of treatment. It should be kept in mind that infancy is a critical course of the disease, where pharmacologic interventions may also lead to long-lasting unfavorable changes [103]. Therefore, determining the optimum therapeutic window is crucial [88]. A randomized clinical trial of early intervention with vigabatrin to prevent seizure development in TSC (EPISTOP) recently concluded in Europe, and an National Institutes of Health-funded trial to prevent epilepsy and improve neurocognitive outcomes in infants with TSC (PREVeNT) is currently ongoing in the USA.The EXIST-2 trial demonstrated the benefit of mTOR inhibitors for renal AMLs, and everolimus was approved by the FDA for asymptomatic and growing renal AMLs larger than 3 cm [104]. Second-line treatment options in cases with unsatisfactory response or a lack of access to everolimus include selective arterial embolization or radiofrequency ablation, especially for hemorrhaging or compressive lesions [105]. Sirolimus was approved by the FDA for use in patients with LAM in 2015, after obtaining successful results in the MILES trial [106]. Pulmonary function and capacity need to be regularly monitored for signs of clinical improvement. Last, but not least, topical rapamycin may be used for facial angiofibromas in cases of significant disfigurement or psychological stress [107].For most TSC-related hamartomas, lifelong treatment will continue to be necessary, as many lesions tend to regrow and seizures may recur upon the discontinuation of medication . Reported side effects of mTOR inhibitors include stomatitis, menstrual irregularities, acne, hyperlipidemia, infections, and poor wound healing, which are related to suppression of the immune system and changes in cellular metabolism [109–111]. In TSC, an interdisciplinary approach with consultations from neurologists, cardiologists, nephrologists, pulmonologists, psychiatrists, psychologists, social workers, educational specialists, genetic counselors and additional practitioners, based on the needs of the specific individual, is essential. To achieve this goal, TSC clinics have been established in numerous hospitals across the USA. TSC has been one of the great examples in medicine where the bench research has been successfully translated to improve the diagnostic and therapeutic yield in a clinical setting.More recent studies have started to focus on developing alternative strategies for the treatment of TSC. One notable approach is the elimination of TSC-deficient cells through the induction of autophagy or oxidative stress by exploiting the inadaptability of the overactivated mTOR pathway [112–115]. Such research questions will continue to be explored and can be promising for the development of preemptive therapy for TSC. TSC is a rare genetic disorder characterized by hamartoma formation in multiple organs. The pathogenesis is driven by uncontrolled mTOR activation, which is the target of rapamycin and rapalogs to control the disease symptomatology with varying success. TSC serves as a model for epilepsy, autism, and tumorigenesis and many other diseases involving the mTOR pathway. Despite the morbidity and mortality that TSC symptoms are associated with, the medical world is fortunate for the discoveries into the genetic and molecular aspects of this disease. Owing to the immense endeavors of TSC researchers, new therapeutic options targeting vulnerabilities in the TSC-related pathways will continue to be developed that maximize efficacy and minimize toxicity. Biomarkers to demonstrate disease progression and treatment efficacy need to be identified for maximizing the benefit for all patient profiles. An individually tailored multidisciplinary approach, with special attention to cognitive and psychosocial comorbidities, is the key to success in the management of this disease. Mustafa Sahin reports grant support from Novartis, Roche, Pfizer, Ipsen, LAM Therapeutics, and Quadrant Biosciences. He has served on Scientific Advisory Boards for Sage, Roche, Celgene, Aeovian, and Takeda.


Signaling from the synapse to nucleus is mediated by the integration and propagation of both membrane potential changes  and intracellular second messenger cascades. The electrical propagation of postsynaptic potentials allows for rapid neural information processing, while propagating second messenger pathways link synaptic activity to the transcription of genes required for neuronal survival and adaptive changes (plasticity) underlying circuit formation and learning. The propagation of activity-induced calcium signals to the cell nucleus is a major synapse-to-nucleus communication pathway. Neuronal PAS domain protein 4 (Npas4) is a recently discovered calcium-dependent transcription factor that regulates the activation of genes involved in the homeostatic regulation of excitatory–inhibitory balance, which is critical for neural circuit formation, function, and ongoing plasticity, as well as for defense against diseases such as epilepsy. Here, we summarize recent findings on the neuroprotective functions of Npas4 and the potential of Npas4 as a therapeutic target for the treatment of acute and chronic diseases of the central nervous system. Many adaptive changes in brain function such as learning depend on the capacity of individual neurons to transduce changes in postsynaptic membrane potential into longer-lasting changes in gene transcription, leading to stable biochemical and structural modifications  . To achiThe neuronal PAS domain-containing protein 4 (Npas4), a member of the PAS family characterized by a conserved basic-helix-loop-helix motif and PAS domain , acts asNpas4 also regulates the development of glutamatergic and GABAergic synapses in neurons to maintain neurocircuit homeostasis essential for information processing and memory formation . Further2 +) influx, thereby activating distinct sets of early- and late-response genes required for the formation of inhibitory synapses , ubiquitinates and degrades postsynaptic density protein-95 (PSD-95) in rat hippocampal neurons, thereby reducing the strength of excitatory transmission . MoreoveNpas4 also regulates the expression levels of genes that serve to increase excitatory input onto somatostatin (SST)-releasing neurons, such as Stac, Osgin2, Nptx2, Ppm1h, Bach2, and Gpr3, which in turn may enhance feedback inhibition within cortical circuits. Several Npas4 target genes in SST neurons are able to act at postsynaptic sites of excitatory synapses  or are homologous to related molecules (Frmpd3), suggesting that Npas4-regulated gene programs in SST neurons’ function is to promote the development of excitatory synaptic inputs and thus increase inhibitory drive within circuits . AlthougIn response to heightened neuronal activity, the aryl hydrocarbon receptor nuclear translocator 2 (Arnt2) protein recruits Npas4 to activity-dependent regulatory elements, where it induces the transcription of genes that increase somatic inhibitory input . In addiIt is well established that Npas4 expression regulates embryonic stem cells and early postnatal brain development by RE-1 silencing transcription factor (REST) and microRNAs . In addiThe increase in intrinsic activity during maturation of adult-born granule cells in the hippocampal dentate gyrus (DG), a simple cortical region that is an integral portion of the larger functional brain system called the hippocampal formation, is sufficient to alter the synaptic connectivity within hippocampal circuits, and Npas4 is required for activity-dependent spine development . AlternaAs a recent achievement in this regard, it was reported that Npas4 controls neuronal homeostasis in epilepsy through the induction of Homer 1a , and NpaThe neuroprotective functions of Npas4 may also extend to drug-induced effects via regulation of drug-responsive genes. Repeated injections of the psychostimulant amphetamine (AMPH) into the NAc upregulate regional expression of Npas4, suggesting that Npas4 transcriptional activity mediates some of the neurological and behavioral effects of AMPH . SimilarGiven that Npas4 possesses a unique arrangement of localization signals  and has Npas4 expression levels were reduced in mice and rats exposed to different forms of stress, including conditioned fear , sleep dCollectively, these studies indicate pivotal contributions of Npas4 to long-term synaptic and circuit plasticity across multiple brain regions involved in fear, addiction, and spatial memory among other neural functions. In addition, these effects on circuit activity, especially modulation of excitatory–inhibitory balance, also confer protection against a variety of insults in animal models of disease.Npas4 is a recently discovered transcription factor that links neuronal activity to adaptive changes in circuit function, as well as stress to protective responses through modulation of excitatory–inhibitory balance . These aAmong the IEGs, Npas4 possesses a host of unique structural and functional features. It is expressed only in neurons, is activated selectively by neuronal activity, and is critical for sustaining normal circuit activity by orchestrating distinct gene expression programs in different neuronal populations, for review All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. JF and JZ retrieved most of the sources and drafted the initial version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the past years, several theories have been advanced to explain the pathogenesis of Major Depressive Disorder (MDD), a neuropsychiatric disease that causes disability in general population. Several theories have been proposed to define the MDD pathophysiology such as the classic “monoamine-theory” or the “glutamate hypothesis.” All these theories have been recently integrated by evidence highlighting inflammation as a pivotal player in developing depressive symptoms. Proinflammatory cytokines have been indeed claimed to contribute to stress-induced mood disturbances and to major depression, indicating a widespread role of classical mediators of inflammation in emotional control. Moreover, during systemic inflammatory diseases, peripherally released cytokines circulate in the blood, reach the brain and cause anxiety, anhedonia, social withdrawal, fatigue, and sleep disturbances. Accordingly, chronic inflammatory disorders, such as the inflammatory autoimmune disease multiple sclerosis (MS), have been associated to higher risk of MDD, in comparison with overall population. Importantly, in both MS patients and in its experimental mouse model, Experimental Autoimmune Encephalomyelitis (EAE), the notion that depressive symptoms are reactive epiphenomenon to the MS pathology has been recently challenged by the evidence of their early manifestation, even before the onset of the disease. Furthermore, in association to such mood disturbance, inflammatory-dependent synaptic dysfunctions in several areas of MS/EAE brain have been observed independently of brain lesions and demyelination. This evidence suggests that a fine interplay between the immune and nervous systems can have a huge impact on several neurological functions, including depressive symptoms, in different pathological conditions. The aim of the present review is to shed light on common traits between MDD and MS, by looking at inflammatory-dependent synaptic alterations associated with depression in both diseases. Depression is one of the most diagnosed mental disorders in adult patients as well as in children and adolescents. It is a heterogeneous disorder caused by a complex interaction between genetic and environmental factors . Stress How inflammation contributes to depressive symptoms either in chronic inflammatory disease or in the context of MDD has been highly debated  and is sAlthough analogies between a neuropsychiatric disorder such as MDD and the inflammatory autoimmune MS disease are difficult to sustain, neuroinflammation is potentially recognized as a common disease factor. In this regard, it is worth considering the huge effect that small physiologic differences of inflammatory components can have over time if they are consistently skewed in one direction. The aim of the present review is to provide an overview of the several theories advanced to explain depressive symptoms in MDD and in MS , 2, withMajor depressive disorder is one of the most common psychiatric disorder in the world recognized by the World Health Organization (WHO) and affects general population across a broad spectrum of ages and social environmental factors .The one-year prevalence of MDD is 6% representing about twofold greater incidence in women than man . The lifClinical features coincide with the evidence of morphological and functional alterations of gray and white matter in the brain of MDD patients. In the recent years, magnetic resonance imaging (MRI) studies revealed cortical thinning in several brain regions of MDD patients including frontal lobe, temporal lobe, hippocampus, pre-frontal cortex (PFC), anterior cingulate cortex (ACC), orbito-frontal cortex, thalamus, and striatum . Worthy L-tryptophan by the enzyme tryptophan hydroxylase in neurons located in the brainstem raphe nuclei  and project to the dorsal and ventral striatum, respectively. Dopaminergic system is implicated in the motivation, addiction and motor function  inducing hypothalamic release of corticotrophin releasing hormone (CRH). CRH stimulates anterior pituitary gland to release adrenocorticotropic hormone (ACTH) leading to glucocorticoid secretion by adrenal glands. This HPA hyperactivity has been associated with increased levels of glucocorticoids both in the plasma and in the cerebrospinal fluid (CSF). The hippocampus plays a key role in the feed-back regulation of HPA and increased CNS level of glucocorticoids overstimulates hippocampal neurons leading to volume decrement. Two main hypotheses have been proposed to explain hippocampal alterations: the neurogenesis hypothesis, in which glucocorticoids decrease the neurogenesis in the dentate gyrus (DG) of the hippocampus, and the neuroplasticity hypothesis, in which glucocorticoids induce the atrophy of mature neurons reducing the total dendritic spine density and length. These structural and functional alterations seem to be associated to lower levels of brain-derived neurotrophic factor (BDNF) in the hippocampus and have been proposed as the neural basis of cognitive alterations in chronic stress and MDD .Accumulating evidence has associated chronic inflammation to MDD and to chronic stress pathophysiology, but it is still a matter of debate how inflammation may contribute to the pathogenesis of these diseases . As repoThe involvement of cytokines in psychiatric diseases was first postulated through the observation that patients treated with the pro-inflammatory cytokine IFN-α developed several depressive symptoms that disappeared when treatment was interrupted .In 1991, Smith proposed the so-called cytokine theory of MDD, supported by clinical and pre-clinical studies . AccordiClinical studies have also suggested that serum levels of cytokines may provide several information both on the risk to develop the disease and on clinical outcome. For example, a retrospective study demonstrated that children with higher levels of IL-6 have 10% greater risk of developing MDD . MoreoveThe association between mood disturbances and cytokine levels has been explored also in several mouse models. For example, a preclinical study on knock out (KO) mice for TNF-α receptor 1 or 2 (TNFR1-2 KO) showed amelioration of conditional fear response and depressive behavior in comparison to control mice  as well It has been demonstrated that the main T helper phenotypes involved in the productions of pro-inflammatory cytokines in MDD are Th1, Th17, Th22 while reduced activity of Th2, Th3, Th9 has been reported . These oMicroglia cells, resident immune cells of CNS, are potent and far-reaching regulators of the extended neuron-glia network by secreting soluble mediators and by establishing direct contacts with the synaptic compartment . In neurMany preclinical studies have demonstrated that activated microglia lead to an up-regulation of pro-inflammatory cytokines, such as IL-1β, TNF-α, and IL-6, prostaglandins, nitric oxide and glutamate causing functional and structural abnormalities of the synaptic compartment . It has Interestingly, stress-induced activation of LC has been associated to increased NA levels in several projecting brain areas, including PFC and hippocampus , that inN-methyl- D-aspartate (NMDA) receptors, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs)], kainic acid receptors (KARs) and metabotropic glutamate receptors (mGluRs) expressed at both pre- and post-synaptic neurons  and glutamatergic synapse as a potential target for pharmacological treatments. Clinical studies suggest associations between glutamatergic hyperactivity and regional volume reductions in the brains of MDD patients as well as significant decrease in glial cell numbers, density and neuronal atrophy . FurtherIt is important to remind that the glutamate is an active player in synaptic brain plasticity  and cognitive impairment is considered as a core endophenotype of MDD . PreclinThe relevance of glutamatergic synaptic dysfunction in MDD is corroborated by pharmacological evidence that drugs with neuroprotective effects against excitotoxic mechanisms have antidepressant properties. Indeed, riluzole, by inactivating the voltage-dependent calcium and sodium channels exerts an inhibitory effect on glutamate release, has also antidepressant properties likely mediated by an enhancement of glutamate clearance by astrocytes, with consequent neuroprotective effects and synaptic plasticity preservation . SimilarIn addition, there is cumulating evidence that dysfunction of the GABAergic system is associated to the pathophysiology of MDD . By reguFinally, components of the endocannabinoid system (ECS), such as the type-1 cannabinoid inhibitory receptors (CB1Rs), have been linked to MDD . It is wAltogether these findings suggest a complex dysregulation of neurotransmission in MDD , 3 and cA growing body of evidence is now looking at the link between neuroinflammation and neurotransmitter perturbations in MDD.As previously mentioned, cytokines released from activated microglia in the HPA-axis can modulate glucocorticoid receptor signaling and induce alteration of hippocampal synaptic plasticity in MDD and chronic stress .The effects of proinflammatory cytokines (IL-1β and TNF-α) released by activated microglia on synaptic plasticity include alteration of NMDA and AMPA receptor subunit expression and a decrease of AMPA receptor phosphorylation  Figure . Human sMoreover, a recent study investigated the biological mechanism underlying the associations between the proinflammatory cytokine IL-18 and depressive symptoms related to MDD. IL-18 lacking mice showed degenerated presynaptic terminals in the molecular and polymorphic layers of hippocampal DG in association to loss of motivation at motor behavioral tests and altered neurogenesis and apoptosis . OverallCytokines are also able to decrease serotonin synthesis by activating the indoleamine 2,3-dioxygenase enzyme (IDO) that metabolizes tryptophan to kynurenine . It is wAlthough further investigations are needed to fully elucidate the challenging relationship between inflammation and synaptic dysfunction in depressive symptoms, we propose that an inflammatory-mediated synaptopathy might be an integral part of the pathophysiology of MDD as suggested by studies conducted in MS and in its experimental model EAE .MS is an autoimmune disorder of the CNS characterized by loss of myelin sheaths and gray matter damage, followed by neurodegeneration. Although its etiology is still unclear, it is now known that interactions between susceptible genes and environmental factors are involved in disease pathogenesis. MS is mainly driven by an inflammatory cascade in the CNS, triggered by autoreactive immune cells that attack myelin and neuronal epitopes leading to demyelination and axonal degeneration. Blood–brain barrier damage allows the infiltration of immune cells, including activated T and B cells, into the CNS. The local activation of microglia and astroglia cells exacerbates the immune response and causes further damage to the neuronal cell network resulting in the genesis of demyelinating plaques and excitotoxic damage  Figures, 3. The Depression is the main psychiatric feature reported in MS patients  and inclThe main consequence of depressive symptoms is scarce adherence to pharmacological treatments or rehabilitative programs associated to worsening of functional outcomes . AlthougOf note, the risk to develop depression is two or five-times higher for MS patients than general population, with a lifetime prevalence of 50%, a rate of 44,5% during relapses but with no differences in terms of gender . In a reAlong with neuroinflammation, a combination of several predisposing factors, including environmental factors and aspects of individual personality  are involved in the etiology of depressive symptoms in MS patients as reported for MDD . On the Regarding neuroimaging analysis, several MRI studies highlighted specific modifications, such as reduced cortical thickness, in the brain of depressed MS patients when compared to non-depressed MS patients. In overlap with MDD, the most involved areas include temporal pole, PFC, hippocampus with anterior and posterior cingulate cortex . MoreoveThe limited attention toward the mood aspects of MS symptomatology is in part due to an inadequate knowledge of their pathological basis. In this respect, some critical issues have been in part addressed by studies in animal models of MS that, despite several limitations, can reproduce some of the MS clinical and histopathological features . One of As in MDD, monoamines are important determinants in neuroinflammation-based diseases. Low tryptophan serum concentration is observed in infectious, autoimmune, and malignant diseases and disorders that involve immune cells activation due to an increase in tryptophan metabolism . AccordiThe immune-mediated hypothesis of depression in MS patients has been supported by evidence from both preclinical  and clinThe cytokine profile of depressed MS patients is characterized by increased levels of proinflammatory cytokines, including TNF-α, IL-1β and IL-6 in both CSF and peripheral blood  Figures, 3. PartPreclinical studies investigated the link between mood disturbances and cytokines (Il-1β and TNF-α) in EAE . RegardiThe main difference of the immunological profile between MDD and MS is the prominent role of CD8+ T lymphocytes in the genesis of depressive symptoms in MS, where CD4+ T lymphocytes seem to have a primary role . ClinicaIt is well known that activated microglia and infiltrating peripheral monocytes in CNS are crucial neuroinflammatory determinants in the pathophysiology of both MS and EAE. Together with primed T cells, they are the main inflammatory players involved in neuronal and oligodendrocyte alterations associated with synaptic dysfunction, subpial and axonal demyelination in MS/EAE . IncreasGlutamate plays a role of primary importance both in MS and in EAE  and elevex vivo models and TMS studies. In this regard, electrophysiological recordings from brain slices of healthy mice incubated with CSF of MS patients carrying elevated levels of IL-1β, revealed an IL-1β-dependent enhancement of the glutamate-mediated excitatory postsynaptic currents – through modulation of the transient receptor potential vanilloid 1channel (TRPV1) – and also an IL-1β-induced neuronal swelling , such as oral dimethyl fumarate, teriflunomide, fingolimod, and intravenous drugs, such as natalizumab, and alemtuzumab  seem to Positive pharmacological response to SSRI, TCAs and iMAOIs in the treatment of depressive symptoms in MS may confirm that antidepressant drugs act simultaneously on both monoaminergic and glutamatergic system dysregulations. Indeed, recent studies demonstrated that SSRI treatment is effective not only on mood comorbidities related to MS, but also as a potential DMT to treat clinical relapses and progression of disability . MoreoveFinally, non-pharmacological therapeutic approaches may also improve mood symptoms in MS patients. Recent works suggest a beneficial role of physical exercise in patients with progressive MS and depression . In partAltogether, these observations concerning the treatment of depressive symptoms in both MDD and MS show a complex interaction between the immune and neuronal pathways suggesting the needed of further investigations, both at preclinical and clinical levels, to ameliorate the clinical outcomes.In conclusion, based on the several evidences reported here, we point to the relevance of inflammation-dependent neurotransmitter alterations as potential contributors to depressive symptoms in both MDD and MS disease. At a first glance, these complex brain disorders that diverge in multiple aspects (simply by considering MS as an inflammatory neurodegenerative disease and MDD as a neuropsychiatric disorder) seems to share few pathological aspects such as damage of brain areas devoted to mood control and some neuroinflammatory mediators. However, clinical and preclinical investigations performed in both diseases revel common traits, from the monoamine- to the glutamate- and inflammatory theories, which are often interconnected and unbalanced in the diseases , 2. WhilFinally, despite we are aware that the role of inflammation in the etiopathogenesis of depressive disorders is still debated and several questions still remain open, we would like to emphasize that inflammatory cytokines are potent modulators of the synaptic compartment and that even subtle changes in their levels can induce, likely in susceptible individuals, synaptic perturbations responsible of early depressive symptoms. The synaptic glutamatergic perturbation, in different entities and with different outcomes, could represent the keystone that connects the two pathologies, joined in different ways by neuroinflammation. It is remarkable that predisposing factors, especially genetic and epigenetic factors, may play a central role to identify their common role in both diseases.AB, ED, DC, and GM conceived and designed the manuscript. All other authors made significant contributions and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The year 2019 marked the 20th anniversary of the formal recognition of autoinflammatory diseases as a distinct group of rheumatological conditions, following the identification of the gene mutated in patients with a dominantly inherited periodic fever known as familial Hibernian fever (FHF)  or dominantly (FHF) inherited trait. This allowed for linkage mapping, positional cloning and candidate gene screening even before the completion of human genome sequencing project in 2003. These gene-hunting projects were laborious and time-consuming, but nonetheless successful and led to identification of the first three genes associated with autoinflammatory diseases: Familial Mediterranean fever was the first disease to be characterized at the molecular level. FMF, being a common illness in multiple Mediterranean populations, was initially noted in the literature by Galen in the second century AD. Although there were reports of cases with cyclic fevers and pains in the nineteenth century, it remained a mysterious disease and its pathogenesis was attributed to the phases of the moon and other environmental factors. Genetic etiology was not suspected until 1958 when Dr. Harry Heller emphasized the genetic nature of the disease and coined its modern name familial Mediterranean fever . The firTNFRSF1A gene  and Muckle-Wells (MWS) syndromes, both diseases considered cryopyrin-associated periodic syndromes (CAPS) (MEFV) and the NLRP3 protein share the same N-terminal ~92aa domain denoted as pyrin domain (PYD). This finding suggested the existence of common pathways in the pathogenesis of autoinflammation. Subsequently, a whole family of proteins with the pyrin domain was identified by in silico analysis. Many of these proteins function as intracellular sensing receptors to recognize foreign or self-generated danger-associated molecular patterns. They form a molecular complex known as inflammasome that was discovered and characterized in 2002 by the team of late Dr. Jurg Tschopp  . Another Tschopp . The iniThe next chapter began around 10 years ago with the development of new genomic technologies, next-gene sequencing (NGS), bioinformatics, and the completion of human genome project. These strategies provided researchers and clinicians a variety of tools for gene-hunting projects. Genetic discoveries are now often made in a matter of months if not weeks. The list of genes associated with monogenic autoinflammatory diseases has grown rapidly and currently includes more than 30 genes. Advanced sequencing technologies revealed unusual inheritance patterns including somatic mutations as the cause of adult-onset diseases and cases with digenic inheritance. Digenic inheritance refers to presence of pathogenic mutations in two interacting proteins as the cause of a disease. High-throughput sequencing has brought to light a number of variants with uncertain clinical significance (VUS). Attempts to clarify the clinical implication of these low frequency (1–5%) variants have been carried out through international collaborations. Despite of major accomplishments in dissecting the genetic basis of autoinflammatory conditions, the genetic cause of disease for many patients remains unknown. To complicate things, many of these patients are sporadic cases and it may require orchestrated efforts between multiple research groups to find causal genes.The field of molecular diagnostics for autoinflammatory diseases has seen substantial growth, providing physicians with the specific information necessary to diagnose and treat patients. There are close to 100 academic and commercial laboratories across the world that perform genetic by testing through single-gene or targeted gene panel analysis, or whole exome sequencing. An international group of experts has been convened in effort to standardize genetic reports and develop consensus guidelines for interpretation of genetic variants.NLRP3, NLRC4, NLRP12, PLCG2, FXII). About a decade ago an immunological continuum was proposed to designate patients who present with features of autoinflammation and autoimmunity  is case in point: patients may present with fevers, rash, ischemic strokes or with a sole manifestation of pure red cell aplasia. Mutations in the same protein—often in different domains—may give rise to distinct clinical features, therefore these phenotypes need to be referred to in the context of mutant protein e.g., Pyrin-, NOD2-, NLRP3-, or NLRC4-associated diseases. In contrast, a distinct phenotype, such is the case with cold-induced urticaria, could be caused by pathogenic mutations in different genes  syndrome, STING-associated vasculopathy with onset in infancy (SAVI) and Aicardi-Goutieres syndrome (AGS).The term autoinflammation has spread beyond the boundaries of internal medicine and rheumatology, and is now used in disciplines of dermatology, immunology, and neurology. Dysregulation of the innate immune system is increasingly considered to have role in the pathogenesis of many human conditions, including common multifactorial cardiovascular, metabolic, neurodegenerative, autoimmune diseases, and in particular in polygenic or systemic inflammatory diseases, such as Behçet disease and periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis (PFAPA) syndrome. A better understanding of the molecular mechanisms underlying dysregulation of the innate immune system will provide a foundation for developing more affordable and effective treatments. Witnessing these developments has been incredibly rewarding for those of us in the field and it will be exciting to see where it goes from here.In the present issue of Frontiers in Immunology, “Autoinflammatory diseases: from genes to bedside,” the first monographic issue about autoinflammatory diseases, several investigators have contributed with original and review articles covering genetic, pathogenic, epigenetic, clinical and therapeutic aspects of different autoinflammatory conditions.Martorana et al.. review the most common mutations and the evidences of genotype/phenotype correlations of the main monogenic autoinflammatory diseases. The role of NLRP3 and pyrin inflammasomes in the pathogenesis of CAPS, and FMF and pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND), respectively, has been addressed by de Torre-Minguela et al.. Aksentijevich and Zhou describe the latest advances on the pathogenic mechanisms of ubiquitinopathies, a new category of autoinflammatory diseases involved in the NF-κB pathway, which include linear ubiquitin chain assembly complex (LUBAC) and OTULIN deficiencies, and haploinsufficiency of A20. Carta et al.. propose two different pathways of inducing abnormal IL-1β production in autoinflammatory diseases depending on the cell type affected, in which the authors postulate that professional inflammatory cells would cause a direct inflammatory response and non-immune cells may participate indirectly in the inflammatory cascade by releasing stress signals that trigger and propagate inflammation. In the same sense, Gül reviews the concept of autoinflammation and uses it for monogenic and polygenic autoinflammatory diseases associated with seemingly unprovoked inflammatory episodes mediated mainly by the innate immune system. In addition, Gül also proposes and expands nomenclature by using the concept of “hyperinflammatory” state for those disorders characterized by episodes of exaggerated inflammatory response only when triggered by certain factors or situations. Álvarez-Errico et al.. review the recent advances on the contribution of epigenetic mechanisms in the disease expression of some autoinflammatory diseases.The “genes” part of the topic explores relevant genetic, pathogenic and epigenetic mechanisms implicated in autoinflammatory diseases. Özen et al.. wrote a comprehensive overview about what are still considered unsolved problems in FMF, such as the involved mechanism of the disease, inheritance patterns and treatment in colchicine resistant patients. Ruiz-Ortiz et al.. make an original contribution in characterizing clinical manifestations associated with the low-penetrance R92Q variant in TNFRSF1A and differentiating disease phenotypes between patients with pediatric and adult onset. In an article about CANDLE syndrome, Torrelo et al. reviews in depth all the pathophysiological, clinical, and biologic features of this complex monogenic interferonopathy. The cytokine signature in patients with Behçet disease is explored by Lopalco et al., who suggest an increased signature of IL-6, TNF-α, and Th17 in patients with mucocutaneous and uveitis manifestations. Cantarini et al.. propose a set of clinical diagnostic criteria for adult-onset PFAPA syndrome with a high-predictive potential for identifying PFAPA patients among subjects with fever of unknown origin. Finally, in two review articles, Figueras-Nart et al.. analyze the most remarkable dermatologic and dermatopathologic features of monogenic autoinflammatory diseases by using a classification based on the predominant cutaneous lesion, and Soriano et al. describe the current treatment of the most frequent monogenic autoinflammatory diseases and PFAPA syndrome based on the best available evidence and also propose a practical guide to their management.The “bedside” part of the topic reviews important clinical and translational research, and therapeutic contributions in autoinflammatory diseases. This first monography entirely dedicated to autoinflammatory diseases provides stimulating information on many of the past, present and future advances and challenges in this field.All authors have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This maintains the stability of their morphological functions. This dynamic balance is highly susceptible to various stress environments, including viral infection. After viral infection, the dynamic balance of the host cell mitochondria is disturbed, affecting the processes of energy generation, metabolism, and innate immunity. This creates an intracellular environment that is conducive to viral proliferation and begins the process of its own infection and causes further damage to the body. Herein, we discuss the mechanism of the virus-induced mitochondrial dynamics imbalance and its subsequent effects on the body, which will help to improve our understanding of the relationship between mitochondrial dynamics and viral infection and its importance.Mitochondria are highly dynamic organelles that maintain the dynamic balance of split-fusion  A virus is a microbe that parasitizes on cells. It uses the host cell’s metabolic system to synthesize nucleic acids and proteins. By regulating the metabolism and physiological processes of the cell, a virus can change the structure and function of the cells to maintain a living environment that is conducive to virus proliferation . MitochoCurrently, the mechanisms of mitochondrial dynamics and viral interactions are in the preliminary research stage, and there are many questions to be answered. In this review, we summarized the latest research results on viral infections and mitochondrial dynamics and discussed in depth how viruses affect mitochondrial dynamics and the pathogenesis of the body after dynamics imbalance, which will provide new ideas for future antiviral treatment strategies.Mitochondria are highly dynamic in the cell, which maintains the dynamic balance of the mitochondrial network through continuous fission and fusion. This dynamic balance is regulated by a variety of dynamic related proteins, and it changes with the needs of cell life activities or external stimulation.In mammals, mitochondrial fission is mainly mediated by dynamin-like protein 1 (Drp1), which belongs to the GTP enzyme system that is generally located in the cytoplasm. After being activated, Drp1 shifts to the outer mitochondrial membrane (OMM) and forms a polymer, which compresses the mitochondria until they split. Drp1 requires the participation of a variety of accessory proteins to perform its function, including mitochondrial fission protein 1 (Fis1), mitochondrial fission factor (Mff), and mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51), which act as ligands for Drp1 on the mitochondria and thereby mediate Drp1 recruitment into the mitochondria . In addi2+/calmodulin-dependent kinase II (CaMKII) phosphorylate Ser616 residues of Drp1, thus enhancing Drp1 migration to mitochondria and fission  and Mfn2 are GTP enzyme proteins that are located on the OMM, which coordinate to mediate OMM fusion . Mfns coFusion of the inner mitochondrial membrane (IMM) is regulated by optic atrophy 1 (OPA1). OPA1 exists in two isoforms: a long isoform L-OPA1 that is combined with IMM and a short isoform S-OPA1 that is dissociated in the inter membrane space (IMS) where S-OPA1 is formed by shear of L-OPA1 . L-OPA1 Mitochondria are energy factories in cells, which provide ATP for cell survival through oxidative phosphorylation. This process is closely related to respiratory chain complex encoded by mitochondrial DNA (mtDNA). Mitochondrial fusion effectively maintains mtDNA content and enhances mitochondrial respiration and ATP generation by promoting the exchange and mixing of mitochondrial contents and complementing damaged mitochondrial alleles . AlthougMitochondria are involved in many life activities in cells and have important physiological significance. By changing the shape, quantity, and distribution of mitochondria, division and fusion can ensure the stability of mitochondrial function and the normal development of various life activities and maintain the steady state of cells. Mitochondrial dynamics can not only affect bioenergy production but also have physiological functions of regulating apoptosis and mitochondrial antiviral signaling (MAVS) pathway. In the following, we will make a further review combining with the influence of viral infection.Mitochondrial dynamics and autophagy are two important events that maintain mitochondrial homeostasis, which are closely linked, and they interact with each other and cooperate to complete mitochondrial quality control . Mitopha2+ to enter mitochondrial inter-membrane space through coupling mitochondrial calcium uniporter (MCU), thus regulating MMP , a byproduct of respiration, causes mutation and damage to mtDNA and eventually damages the mitochondrial function. Mitochondrial fission can release these damaged mitochondria from the mitochondrial network and quickly clear them through autophagy . In additing MMP . Howeverting MMP . MitochoPrevious studies have shown that mitochondrial dynamics have an important biological significance, including adapting to the needs of cell metabolism, maintaining cell function, and regulating cell differentiation, and it plays an important role in the body’s resistance to viral infection by participating in innate immune regulation and regulating cell apoptosis. Viruses have developed a variety of strategies to interfere with the dynamic balance of the mitochondria throughout evolution and promote their own infection and proliferation. In this section, we review the mechanisms of different viruses that regulate mitochondrial dynamics and their subsequent effects on the body. These mechanisms are summarized in Apoptosis is an active death process under physiological or pathological conditions, and it is strictly regulated by a variety of genes to maintain the homeostasis of the internal environment. It can be caused by a variety of stimuli. Based on the different initiation stages, apoptosis can be divided into three pathways. In this article, we mainly discuss the pathway that is closely related to mitochondria, i.e., the cytochrome c (Cyt C)-mediated apoptosis pathway. When cells are induced to undergo apoptosis, pro-apoptotic Bcl-2 family members (Bax and Bak) form an oligomer complex that is inserted into the OMM, triggering mitochondrial outer membrane permeability (MOMP), accompanied by the release of apoptotic factors such as CytC from the mitochondria. This Cyt C binds to apoptosis protease activating factor 1 (APAF1) to form an apoptosis complex that activates the caspase-9 precursor, which then cleaves caspase-3 and caspase-7 to activate them. These two proteases selectively degrade proteins in cells, thereby inducing apoptosis . The sigIn the early stage of viral infection, cells block virus replication and transmission through apoptosis, which is a cellular defense mechanism . TherefoIn HCV-related studies, many viral proteins were found to play important roles in inducing mitochondrial fission and autophagy. For example, envelope glycoprotein 2 (E2), core protein, and non-structural protein 3/4A (NS3/4A) can all induce phosphorylation of Drp1S616 , and nonIn the later stage of infection, viruses release virus particles by inducing apoptosis. Rotavirus (RV) is a typical case of promoting apoptosis in the late stage of infection. Mitochondrial fission was shown to be a prerequisite for RV to induce apoptosis, and the level of Drp1 has a significant regulatory effect on apoptosis . In the Above, mitochondrial fission has a different regulatory effect on apoptosis in different infection periods, which may be the result of the interaction of viral factors and intracellular signaling molecules, and it is also related to different apoptotic signaling pathways. With research advancements, the relationship between viral infection and apoptosis and the role of mitochondrial division are gradually being clarified. Researchers have applied viral products that promote apoptosis or inhibit apoptosis at different stages of viral infection to treat the infection based on the mechanism of action of the virus studied. Future research can take this as a direction to further study the relationship between viral infection and apoptosis, as well as the interaction between viral products and intracellular molecules to develop new antiviral agents.When the body is infected by viruses, RIG-I-like receptors (RLRs) recognize the viral RNA in the cytosol and undergo conformational changes to interact with the caspase recruitment domains (CARD) of the MAVS protein, which recruits TANK-binding kinases (TBK1) and IκB kinases (Iκk) to form complexes that activate interferon regulatory factor 3 (IRF3) and nuclear transcription factor κB (NF-κB), respectively. This signaling further initiates the downstream interferon (IFN) response and inflammatory response to increase the expression of INF and various proinflammatory factors, which results in a highly effective antiviral immune response . In recevia the translocase of the outer mitochondrial membrane 40 (TOM40), activates OMA1 located in the inner membrane, and then shears L-OPA1, resulting in fragmentation of mitochondria  is a member of the herpes virus family, which can infect many animals and cause serious damage to many systems in the body. CMV has been shown to induce mitochondrion fission and block the antiviral signaling pathway by targeting MAVS and stimulator of interferon genes STING; . STING ichondria ; PB1-F2 chondria , but it It has been found that some viruses can upregulate intracellular microRNA (miRNA) after infection so as to regulate the process of virus infection. miRNA is a non-coding RNA with a length of about 20–25 nucleotides, which influences various cell activities by regulating the expression of genes in eukaryotic cells. When Sendai virus (SeV) was used to attack cells, the expression of various miRNA was upregulated, among which miR-302b and miR-372 both inhibited the expression of phosphorylated Drp1 at S637 site and caused abnormal mitochondrion fission, thus inhibiting part of RLR signaling . SubsequAlthough mitochondrial elongation is generally believed to promote MAVS-mediated antiviral immune responses, mitochondrial fusion can also disrupt this innate immunity in the context of certain viral infections. Dengue virus (DENV) is an RNA virus. When DENV invades cells, it uses ER-derived structures in host cells to translate various viral proteins for assembly of its own viral particles or replication of viral genes . Convoluvia the microtubule structure to the dendrites and axons that have the highest energy demand to ensure the normal function of neurons  invades the central nervous system of the body after infection, causing neuronal damage and eventually a series of neurological syndromes . After HAbnormalities in mitochondrial dynamics have been observed in many neurodegenerative diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease PD; , suggestCell migration is the movement of cells after receiving certain migration signals. It is a common form of cell movement and is closely related to a variety of physiological activities. Malignant migration of cells is the main cause of cancer death, and some studies have found that mitochondrial dynamics and peripheral distribution will affect cell migration . In PRCCEpstein-Barr virus (EBV) is a carcinogenic virus that is associated with a variety of epithelial and lymphoid malignancies. Its latent membrane protein upregulates Drp1 expression through the Notch pathway. Additionally, the increase in Drp1 expression leads to an increase in cell migration and epithelial–mesenchymal transformation, thus increasing the invasion force of the virus . AnotherSyncytium is a multinucleated giant cell that is produced by the fusion of two or more cells under natural conditions or artificial induction. The formation of syncytium causes pathological changes in normal cells or tissues. Some viruses, which are called syncytial viruses, can cause this cell fusion.in vivo and in vitro studies.Newcastle disease virus (NDV) is a syncytial virus, and its fusion protein (F) and hemagglutinin-neuraminidase (HN) induce syncytium formation, thus causing a series of cytopathic effects (CPE) and cell death . Ren et Extracellular microvesicles (EMVs) are small vesicles that fall off the cell membrane after activation, injury, or apoptosis. They can encapsulate proteins, lipids, nucleic acids, and other substances and transport them to other cells through release to mediate information transmission between cells. Many envelope viruses take advantage of EMVs to pack virus proteins and genomes into them and release them from cells, thus promoting viral spread and persistent viral infection .Drp1 expression, CBV transmission was well controlled  post-translational modification of dynamic proteins, (2) regulation of dynamic protein expression, (3) cleavage of mitochondrial dynamic proteins, and (4) changes in the physiological environment within cells to interfere with mitochondrial dynamics. Through the above effects, the virus indirectly regulates cell apoptosis, inhibits the RLR antiviral signaling pathway, promotes tumor cell migration or promotes syncytium formation to upregulate viral infection processes and cause damage to the host’s body. In recent years, several studies have reported the association of cGAS-STING pathway between mitochondrial homeostasis and antiviral activity: after excessive mitochondrion fission or inhibition of autophagy leads to a large accumulation of damaged mitochondria, mtDNA in cytoplasm increases abnormally and is recognized by a DNA receptor cyclic GMP-AMP synthase (cGAS), and then cGAS catalyzes the synthesis of cyclic GMP-AMP (cGAMP), which further activates STING, then STING recruited and activated TBK1, thus inducing interferon production . HoweverAlthough much progress has been made in this field, there are still many unsolved problems. These unsolved problems include few studies that mention the effects of the mitochondrial dynamic balance that is manipulated by viruses on the mitochondria and cellular physiological functions and the mechanism of interaction between mitochondrial dynamic proteins and viral factors. Understanding these problems may improve our understanding of viral pathogenesis. In future studies, abnormal changes in mitochondrial dynamic proteins may be used as an indicator to predict damage to the body after viral infection, and the corresponding treatment may be given. In addition, the functional correlation between mitochondrial dynamics and viral pathogenesis should be further elucidated to develop new antiviral treatment strategies and new and efficient antiviral drugs.ZR: conceptualization, writing-review and editing, and revised the manuscript. XZ: writing-original draft and revised the manuscript. TD: writing-review. ZZ: software. HH: validation. ZX, JD: supervision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Polycystic ovary syndrome (PCOS) is an oxidative state resulting in ovarian dysfunction. Licorice is one of the natural antioxidants used for the treatment of infertility.To evaluate the effect of licorice extract on ovarian morphology, oocyte maturation, and embryo development in PCOS-induced mice.thirty-two female NMIR mice were divided into four groups (n = 8/each): control group receiving no treatment (group I); PCOS group injected with estradiol valerate once daily for 21 days (group II); and experimental groups receiving either 100 mg/kg (group III) or 150 mg/kg (group IV) licorice by gavage along with estradiol valerate once daily for 3 wk. Serum levels of the testosterone and estrogen were measured using ELISA kit. Histological study of ovaries was evaluated, and oocyte maturation, fertilization rate, and embryo development were determined after in vitro maturation.Experimental groups  had significantly higher testosterone and estradiol levels compared to the PCOS group (p It appears that the two doses (100 mg and 150 mg) of licorice could decrease ovarian cyst and improve the fertilization rate of oocyte and embryo development in PCOS mice. However, there was no statistically significant difference between the two experimental groups. Polycystic ovary syndrome (PCOS) is a hormonal imbalance and metabolic disorder affecting about 8-13% of all women during their reproductive age. Anovulation, hyperandrogenism, polycystic ovary, hyperinsulinism, hirsutism, and elevated concentrations of LH are some of the major implications of PCOS . It is seen that the increased enzymatic activities of 17αhydroxylase/17, 20 lyase, 3β-hydroxysteroid dehydrogenase, as well as the side chain cleavage enzyme lead to hyperandrogenism in patients with PCOS . In addition, the biosynthesis of estrogens from androgens is catalyzed by aromatase; hence, a deficiency in aromatase activity may lead to intraovarian disturbances and hormonal imbalance (5).An ovulatory cycle may lead to infertility resulting in a need for assisted reproductive techniques (6). Moreover, in vitro maturation (IVM) is a relatively new method in assisted reproductive techniques, in which immature oocytes are extracted from the ovaries without gonadotropin stimulation, thus limiting the risk of ovarian hyperstimulation syndrome compared to in vitro fertilization (IVF) technique (7). Currently, using various herbal remedies as natural antioxidants is suggested for improving all clinical features of PCOS to recover the menstrual cycle and normal serum hormones levels. Therefore, antioxidant supplementation are known to be effective in reducing testosterone, polycystic ovaries, and improving reproductive cycles .Glycyrrhiza glabra) has been used to treat different diseases for many years (10). Multiple biological activities such as the powerful antioxidative, antifatigue, antibacterial, antiviral, antiproliferative, and estrogenic activity of licorice have been demonstrated in several experimental evidence . Therefore, some studies have suggested licorice extract as a possible candidate for the treatment of infertility as well as PCOS (13). Previous studies have revealed that blocking the activity of 3β-hydroxysteroid dehydrogenase (3HSD), 17-hydroxysteroid dehydrogenase (17HSD), and 17-20 lyase, and stimulating the activity of aromatase, while also affecting the 5a- and 5b-reductase activity, are some effects of licorice extract . All these enzymes are involved in the synthesis and/or metabolism of androgens and estrogens (14). Lakshmi and colleagues demonstrated that inhibiting 17HSD, licorice could decrease serum testosterone in PCOS women, indicating that licorice has an anti-testosterone activity that may be beneficial in treating women with PCOS (16). Additionally, licorice exhibits estrogenic properties that could result in aromatase stimulation activity (17), and currently supplements of licorice are used for overweight PCOS patients (18). It was recently demonstrated that licorice extract can result in an improvement in IVF results in mice (19). Also, data show an improvement of PCOS symptoms such as imbalance in the hormonal levels and irregular ovarian follicles in letrozole-induced female rats (20).licorice , unless otherwise mentioned.Adult Female (6-7 wk old) and male (8-12 wk old) Naval Medical Research Institute (NMRI) mice were obtained from the breeding center at the Faculty of Science, Urmia University . The mice were housed in a 12-hr light/dark cycle, at 25 The female mice were randomly assigned into four groups (n = 8/group) as follows:Additionally, in the present study, 0.2 mg/kg body weight (BW) single dose of EV  was injected subcutaneously for 21 days (21). Studies have shown that the administration of estradiol valerate leads to anovulation and cystic ovarian morphology (22).The licorice was collected from a local farm in Urmia, north-west of Iran. The collected samples were identified by the Plant Botanical Laboratory, Faculty of Agriculture Science, Urmia University of Iran. The plant was washed with water and dried in the shade. Then, they were pulverized into a powder using a mixer grinder. To prepare the hydro-alcoholic extract, the powder of the dried licorice was extracted for 72 hr in ethanol 80%, following a solvent evaporation using a rotary evaporator. Next, it was completely dried and collected. Finally, the extract of the licorice was dissolved in normal saline to achieve an appropriate concentration.After the injection, the female mice were sacrificed under anesthesia, before drawing blood (2 mL) from the heart. Blood serum was separated by centrifuging the blood samples at 6000 g for 5 min. Serum samples were stored at -70°C for future analysis. Serum testosterone and estrogen concentrations were assessed through immune-radiometric techniques using commercially available Elisa kits  according to the manufacturer's guidelines (23).Ovarian tissue was removed for histological examination following fixation in 10% formalin. Afterward, the samples were cut serially at 5 μm and stained by hematoxylin and eosin. In the final stage, the stained slides taken from the ovaries were observed under a light microscope. The observed follicle was defined in six groups based on the morphology and diameter: primordial follicles (PRIF), in which the oocyte was closely surrounded by one layer of flat granulosa cells; Primary follicles (PF), where the growing oocyte was surrounded by one layer of cuboidal granulosa cells; preantral follicles (PAF), where several layers of granulosa cells surround the oocyte with no cavity within; Antral follicles (AF), where the oocyte is surrounded by cumulus oophorus cells within the cavity of antrum forming; Cystic follicles (CF); and corpus luteum (CL). Thereafter, all follicles were classified as healthy or atretic. The characterization of follicular atresia was done by the presence of pyknotic nuclei in the granulosa cells, degeneration of the granulosa cell layer from the basement membrane, and change in the oocyte morphology. Follicles were then counted in each section and classified according to the work of Radavelli-Bagatini .The NMRI female mice were superovulated by an intraperitoneal injection of 7.5 IU of pregnant mare serum gonadotropin . After 48 hr, they were euthanized by cervical dislocation, and ovaries were dissected and washed several times before being transferred into a preheated dissecting medium containing α-MEM supplemented with 5% FCS, 100 μg/ml penicillin, and 50 μg/ml streptomycin. under a stereomicroscope Germinal vesicle (GV)-stage oocytes of the ovarian follicles were aseptically harvested by aspiration from follicles with a 26-gauge needle in a sterile petri dish, and released in the culture medium and washed several times .The IVM medium consisting of complete MEM containing 100 IU/ml recombinant human follicular-stimulating hormone (rhFSH), and 7.5 IU/ml human chorionic gonadotrophin (hCG). The GV was transferred to 25 µl drops covered with mineral oil and cultured for 16-18 hr at 37°C in 5% COThe oocytes in the MII stage were obtained from the different groups and fertilized by sperm acquired from the cauda epididymides of 8-10 wk-old male mice with proven fertility. The epididymis was isolated from testes and placed in a petri dish containing 1 ml of equilibrated HTF medium supplemented with 4 mg/ml BSA. The epididymides were then cut and gently pressed allowing the spermatozoa to swim out into the culture media, and then allowed to incubate at 5% COFinally, the number of oocytes at GV, MII stages, and the number of two-pronuclear (2PN) formations were recorded using a stereo microscope. While the two-cell embryos were evaluated 24 hr after fertilization, the percentage of blastocyst-stage embryos was calculated on days 4 and 5 of fertilization (28).All animal experiments and study protocols were approved by the educational assistant of the Faculty of Science, University of Urmia, Urmia, Iran.Data presented as mean As shown in Table I, testosterone and estrogen levels increased significantly in the PCOS group compared to the control group . The number of healthy PRIF was not significantly different between the groups. While in the PCOS group, the number of healthy follicles were lower than the control group (PF (p = 0.002), PAF (p = 0.02), AF (p = 0.01)) (Table II). In the experimental groups, the number of healthy follicles PF , PAF (p = 0.01) were higher than the PCOS group. Moreover, there were no significant changes in the number of healthy follicles in the experimental groups (Table II). Morphological studies in the PCOS group showed that the number of atretic follicles PRIF (p = 0.01), PF (p = 0.002) in this group were significantly higher compared to the control group (Table III). Also, an evaluation of the number of atretic follicles PF . Follicular development and morphology formation in the ovaries were a result of elevated levels of testosterone and estrogen in the PCOS group (30). This refers to hyperandrogenism that leads to the generation of CF, reduction of healthy follicles, and increase in the atretic follicles (31), thereby consequently reducing the progesterone and estrogen levels due to regression of the CL (32). Licorice is a medicinal herb that is used extensively as it is regarded as an important and safe medicine (32). Nearly 500 components have been identified in the licorice root, and glycyrrhizin and several other flavonoids are the major components and most abundant constituents of licorice (32).Treating overweight women and alleviating metabolic syndrome using licorice were examined in clinical studies (33). In addition, licorice's effect on premenopausal syndrome and menopause syndromes have also been investigated and it showed promising and effective results in alleviating these syndromes (34). Our findings show that the exposure to licorice, both in doses of 100 mg/kg/day and 150 mg/kg/day, significantly decreased the testosterone and estrogen in PCOS-induced mice. In addition, licorice could increase the number of normal follicles and CL and reduce the number of atretic follicles compared to the PCOS group. There were no significant differences in the testosterone, estrogen, number of follicles among the experimental groups. Similar to these results, a study demonstrated that licorice extract improves the adverse effects resulting from hyperandrogenism due to PCOS in female mice and also has a positive effect on fertility (35).Several studies showed that licorice extract will lead to a reduction in intraovarian androgen concentration when taken orally. Subsequently, this results in decreasing the levels of androgen synthetized from estrogen, causing positive feedback on the LH secretion (36). Catalyzing the biosynthesis of estrogens from androgens is done by aromatase, and deficiency in the activity of this enzyme can be expected to result in increased ovarian androgen production and development of PCOS (37). Armanini and colleague indicated when 17-hydroxysteroid and 17-20-lyase are blocked and the activity of aromatase increases, there is a significant reduction in serum testosterone levels suggesting that licorice could be considered as adjuvant therapy of PCOS (36). A major finding of this research was that licorice positively affected IVM and IVF demonstrating a significantly higher percentage of oocytes reaching MII and blastocyst stages in both experimental groups as compared to the PCOS group. This is in accordance with the results obtained from Esmailii and colleagues, who showed that licorice consumption improved the oocyte maturation and number of follicles. They also studied the beneficial effects of licorice extract on oocyte maturation and infertility due to its phytoestrogen properties (38).Previous studies have shown oxidative stress to be a prominent pathological feature of PCOS, and women with PCOS have shown a decrease in their total antioxidant status (37). Additionally, antioxidants and reactive oxygen species (ROS) play a physiological role in the reproductive processes including oocyte maturation and fertilization (39). Several previous investigations have revealed that antioxidants can enhance the maturation of oocytes and have a positive effect on embryo development in mice (38). Ju and colleagues have also demonstrated that licorice flavonoids present a powerful antioxidant activity and are capable of scavenging more free radicals (40). Also, licorice is a source of phytoestrogen containing herbal estrogen that is attributed to the presence of isoflavones that are the key components of the plant (41).Some articles have reported that phytoestrogens decrease the serum level of androgens by increasing the level of sex hormone-binding globulin (SHBG) (42). The isoflavones glabridin and glabrene have estrogen-like activity (12). Also, a study investigated the role of estrogen in modulating sperm during fertilization (43). Sexual development, such as pubertal commencement, impaired estrous cycling, ovarian function, and that functions of the hypothalamus and pituitary can be regulated by isoﬂavones, including formononetin (44). Additionally, a study by Hajirahimkhan and colleague indicated that liquiritigenin is an active ﬂavonoid isoliquiritigenin that may be cyclized of liquiritigenin (34).Tung and colleague identified that formononetin and isoliquiritigenin are two active components in licorice root that enhance IVF (19). Although Tung and co-workers have reported a not entirely clear relationship between estrogen and fertilization, the two phytoestrogens described here may promote fertilization (45). It is possible that some of the 500 components of licorice extract may act synergistically to stimulate fertilization (45). We observed that two different doses of licorice as an antioxidant has numerous beneficial effects on the hormonal changes and improved ovarian morphology in mice with PCOS. However, different doses of licorice did not differ in the result. Our findings also showed that antioxidants and phytoestrogen activities of licorice might be useful in the promotion of IVM and fertilization. Therefore, we believe that the licorice extract could be useful for managing PCOS in women. Nevertheless, a limitation of this study was the evaluation of oxidative stress markers.According to the results of the present study, both doses of licorice may act as a useful treatment for improving PCOS. Our findings support that oral licorice extract intake can cause a reduction in testosterone and estrogen levels, and significantly enhance oocyte maturation, fertilization, and embryo developmental rates in mice. Also, licorice extract can improve ovary morphology in PCOS-induced mice. However, there was no statistically significant difference between the two experimental groups. Since no difference was found between the doses of 100 mg and 150 mg, these conditions will determine the final dose of atorvastatin for each mouse.The authors declare that there is no conflict of interest.