In the past years, we have learnt that tumors co-evolve with their microenvironment, and that the active interaction between cancer cells and stromal cells plays a pivotal role in cancer initiation, progression and treatment response. Among the players involved, the pathways regulating mitochondrial functions have been shown to be crucial for both cancer and stromal cells. This is perhaps not surprising, considering that mitochondria in both cancerous and non-cancerous cells are decisive for vital metabolic and bioenergetic functions and to elicit cell death. The central part played by mitochondria also implies the existence of stringent mitochondrial quality control mechanisms, where a specialized autophagy pathway (mitophagy) ensures the selective removal of damaged or dysfunctional mitochondria. Although the molecular underpinnings of mitophagy regulation in mammalian cells remain incomplete, it is becoming clear that mitophagy pathways are intricately linked to the metabolic rewiring of cancer cells to support the high bioenergetic demand of the tumor. In this review, after a brief introduction of the main mitophagy regulators operating in mammalian cells, we discuss emerging cell autonomous roles of mitochondria quality control in cancer onset and progression. We also discuss the relevance of mitophagy in the cellular crosstalk with the tumor microenvironment and in anti-cancer therapy responses. Mitochondria are double-membrane organelles deputed at cell energy supply; defects in mitochondrial functions not only affect cell homeostasis, bioenergetics and redox control but also are decisive for cell death. In the particular case of cancer cells, mitochondrial-harbored metabolic pathways are rewired to meet the increased bioenergetics and biosynthetic needs of the cancer cells and to handle oxidative stress. Therefore, a tight control of the mitochondrial network homeostasis is essential for cancer cells.Several highly interrelated mechanisms, including mitochondrial dynamics (fusion and fission) as well as macroautophagy (mitophagy), operate in mammalian cells as key mitochondrial quality control processes, and their implication in tumor development and progression has recently been elucidated. In particular, the selective removal of mitochondria through the process of mitophagy has been recently implicated in reshaping the metabolic landscape within cancer cells and the interaction between cancer cells and other key components of the tumor microenvironment (TME), to foster the adaptive and survival ability of cancer cells. Moreover, and considering the tight relationship between mitochondrial homeostasis and susceptibility to cell death, mitochondria quality control and mitophagy in primis are critical in anti-cancer therapeutic response as well as cancer-related off target effects. In this review, after a brief introduction of the main mitophagy pathways, we discuss the interplay of mitophagy with the key pathways involved in tumorigenesis, its coordination of the TME and its implication in the success (or not) of current anti-cancer therapies.Macroautophagy (hereafter referred to as autophagy) is a self-degradation process which is typically stimulated under conditions of nutrient deprivation or cellular stress. During autophagy, proteins, macromolecules and/or organelles are engulfed in a double-membrane vesicle called autophagosome, which eventually fuses with the lysosome where cargo degradation takes place . The Mitophagy is a specialized form of autophagy in which damaged, dysfunctional or obsolete mitochondria are recognized by the autophagy machinery and eventually degraded by the lysosome. Damaged mitochondria are, in general, those mitochondria which are not able to execute oxidative phosphorylation (OXPHOS) efficiently. This is mainly because of the dissipation of their transmembrane potential and consequent accumulation of reactive oxygen species (ROS) causing an increase in the overall cellular oxidative stress levels, precipitating mitochondria-mediated cell death . Since mBox 1Fusion is the union of two separate mitochondria into a single entity to guarantee at least one copy of mitochondrial DNA (mtDNA) per mitochondrion as well as efficient OXPHOS . Fusion In contrast, fission refers to the excision of one part of the mitochondrion from the main organelle. This separation can occur either symmetrically, where the two resulting mitochondria have similar respiratory capacity and membrane potential, or asymmetrically, where fission will result in one healthy mitochondrion and one damaged, the latter being targeted for degradation by mitophagy . FissionBelow we provide an overview of some of the best characterized  or emerging mitophagy pathways described to date in mammalian cells. The PINK1/Parkin pathway is the most studied pathway of mitophagy  32,33].,33.32,33BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and BNIP3-like (BNIP3L/NIX) are LIR-containing proteins located at the OMM which are able to directly bind LC3/GABARAP and trigger mitophagy without requiring protein ubiquitination or additional adaptors  45,46,4,446,47. FUN14 Domain Containing 1 (FUNDC1) is another OMM protein capable to bind directly LC3 and trigger mitophagy  62,63].,63.62,63CL is a phospholipid which is particularly enriched in the IMM, which is able to directly interact with GABARAP upon translocation from the IMM to the OMM ,71 in reActA  exacerbates mitophagy and suppresses oxidative stress and apoptosis induced by mitochondrial poisons [Another mitophagy receptor is Autophagy And Beclin 1 Regulator 1 (AMBRA1), a Parkin interactor  that dir poisons , indicatThe mammalian ortholog of the only mitophagy receptor in yeast (atg32) is BCL2L13/BCL-RAMBO, an OMM protein able to bind LC3 and able to induce DRP1-independent mitochondrial fragmentation . Not onl a known inhibitor of the mammalian target of rapamycin (mTOR), is a protein anchored to the OMM which is able to bind lipidated LC3A/GABARAP; its overexpression promotes mitochondrial fission in a similar fashion as BNIP3 or NIX overexpression [The recently described mitophagy receptor FK506-binding protein 8 (FKBP8/FKBP38),pression . Howeverpression ,84. Endosomal trafficking and recycling governed by the Rab family of small GTPases contributes to the mitophagic removal of mitochondria. Recent studies have highlighted mechanisms for the removal of mitochondria that are dependent on the activity of several Rab-GTPases. A recently described pathway, independent of the classical ATG5/ATG7-LC3A/B/C autophagosome axis but regulated by the Beclin1 and ULK1 complex, involves a Rab9-mediated vesicular mechanism where autophagosomes are generated by the fusion of isolation membrane with vesicles derived from the trans-Golgi and late endosomes . Recent Mitochondria can also be sequestered into Rab5-decorated endosomes through the endosomal sorting complex (ESCRT) and delivered to the lysosomes for degradation . For thiMost of the above described proteins involved in the mitophagic processes have been shown to be dysregulated in cancer patients , but wheMetabolic reprogramming is one of the hallmarks of cancer . Cancer In the following subsections, we will discuss emerging links connecting oncogene-driven metabolic pathways and key modulators of the mitophagic machinery.2-independent mechanisms to stabilize HIF-1α under normoxia and drive tumorigenesis, highlighting the essential pro-glycolytic role of this transcription factor. Moreover, changes in the expression levels of the mitophagy receptors BNIP3 and NIX can feedback on HIF-1α stability. For instance, loss of BNIP3 in a mouse model of mammary tumorigenesis reduces mitophagy and increases mitochondrial ROS levels, which results into increased normoxic HIF-1α stabilization, eventually promoting the Warburg effect and subsequently tumor progression [A main metabolic phenotype observed in cancer cells is driven by the Warburg effect, which consists in the shift from ATP generation through OXPHOS to ATP generation through glycolysis, even in the presence of oxygen , being tgression  generation of mitochondria that are metabolically adapted to cope with the metabolic and nutrient stress from the TME. An interesting conjecture proposes that the coordinated induction of mitochondria biogenesis and mitophagy may be used to generate pools of mitochondria that are better suited to catabolize fatty acids through fatty acid oxidation (FAO) . Given tThe role of mitophagy, as for autophagy more in general, might be also modulated during the various phases of tumorigenesis . NotablyTransformation mediated by c-Myc or RAS-v12 overexpression increases AMP-activated protein kinase (AMPK) phosphorylation, favoring the activation of FoxO3, which upregulates the expression of genes such as BNIP3 and LC3 . In addiAltogether, these studies reveal that mitophagy either directly or indirectly impacts the metabolism of cancer cell, but if tumor progression is favored by mitophagy or not, will depend on the mitophagy pathway involved, type of tumor and possibly on the stage of tumor development. Finally, it should be considered that beyond the role of mitophagy in cancer cells, stromal cells’ autophagy and perhaps mitophagy (as discussed further below) are emerging as key contributors to tumor progression, by providing essential amino acid fueling metabolism in cancer cells ,239. Pink1- or Park2-deficient mice. Furthermore, in a STAT3 deficient model of colorectal cancer, elevated mitophagy in intestinal epithelial cells (IECs) caused an accumulation of iron (II) in lysosomes, provoking lysosomal membrane permeabilization. This enabled antigen processing and stimulation of dendritic cells-mediated CD8+ T cells-induced anti-tumor immunity [Iron homeostasis is essential for numerous cellular processes, and either too much or too little iron can be detrimental for cell survival. Iron is fundamental for cell growth, but excessive iron accumulation induces the production of ROS and oxidative injury . Iron trimmunity . Hence, these studies support the view that mitophagy or mitophagy players take part in the control of iron trafficking at organellar level in cancer cells and stromal cells, affecting tumor progression at different molecular and cellular levels; from metabolic reprogramming to anti-tumor immunity regulation. Considering the emerging role played by mitochondria during iron-dependent ferroptosis—a regulated form of necrosis to which drug-resistant cancer cells are particularly vulnerable —it is teTumors are complex cellular systems where different subpopulations of cells coexist. Cancer stem cells (CSCs) constitute one of these subpopulations, which is characterized by their ability of self-renewal, dedifferentiation, generating the bulk tumor cells, and metastatic potential. CSCs are not a fixed entity and phenomena of dedifferentiation of mature tumor cells to CSCs can occur . TherefoHence, it seems that the role of mitophagy in CSCs may largely depend on the affected regulators and signaling pathways that control the differentiation of mature tumor cells to CSCs or the maintenance of stemness.Although mitophagy is crucial to preserve cell homeostasis, under certain conditions, either healthy or damaged mitochondria can be exchanged between cells to improve the OXPHOS capacity of the receiving cells or be degraded by the mitophagy machinery of the receiving cell, in a process called transmitophagy ,253. ForAlthough several studies suggest that mitochondrial transfer is an active process, the mechanistic underpinnings of this process remain highly controversial. Mitochondrial transfer could require direct cell-cell contact between the donor and acceptor cell , mainly Together, these studies highlight that intercellular mitochondrial transfer is an emerging mechanism of mitochondrial quality control as well as a crucial mechanism of interaction between cancer and stromal cells. However, the mechanistic underpinnings of this process are still largely elusive and more studies are needed to fully appreciate how and when mitochondrial transfer between cancer cells and their stroma contributes to cancer progression.Mitochondria are emerging as key modulators of cellular danger signaling and systemic immunity responses aiming to restore cellular or tissue homeostasis. This is perhaps not surprising since mitochondria are ancestral remnants of a-proteobacteria (endosymbiotic theory) and, as such, the mitochondrial genome (mtDNA) harbors CpG DNA repeats and encodes for formylated peptides . Given the pleiotropic effects in inflammation, immunosurveillance and therapy responses of IL-1β and IL-18, the activation of the inflammasome in cancer may have contextual pro-tumorigenic or anti-tumorigenic roles . On the other side, mitophagy has also been shown to modulate the adaptive immune response in terms of dendritic cell-T cell synapse , activatHistorically, mitophagy has been considered a protective mechanism used by the cancer cells against the onset of mitochondrial apoptosis, the main cell death pathway driven by the metabolic stress of the TME in cancer cells. Considering that several classes of anti-cancer drugs or treatments either directly or indirectly cause mitochondrial dysfunction, ROS production and cytochrome c release launching caspase activation, targeting these dysfunctional mitochondria by mitophagy could hamper the initiation of the apoptotic cascade. Therefore, the induction of mitophagy by anti-cancer therapies may modulate their cytotoxic ability and contribute to therapy resistance. This assumption is supported by several examples where the genetic inhibition of mitophagy pathways sensitize cancer cells towards cell death induced by anticancer treatments ,294. In Considering this pro-survival role of mitophagy after anti-cancer treatment, it could be expected that therapy-resistant cells would display higher mitophagy levels. Indeed, enhanced mitophagy contributes to cisplatin and etoposide resistance in cancer cells  and mitoHowever, different mitophagic pathways may be engaged to counteract therapy-induced mitochondrial damage and contribute to the therapy resistance of cancer cells. For example, in docetaxel-treated prostate cancer cells, trehalose induces ULK1-independent mitophagy and reduces their sensitivity to their treatment . MoreoveMitochondrial clearance  could also be involved in modulating cancer cell non-autonomous response to therapy-mediated cell death. Mitochondrial transfer from surrounding cells via microtubules could also contribute to cancer cell therapy resistance, since mitochondria from endothelial cells or stromal cells have been reported to increase the chemoresistance of breast cancer cells  or leukeAlthough the involvement of mitophagy has not been formally proven, blocking the release of the mitochondrial DAMP N-formyl peptides during immunogenic cancer cell death elicited in response to anthracyclines and reduces anti-tumor immunity , suggestWhen talking about therapy resistance, CSCs deserve special mention. CSCs are a subpopulation within cancer cells which is particularly resistant to therapy and which is responsible for the tumor repopulation after treatment . IntereAltogether, preserving mitochondrial fitness by mitophagy provides cancer cells a mechanism to resist anti-cancer therapy ; neverthLastly, mitophagy is also relevant for off-target toxicities derived from anti-cancer therapies as well as from cancer itself. In terms of chemotherapeutics, one of the major side-effects related to doxorubicin treatment is its cardiotoxicity caused by enhanced Parkin-mediated or BNIP3-mediated mitophagy in cardiomyocytes ,316,317.Nonetheless, the most life-threatening side effect of cancer is cancer-associated cachexia: it occurs in more than 80% of the late-stage patients, it is directly associated to 20% of cancer-related deaths , and it In different cancer mouse models (and independently of chemotherapeutic treatment), cachectic muscle displayed alterations in mitochondrial homeostasis   functions of known mitophagy modulators and their link with oncogenic signals must be deepened in order to be able to advance more effective cancer treatments.


We present the case of a 23-year-old female with a subacute history of complex additive neurology which consisted of progressive unilateral visual impairment and subsequent blindness of the right eye, in conjunction with distal lower motor neuron symptoms of weakness and sensory loss from T4 level down. Special investigations performed, included serology and an urgent magnetic resonance imaging (MRI) of the brain and spinal cord, which exhibited a diffuse demyelinating disease of the brain and spinal cord without the typical features of multiple sclerosis (MS) and laboratory findings, which were positive for the AQP-4 antibody, confirming the diagnosis of neuromyelitis optica (NMO). Pulsed methylprednisolone was initiated urgently with good effect and immunosuppression with cyclophosphamide was added after the exclusion of additional pathology. She experienced a complete resolution of her weakness and sensory impairment upon discharge; however, her unilateral visual loss remained. The recent advances in the identification of autoimmune biomarkers and the widening spectrum of imaging findings in NMO necessitate that the clinician and radiologist keep abreast of the current diagnostic tools and criteria that distinguish NMO from other demyelinating conditions. This clinical entity was previously considered a variant of multiple sclerosis (MS), but recent developments in clinical, radiological and immunological characteristics of the disease have paved the way for a more detailed definition of this condition as a separate neurological affliction.5 The spinal cord and optic nerves are occupied by aquaporin channels and appear to be the preferential targets, probably because of the increased permeability of the blood-brain barrier in these regions.4The aetiology of this condition is driven by the action of the autoantibody, namely, NMO-immunoglobulin G (IgG), against aquaporin-4 (AQP4) water channels. Aquaporin-4 channels are highly populated in the astrocytes which facilitate the transportation of water through cell membranes.1The previous classification of NMO regarded the condition as a clinical subtype or variant of MS, and this resulted in delayed detection and misrecognition of NMO. The early differentiation between NMO and MS is particularly important because the course of NMO is more severe and the treatment strategies for attack prevention differ. Immunotherapies approved for the treatment of MS have proven ineffective and even appear to aggravate NMO relapses.The diagnostic limitations of complex neurological findings in patients who present with overlapping upper and lower motor neuron signs remain a challenge for health care practitioners, especially in resource-limited centres. The cohesive application of clinical, radiological and immunological findings to reach a definitive diagnosis is of the utmost importance in such settings.We present the case of a 23-year-old female who was referred to Livingstone Tertiary Hospital with a 1-year history of Coombs-positive autoimmune haemolytic anaemia secondary to Systemic Lupus Erythematosus (SLE). She was previously on corticosteroid therapy which was discontinued and she had no other established organ involvement.She developed progressive loss of vision in the right eye, in association with left hemiparesis and subtle sensory deficit extending inferiorly from the level of T4. On clinical examination, she was fully conscious and oriented; her vision was limited to light perception in the right eye and fundoscopy revealed a clinically pale right optic disc.Further neurological assessment revealed that she had motor weakness with 4-/5 power in the left upper and lower limbs and sensory impairment predominantly affecting the left side with a T4 sensory level. In addition, she had bilateral sensorimotor weakness superimposed on her left hemiparesis and features of chronic distal lower motor neuron polyneuropathy.Initial investigations included serology and urgent magnetic resonance imaging (MRI) of the brain and spinal cord with contrast –6.Magnetic resonance imaging of the cervical spine revealed a T2 hyperintense central grey matter lesion extending longitudinally and contiguously across three vertebral body lengths. Brain MRI demonstrated similar T2 hyperintense lesions involving the splenium of the corpus callosum, the midbrain and centrum semiovale. There were no perivenular radially oriented lesions (Dawson fingers) that are typical of MS.Cerebrospinal fluid analysis demonstrated no lymphocytes, normal protein and negative oligoclonal bands. Serum studies were positive for AQP4-antibodies (NMO-IgG), confirming the diagnosis of NMO. Treatment with high-dose corticosteroid therapy and cyclophosphamide was initiated and this led to a complete resolution of the patient’s weakness and sensory deficit; however, the visual impairment in the right eye persisted.4There are limited studies on the prevalence of NMO in the Southern African population, especially in human immunodeficiency virus-(HIV) negative subjects, and this condition is likely underdiagnosed. North American studies depict ranges from 0.5 to 4.4 per 100 000, with a higher incidence in Asian populations, Indian populations and African populations, with women being more commonly affected with a ratio of 6.5–1.5Acute attacks are characterised by progressive loss of vision in one or both eyes, varying degrees of weakness or paralysis in the legs or arms, sensory deficits as well as bladder and/or bowel dysfunction. The onset of optic neuritis can be unilateral or bilateral and may be confounded by concurrent myelitis; hence, serological findings can serve to be a valuable diagnostic tool. NMO-immunoglobulin G offers a specificity of more than 90% and sensitivity of 70% – 90% for this condition.Conventional magnetic resonance (MR) imaging changes have traditionally been recognised in the spinal cord and optic nerves. However, recent studies documenting cerebral involvement have mandated the inclusion of cerebral involvement as one of the major supportive criteria.5The lesions in NMO are typically hyperintense on T2-weighted images. The distribution is observed in the regions of the central nervous system known to be rich in AQP4, typically in a periependymal distribution in the periventricular white matter, corpus callosum and central grey matter of the spinal cord.6 In contradistinction, the lesions in NMO occur along the ependymal lining of the lateral ventricles, the regions around the third ventricle, cerebral aqueduct and fourth ventricle. These are heterogeneous, oedematous and form large confluent lesions creating a ‘marbled pattern’. There may be notable involvement of the splenium presenting as a unique ‘bridging arch pattern’ as was demonstrated in this case .his case .2,5,6 Si4 LESCLs are, however, not exclusive to NMO and appear to be less disease specific in children than in adults. They are frequently observed in children with acute disseminated encephalomyelitis, as well as in 17% of those with MS and in 67% – 88% of those with monophasic transverse myelitis.6 In NMO there is a preferential involvement of the central grey matter which is abundant with AQP4 in the glial cell processes adjacent to the ependymal cells of the central canal with lesions frequently affecting the cervical and thoracic spinal cord.5 Lesions in NMO exhibit high signal intensity on T2-weighted images and low signal intensity on T1-weighted images.Spinal cord imaging features in NMO demonstrate the presence of longitudinally extensive spinal cord lesions (LESCLs), which extend contiguously across at least three vertebral segments.6In MS, however, hypointense lesions on T1-weighted images are very rare. The typical spinal cord features of MS are of multiple, short lesions which have a peripheral, asymmetric and often posterior distribution.6Optic nerve changes occur in both NMO and MS. The associated imaging features of both depict acute nerve sheath thickening and enhancement; however, long-segment inflammation that extends posteriorly to the optic chiasm should rouse the suspicion of NMO, especially when findings are simultaneous and bilateral.The current diagnostic criteria for NMO mandate the occurrence of: (1) optic neuritis, (2) myelitis and (3) at least two of the following three supportive criteria:longitudinally extensive spinal cord lesionsseropositivity for IgG antibodies that bind AQP4 on astrocytes5brain imaging findings non-diagnostic for MS.5 In presenting this case, we would like to draw special attention to the imaging features and the diagnostic biomarkers that assist in defining NMO as a clinical entity.The fulfilment of the current diagnostic criteria differentiates NMO from other demyelinating disorders, which is crucial as specific therapeutic interventions are required to reduce the frequency of flare-ups. MR imaging in patients with NMO has revealed an expanding array of lesion subtypes with which the radiologist must be familiar.Novel immunological investigations (NMO-IgG/AQP4-antibody) have had a tremendous impact in achieving greater diagnostic accuracy. With this case we hope to highlight the diagnostic complexity encountered when analysing the imaging findings in NMO and encourage a high-index of suspicion when met with the above-mentioned imaging features.


The opaque endosperm phenotype is sometimes accompanied by a soft kernel texture and increased nutritional quality, including a higher lysine content, which are valuable agronomic traits that have drawn attention of maize breeders. Recently, an increasing number of genes that underlie opaque mutants have been cloned, and their characterization has begun to shed light on the molecular basis of the opaque endosperm phenotype. These mutants are categorized by disruption of genes encoding zein or non-zein proteins localized to protein bodies, enzymes involved in endosperm metabolic processes, or transcriptional regulatory proteins associated with endosperm storage programs.The endosperm of angiosperms is a zygotic seed organ that stores nutrient reserves to support embryogenesis and seed germination. Cereal endosperm is also a major source of human calories and an industrial feedstock. Maize  Endosperm is a product of double fertilization in the female gametophyte (embryo sac), and it functions as a nutritive organ to support embryogenesis and seedling development , recessive], “floury” , or other assorted names   and genes encoding non-zein proteins associated with PBs ; (2) genes encoding enzymes involved in endosperm metabolic processes ; and (3) transcriptional regulatory genes  at the 3′ end of intron 6 , which is required for normal amyloplast and chloroplast functions and normal morphology of starch granules ] mutant has been reported to block biogenesis of proline, resulting in a general reduction in protein synthesis, an inhibition of cell proliferation, and an associated down-regulation of cyclin gene expression  involved in tyrosine synthesis, has been shown to affect accumulation of all families of zeins . O2 is specifically expressed in the endosperm as early as 6 days after pollination and encodes a bZIP-family TF , most of these proteins are encoded by genes that are ubiquitously expressed throughout the plant life cycle  gene whose loss-of-function mutants can produce an opaque phenotype is  TF Fig.   show a reduction of zein synthesis, alleviation of the opaque phenotype, and display additive or synergistic defects in combination with o2 mutants  and co-regulates a subset of target genes with O2  exhibit defects in SE and AL cell differentiation  and cyPPDK2  as direct target genes of O11  showed O2-network genes exhibit diverse spatial and temporal patterns of expression and functionalities ] or indirectly  activated by O2 , and nutrient reservoir activity  , a number of genetic suppressors of o2 (o2 modifiers) have been discovered that enabled development of “quality protein maize (QPM),” which manifests a high lysine content and vitreous endosperm , transcriptional regulators , and others   morphology , many opaque mutants also show defects in developmental processes of both seed and non-seed tissues. First, the coincident perturbation of SE/AL/ESR differentiation with storage compound accumulation in the Viviparous-1 (VP1) gene that encodes an ABI3-VP1 TF family required for proper seed maturation and germination  gene that is involved in response to hypoxia  that can modify the opaque, starchy endosperm phenotype.Recent studies of TFs  with corresponding mutants that show an opaque phenotype led to identification of additional regulatory proteins that function upstream, downstream, or as partners with the TFs. Some of these proteins are implicated in regulation of storage programs and/or other key programs of seed development or the endosperm’s response to abiotic stress. For example, NKDs regulate the


The nuclear factor-erythroid 2-related factor 2 (NRF2) transcription factor plays a central role in mediating the cellular stress response. Due to their antioxidant properties, compounds activating NRF2 have received much attention as potential medications for disease prevention, or even for therapy. Accumulating evidence suggests that activation of the NRF2 pathway also has a major impact on wound healing and may be beneficial in the treatment of chronic wounds, which remain a considerable health and economic burden. While NRF2 activation indeed shows promise, important considerations need to be made in light of corresponding evidence that also points towards pro-tumorigenic effects of NRF2. In this review, we discuss the evidence to date, highlighting recent advances using gain- and loss-of-function animal models and how these data fit with observations in humans. As the organ continuously exposed to the outside world, the skin is charged with many important functions, including maintaining body temperature, hydration, and sensing the environment. Among its most important functions is to provide a barrier to an external environment rich in pathogens and toxins. Furthermore, it is frequently exposed to harmful ultraviolet (UV) radiation. Given the importance of maintaining the integrity of the skin, mammals have evolved a complex and sophisticated wound healing response, whereby damaged tissue is efficiently repaired following injury. While the complex nature of this response contributes to its effectiveness, it also leads to challenges when attempting to remedy pathological scenarios where wound healing goes wrong, such as in chronic, non-healing wounds/ulcers and in hypertrophic scars and keloids ,2.The normal wound healing response involves a series of overlapping phases beginning with haemostasis and the formation of a fibrin clot. Haemostasis is initiated by platelets immediately after injury and functions critically to reduce blood loss from damaged vessels. It results in the deposition of a provisional matrix made primarily of fibrin that provides a rapid seal of the wound. This temporary fibrin mesh also functions as a scaffold for cells to migrate into the wound and as a reservoir of cytokines and growth factors. The inflammatory phase follows soon after, with neutrophils being the first immune cells to arrive at the wound site due to their abundance in circulation. They function as an innate response to invading pathogens through their production of proteinases and reactive oxygen species (ROS), which, however, can also damage the cells at the wound site. In addition, neutrophils assist in the recruitment of other immune cell types, such as macrophages and lymphocytes, which accumulate in the following days. Inflammatory cells also function critically to produce cytokines and growth factors, which serve to attract fibroblasts, stimulate keratinocyte migration and proliferation, and initiate the formation of new blood vessels ,4. This The complex gene expression pattern during wound healing is controlled by various key transcription factors, which orchestrate different processes at the wound site ,17. One Nuclear factor-erythroid 2-related factor 2  is a member of the cap‘n‘collar family of transcription factors, which also includes NFE2, NRF1 (NFE2L1), NRF3 (NFE2L3), BACH1, and BACH2 . NRF2 isThe benefits of activating the NRF2 pathway have generated intense interest in recent years, and NRF2 activating compounds continue to be tested for their health benefits and prevention of disease ,27,28. HNrf2 gene expression increases early after full-thickness wounding of mice along with ROS production, followed by a gradual reduction as wound healing progresses [Nrf2, but expression of this gene was also seen in cells of the granulation tissue [Wounds produce large amounts of ROS to combat invading pathogens , to attrogresses . Keratinn tissue . NRF2 han tissue .A number of studies have tested NRF2-activating compounds for their impact on wound healing and have shown interesting results. These include purified molecules (including nitric oxide (NO) or NO donors), as well as bee venom, olive oil, and various plant extracts that include these compounds ,36,37,38Global NRF2 knockout (KO) mice have no obvious skin phenotype under normal conditions and were first used in wound healing studies nearly two decades ago. Surprisingly, NRF2 KO mice healed wounds at normal rates and displayed no obvious histological differences compared to control animals . SimilarDespite the observed differences in cytokine production, the lack of a difference in wound healing kinetics in NRF2 KO vs. control mice may be due, at least in part, to compensatory actions of other transcription factors  . To addrIt is likely that certain actions of NRF2 differ substantially depending on cell type, as cells located in different organs and tissues have diverse functions, specific NRF2 targets ,23, and Nrf2 and cytoprotective NRF2 target genes . Nevertheless, and similar to other cell types, myeloid-specific NRF2 KO mice showed no difference in wound healing as revealed by morphometric analysis of histological wound sections and analysis of the wound immune cell content [The prolonged wound inflammation in NRF2-deficient mice suggested a possible role for NRF2 in immune cells. Immune cells of the myeloid lineage have important functions during wound healing in fighting off invading bacteria through the production of ROS and in the production of healing-promoting factors . In ligh content . One cavIn contrast to normal wound healing, pathological wound healing represents a situation where the normal wound healing process has failed, leading to both chronic wounds, ulcers, or hypertrophic scars and keloids. Due to the increased challenge faced by the skin in these scenarios ,2, it isdb/db mice) and in streptozotocin-induced diabetic mice was significantly improved through the use of NRF2 activating compounds when applied either systemically or topically [Typically, it is in pathological scenarios where NRF2 activators continue to gain attention as possible therapeutics due to their antioxidant properties. As shown in diabetic mice, NRF2 activity appears to be insufficient during impaired wound healing, resulting in excessive ROS levels, which can lead to severe tissue damage and uncontrolled inflammation. Consistent with this assumption, delayed wound healing observed in mice that are diabetic as a consequence of a mutation in the leptin receptor  gene expression signature, capable of promoting tumor growth in vivo in a xenograft model of squamous cell carcinoma (SCC) . Here, SThe last three decades have seen an explosion of interest in NRF2 and its antioxidant role, cementing its importance in maintaining the cellular redox status. During this time, many researchers have investigated the use of NRF2 activating compounds as a means of cancer prevention and for preventing, or even treating, chronic inflammatory and degenerative diseases ,27,28. HKEAP1, also undergo senescence at an accelerated rate compared to untreated cells [In addition to the promising effects on wound healing observed in mice, many of these findings may be translated to humans . NRF2 aned cells . Howevered cells . For othed cells , the impTogether, these findings demonstrate the multifaceted actions of NRF2 during tissue repair, and have helped uncover important mechanisms that may benefit humans suffering from chronic wounds. However, it is also evident from multiple studies that pro-tumorigenic actions and other potentially detrimental activities should remain an important consideration prior to employing NRF2 activating compounds to wounds in the clinic. Therefore, application of such compounds should be limited to short treatment periods and restricted to patients in which malignancy at the wound site is excluded. Future work aimed at unravelling beneficial vs. deleterious actions of NRF2 and identification of the relevant target genes and their regulation will be of particular importance. A better understanding of these matters will potentially lead to the development of more potent and safer NRF2 activators that may reliably be used to promote wound healing in patients.


A wide range of potential congenital myopathy therapies have been investigated on the basis of improved understanding of disease pathomechanisms, and some therapies are in clinical trials. Although large hurdles remain, promise exists for translating treatment benefits from preclinical models to patients with congenital myopathy, including harnessing proven successes for other genetic diseases.By definition, congenital myopathy typically presents with skeletal muscle weakness and hypotonia at birth. Traditionally, congenital myopathy subtypes have been predominantly distinguished on the basis of the pathological hallmarks present on skeletal muscle biopsies. Many genes cause congenital myopathies when mutated, and a burst of new causative genes have been identified because of advances in gene sequencing technology. Recent discoveries include extending the disease phenotypes associated with previously identified genes and determining that genes formerly known to cause only dominant disease can also cause recessive disease. The more recently identified congenital myopathy genes account for only a small proportion of patients. Thus, the congenital myopathy genes remaining to be discovered are predicted to be extremely rare causes of disease, which greatly hampers their identification. Significant progress in the provision of molecular diagnoses brings important information and value to patients and their families, such as possible disease prognosis, better disease management, and informed reproductive choice, including carrier screening of parents. Additionally, from accurate genetic knowledge, rational treatment options can be hypothesised and subsequently evaluated Congenital myopathies are typically characterised by hypotonia, skeletal muscle weakness, and specific pathological hallmarks on skeletal muscle biopsy  are also increasingly recognised3.Genetic diagnosis has improved with many novel disease-causing genes and variants causing congenital myopathies identified following the widespread adoption of massively parallel sequencing, and there was a peak in gene discovery in the early 2010sAlongside the advances in understanding the genetic basis, there has been a recent focus on genetic therapies for congenital myopathies, including exon skipping, RNA interference, and adeno-associated virus (AAV)-mediated gene replacement. The drive for therapies is complemented by increasing interest in the introduction of population-based carrier screening for recessive and X-linked diseases, including relevant congenital myopathies.Here, we discuss the recent advances made toward understanding the molecular basis of and potential therapies for congenital myopathies, together with the current and future challenges for the field.4. It is difficult to be certain of the overall diagnostic rate for congenital myopathies, but Agrawalet al. suggested that 60% to 80% of centronuclear myopathies were genetically resolved5, and in a recent Danish study of 107 national congenital myopathy cases that were older than five years of age, 56% received a genetic diagnosis6. Interestingly, the rate of genetic diagnosis was 83% in cases with specific features on skeletal muscle biopsy but only 29% in cases with non-specific histology6.RYR1 mutations are the most frequent culprit in congenital myopathy7. However, despite targeted gene panel and whole exome sequencing, many patients with congenital myopathy remain without a genetic diagnosis.The extensive gene discovery success over the last eight years allows the provision of a molecular diagnosis to many more patients with congenital myopathy than was previously possiblePPA2 andPYROXD1 adds altered redox regulation as a primary disease mechanism in the congenital myopathies. Despite these recent discoveries, the identification of novel disease genes has waned, suggesting that genetically undiagnosed families may harbour variants in known congenital myopathy disease genes that are not currently recognised as pathogenic or are in genes that represent very rare forms of disease. This renders the ability to identify and confirm further novel disease genes more difficult, since researchers typically rely on the identification of additional families with a similar phenotype and variants in the same gene to confirm the diagnosis in the initial family.In the last couple of years, six new congenital myopathy disease genes have been identified . In addiIn addition to the discovery of novel disease genes, our ability to perform massively parallel sequencing via whole exome or targeted gene panels has resulted in unexpectedly large expansions of the genotype–phenotype correlations for some myopathy genes .1. This, coupled with the decreased use of skeletal muscle biopsies and corresponding identification of distinct pathological features, means that it is becoming increasingly common to refer to diseases by the causative gene .Therefore, the clearly defined entities are blurring into a continuum of myopathic phenotypesSCN4A expanding the phenotype to sudden infant death syndrome (SIDS)22 makes it tempting to speculate that functional variants inRYR1 andCACNA1S are also responsible for a proportion of SIDS cases.Mutations in key excitation–contraction coupling proteins have long been known to result in severe congenital myopathies ; minimising incidental findings; reduced data handling and storage requirements; and better copy number variation calling compared with whole exome sequencing. Critical to accurate molecular diagnosis is the curation of variants by expert diagnosticians with an intimate knowledge of the group of diseases and their causative genes and proteins. Diagnostic centres where this is not the case sometimes miss pathogenic variants.Definitive genetic diagnosis for the congenital myopathies is critical to the family for reproductive planning and for optimal care of the patient. Molecular diagnostics through massively parallel sequencing—whether by targeted gene panels or whole exome or genome sequencing—is becoming commonplace. While many extol the virtues of exome or genome sequencing, it is our opinion that currently targeted gene panelset al. obtained a 35% diagnostic rate by transcriptome-sequencing a cohort of patients that did not have a diagnosis following massively parallel sequencing of genomic DNA35. We believe that inclusion of RNA sequencing (RNA-seq) in diagnostic workflows, for unsolved cases after whole exome or gene panel sequencing, is likely to result in improved diagnostic rates for congenital myopathies. A muscle biopsy can be readily collected for unsolved patients and therefore RNA-seq likely represents a better use of resources than moving to whole genome sequencing at this time.In support of the hypothesis that for many patients the causative mutation is an unrecognised mutation in a known disease gene, Cummings36. Given the high rate ofde novo variants within the congenital myopathies, cases will continue to be encountered within ICUs despite the best preventative strategies . The high rates of genetic diagnosis in very sick babies in these ICU settings compared with cohorts with later-onset or milder diseases (or both) suggest that the underlying causes of disease in later-onset cohorts may not be purely Mendelian.A number of tertiary centres have investigated the place of rapid whole exome or genome sequencing within neonatal and paediatric intensive care units (ICUs). One such study recently abandoned the randomised standard testing arm because of loss of equipoise when it became apparent that rapid whole genome sequencing of trios resulted in more timely accurate genetic diagnosis of critically ill newbornsPPA213 andPYROXD115 using yeast and both yeast and zebrafish, respectively. Advances in CRISPR-Cas9 gene editing will allow the generation of sophisticated cell and animal models precisely mirroring changes observed in patients and will be of particular use in the analysis of large proteins , where transgenic approaches are not feasible. While CRISPR-Cas9 gene editing is able to delete genes of interest relatively easily, generating specific point variations is much less efficient and takes considerable time and resources. In addition, it has been suggested that the best preclinical model for the evaluation of CRISPR-Cas9 gene therapies likely consists of patient cell lines, since the genomic background is identical to that of the patient. Thus, there will always be a place for patient biopsies and cell lines in research, and biobanking should continue to be supported for the congenital myopathies.The use of animal models remains critical to the functional evaluation of novel variants and genes. However, with the increasing rate of variant discovery, functional analysis is becoming a bottleneck in both gene discovery and diagnosis. Selection of the most suitable models can reduce the time required. Examples include the recent analysis of variants inWhilst functional analysis of variants in novel disease genes is of great interest both diagnostically and for fundamental research, “variants of unknown significance” (VUSs) in known disease genes are a significant issue for diagnostic laboratories. However, the analysis of VUSs is less advantageous for research-focused laboratories. In our opinion, there is an urgent need for high-throughput functional genomics to be built into diagnostic pipelines and included in the cost of diagnosis. Diagnostic pipelines cannot continue to rely on research funding in research laboratories to perform functional analysis of VUSs in known disease genes.37 and of the excitation–contraction coupling basis of the core and centronuclear myopathies28. A 2015 review by Ravenscroftet al.38 explored the overlap and blurring of the boundaries of the pathobiology of the different congenital myopathies, suggesting possible treatments. However, overall, our understanding of pathophysiology is not keeping pace with the discovery of new genes. Therefore, in this age in which journals demand more and more functional analysis in order to publish novel gene–disease relationships, it is prudent to remember that, after decades of research, sometimes the precise pathophysiological basis of a disease remains obscure. For example, the exact pathological mechanism of how superoxide dismutase 1 (SOD1) mutations cause familial amyotrophic lateral sclerosis is not known a quarter of a century after the association was published39. Similarly, the pathobiology by which mutations in the slow skeletal muscle/beta cardiac myosin (MYH7) tail that breaks the coiled-coil rule can cause a distal myopathy is not known 14 years after the initial publication40. However, even in the absence of a complete understanding of the pathophysiology, the identification of causative mutations has immediate benefits for diagnosis, counselling, and family planning. For genes in which the pathophysiological basis of the disease is identified, evidence-based approaches to therapy can be researched.There have been significant increases in the understanding of the pathophysiology of the major classes of the congenital myopathies, core myopathies, nemaline myopathies, and centronuclear myopathies. Recent large comprehensive reviews should be accessed for the current state of knowledge. These include reviews of the sarcomeric pathobiology that is the basis of the nemaline myopathieset al.41. Genetic therapies for neuromuscular diseases are gaining much attention, most notably with the controversial US Food and Drug Administration (FDA) approval of exon skipping therapy for Duchenne muscular dystrophy43 and FDA approval of Spinraza (nusinersen) for spinal muscular atrophy. However, as the costs of these treatments are in the order of hundreds of thousands of dollars per patient per year, this renders them unaffordable for most families and leaves healthcare systems with difficult decisions to make29. As such, the need for treatments that are more affordable persists.A range of therapeutic options is under investigation and showing great promise for neuromuscular disease. These were recently reviewed by Dowling48. However, in these studies, splice sites were disrupted, resulting in exon skipping and restored function. Though highly encouraging, this approach is not necessarily suited to the underlying molecular cause of most congenital myopathies, and the ability to efficiently correct point mutationsin vivo is a significant hurdle.A longer-term alternative to exon skipping would be genetic correction, but, to date, no studies have been published testing CRISPR-Cas9 gene editing approaches for the treatment of any congenital myopathy animal model. Success has been reported in models of muscular dystrophyet al. delivered atrial/embryonic myosin light chain 1  using AAV to the tibialis anterior of KIActa1H40Y mice in an attempt to improve muscle force49.MYL4-treated muscles were hypertrophic and had a marked increase in steady-state isometric maximal force production49. It remains to be seen whether systemic delivery ofMYL4 in males of this line can rescue the early lethality caused by urethral obstruction50.Whereas genome editing is not likely to be clinically applied in congenital myopathy in the near future, other genetic approaches are perhaps closer, and there has been proof of principle in animal models. Lindqvist52, the efficacy of systemic intravenous AAV delivery of canine myotubularin (Mtm1) to dogs at 10 weeks of age  was assessed. Not only was the intervention well tolerated but also it corrected skeletal muscle pathology body-wide; improved neurological function, respiratory function, gait, and limb strength; and prolonged the usually shortened lifespan53. Moreover, follow-up at four years post-treatment for two dogs demonstrated findings similar to those of unaffected littermates for multiple parameters54. Most excitingly, a phase I/II clinical trial is under way for XLMTM using a single intravenous dose of AAV8hMTM1 . These advances for AAV approaches are also significant because of possible translation to other congenital myopathies.Building on previous positive results with mouse and dog models of X-linked myotubular myopathy (XLMTM)55 or an antisense oligonucleotide56; (b) silencing mutant dynamin 2 in autosomal dominant centronuclear myopathy using allele-specific small interfering RNA (siRNA) sequences57; (c) a spliceosome-mediated RNA trans-splicing strategy for dynamin 258; (d) viral delivery ofMtmr2, a close homologue of the causative disease geneMTM160; and (e) lowering phosphatidylinositol-3-phosphate accumulation through PIK3C2B inhibition61. Centronuclear myopathy or other congenital myopathy patients with a confirmedRYR1 mutation are being included in a current clinical trial to evaluate the clinical benefit of antioxidant therapy in RYR1 myopathy via thrice-daily oral/G tube n-acetylcysteine . This follows promising preclinical data inRYR1 cell lines and a zebrafish model62.Additional encouraging approaches for the centronuclear myopathies include (a) targeting dynamin 2  with a short hairpin RNAActa1H40Y nemaline myopathy mouse line using an activin type IIB receptor monoclonal antibody  extended the usually shortened lifespan of male mice but did not improve other disease features50. Myostatin inhibition treatment using mRK35  was investigated in the TgACTA1D286G mouse model of nemaline myopathy and was found to be efficacious in normalising body weight, myofibre force, and grip strength63.Alongside the exploration of genetic approaches, more traditional and rapidly translatable approaches have been evaluated in animal models for nemaline myopathy. Although nemaline bodies are the hallmark pathological feature of nemaline myopathy, small myofibres are also common, suggesting that promoting muscle growth could be beneficial. Inhibiting myostatin in the KI65 and a previous study with mice66 were supportive). However, supplements did not improve muscle strength in animal models of nemaline myopathy: L-tyrosine in mouse and zebrafish skeletal muscle alpha-actin models67 and creatine, L-tyrosine, L-carnitine, and taurine in a zebrafish nebulin model68.Patients with nemaline myopathy, their families, and clinicians have reported benefit for certain dietary supplements  retained high levels of the cardiac  actin isoform (ACTC1) in skeletal muscle and that the degree of ACTC1 expression determined the level of severity72. Carrier screening aims to facilitate informed reproductive decision making by identifying those couples at risk of having an affected child with an  recessive disorder73. As congenital myopathies are often severe diseases, they can be included in carrier screening programs 74. Recommendations on carrier screening from professional associations have been evolving toward recommendations for broader screening. The American College of Obstetricians and Gynaecologists (ACOG) 2017 Opinion 690 on carrier screening recommends that a carrier screening approach be “offered to and discussed with each patient, ideally before pregnancy”75. Israel has a nationwide panethnic carrier screening program in place, screening over 60,000 individuals a year for multiple conditions76. In other countries, such as Australia, community interest in carrier screening is being investigated77. The Australian Federal Government, in its 2018 budget, announced a $20 million research project into population-based reproductive carrier screening . Although these programs will not remove the need for therapies, they could have profound impacts on the frequency, morbidity, and mortality of congenital myopathy.Reproductive carrier screening programs for recessive diseases have been in place for nearly 50 years, starting with Tay–Sachs disease in the 1970sCongenital myopathy genetics, molecular diagnostics, pathophysiology, treatments, and prevention are advancing rapidly. The next few years, moving from gene discovery toward understanding pathophysiology and developing therapies, will see a need for changing skill sets and even broader multi-disciplinary teams to be involved in congenital myopathy research.


Polycystic ovary syndrome (PCOS) is a widespread endocrine disorder, affecting approximately 20% of women within reproductive age. It is associated with hyperandrogenism, obesity, menstrual irregularity, and anovulatory infertility. Melatonin is the main pineal gland hormone involved in the regulation of the circadian rhythm. In recent years, it has been observed that a reduction in melatonin levels of follicular fluid exists in PCOS patients. Melatonin receptors in the ovary and intra-follicular fluid adjust sex steroid secretion at different phases of ovarian follicular maturation. Moreover, melatonin is a strong antioxidant and an effective free radical scavenger, which protects ovarian follicles during follicular maturation.In this paper, we conducted a literature review and the summary of the current research on the role of melatonin in PCOS.Electronic databases including PubMed/MEDLINE, Web of Science, Scopus, and Reaxys were searched from their inception to October 2018 using the keywords “Melatonin” AND “Polycystic ovary syndrome” OR “PCOS.”Based on the data included in our review, it was found that the administration of melatonin can improve the oocyte and embryo quality in PCOS patients. It may also have beneficial effects in correcting the hormonal alterations in PCOS patients.Since metabolic dysfunction is the major finding contributing to the initiation of PCOS, melatonin can hinder this process via its improving effects on metabolic functions. Polycystic ovary syndrome (PCOS) is a complex disorder arising from the interaction of genetic and environmental reasons that affects up to 20% of women at reproductive age (1). According to the ESHRE/ASRM consensus, at least two of the following three features should be present for proper PCOS diagnosis: 1) ovulatory dysfunction (oligoanovulation and/or anovulation); 2) hyperandrogenemia  or hyperandrogenism ; 3) polycystic appearance of ovaries in ultrasonography (2), together with the exclusion of other etiologies (3). In addition to reproductive and cosmetic sequelae, PCOS syndrome is associated with a higher risk of metabolic disorders including insulin resistance, increased oxidative stress (4), cardiovascular disease, type 2 diabetes mellitus, liver disease, and endometrial cancer .Women with PCOS often seek treatment due to their complaints of infertility and menstrual cycle irregularities which are the results of chronic oligo/anovulation (7). Changing lifestyles, such as nutritional counseling and weight loss are the necessary step of all treatment plans (8). Despite the many advances in the understanding of the Pathobiology and treatment strategies of PCOS over the past decades, many questions remain to be answered and the treatment of the syndrome remains empirical.MTNR1A) and melatonin receptor 2  (18).Melatonin (N-acetyl-5-methoxytrypamine) is an indolamine hormone that was firstly recognized in the 1950s (9). Melatonin levels are regulated by photoperiod as its production and secretion are promoted at night in response to darkness since light can suppress its secretion (9). Melatonin is also produced in other organs such as the gastrointestinal tract, skin, retina, bone marrow, and lymphocytes . It seems that mitochondria are the site of melatonin synthesis within cells. Moreover, the female reproductive organ, including the follicular cells, oocytes, and cytotrophoblasts are also among the melatonin production sites (12). Several studies have shown the involvement of melatonin in the pathogenesis of diabetes, cancer, Alzheimer's disease, immune and cardiac diseases (13-15). Melatonin has been identified to have different pharmacological properties such as antioxidant, immunomodulatory, anti-angiogenic, and oncostatic effects (16). Melatonin acts as an inhibitory factor on the hypothalamic pituitary gonadal axis (17). Melatonin receptors are transmembrane G-protein-coupled receptors including melatonin receptor 1 . Melatonin is also a potent free radical scavenger that exerts protective effects in female reproductive organs; for instance, it is involved in the protection of the oocyte against oxidative stress, particularly at the time of ovulation. It can also be used to protect the developing fetus from oxidative stress can be happened by melatonin (20). The levels of melatonin in follicular fluid are higher than its levels in the blood (21). The concentration of melatonin in follicular fluid significantly rises as the follicles become mature (22).In this paper, we have made an effort to review the possible roles of melatonin in the pathogenesis of PCOS as well as the potential melatonin-centered therapeutic measures, which can be recruited herein.Related published articles were searched in the following electronic databases: PubMed/MEDLINE, Web of Science, Scopus and Reaxys. All the sources were searched from their inception to October 2018 using the keywords “Melatonin” AND “Polycystic ovary syndrome” OR “PCOS.”Titles and abstracts from the electronic databases were scrutinized following the search for the keywords, and the full-text papers which were expected to match with our inclusion criteria were obtained. The following specified inclusion criteria were used: (1) studies that used melatonin in context of PCOS or those in which melatonin was somehow shown to be related to any of the mechanisms involved in the pathogenesis of PCOS; (2) papers with available full texts; (3) Papers in English language only. There was no limitation regarding the inclusion of any human or animal models for PCOS or cell lines researches. Furthermore, the following exclusion criteria were applied: (1) no report on the treatment with melatonin; (2) no report with the PCOS disease; (3) no report on the use of melatonin for treating PCOS in clinical or animal models or cell lines researches; (4) no report on the melatonin level alternation in the different body biological fluids; (5) review articles; and (6) non-English language articles .The papers, meeting all the aforementioned criteria were reviewed separately to confirm the reliability of the extracted data. All characteristics of the articles such as author name, year, study location, study population, study period, treatment method, dose of treatment, and outcomes were extracted from the papers. The extracted data of the selected studies are shown in Tables I and II.After searching the aforementioned databases with considered keywords, the papers were reviewed by two authors. In the first step, titles and abstracts were screened and 121 papers that had enough connectivity with our keywords were included. In the second step, full texts were reviewed for the eligibility and relevance of their findings, and 99 articles were excluded due to duplicate data, non-English languages, review articles, and insufficient relevance. Finally, 22 studies were selected, including studies that have used melatonin administration for treating PCOS in clinical and animal models of PCOS and also the studies that have shown alternation of melatonin levels involved in the pathogenesis and diagnosis of PCOS; 22 articles were included in the current review, including 15 clinical human studies and 7 animal studies. Table I is related to the studies that indicate the alternation of melatonin levels in different biological fluids such as blood, serum, urine, saliva, and follicular fluid in PCOS patients. Table II is related to the studies that have used exogenous melatonin as a drug for curing PCOS in sick women, animal models, or cell lines researches. And, the remaining studies are related to the melatonin receptor gene polymorphisms in PCOS patients.PCOS may be initiated by some specific abnormalities in the hypothalamus-pituitary compartment, ovaries, adrenal gland, and the peripheral compartment like adipose tissue (23). In PCOS patients, an increase in gonadotropin-releasing hormone (GnRH) pulse frequency enhances the luteinizing hormone (LH) pulse frequency and amplitude (24). In spite of elevated LH secretion, follicle-stimulating hormone (FSH) levels remain in the lower follicular range due to the negative feedback of enhanced estrogen levels and follicular inhibition (24). As a result of altered LH:FSH ratio in PCOS women, the androgen production by the theca cells in the ovaries is increased; however, due to the low FSH levels, the follicular maturation is dramatically impaired (25). In addition, ovarian dysregulation of cytochrome-17, defects in the aromatase activity of the ovarian granulosa cells (GC), as well as the stimulation of the ovarian theca cells by high levels of insulin-like growth factor 1 (IGF-1) are among other mechanisms involved in androgen overproduction (26). Excessive adrenocortical secretion of dehydroepiandrosterone is observed in approximately 50-70% of PCOS patients (27). Increased peripheral 5 alpha-reductase activity and enhanced peripheral aromatization of androgens to estrogens that induces the reversal of estrone to estradiol ratio and a chronic hyperestrogenic state are also seen in PCOS patients (28). There is general agreement that insulin resistance and hyperinsulinemia play a major part in the pathogenesis of PCOS (29). Hyperinsulinemia stimulates ovarian theca cells and leads to androgen overproduction. Hyperinsulinemia along with hyperandrogenemia and enhanced levels of IGF-1 inhibit sex hormone-binding globulin secretion, which surges the levels of bioactive androgens and worsens the clinical manifestations of androgen excess in PCOS patients (29). Also, it has been discovered that insulin resistance and hyperandrogenism can affect normal function of adipocytes (30). Previous researchers have found that adipose tissue dysfunction are associated to metabolic and reproductive dysfunction including insulin resistance and androgen excess secretion in most PCOS patients (31) .Melatonin synthetic enzymes such as arylalkylamine N-acetyl-transferase and hydroxyindole-O-methyltransferase have been identified in most tissues including ovaries (10). Measurable levels of melatonin have been identified in most biological fluids, interestingly in the follicular fluid. The concentration of melatonin in preovulatory follicles is higher than that of plasma suggesting its possible direct effects on ovarian function (32).In several studies, melatonin levels in PCOS women have been measured to find its role in the PCOS pathogenesis (Table I) . The most important metabolite of melatonin, urinary 6-sulfatoxymelatonin (aMT6s), is considered to be a good marker for melatonin production in the body (35). The plasma concentrations of aMT6s in hyperandrogenic and PCOS women were significantly higher than healthy women possibly due to higher melatonin production (35). It has been demonstrated that testosterone and estrogen can regulate melatonin secretion. Melatonin secretion has been shown to decrease in castrated rats due to the reduction of testosterone levels (36). Ovariectomized rats exposed to 17β-estradiol had reduced numbers of α/β-adrenoreceptors, responsible for the stimulation of melatonin production, in their pinealocytes (37). Sex hormones can adjust the human biological clock by affecting the hypothalamic suprachiasmatic nucleus and pinealocytes (38). In a study by Luboshitzky and colleagues, PCOS women had higher levels of αMT6s, LH, and testosterone than patients with idiopathic hirsutism or the control groups. The results of this study showed that αMT6s level inversely related to testosterone level in PCOS disease. However, this result seems contradictory because other studies in PCOS introduced the direct relationship between αMT6s and testosterone (33). Treatment with estradiol-cyproterone acetate normalized the αMT6s levels in PCOS patients by inhibiting androgens and gonadotropins (33). In another study by the same group, women with PCOS had higher aMT6s, testosterone, LH/FSH ratio, and insulin values than women in the control groups. Higher aMT6s levels were due to the amplification of melatonin production. In some studies, testosterone was introduced as a determinant of aMT6s level in PCOS patients (35). Melatonin has been found to increase the secretion of progesterone and androgen in pre-antral follicles after incubation for two weeks and in antral follicles after a 30-hr incubation (39). In other studies, the elevated melatonin levels in serum of PCOS patients was found to be positively associated with testosterone levels (40). Results from studies show that melatonin levels in blood and saliva along with the level of 6-sulfatoxymelatonin in urine were significantly higher in PCOS patients compared to the healthy women (41). The levels of 6-sulfatoximelatonin in urine, nocturnal melatonin levels in saliva (at 3:00 am), and melatonin in the blood had a significant correlation with the degree of sleep disorders (41). 8-hydroxy-2'-deoxyguanosine (8-OHdG) is a marker of oxidative damage to DNA that can be detected in urine (42). In one study, the daytime urinary aMT6s and 8-OHdG levels were similar in PCOS patients and the control group, while the night-time levels of these molecules were significantly higher in PCOS patients than those of the control group (42). During the night, PCOS women with raised oxidative stress markers had higher levels of 8-OHdG and aMT6s. The production of higher amounts of melatonin was probably for neutralizing reactive oxygen species (ROS) at the night time. Melatonin level in PCOS cases was shown to have a significant correlation with the serum LH:FSH ratio (40). In patients with higher LH:FSH ratios, melatonin levels were significantly lower than the patients with lower ratios, indicating the inverse correlation of LH:FSH ratio and melatonin secretion (40).Different studies have indicated that PCOS patients have higher serum levels of melatonin , therefore melatonin could be used as a valuable marker for the prediction of PCOS. It was suggested that the elevation of serum melatonin in PCOS patients is due to the reduction in its follicular fluid concentration (32) The role of melatonin in oocyte maturation has been also approved. Concentration of melatonin in the preovulatory follicles is higher than smaller immature follicles, resulting in higher antioxidant capacity of larger follicles (43). In PCOS patients, the decline in the follicular concentration of melatonin is due to the reduction in the uptake of melatonin from circulation and an increase in the numbers of atretic follicles (32). Follicular atresia could be seen in PCOS patients due to increased oxidative stress and follicular damage which occurs as a result of the reduction in intra-follicular melatonin levels (32). A great deal of research has shown that ROS generation and lipid peroxidation are meaningfully higher in PCOS cases . Along with these changes, levels of superoxide dismutase, catalase, and glutathione peroxidase are reduced, which causes intense oxidative stress in ovarian follicles (46). Melatonin is shown to be capable of regulating the gene expression of antioxidant enzymes, in addition to preventing apoptosis by increasing Bcl2 and reducing Caspase 3 . Terzieva and colleagues reported that melatonin levels in women with PCOS in the morning were significantly higher than at night time, and the night-day difference of melatonin levels in PCOS cases was lower than that of the healthy group (49). However, a study reported conflicting results stating that the total serum melatonin levels were significantly lower in women with PCOS (50).MTNR1B gene were determined and no differences in genotype and allelotype frequencies for these SNPs were found neither in the PCOS nor in the healthy women. In addition, they found a significant association between the rs10830963 SNP and the concentration of testosterone in PCOS patients. The amount of testosterone, glucose, and insulin in the serums of women with cytosine/guanine allele (CG) and guanine/guanine allele (GG) genotypes were considerably higher than the cytosine/cytosine allele (CC) genotypes, which describes the possible effects of DNA sequence variations of melatonin receptor genes on the occurrence of PCOS. In subsequent research that was performed on Chinese women, the relationship between the pathogenesis of PCOS and rs2119882 SNP, which is located in the MTNR1A gene was evaluated. Genotype and allele frequencies of rs2119882 were significantly different between the PCOS cases and the controls; the C allele frequency in the PCOS patients was significantly higher than the control group which confirms the association of SNP rs2119882 with PCOS (52). In another study, an association of two SNPs, rs10830963 and rs10830962 located at the MTNR1B gene with PCOS, was examined. Both genotypes and allelotypes occurrences of the rs10830963 SNP in PCOS women were significantly different compared to healthy women. In addition, the occurrences of GG and GC were higher among PCOS women. According to the results of the mentioned study, rs10830963SNP is associated with the predisposition of women to PCOS. No suggestive differences were observed in the genotypes and allele distributions of the other SNPs (rs10830962) between the PCOS and the healthy women (53). Recently, Song and colleagues reported that there is a significant association between rs2119882 and the prevalence of PCOS, although no association was found between rs10830963 and PCOS (54). They reported that the clinical and metabolic features of PCOS manifest largely in CC genotype carriers than the TC and TT genotypes. They also showed that there exists a considerable difference in the transmission of allele C of rs2119882 between obese and non-obese PCOS patients.The positive effects of melatonin on different tissues of the body are mediated by the melatonin receptors 1A and 1B (18). The earliest study on the association between common genetic variations of melatonin receptors and the prevalence of PCOS was conducted by Wang and colleagues (51). In this study, four single nucleotide polymorphisms (SNPs)  in An improvement of metabolic function in PCOS patients may enhance ovarian function. There has been growing evidence suggesting the relation of melatonin with glucose homeostasis and insulin secretion. Peschke and colleagues have shown a negative correlation between melatonin and insulin levels in patients with type 2 diabetes (55). Furthermore, several studies have shown that melatonin administration can improve glucose hemostasis, exert antihyperglycemic effects, and improve endothelial vascular function in experimental models of metabolic syndrome and type 2 diabetes (56). Faria and colleagues showed that melatonin, through melatonin receptors 1 and 2, activates a brain-liver communication and suppresses hepatic gluconeogenesis via peripheral muscarinic receptors in rats (57). Evidence from in-vitro studies have shown that glucose uptake in adipocytes and skeletal muscle cells can be increased by melatonin (58).Letrozole is an aromatase inhibitor which is largely used for treatment of breast cancer (34). In 2001, Letrozole, for the first time, was described as an ovulation-inducting agent (35). Letrozole inhibits androgens-to-estrogens conversion at the GC, resulting in the reduction of estrogen levels, which consequently releases the hypothalamus-pituitary axis from its negative feedback and increases the FSH secretion by pituitary stimulation (35). In addition, an inhibition of aromatase activity at the ovarian level increases intraovarian androgens that improve follicular sensitivity (36). A meta-analysis including 26 randomized controlled trials  concluded that letrozole therapy appears to improve live birth and pregnancy rates in anovulatory PCOS patients compared with clomiphene citrate . Chottanapund and colleagues (59) have evaluated the aromatase suppressive effects of melatonin on hormonal positive breast cancer cells and have shown that melatonin was as potent as letrozole in inhibiting aromatization of androgen to estrogen. Furthermore, in several studies, it has been demonstrated that melatonin behaves as a selective estrogen enzyme modulator (SEEM) and a selective estrogen receptor modulator (SERM) (40). Moreover, the aromatase-suppressive effect of melatonin has been also shown in various cells, such as breast cancer, glioma, and endothelial cells (42). Several studies have demonstrated that melatonin reduces obesity and restores adipokine patterns and ameliorates the proinflammatory state, which underlies the development of insulin resistance (60). These findings all together show that the use of melatonin due to its aromatase-modulating activity, as well as its reducing effects on hepatic gluconeogenesis, ameliorating the pro-inflammatory state present in PCOS, improvement of glucose uptake by peripheral tissues, and the subsequent reduction in insulin levels, may be effective in the management of PCOS patients (Table II).PCOS affects ART outcomes and controlled ovarian hyperstimulation (COH) with conventional protocols leads to a higher risk of ovarian hyperstimulation syndrome (OHSS) (61). Several studies have demonstrated that the poor fertilization, low oocyte, and embryo quality adversely influence the clinical outcomes in PCOS patients undergoing ART treatment (62). Insulin resistance of ovarian GC and overexpression of vascular endothelial growth factor (VEGF) due to insulin stimulation are proposed to be the underlying mechanisms of poor clinical outcomes (63). Melatonin may affect ovarian microenvironment by improving insulin resistance. In a randomized double-blind trial of PCOS patients, the administration of melatonin with myo-inositol enhanced the oocyte and embryo quality (64). The main goal in COH of PCOS patients is to prevent OHSS. The only way to prevent all type of OHSS in ART treatment is in-vitro maturation (IVM) followed by in-vitro fertilization (IVF) since this approach avoids the activation of VEGF-mediated processes (65). However, despite the extensive research conducted so far, IVM is still a suboptimal procedure and the application of IVM in infertility treatment remains immature (66). The IVM involves additional procedures that increase oxidative stress in oocytes and embryos and reduce the developmental competence of oocytes, in comparison to the conventional IVF (67). The effects of melatonin on oocyte and embryo quality and ART outcomes are discussed in the following sections of this review.Recently, several studies evaluated the effects of melatonin in the treatment of PCOS to improve fertility and hormonal alterations. Lemos and colleagues have shown that rat models of PCOS have lower weight gain during pregnancy and show reduced numbers of implantation sites which result in a decreased number of offspring. Also, rat models of PCOS have higher collagen content in the uterine horns which hinders the blastocyst-endometrial interactions and reduces the implantation rate (68). Co-treatment of melatonin and metformin can reverse the reduced weight gain and the high collagen fiber content of uterine horns in the PCOS group, compared to the control group. Moreover, the number and weights of the offspring, the blastocyst-endometrium interactions, and the fetal development were increased while the time required for pregnancy was decreased (68). In some recent studies, it has been reported that oral administration of melatonin has no significant effects on clinical pregnancy rate or oocyte and embryo qualities (69).Follicular fluid melatonin produced by the luteinizing GC in the late folliculogenesis period has a substantial function in the growth and maturation of mammalian oocytes (47). In the ovary, melatonin receptors in granulosa-lutein cells can regulate the function of this organ by controlling progesterone secretion in addition to LH and GnRH receptors gene expression via pathways such as mitogen-activated protein kinase and activation of Elk-1 (22). Melatonin concentrations are significantly higher in larger follicles, particularly in preovulatory follicles, due to participation in the secretion of progesterone and maturation of oocyte along with subsequent ovulation and luteinization events (70). Interestingly, intrafollicular melatonin level is considerably lower in PCOS women than the healthy women which may be the reason for the anovulation and low oocyte quality (32).The supplementation of culture medium with different substances can increase oocyte maturation and IVF, as a remedy for reproductive problems (71). Based on these findings, a number of studies were designed, using supplementation of culture media by melatonin to enhance oocyte maturation and embryonic development. In a study by Kim and colleagues, melatonin concentration gradually increased in the culture media of GC due to its production by GC. Also, the addition of melatonin to IVM media improved the cytoplasmic maturation of immature oocytes and increased the rates of implantation (72). The amplification of mRNA expression of the enzymes that participate in melatonin production in cultured GC including acetylserotonin O-methyltransferase (ASMT) and aralkylamine N-acetyltransferase (AANAT) has also been shown. PCOS results in anovulation and is considered an important cause of IVF failure. Melatonin along with other substances such as myo-inositol was identified as a good predictor for IVF outcomes, and their high concentrations were an indicator of appropriate quality of oocytes (32). Oral administration of melatonin in combination with myo-inositol in PCOS patients enhanced the quality of the oocyte and embryo, increased the number of mature oocytes, and finally resulted in an increased concentration of follicular fluid melatonin (64). In another study, the beneficial effect of melatonin on the quality of oocytes in PCOS patients during IVM with different melatonin concentrations was investigated. Also, melatonin proved to be effective in the stimulation of nuclear maturation of oocytes as well as in increasing the cleavage rate via regulating free radicals to a certain level required for increasing maturation rate (73). This effect of melatonin on oocyte maturation had a dose-dependent pattern in a manner that lower doses were more effective on maturation rate.In one study, the histopathological inspection of the ovary and uterus showed reductions in neoplastic endometrial glands and cystic follicles in PCOS rats following melatonin treatment (56). In another study, the administration of melatonin reduced cystic follicles and thickness of the theca layer and increased the number of corpus luteum and the granulosa layer thickness (74). The reduction in the thickness of theca interna is due to the action of melatonin on the reduction of androgen production in the ovary (75). Melatonin showed protective effects on corpus luteum against ROS via its antioxidant effects (76). The absence of melatonin in pinealectomized animals causes the development of ovarian cysts due to the modification of the synthesis of LH and FSH. Increase in the LH levels is a major abnormality detected in PCOS (77). Similarly, constant illumination, an induction model of PCOS in rats, have shown permanent estrous condition and polycystic ovaries aspect (78). In one study, pinealectomy and continuous light were used separately in female rats to induce PCOS. In both of these methods, the production of melatonin was diminished and PCOS condition was observed. In the group treated with melatonin, a significant reduction in the number and size of cysts were observed, probably as a result of the antigonadotrophic effects of melatonin (79). Melatonin has shown protective effects against metabolic and reproductive abnormalities in animal models of PCOS . Melatonin treatment in PCOS patients significantly affects body characteristics including reduced body weight, body mass index, and intra-abdominal fat (56).Several studies revealed that oxidative stress is one of the main reasons for female reproductive system disorders such as infertility, endometriosis, and PCOS (81). This has been supported by the fact that PCOS patients have higher levels of oxidative stress compared to healthy women . It has been demonstrated that PCOS patients are at risk of cardiovascular diseases due to their higher exposure to oxidative stress and the following undesirable effects such as blood pressure and insulin resistance (45). Lemos and colleagues showed that animal models of PCOS induced by constant illumination had higher levels of lipid peroxidation, which leads to increases in oxidative stress, pro-oxidant enzymes, and pro-inflammatory cytokines (83). Treatment with a combination of metformin hydrochloride and melatonin was shown to be advantageous in the PCOS group by regulating plasmatic variables of oxidative stress, for example, reduction of nitric oxide and total glutathione levels. Furthermore, treatment with melatonin apparently reduced liver toxicity, pro-inflammatory cytokines, TNF-α and IL-1 and the pro-inflammatory enzymes iNOS (83). Similarly, in different studies, the antioxidant effect of melatonin along with its inhibitory effects on pro-oxidant enzymes and pro-inflammatory cytokines were reported (84).Melatonin supplementation to the culture medium has been shown to increase the oocyte maturation rate and reduce ROS production (85). It can promote the expression of superoxide dismutase and glutathione peroxidase (86); also, melatonin is able to quench ROS and reactive nitrogen species (RNS) (76). Scavenging action of melatonin plays a valuable role during ovulation, because ovulation is an inflammatory process, and reactive species are generated and released in the follicular fluid (87). Therefore, melatonin quenches ROS and RNS and protects GC and oocyte during ovulation (32). Studies have also displayed that oxidative stress is detrimental to oocyte maturation, since the activation of oxidative stress pathway in follicular and IVM medium is unavoidable due to cellular metabolism and lack of antioxidant mechanisms, melatonin is a suitable candidate to be added to the IVM medium .Menstrual cycle irregularities are among the major complications of PCOS that affect the quality of life of patients leading to infertility (89). The altered steroid sex hormones in PCOS patients affect the hypothalamic-pituitary-ovarian axis and lead to the failure of follicular maturation and ovulation (89). Furthermore, hyperinsulinemia and insulin resistance cause ovarian dysfunction (90). These events result in anovulation and inadequate hormonal levels leading to irregularities in menstrual cycle.Melatonin seems to promote follicular maturation and ovulation through the protection of follicles against oxidative stress and their rescue form atresia (40). After six months of melatonin therapy in 40 normal-weight PCOS patients, menstrual irregularities and hyperandrogenism were improved. The lack of significant alterations in the secretion of insulin and insulin sensitivity suggests that melatonin may act on the ovary through an independent mechanism (91). However, the effect of long-term melatonin therapy with the aim of menstrual cyclicity improvement needs to be evaluated in large-scale prospective randomized studies.Thanks to the highly selective effects of melatonin on its receptors and therefore an excellent safety profile, it is well tolerated (92). However, more studies are required to be carried out in order to shed light on the possibility of its application in the context of PCOS.The current review summarizes the role of melatonin in the pathogenesis of PCOS and the protective effects of exogenous melatonin administration in the regulation of reproductive function in the context of PCOS. Melatonin levels in serum and follicular fluid of PCOS patients are different from healthy women. In PCOS patients, melatonin level in serum is usually higher than in healthy women, which is considered as a sign of diagnosing PCOS (32). But a reverse condition occurs in melatonin level of follicular fluid. Due to fewer uptakes of melatonin in ovarian follicle in PCOS patients against healthy women, follicular fluid contains lesser melatonin level compared to the healthy condition (32). Melatonin seems to promote follicular maturation and ovulation through the protection of follicles against oxidative stress and their rescue form atresia (40). Furthermore, melatonin showed protective effects on corpus luteum against ROS via its antioxidant effects (76). Melatonin level in follicular fluid of PCOS women is notably lower than in healthy women, which is related to the ovulation problems. It has been reported that melatonin administration can compensate the reduction of this hormone in follicular fluid and can halt ovulation problems (32). Melatonin treatment indicated protective effects against metabolic and reproductive abnormalities in PCOS patients. Melatonin administration in PCOS patients significantly affects body characteristics including reduced body weight, body mass index and intra-abdominal fat (56). During the ovulatory process, ROS are produced within the follicles; for this reason, the scavenging activity of melatonin plays an important role during ovulation. Melatonin reduces oxidative stress and causes oocyte maturation and luteinization of GC making it as an effective treatment for PCOS patients. Intra-follicular melatonin concentration was considerably lower in PCOS patients giving rise to anovulation and poor oocyte quality in these patients. The administration of melatonin alone or in combination with other drugs in PCOS women has been shown to increase the intrafollicular melatonin concentration, reduce the intrafollicular oxidative stress, and also increases the fertilization and pregnancy rates. Melatonin also improves the production of progesterone from corpus luteum in PCOS patients. The deficiency of melatonin alters gonadotrophin secretion, reduces the synthesis of FSH, and increases the synthesis of LH, the latter being the major change detected in PCOS patients (77). Melatonin can adjust the hypothalamic axis by inhibiting the release of hormones such as FSH, which leads to the reduction of cystic follicles. Melatonin can also regulate the synthesis of GnRH through its receptors in the granulosa-luteal cells via inhibiting GnRH receptor expression and sustaining the corpus luteum which maintains progesterone secretion (93).In summary, metabolic dysfunction is the major finding contributing to the initiation of PCOS, melatonin can hinder this process via its improving effects on metabolic functions. Melatonin treatment in PCOS patients can enhance the quality of the oocyte and embryo, increase the number of mature oocytes, reduce obesity, and ameliorate the proinflammatory state, which underlies the development of insulin resistance.All authors declare no conflicts of interest.