Understanding how the packaging of chromatin in the nucleus is regulated and organized toguide complex cellular and developmental programmes, as well as responses to environmentalcues is a major question in biology. Technological advances have allowed remarkableprogress within this field over the last years. However, we still know very little abouthow the 3D genome organization within the cell nucleus contributes to the regulation ofgene expression. The nuclear space is compartmentalized in several domains such as thenucleolus, chromocentres, telomeres, protein bodies, and the nuclear periphery without thepresence of a membrane around these domains. The role of these domains and their possibleimpact on nuclear activities is currently under intense investigation. In this review, wediscuss new data from research in plants that clarify functional links between theorganization of different nuclear domains and plant genome function with an emphasis onthe potential of this organization for gene regulation. We review recent discoveries that have elucidated how the spatial organization of thenuclear DNA in functional domains is relevant for the regulation of the genome inplants. PcBs concentrate distant transcriptionally inactive PcG-targeted genomicregions that form intra- or interchromosomal interactions also detectable by chromatinconformation capture techniques . A dispeCajal bodies  are dynamic substructures, which are found in animalas well as plant cells . CBs arede novoformation of the PBs  which surrounds the nucleoplasm consists of the nuclear envelope(NE), a double membrane layer with a continuum to the endoplasmatic reticulum (ER), nuclearpore complexes (NPCs), and the membrane adjoining the NPCs. The NP ensures the regulatedtransfer of mRNAs from the nucleus to the cytoplasm and of proteins in the reciprocaldirection, and has essential roles in gene regulation and genome organization in providingattachment sites for genomic regions and chromatin. While the cell biology of the NP and theassociated chromatin has been extensively reviewed (e.g. Daucus carota as the nuclear matrixconstituent protein (NMCP) family , and maize with two orthologues called NMCP/CRWNhomolog (NCH1 and 2) genes  in Arabidopsis results innuclear morphology changes such as reduced nuclear size and increased circularity, but doesnot lead to obvious growth defects  results in de-regulation of many genes which are involved in stressand defence responses, but also include other Gene Ontology (GO) terms  complex spanning the NE. The nuclear lamina in animals consists oflamins, type-5 intermediate filament proteins, and lamin-associated proteins. While some ofthe LINC complex members are conserved in plants, strictly defined lamins andlamin-associated genes are not present in plant genomes (2) genes . Anothercrwn4mutants, chromocentres are dispersed, while the number of chromocentres is reduced incrwn1 crwn2 double mutants  and of active chromatin (H2A.Z and H3K4me3)   (revieweH3K4me3) .PWO1 lead to a reduction innuclear size and misexpression of a set of genes which are also misregulated incrwn1 crwn2 mutants  in Arabidopsis to200 kb in tobacco  proteins, members of the Single-Myb-Histone proteinfamily . The TRBDvorackovaThe telomeric epigenetic pattern depends neither on telomere sequence composition nor ontelomere lengths, and shows large variability . ConsistThe impact of plant telomeres on long-distance interactions, the telomere gene silencing inother plant species, or participation of telomeres in formation of NBs also represent openquestions.Progressive approaches including CRISPR-based telomere labelling  describes chromatin regions that appearas ‘self-organized’ structures, formation of which is associated with chromatincompartmentalization, chromatin looping, and chromatin insulation (reviewed in Drosophila genome causing changes in chromatintopology (including TADs) showed that they are not predictive of changes in gene expression.These results suggest that the expression of a gene can endure alternative TAD patterns aslong as it maintains the same enhancer–promoter contact profile  showed thattissue-specific TAD borders in rice and maize often overlap with gene up-regulation. Anotherexample comes from comparisons of TAD patterns in diploid and subgenomes of tetraploid(formed via polyploidization) cotton genomes, which shows that conserved TAD boundaryregions tend to have a higher level of chromatin accessibility and the euchromatin histonemark H3K4me3 than do non-conserved regions  and bZIP (basic leucine zipper) proteins, which belong to twolarge plant transcription factor families  or liquid phase condensation is the de-mixing of ahomogeneous solution of proteins into two liquid phases where one is enriched for theprotein  providingthe biomolecules with an intrinsic capacity for LLPS required to form nuclear bodiesinvolving chromatin. The extensively studied non-histone chromatin protein family,Heterochromatin Protein 1 (HP1), first described in osophila , is a prDrosophila and a component of PRC1. CBX2 undergoes phase separation,thereby compacting PcG-associated chromatin  via LLPS.CBX2 is the homologue of chromodomain-containing Polycomb (Pc) protein oftin , is a clear example of a nuclearcompartment formed by LLPS. Fibrillarin (FIB1) in the dense fibrillar component andnucleophosmin (NPM1) in the granular component of the nucleolus can phase separateThe list of studies on LLPS-mediated formation of NBs is still growing, indicating thatphase transitions are key mechanisms for nuclear organization. For example,phosphatidylinositols, in particular phosphorylated PIP2, can play an important role innuclear compartment formation since they have been detected in the nucleoplasm, nuclearspeckles, and nucleolus. They interact with >300 nuclear proteins  and Bromodomain-containing protein 4 (BRD4) areenriched at clusters of enhancers and transcription machinery components, so-called superenhancers  to decipher in more depth the genetic andepigenetic information of an increased number of plant species, have allowed the existenceof several compartments in the 3D nucleus to be highlighted and are helping to uncoverconnections between regulatory domains of the 3D genome. Nevertheless, how spatial andstructural organization of the nucleus influences its function is far from being understood.Recent discoveries in this area have indeed proven to be key in depicting the complexity ofgene expression regulation within the reduced nuclear space as we have discussed. However,caution needs to be present in attributing functions to 3D nuclear organization. Theintrinsic capacity of chromatin to be plastic allied to adjustments of chromatinorganization and nuclear domain arrangements in relation to plant species, specifictranscriptional requirements, developmental stages, cell and tissue type, and epigenetic andstress factors can aid in allowing fast changes in gene expression and thus to inducequicker responses to specific requirements .LLPS is increasingly emerging as an important player in 3D genomics. Understanding theformation and organization of highly conserved and plant-specific nuclear domains inresponse to external and internal cues will contribute to our understanding of theregulation of these processes and plant phenotypes but also, most probably, to the discoveryof future molecular tools. On the other hand, this complex nuclear dynamism illustrates wellthe difficulty in finding universal rules for making direct connections between 3D nuclearorganization and predictable gene expression. Furthermore, chromatin-mediated regulation ofgenome accessibility may also be involved in molecular memories of responses given todifferent types of stimuli, but very little is known about perpetuating chromatin states andeven less about inheritable gene expression patterns between generations as a way to ensuremore prompt responses to challenges  (reviewe


Despite the important evolution of immunotherapeutic agents, brain tumors remain, in general, refractory to immune therapeutics. Recent discoveries have revealed that the glioma microenvironment includes a wide variety of immune cells in various states that play an important role in the process of tumorigenesis. Anti-tumor immune activity may be occurring or induced in immunogenic hot spots or at the invasive edge of central nervous system (CNS) tumors. Understanding the complex heterogeneity of the immune microenvironment in gliomas will likely be the key to unlocking the full potential of immunotherapeutic strategies. An essential consideration will be the induction of immunological effector responses in the setting of the numerous aspects of immunosuppression and evasion. As such, immune therapeutic combinations are a fundamental objective for clinical studies in gliomas. Through immune profiling conducted on immune competent murine models of glioma and ex vivo human glioma tissue, we will discuss how the frequency, distribution of immune cells within the microenvironment, and immune modulatory processes, may be therapeutically modulated to lead to clinical benefits. Gliomas are the most common primary brain tumors and are classified by the World Health Organization (WHO) as grade I–IV tumors based on molecular and genomic features, allowing a more accurate classification of patients. Given the transition from histological characterization, this newer molecular classification system enables precision medicine therapeutic selection and leads to a more accurate prediction of prognosis ,2. One oDespite the beneficial effects of immunotherapies in multiple types of cancers including brain metastases from several solid tumors ,7, the vSeveral criteria need to be met in order to induce effective clearance of cancer, including: the presence of the immunological target, capacity to induce immune activation, homing and dispersal of effector immune cells throughout the tumor microenvironment (TME), and the capacity to maintain an effector response. In the context of glioblastoma, multiple barriers exist for each criterion that are further confounded by an overwhelming recruitment of immunosuppressive cells, inhibition and exclusion of effector anti-tumor cells, secretion of immunosuppressive factors (like TGF-β) and expression of inhibitory checkpoint markers .Gliomas, especially glioblastoma, have not been responsive in general to immune checkpoint inhibitors and one reason is that the biomarkers of response such as PD-L1 expression, tumor infiltrating lymphocytes (TILs), and neoantigen creating defects, are relatively lower compared to other responsive cancers ,15,16. TAlthough chimeric antigen receptor (CAR) T cell immunotherapy would be able to overcome several of the aforementioned limitations, in contrast to the clonal expression of tumor antigens on leukemia that provides a more uniform and durable response, no ubiquitous homogenously expressed antigen in GBM has been identified to date and as such CAR therapy targeting a tumor-specific antigen, epidermal growth factor variant III (EGFRvIII), did not result in durable responses and antigen escape resulted  similar Gliomas, especially glioblastomas, are typically described as having a “cold” microenvironment given the paucity of TILs displaying an effector response. Molecularly distinct gliomas, depending on their IDH mutation status, have different immune compositions and landscape that defines its TME ,25,26. IIn addition to the different immune populations that populate different cancer lineages within the CNS, unbiased transcriptional profiling is beginning to reveal distinct subpopulations and states that further increase immunological complexity and heterogeneity even within the same immune cell lineage in the TME. Based on transcriptomics, there may be many different microglia subtypes that demonstrate different functional signatures such as phagocytosis, antigen presentation and lytic functions . AlthougAdditional evidence that the glioma TME is distinct from other cancer lineages is the lack of tumor mutational burden (TMB) being a predictive response biomarker to immune checkpoint inhibitors. McGrail et al. recently showed in an analysis of over 10,000 patients that not all tumors with high-TMB developed effector immune responses to immune checkpoint inhibitors therapy . SpecifiThe heterogeneity of the immune microenvironment of gliomas is not only defined by a different immune compositions within a tumor, but also the localization throughout the tumor and its surrounding. Most studies focused on the immune profiling within the tumor itself, neglecting its surrounding. A chemokine analysis from the Ivy Atlas suggests differential expression of immune populations at the infiltrating edge . The CNSCurrent dogma is that antigen presentation by antigen presenting cells (APCs) for T cell activation occurs in the lymph node with the effector response migrating to the TME. Under certain treatment scenarios, such as radiation and signal transducer and activator of transcription 3 (STAT3) blockade, dendritic cells are drawn into the TME forming immune activating immune cluster interactions that ultimately result in tumor clearance from the CNS . AdditioThe BBB represents an important factor of treatment failure at the infiltrating edge and in low-grade gliomas. By limiting the access to the tumor, therapies fail to achieve sufficient concentrations in vivo and to engage target limiting therapeutic effect . There aTo induce better drug delivery to the tumor, direct intracranial administration strategies have been conducted including clinical trials of convection enhanced delivery that have only shown modest prolongation of survival . This ap+ T cells, NK cells and macrophages associated with a decrease in regulatory T cells (Tregs) in the brain TME [Immunotherapy molecular conjugates are also being developed as a way to enhance delivery across the BBB. The coupling of CTLA-4 and PD-1 to targeted nanoscale immunoconjugates on a natural biopolymer scaffold  triggers prolonged survival in preclinical glioma models with better delivery of the drugs, and induced adaptive immune response including an increase of CD8rain TME . Anotherrain TME ,69,70,71+ tumor infiltrating T cells in murine models of glioma [Multiple immune suppressive mechanisms and pathways are utilized by the tumor cells and their environment to inhibit anti-tumor eradication. T cells are usually the effectors of the cytotoxic anti-tumor immune response in many cancers and if found in the glioblastoma TME, they are under a dysfunctional state and exhausted. Exhaustion occurs after repeated antigen exposure that leads to the expression of multiple immune checkpoints on the T cell surface . Brain mf glioma . More ref glioma . The assA frequently expressed immune suppressive pathway in glioblastoma is the A2aR-adenosine pathway ,100. Rel+ effector T cells mediated immune responses [TAMs, the most abundant immune cells present in the TME, express a wide variety of immunosuppressive phenotypes . Gliomasesponses ,106. Howesponses ,108. TheStimulator of interferon genes (STING) is an important component of the innate immune response to pathogenic DNA. It is a widely expressed sensor of cellular stress, activated by cytosolic cyclic dinucleotides, which may be released by bacteria or created through cytosolic self- or viral-DNA interaction with cyclic GMP-AMP synthase (cGAS) ,110. STI+ T cell activation by increasing the secretion of INF-γ [The reciprocal immune modulatory strategy of TAMs is to block a key deactivating pathway the signal transducer and activator of transcription 3 (STAT3). STAT3 is an important mechanism of suppression of both innate and adaptive components of the immune system . STAT3 eof INF-γ . STAT3 aof INF-γ ,136,137.of INF-γ ,138.The combination of the standard of care radiation therapy with a STAT3 inhibitor, showed improvement in the overall survival of mice harboring intracranial gliomas . This waTherapy with a single modality is unlikely to achive meaningful improvements in the outcomes of patients with glioblastoma. In order to maximize the effect of current immunotherapies, several complimentary potential solutions will need to be deployed such as cytoreductive surgery and other measures to reduce immune suppression . Immune


Erectile dysfunction (ED) is an important cause of reduced quality of life for men and their partners. Recent studies have found that cavernous nerve injury (CNI) during prostate cancer surgery and other pelvic surgery results in medically induced CNIED in more than 80% of patients. The efficacy of first- and second-line treatment options for ED is poor. A great deal of research has been devoted to exploring new methods of neuroprotection and nerve regeneration to save erectile function in patients with CNIED, especially in patients with cavernous nerve injury after prostate cancer surgery. In addition, such as neuromodulatory proteins, proimmune ligands, gene therapy, stem cell therapy, and the current cutting-edge low-energy shock wave therapy have shown advantages in basic research and limited clinical studies. In the context of today's modern medicine, these new therapeutic techniques are expected to be new tools in the treatment of cavernous nerve injury erectile dysfunction. This article presents the main causes, mechanisms, and treatment of cavernous nerve injury erectile dysfunction and combines them with new treatment strategies. Erectile dysfunction (ED) is a male sexual disorder in which there is repeated or persistent difficulty achieving or maintaining an erection with a prevalence of 30-64% in men aged 40 to 79 years. Erectile dysfunction has a variety of causes, and with the widespread introduction of pelvic surgery in recent years, a type of neurologically injurious erectile dysfunction is becoming a focus of urological research, namely, cavernous nerve injury erectile dysfunction (CNIED). A global multicentre study conducted by the European Male Ageing Study (EMAS) has shown that CNIED accounts for over 14% of the total number of patients with ED .The cavernous nerve (CN) is the main autonomic nerve regulating penile erection and is involved in the voiding reflex by innervating the urethral transverse muscle on the anterior aspect of the prostate through branches. Han et al. first probed the course of the CN by evoking electrical stimulation during RP and detailed the male fetal and neonatal cavernous innervation pathways, demonstrating that disruptions in the conduction pathways of the erectile reflex arc are a direct contributor to ED . The CN Medically induced injury is the most common cause of CN injury, with radical prostatectomy (RP) being the cause of increasing interest in recent years. Prostate cancer (PCa), the second most prevalent malignancy in men, has seen recent advances in diagnostic techniques, yet RP remains the preferred strategy for early treatment . Due to Nonmedical injuries leading to cavernous nerve injury erectile dysfunction are mainly caused by pelvic trauma, including pelvic nerve injury and posterior urethral injury-causing CN disuse, in which the proportion of patients with deep abdominal nerve injury complicating ED is as high as 95.12% .It is generally accepted that the neurotransmitter nitric oxide (NO) and the constitutive NO synthase (NOS) isoforms are important mediators of penile erection . Combinaβ1) is upregulated, and prostaglandin E1 (PGE1) and cyclic adenosine monophosphate (cAMP) are inhibited. TGFβ1 and prolonged hypoxia increase the synthesis of endothelin-1 (ET-1) [Antifibrotic compounds within smooth muscle cells inducible nitric oxide synthase (iNOS) are downregulated, transforming growth factor beta 1 (TGF1 (ET-1) . This vi1 (ET-1) . Followi1 (ET-1) .Phosphodiesterase type 5 inhibitors (PDE5i) have been the mainstay of treatment for CNIED for many years, and PDE5i has been used in various types of ED by increasing cGMP levels to counteract cavernous tissue fibrosis. Although they have been shown to be effective in some populations, patients with CNIED are generally complex and PDE5i is extremely dependent on the NO pathway and only works in those with preserved nerves, resulting in poor efficacy . In a stBenjamin et al. first found in a rat model of CN injury that nNOS-positive nerve fibres recovered significantly when given brain-derived nerve growth factor (BDNF) or vascular endothelial growth factor (VEGF) injections and that the combination of these neurotrophic factors (NTFs) also promoted the formation of myelin in Schwann cells . In anotβ1 (TGFβ1). Histone deacetylase (HDAC) hyperactivates the extracellular signal-related kinase (ERK) and phosphatidylinositol 3 kinase (PI3K) pathways via TGFβ1, which nullifies the deacetylation of HDAC, leading to vascular remodelling and fibrosis [In contrast, the dominant negative effector cytokine in the pathological process following CN injury is transforming growth factor fibrosis , 25.β1, for example, are key in the regulation of neural repair, cytokine targets are broad and nonspecific, and the risk of adverse effects of directly applied related agents is extremely high, and further validation in the ED therapeutic field is lacking. At the same time, these growth factors can be induced by a variety of interventions to circumvent the potential risks of direct application, setting the stage for various CNIED treatment modalities to be investigated.Although NTFs and TGFNinj1 expression is low in healthy organisms, and in human peripheral blood, Ninj1 is mainly regulated by monocytes. Ifergan et al. identified an important role for Ninj1 in the infiltration of antigen-presenting cells (APCs) into the central nervous system . It was The current research on Ninj1 antibodies is mainly focused on DM neuropathic ED, and no studies have been reported on its direct application to CNIED. Due to its neurotrophic and restorative capacity, it can be considered that Ninj1 antibodies have the potential to become a new treatment modality for CNIED when the research is refined at a later stage.2+ sensitization pathway, an erection inhibition process that was thought to be regulated independently of NO signaling in earlier studies [RhoA is a small molecule monomer of the Ras-GTPase family of G proteins with bioregulatory effects. RhoA signaling in the penis activates Rho-associated protein kinase (ROCK), which inactivates myosin light chain phosphatase (MLCP) and regulates cavernous smooth muscle contraction by mediating the Ca studies . However studies . Sopko e studies . This co studies , but man studies –35. Hann studies . The non studies .Smooth muscle diastole was evident in Y-27632 in human cavernous  applications, and the effect was even more pronounced with the combination of vardenafil. Because RhoA/ROCK inhibitors act on a wide range of upstream sites, they have been used in current clinical studies for the treatment of cardiovascular disease. For the treatment of CNIED, Löhn et al. selected the more selective ROCK inhibitor SAR407899 for evaluation, and clinical phase II trials have been completed, but the results have not yet been published . CombineImmunophilin is a specific receptor protein for immunosuppressants and was first recognized in the immune system as the receptor protein that binds cyclosporine, rapamycin, and the novel immunosuppressant tacrolimus (FK506). At the end of the last century, FK506 began to be used in animal models of peripheral nerve injury, and its ability to enhance nerve recovery was demonstrated . SubsequDespite mostly positive results in animal studies, in 2010, the American Urological Association published the results of a multicentre randomised controlled phase IV clinical trial showing that patients with CN injury after RP had no significant improvement in erectile function during tacrolimus treatment and two years of follow-up but instead experienced serious adverse effects such as nephrotoxicity and neurotoxicity due to long-term immunosuppression . In anotIn addition to medical techniques such as PDE5i and maturing prosthetic implants, vacuum negative pressure devices and prostilbestrol combined with extracorporeal injections all play an important role in the adjuvant treatment of CNIED, which are mainly involved in the repair of damaged cavernous oxygenation, smooth muscle antiapoptosis, and antifibrotic changes. Regenerative medicine research focuses on promoting functional nerve repair and regeneration through the endogenous regenerative capacity of damaged tissues, and the available regenerative medicine tools for CNIED include gene therapy, stem cell therapy (SCT), tissue engineering, and low-intensity Extracorporeal Shockwave Therapy (LESWT).SCT is an emerging treatment for CNIED. The use of different tissue-derived stem cells such as adipose tissue-derived stem cells (ADSCs) , urogeniClinical studies with stem cells in CNIED have shown that stem cells have the ability to reverse structural damage and apoptosis in cavernous tissues, thereby reducing patients' dependence on the transient effects of PDE5is and reducing drug resistance –54. VariStructural changes seen in the cavernous body after SCT include increases in endothelial and smooth muscle cell markers, increases in neuronal cell markers, decreases in collagen content, and decreases in CN cell apoptosis. Importantly, preclinical studies have confirmed that treatment with intracavernous SCT in the cavernous body is safe for rats and can serve as an appropriate model, thus facilitating further studies. Recent advances in SCT studies have facilitated clinical trials in men after RP. Haahr et al. obtained autologous ADSC by liposuction in 17 patients with refractory ED after RP liposuction . IntracaTissue engineering is widely and maturely used in areas such as burn skin vascular transplantation, artificial bladder replacement, and some of these stem cell therapy applications are also part of the modality. Tissue engineering is currently used in the field of erectile dysfunction treatment mainly for artificially constructed structural scaffolds seeded with cell implants. Kershen et al. were the first to use polyethanol polymers to grow endothelial cells and cavernous smooth muscle cells for implantation in vitro, and the grafts were shown to form vascular tissue in vivo, significantly increasing the maximum ICP in experimental animals . Song etPast studies have demonstrated that LESWT can repair endothelial damage and improve tissue blood supply in rat cavernous tissue . ConsistThe cavernous nerve structure is complex and prone to injury, and today, cavernous nerve injury erectile dysfunction has become a nonnegligible clinical problem after RP surgery that requires attention. Its prevalence remains high with the development and promotion of the procedure. For many years, PDE5i has been the first-line treatment modality for CNIED. Although studies have demonstrated the efficacy of these conventional drugs, which are primarily used for vascular erectile dysfunction, cavernous tissue damage is more complex in patients with CNIED who have nerve damage, resulting in poorer efficacy and resistance to these drugs. The discovery and breakthrough of pathways represented by RhoA/ROCK signaling continue to provide CNI-specific targeted targets for alternative treatment modalities to conventional PDE5i interventions.Despite the lack of adequate clinical trial reports, initial basic experimental exploratory work on these therapies has been completed or utilised in neurogenic pathology in other tissues, bringing hope for urological applications. It is believed that conventional treatments for cavernous nerve injury erectile dysfunction will be severely challenged under current therapeutic thinking and that stem cell-related regenerative medicine techniques will play a greater role.


Awareness of pectus carinatum has increased among the medical community over the last several decades, as innovative options for nonsurgical treatments have become more widely known. Management alternatives have shifted from open resective to minimally invasivestrategies, and finally, to reshaping the chest using both surgical and non-surgical modalities. We aim to review the evolution of the diagnosisand treatment of pectus carinatum up to its current management. Severalimportant developments in the diagnosis andtreatment of pectus carinatum have takenplace since these initial steps. Our aim is todescribe these events in a way that enablespaediatric and general thoracic surgeons tounderstand the origins and evolution of thisdisease and its treatment.Although pectus carinatum has classicallybeen seen as an isolated protrusion of thesternum, the condition comprises a deformityof the entire thoracic cage, both the sternumand the adjacent chondrocostal structuresA Medline literature search was undertakenusing the MeSH term ‘pectus carinatum’. Atotal of 382 articles were retrieved. A subsetwas chosen according to the authors’ viewof the best description of the evolution ofdiagnosis and treatment of pectus carinatum.The incidence of pectus carinatum is verydifficult to estimate because the condition isoften underdiagnosed. The development andspread of non-invasive treatment methods has raised interest in this malformation, andreferral has increased exponentially in thelast 20 years.6] It is moreprevalent in males, at a ratio of 3 or 4 to 1.6]Pectus carinatum is considered thesecond-most common cause of thoracicmalformations. Its reported incidence rangesfrom a fifth of that of pectus in some centres,to an equal incidence in others.[7] and secondary arethose malformations that occur after largesternotomies for cardiac surgeries,or resectiveoperations for pectus excavatum repair.9]Aetiology may be primary or secondary.Primary malformations are those with nopredisposing factors except for familialaggregation of 25%,Pectus carinatum may be classified as of eitherchondrogladiolar or chondromanubrialtype, depending on the site of the sternalangulation [4]Special malformations include pectusarcuatum Diagnosis is made by physical examination;the deformity can be characterised withoutcomplementary studies such as X-ray orcomputed tomography, which can addno information and in addition requireradiation. Systematic photography is used toassess the shape of the chest for the durationof the follow-up period. The elasticity of the sternum and cartilages should always beassessed; this can be achieved subjectively bymanual compression or by means of pressure-measuring devices. Only in special cases should furtherstudies be done, such as in associated skeletaldeformities, or when associated syndromessuch as von Recklinghausen, Poland orMarfan, or cardiac conditions, are suspected.11] Three-dimensional virtualreconstruction allows the attendingphysicians to measure the severity, thesymmetry and the evolution of thedeformity with treatment, both operativeand non-operative, at outpatient clinics.This technology seems promising and avoidsradiation, but it still requires validation.Recently, the use of 3D scanners has beenreported for the diagnosis, characterisationand follow-up of patients with pectus carinatum and those with excavatum[12] Since then, surgeons have reportedseveral different variations on the originaltechnique. Following Nuss et al.’s[13] reportof a new non-resective operation for chestremodelling with a high level of success, manysurgeons began exploring and publishingtheir experiences with non-resective surgeries, which included bar implants for pectusboth excavatum and carinatum, both intra- and extrathoracic.As is the case for other thoracic deformities,treatment in the modern era began in themid-20th century, with the first report of anattempt at surgical correction by Ravitch in1952.[14] as earlyas 1979, this modality did not gain popularityamong surgeons until the Nuss techniqueof chest remodelling (instead of resecting)completely changed the conception oftreatment.Although dynamic chest compression wasdescribed by Haje and Raymundoet al.[6]reported the creation of a pressure-controlledbracing system, and described its applicationin patients, introducing new concepts such aspressure of initial correction and pressure oftreatment, as well as the use of pounds persquare inch (psi) as a unit of measure forrigidity of the thoracic cage.This change of paradigm inspired surgeonsto explore the concept of reshaping the thoraxfrom the outside, avoiding the need for anoperation. In 2008, Martinez-Ferro In summary, the treatment modalities ofpectus carinatum may be either surgical ornon-surgical, and surgical treatments areeither resective or non-resective [16] Asphyxiatingchondrodystrophy has been reported as aresult of open resective surgeries done tooearly in childhood in pectus excavatumpatients (though this is less likely in pectuscarinatum cases).18] Far from beingabandoned, these surgeries, or variations ofthem, are still the first choice in cases of verydeformed, rigid, non-articulated sternumswith associated malformations, or after otherapproaches have failed. Open resective surgeries of the affectedcartilages associated with sternal osteotomieswere the first operations performed forthis thoracic deformity. They consistof large incisions on the chest that areusually submitted to considerable strain,with potential keloid formation, and ample resection of several cartilages, withperichondrium preservation and sternalosteotomies fixed with small bars thatrequire subsequent removal. Disadvantagesof this operation include significant bloodloss, long operating time and considerabledamage to soft tissues.Open resective techniques: The Ravitch,[12]Robicsek,[19] Willital[21] and Welch[20] werethe most commonly used open resectivetechniques prior to Nuss et al.’s[13] first reportof minimally invasive surgery for pectusexcavatum.[12] first report of an open correctionof a pectus carinatum deformity established aseries of steps, including cartilage resectionof the ribs, sternum osteotomies andfixation. Robicsek et al.[19] reported an uppersternal osteotomy and resection of its lowerangulated portion and the xiphoid process.Shamberger and Welch[20] reported on theirextensive experience using bilateral cartilageresection, even in cases of pectus carinatum,as well as various osteotomies, dependingon the type of sternal deformity. Saxenaand Willital[21] reported extensive chondraland distal segments of ribs, detorsion ofthe sternum with retrosternal mobilisationand fixation with trans-sternal and lateralparasternal metal struts. It is now generallyagreed that chondral resection must ensurepreservation of the perichondrium to allowfor cartilage regrowth Ravitch’sMinimally invasive resective techniques:Following the introduction of minimallyinvasive surgery, thoracoscopic optionsappeared. One of these was the Nussprocedure for pectus excavatum. Thoracoscopic resective correction is further classifiedas intra- or extrathoracic.[22] reported multiple short chondral and ribsegmental resection with preservationof the anterior perichondrium from anintrathoracic aspect under thoracoscopicvision. Varela and Torre[23] extended Kim’sresection, and excised the affected cartilagescompletely using three ports.The Kim and Varela techniques aretwo examples of thoracoscopic resectiveintrathoracic surgeries for the correction ofcarinatum deformities. Kim and Idowuet al.[24] reported anextrathoracic resective thoracoscopicapproach to pectus carinatum by meansof sub-pectoral carbon dioxide dissection.Carbon dioxide insufflation was used todissect the pectoral muscles from the entireanterior thoracic wall. Through a smallincision of 2.9 - 4.7 cm, and the port holes,wedge resections of the cartilages andosteotomies of the sternum were performedas necessary, with subsequent fixation withbars that were removed later.Schaarschmidt et al.’s[13] introduction of chestremodelling. The avoidance of resectionimplies thoracoscopy, with smaller lateralincisions resulting in better cosmesis, lessblood loss and shorter operative time.Dystrophy of chondral rib segments is nolonger a concern, since growth plates areunaffected. However, these techniques aremore effective in flexible thoracic cages thatare also amenable to non-operative bracing,so their use in pectus carinatum is graduallydecreasing.Non-resective techniques were the result ofthe fundamental conceptual change broughtabout by Nuss Non-resective modalities can be furthersub-classified into extrathoracic andcombined intra- and extrathoracic.Non-resective extrathoracic: The firstreport on non-resective extrathoracictreatment of pectus carinatum, or ‘reverseNuss’, was by Abramson.26] The techniqueconsisted of the placement of a subcutaneoussteel bar, introduced through thoracic lateralincisions, and stabilised with two lateralsteel devices and steel wire at the incisionsite et al.[27] that uses newly designed implants,the aim of which is to reduce dislocation,one of the most relevant complications of thetechnique et al.[28]reported his experience with 172 patientsover 10 years, and four generations ofchest bars, with excellent results in 93.8%of the cases.Modifications of this techniquehave been published, such as one by Yükselet al.[29] have reportedthe zip-back technique. This approach avoidsthe use of lateral stabilisers, fixing the implantdirectly to the ribs by means of speciallydesigned polymer zip-ties Recently, Bellia-Munzon In addition to avoiding resection of cartilageand sternal osteotomies, the advantages ofnon-resective extrathoracic techniques includesmaller scars at the side of the chest and acomplete reshaping of the chest wall, insteadof the local correction of the deformity offeredby open resective approaches. This completeremodelling of the chest includes the wideningand flattening of the whole anterior chest, withconsequent improvement of shoulder andspine posture Non-resective combined intra-/extrathoracic: Several authors, such asKálmán,[30] Pérez et al.[31] and Park andKim,[32] have reported variations of nonresective combined intra-/extrathoracic surgeries. Kálmán[30] used a modelled barintroduced into the thoracic cavity from theleft aspect of the thorax, extruded into thesubcutaneous tissue at the point of maximumprotrusion of the sternum and reintroducedinto the thorax parasternally to the right,fixed with absorbable sutures and meant toavoid the use of stabilisers. Pérez et al.[31] useda steel bar 2 cm longer on each side than theinterareolar distance, and introduced it fromthe right side into the thorax, exited out of thepleural space and advanced it anteriorly to thesternum at the site of maximum protrusionand then reintroduced into the thorax,leaving the tip of the bar inside the pleuralspace. Finally, Park and Kim[32] developed the‘sandwich technique’ for asymmetric pectuscarinatum or carinatum/excavatum complex.This operation requires two bars, an internalimplant for excavatum correction and anexternal for carinatum, with excellent results.Bracing therapy has gained popularity as anon-operative alternative that has proven tobe as good as operative strategies to correctpectus carinatum in patients with flexiblethoracic cages and high compliance. This isa good choice as the first line of treatmentin almost all cases, and the only solutionnecessary in many cases with non-complexdeformities, flexible thoracic cages and ahigh level of compliance. This last aspectis the major cause of failure of treatmentand abandonment, so a multidisciplinaryapproach is recommended, with theparticipation of rehabilitation physiciansand physical therapists. There is still a lack ofconsensus regarding the minimum numberof hours patients should wear the brace, andwhether bracing may play a role in adultpatients. Non-surgical treatments may beclassified as classic bracing or pressurecontrolled bracing.[33] and Bianchi[34] in the 1960s.Haje et al.38] reported the use of an orthosisreferred to as the dynamic chest compressor,and later their extensive experience using thedynamic brace in mild to moderate cases.Since 2000, there have been many reports on the use of classic braces for the treatment of thecarinatum deformity.47]The first reports on bracing for pectuscarinatum were published by Jaubert deBeaujeuet al.[6] publishedan 8-year review on the use of a speciallydesigned dynamic compression system (theFMF Dynamic Compression System) In 2008, Martinez-Ferro et al.[48] reported its use inpatients with PIC as high as 14 psi, with goodresults in 61 of 68 cases in 17 months; andCohee et al.[49] created an algorithm designedto determine the chances of the conditionbeing resolved with a non-operativeapproach. De Beer et al.[52] validated the useof PIC to predict the duration of treatment,and Poola et al.[51] found that a low PIC at thebeginning of treatment was a good prognosticfactor. In 2018, de Beer et al.[50] published asystematic review of the measured dynamiccompression system. They found 14 studiespublished between 2008 and 2018, andselected 8 for further analysis. A total of 1 185patients were included, of whom 44% werestill under treatment, 29% had completedtreatment successfully, 6% had dropped outand 21% were lost to follow-up. Strategies areneeded to decrease rates of abandonment byincreasing compliance, which will probablyimprove results.This system comprises multiple adjustableelements and a special device that monitorspressure. The concept of pressure of initialcorrection (PIC) was developed to describethe pressure necessary to completely correct the deformity, by means of measuring therigidity of the thoracic cage. This permitsthe doctor to predict prognosis. Pressureof treatment (POT) was also developedto calculate the pressure needed at eachadjustment when the patient sees the doctoragain. Pounds per square inch (psi) is usedas the unit of measure, and pressure under2.5 psi was considered safe in order to avoidskin lesions. Subsequent authors have alsoreported their experience with the use of this device: Lopez Overall, the results obtained with bracingare considered better than those obtainedwith surgical procedures We have described the history of the treatmentof pectus carinatum, which has changeddramatically over the last 70 years, from thefirst descriptions of large incisions and wideresections, to minimal access strategies and,finally, to highly efficient non-operativesolutions, in a trend towards minimisinginvasiveness. Today, with a wide variety ofchoices, treatment modalities are transitioningtowards a tailor-made strategy for each patient.


Despite many recent advances in imaging and epidemiological data analysis, musculoskeletal injuries continue to be a welfare issue in racehorses. Omics studies describe the study of protein, genetic material  and metabolites that may provide insights into the pathophysiology of disease or opportunities to monitor response to treatment when measured in bodily fluids. As these fields of study are scientifically complex and highly specialised, it is timely to perform a review of the current literature to allow for the design of robust studies that allow for repeatable work. Systematic reviews have been introduced into the medical literature and are a methodological way of searching for relevant papers followed by critical review of the content and a detection of biases. The objectives of the current systematic review were to identify and critically appraise the literature pertaining to microRNA (miRNA) and their target genes that are correlated with stress fractures in racehorses and humans. The object was to define a panel of miRNAs and their target genes as potential biomarkers in either horses or human subjects. The online scientific databases were searched and a reviewed was performed according to preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines. MicroRNA profiling studies in horses continue to emerge, but as of yet, no miRNA profile can reliably predict the occurrence of fractures. It is very important that future studies are well designed to mitigate the effects of variation in sample size, exercise and normalisation methods.Despite many recent advances in imaging and epidemiological data analysis, musculoskeletal injuries continue to be a welfare issue in racehorses. Peptide biomarker studies have failed to consistently predict bone injury. Molecular profiling studies provide an opportunity to study equine musculoskeletal disease. A systematic review of the literature was performed using preferred reporting items for systematic reviews and meta-analyses protocols (PRISMA-P) guidelines to assess the use of miRNA profiling studies in equine and human musculoskeletal injuries. Data were extracted from 40 papers between 2008 and 2020. Three miRNA studies profiling equine musculoskeletal disease were identified, none of which related to equine stress fractures. Eleven papers studied miRNA profiles in osteoporotic human patients with fractures, but differentially expressed miRNAs were not consistent between studies. MicroRNA target prediction programmes also produced conflicting results between studies. Exercise affected miRNA profiles in both horse and human studies . MicroRNA profiling studies in horses continue to emerge, but as yet, no miRNA profile can reliably predict the occurrence of fractures. It is very important that future studies are well designed to mitigate the effects of variation in sample size, exercise and normalisation methods. Musculoskeletal injuries in racehorses are common and associated with potential welfare issues. In a recent meta-analysis, the pooled incidence of catastrophic musculoskeletal injury was 1.17 per 1000 race starts . The majStress fractures arise because of an imbalance between damage accumulation and targeted repair at predictable sites of repetitive bone injury. Despite the progress in advanced imaging, micro-CT  and MRI Omics technologies, genomics, transcriptomics, proteomics and metabolomics represent the study of the molecules, DNA, RNA, protein and metabolites, respectively. Genetic association studies aim to identify if a certain gene (DNA) controls the phenotype of a particular disease. Transcriptomic studies use next generation sequencing to identify if messenger RNA (mRNA) influences a particular disease. MicroRNAs (miRNAs) are small non-coding RNA molecules that are able to influence post-transcriptional gene expression. MicroRNAs have been proposed as potential diagnostic and prognostic biomarkers ,9 in manAs circulating miRNAs have been identified as potential biomarkers of fractures in osteoporosis ,16,17,18Systematic reviews use explicit methods to identify, select, appraise and synthesise results from similar but separate studies to identify high-quality evidence and highlight gaps in the current research. Scoping reviews are essential where there is a large and diverse evidence base, to provide a broad overview of the current evidence and to identify areas suitable for more detailed evaluation in a systematic review. Currently there are only two narrative reviews of miRNA relating to veterinary orthopaedics ,28, and It has been recommended that miRNA–RNA interactions should be validated in appropriate biological systems . AnalysiThe objectives of this review are to (1) make recommendations regarding blood sampling for miRNA studies with respect to the timing of feeding, exercise and haemolysis; (2) summarise the relevant omics data pertaining to stress fractures in horses and humans; (3) define and compare the panels of miRNA biomarkers and their miRNA targets identified in studies of osteoporotic fractures in humans to provide methodological insights for equine research.The review followed the methodology described in the Cochrane handbook for systematic reviews and the PRISMA statement for reporting systematic reviews and meta-analyses ,30. ThisThe three primary outcomes were to identify (1a) changes microRNAs/peptide analysis/gene expression related to musculoskeletal injuries in horses, (1b) microRNAs and their targets in response to exercise and mechanical loading in horses and humans and (1c) genetic association studies related to stress fractures in horses and young adults  equivalent to equine stress fractures. The secondary outcomes were to analyse miRNA and their targets related to osteoporotic fragility fractures in humans.We included randomised controlled trials (RCTs), and cohort, case-control and cross-sectional studies. We excluded case series, case reports, narrative reviews and textbook chapters. Study definition and categorisation were based on the Joanna Briggs Institute (JBI) reviewer’s manual and methods for the development of the National Institute for Health and Care Excellence (NICE) public health guidance. A randomised controlled trial (RCT) is a study where participants are randomly allocated to receive either the intervention or the control. A quasi-experimental study or non-randomised controlled trial is a study where participants are allocated to receive either the intervention or the control, but the allocation is not randomised, an approach often called a controlled before-and-after or a time-series study. A cohort study is an observational study in which a group of people or animals (cohort) are observed over time in order to see who develops the outcome longitudinally. A case-control study is a study where the investigator selects people or animals who have an outcome of interest  and others who do not (controls), and then collects data to determine previous exposure to possible causes. A cross-sectional study is an observational study in which the source population is examined to see what proportion has the outcome of interest, or has been exposed to a risk factor of interest, or both ,37. A stMedline In-Process and Non-Indexed Citations and Ovid MEDLINE: 1946–present;WEB of Science (Core Collection: Citation Indexes): 1950–present;SCOPUS: 1966–present.The following databases were searched for this study:For the horse studies, the following search terms were utilised for each of the respective databases:Medline:horse* OR equine* OR equus* OR racehorse* OR Thoroughbred* AND Fracture* OR fractures* OR catastrophic* OR stress fracture* OR injury* OR orthopaedic* OR bone* OR musculoskeletal* AND miRNA* OR microRNA* OR small RNA* OR RNA-seq* OR GWAS*.WEB of Science:TS = (horse* OR equine*) AND TS =  AND TS = .SCOPUS:TITLE-ABS-KEY  AND ).For the human studies, the following search terms were utilised for each of the respective databases:Medline:Human* OR athlete* OR military recruit* AND fracture* OR stress fracture* OR fatigue fracture* AND miRNA* OR microRNA* OR small RNA* OR RNA-seq*.WEB of Science:TS = (human*) AND TS = (fracture* OR stress fracture* OR fatigue fracture*) AND TS = .SCOPUS:TITLE-ABS-KEY (human* OR athlete* OR military recruit* AND stress fracture* OR fatigue fracture* AND miRNA* OR microRNA* OR RNA-seq* OR GWAS*) AND ).A primary literature search of the databases was conducted using the search terms outlined previously. The results of each search were downloaded into bibliographical software EndNote X9 (Thomson Reuters). Duplicates were deleted within EndNote. Publications were then assessed through three stages: review of titles for suitable publications, review of abstracts against inclusion and exclusion criteria and review of the full publications. All titles within the EndNote library were examined and their abstracts were reviewed. Ambiguous titles were retained for further review at the next stage (review of abstract).Abstracts from these publications were then independently assessed by reviewers for agreement with the inclusion and exclusion criteria for each of the primary and secondary outcomes . Any pubThe final full publications and methodological features were recorded on the detailed data extraction form  based onIncluded studies were qualitatively assessed by two independent reviewers based on the JBI critical appraisal checklist. Cumulative evidence of each paper was assessed by two independent reviewers (SL and MB) based on the grading of recommendations, assessment, development and evaluations (GRADE) system  and vetGThe bibliographic searches performed between 24 April 2020 and 7 May 2020 identified 201 and 153 publications in horse and human studies, respectively, after the removal of duplicates. A review of the titles and abstracts was performed against the inclusion and exclusion criteria. Overall, 51 horse and 40 human studies were selected on the basis of their relevance. A further full text review was carried out, which excluded articles for various reasons according to our inclusion/exclusion criteria .A total of 21 horse studies and 19 human studies met the defined inclusion/exclusion criteria and were assessed with the JBI critical appraisal tools. The PRISMA 2009 flow diagram of the study selection is reported in The data extracted from the study characteristics consisted of the year of the study, study design , inclusiThe horse study population recruited included horses who visited veterinary hospitals (11 studies), healthy horses (8 studies) and post-mortem material (2 horse studies). The sample size of horse studies varied; seven studies had sample sizes of less than 30, nine studies ranged from 30 to 100 and five larger studies had sample sizes ranging from 111 to 529. The mean or median reported age for horses was most commonly from 2 to 7 years old (13 studies), 2 studies did not provide data, 5 studies included horses older than 7 years of age and 1 study reported a mean age of 2 or less. Information of breed or type of horses involved was reported for all studies, with Thoroughbred being the most common type of breed (nine studies). Horse sex was not reported in 5 studies, was balanced in 15 studies and 1 study included only male horses. There were specific exclusion reasons relating to technical issues or confounding disease in 12 horse studies .The human patients were referred to hospital (17 human studies) and healthy participants (2 human studies). Human studies also varied in scale; four studies had sample sizes less than 30, seven studies ranged from 30 to 100 and eight studies had samples sizes greater than 100. The mean or median reported age for 8 human studies was relatively young, ranging from 20 to 30 years old, while 11 osteoporotic fracture studies involved older individuals ranging from 40 to 80 years old. Human studies revealed more sex bias due to the study population ; 6 studies included only male or majority male subjects, 10 studies had a majority of female subjects and 3 studies were of mixed sex. There were specific exclusion reasons relating to technical issues or confounding disease in 14 human studies .All included studies met at least 60% criteria out of total criterial for each study. For primary outcome 1a there was one horse randomised controlled trial study , when asFor primary outcome 1b, there was one human case-control study, reported by Sansoni et al. 2018)  that met  that meReviewing miRNAs and their targets to equine musculoskeletal injuries identified 12 publications including 2 miRNA candidate studies, 1 miRNA profiling study, 2 mRNA profiling studies and 7 biomarker studies (horse peptide biomarker studies prior to 2008 were not reviewed) . The thrIn terms of peptide biomarker candidate  studies A review of miRNAs and their targets in response to exercise and mechanical loading in horses and humans identified 11 publications including 5 horse miRNA profiling studies ,55,56,57The three horse miRNA studies ,56,57 anReviewing genetic association studies related to stress fractures in young adults (18–35 years) identified 5 human publications and 1 horse paper . All theReviewing miRNAs related to osteoporotic fragility fractures in humans identified 11 publications, including 7 miRNA candidate studies ,69,70,71Profiling studies frequently utilise a panel of selected potential differentially expressed miRNAs as biomarkers in order to increase the sensitivity and specificity to detect bone fragility fractures. One of the earlier validation studies  that usehttps://bio.tools/DIANA-microT (accessed on 28 March 2021)), they identified different potential target genes for this miRNA. Five studies attempted to correlate circulating miRNAs and bone turnover markers [Of the 10 studies that evaluated osteoporotic fragility factors, 4 used miRNA target prediction programmes ,17,18,69 markers ,68,69,70 markers  and one This review assessed the current evidence on, and feasibility of, using reported miRNAs and mRNA targets for potential diagnostic biomarkers to predict fractures. Very few high-quality studies exist in the equine and human literature. In total, 21 equine studies and 19 human studies were selected for this systematic review.Objective (1): MiR-21 and miR-125b were frequently reported miRNAs affected by exercise and mechanical loading in horses and humans. Blood for circulating miRNA analysis should be collected from fasted animals before exercise and samples with evidence of haemolysis should be discarded. Objective (2): There were differentially expressed miRNA, mRNA, peptide biomarkers or genetic associations reported as statistically significant across the 40 papers. There was no consensus of miRNAs to predict musculoskeletal injury in horses. Genetic association studies were often underpowered, were subject to bias and were not sex specific. Objective (3): differentially expressed miRNAs and predicted targets were not consistent between human osteoporotic fragility fracture studies.The two blood fractions, plasma and serum, are valuable sources for the investigation of potential biomarkers in various pathological conditions. Although they are usually considered to be equivalent, several studies addressed how the type of blood fractions (serum or plasma) influences the analysis of circulating miRNA and how their miRNA content may significantly differ ,75. PlasAll of the post-exercise plasma samples and the majority of the pre-exercise plasma samples underwent haemolysis in one study , highligThere is increasing work demonstrating the influence of miR-21 on bone homeostasis, the miR-21 knockout mouse showed impaired formation of the calvarial bone and in vitro work has demonstrated a dual action of miR-21 on both osteoblasts and osteoclasts . MicroRNThere was no common miRNA reported in stress fracture/musculoskeletal injuries in horses, possibly because of the limited number of studies. The plasma- and serum-derived miRNA profile pattern in laminitic horses differed from the control; however, their miRNA targets were related to pain-related genes in the glutamatergic pathway rather than bone-related genes . InjuredDifferentially expressed miRNAs and genes were defined on the basis of the results of statistical analysis. Only significantly differentially expressed miRNAs and their targets were highlighted in cross-comparison studies. The miRNA target was validated using a qPCR of tissues to detect levels of mRNA or combination of transcriptomic data, metabolomic profiles or measurement of metabolites ,56,57. IOverall, 10 studies out of 23 miRNA studies including, 12 non-biased miRNA profiling studies and 11 targeted miRNA candidate studies, carried out the validation of the findings with an independent cohort. Of these, a non-biased miRNAs profiling study in humans by Sun et al.  performeZarecki et al.  showed mCastanheira and their colleagues  revealedBlott et al. (2014)  reportedRaw data or omics data  from most of recent studies in this review were deposited in online databases, allowing for free sharing and open exchange data. However, for best practice, establishing the relevant data standards including clearly stated experiment descriptions, data exchange, terminology standards and experiment execution standards must also be shared to maximise the utility of existing data.The main limitation of this systematic review is the heterogeneity of the included publications due to the lack of equine fracture omics research. Therefore, the search terms were extended to include musculoskeletal injury, gene expression studies and human studies. Stress fractures in equine athletes are thought to be equivalent to fatigue fractures or stress fractures in elite human athletes or military recruits. There has been extensive research on osteoporotic fractures in humans, particularly in the ageing population, and a large number of studies have investigated miRNAs as potential biomarkers to predict fractures. Within the 11 osteoporotic fracture publications reviewed, variations in specific miRNAs analysed and different normalisation methods prevented a meta-analysis. Only a small number of miRNAs and targets were consistently identified as affected by disease; therefore, assessment of the potential of miRNAs as biomarkers for detection of fractures requires further research. The reviewed work was not substantial enough to identify an effect of age on miRNA expression and detection in horses, as many of the studies had too small a sample size to detect the influence of age, as has been reported subsequent to the literature search for this review ,23,24.Following review of the current literature, blood for circulating miRNA analysis was most frequently collected from fasting animals, pre-exercise. The degree of haemolysis should be closely monitored such that any samples with haemolysis can be discarded. Comparisons of recommended miRNA profiles between studies reported conflicting results, suggesting there is currently no miRNA profile that can accurately predict fracture. Direct study comparisons remain difficult due to differences in the sample size, study population and molecular analysis techniques used.This review highlighted the complexities of circulating miRNA analysis and genetic association studies. The key considerations when planning a genetic association study include a power calculation, sex-specific analysis and consideration of the effects of nutrition. In order to achieve high-quality, reproducible miRNA results, researchers must strive to minimise differences caused by pre-analytical factors , analytical factors  and post-analytical factors (consideration of the most stable reference gene(s) for individual studies). Despite a robust study design, including power calculations and a large sample size of several human studies, a reliable panel of miRNAs to detect and/or predict fracture occurrence was not identified.


We read with interest the study by Biccler et al. on the public health impact of low-dose aspirin on colorectal cancer (CRC), cardiovascular disease (CVD) and safety in the UK Significant questions and concerns have been raised after publishing the outcomes Aspirin in Reducing Events in the Elderly (ASPREE) trial vs 0.2%, p = 0.15). Similarly, the frequencies of haemorrhagic stroke, fatal bleeding, and peptic ulcer disease were low and did not differ significantly between groups.An important metanalysis of three large, randomized trials  with a total of 18,162 participants was published at Lancet The recent study by Biccler et al. and other recent high impacted trials on the effect of aspirin on CVD and CRC risks have high translational values into the daily management of CVD. Results like that could be easily translated and implemented into daily protocols of cardiovascular disease management, tempting healthcare practitioners to advocate dietary changes combined with polypill pharmacotherapy as evidence-based medicine among patients without cardiovascular disease with intermediate cardiovascular risk. Moreover, in the pandemic era with an increase in non-communicable diseases, healthcare professionals are tempted to advocate lifestyle and dietary changes, frequently combined with aspirin administration in their patients, including those without CVD and with intermediate cardiovascular or cancer risk.1.the complexity of food-drug-microbiome interactions and potential ability of certain foods combined with polypill and aspirin to alter the gut microbiome and intestinal mucosa triggering yet undesired or unknown metabolic effects2.higher all-cause mortality attributed to cancer deaths in healthy older adults receiving daily aspirin3.association between the NSAIDs use and various dietary regimens with increased risk of Clostridium Difficile diarrhoea, at least in individuals at intermediate cardiovascular risk in the Covid-19 eraHowever, several key issues remain to be addressed before announcing the full safety of these approaches:In conclusion, Biccler et al. study is a forward stride in developing a safe approach to minimize further CVD and CRC risks. However, considering recent findings, full implementation of low-dose aspirin supplementation in selected groups of individuals requires multidisciplinary efforts supported by innovative research.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Using this experimental setup, we determined the impact of the microbiome on the transcriptional response to intermittent hypoxia and hypercapnia in the left ventricle of the mouse heart. We identified significant changes in gene expression in both conventionally reared and germ-free mice. Following intermittent hypoxia and hypercapnia exposure, we detected 192 significant changes in conventionally reared mice (96 upregulated and 96 downregulated) and 161 significant changes (70 upregulated and 91 downregulated) in germ-free mice. Only 19 of these differentially expressed transcripts (∼10%) were common to conventionally reared and germ-free mice. Such distinct transcriptional responses imply that the host microbiota plays an important role in regulating the host transcriptional response to intermittent hypoxia and hypercapnia in the mouse heart.The microbiota plays a critical role in regulating organismal health and response to environmental stresses. Intermittent hypoxia and hypercapnia, a condition that represents the main hallmark of obstructive sleep apnea in humans, is known to induce significant alterations in the gut microbiome and metabolism, and promotes the progression of atherosclerosis in mouse models. To further understand the role of the microbiome in the cardiovascular response to intermittent hypoxia and hypercapnia, we developed a new rodent cage system that allows exposure of mice to controlled levels of O It has been demonstrated by experimental and clinical studies that OSA may lead to a number of health consequences including cardiovascular diseases ranging from cardiometabolic disorders and arrhythmogenesis to heart failure (Obstructive sleep apnea (OSA) is the most common type of sleep disorder in humans, and OSA is very prevalent in adults and children. It is characterized by repeated episodes of complete or partial obstructions of the upper airways during sleep, which is associated with a reduction in blood oxygen saturation and an increase in blood CO failure .In addition, evidence from both animal and human studies supports the notion that the gut microbiota plays a critical role in the onset and progression of various cardiovascular diseases . PreviouBesides impacting cellular signaling, the microbiome has been demonstrated to modify the epigenetic landscape of the host genome, which may influence cellular differentiation and proliferation, leading to new phenotypic traits throughout the age spectrum . For exaSince gut microbiota plays an important role in regulating host gene expression , in the GF and CONV C57BL/6 mice that were 8–12 weeks old were used for this experiment. GF status was confirmed weekly by standard microbiological cultures of fecal extracts, using thioglycollate broth and potato dextrose broth for facultative anaerobes including fungi, and cooked meat broth, heart infusion broth, and Columbia blood agar for obligate anaerobes . All ani2/CO2 analyzer . The IHH condition was maintained in a computer-controlled atmosphere chamber system  as previously described (2) (from 21 to 8%) and increase in (CO2) (from ∼0.5 to 8%) separated by alternating ∼4-min periods of normoxic room air [(O2) = 21% and (CO2) ≈ 0.5%] with 2 ramp intervals (∼1 min each) for a total of 10 IHH events per hour for 10 h per day during the light cycle. Controls received a flow of room air throughout the entire experimental period. Prior works have shown that the oxygen saturation level (SaO2) ranged from 60 to 80% when mice were subjected to 5–10% hypoxic treatments  . These sescribed . IHH expeatments .n = 3/group). A 150 nt paired-end sequencing on the Illumina HiSeq 4000 platform  was performed. Rigorous quality controls of the paired-end reads were assessed using FastQC tools, and the adapter sequences and low-quality bases were trimmed using Cutadapt . RNA from the LV was extracted using the RNeasy Mini Kit  and sent to Novogene  for QC and sequencing .The paired-end RNA-seq data of The microbiome, particularly in the gut, plays an important role in maintaining systemic homeostasis and contributes to a number of human diseases  that are involved in “DNA-templated transcription”  and “regulation of DNA-templated transcription”  . The topFollowing IHH exposure, “regulation of DNA-templated transcription” was one of the most significantly altered biological processes represented by 24% of the significantly altered transcripts in CONV mice. These results suggested a fine-tuning of the transcriptome, at least in part, through epigenetic modifications and rebalance of transcription regulators. Indeed, as shown in Substantial evidence has been reported that OSA significantly increases the risk of stroke, coronary heart disease, and heart failure in humans . InteresIn summary, our study demonstrated that the microbiome regulates the cardiac transcriptome at baseline and in response to IHH stress. A total of 117 DEGs were identified in the heart of GF mice under normoxic condition. These DEFs encode proteins involved in transcriptional regulation, alpha-actinin binding, and sarcolemma organization. Lack of microbiome also altered cardiac transcriptional responses to IHH stress, since we found 192 IHH-induced DEGs in CONV mice and 161 DEGs in GF mice with little overlap, suggesting that IHH exposure leads to different cardiac responses between CONV and GF mice. Indeed, 24% of the DEGs detected in CONV mice are involved in the “regulation of DNA-templated transcription,” while in GF mice, IHH-induced cardiac DEGs are mostly involved in functional or activity adjustments, such as circadian rhythm and cell cycle. Future functional studies will be needed to define the functional implications of the observed DEGs. For example, IHH-induced DEGs suggest distinct risks of cardiac disorders in GF and CONV mice, since disease-related DEGs mainly affected cardiac necrosis and cell death in CONV mice, while regulators of cardiac hypertrophy were mostly observed in GF mice. Additional studies are also warranted to explore the function of specific microbial species and their metabolites on regulating cardiac gene expression under baseline and stress conditions. Gnotobiotic mouse models are uniquely suited to unravel such microbial contributions to specific physiologic responses.Even though it has been shown that OSA or IHH exposure may lead to the development of various types of cardiac disorders [for selected reviews, see The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The animal study was reviewed and approved by the Animal Care Committee of the University of California, San Diego.DZ, JX, LE, and GH conceptualized the study, designed the experiments, and wrote the manuscript. DZ, JX, YM, and OP performed the experiments. DZ and JX performed data curation and analysis. LE and GH acquired funding and facilitated access to resources for the study. GH supervised the study. 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.


The N3 and N6 long chain polyunsaturated fatty acids (LCPUFA) docosahexaenoic acid (DHA) and arachidonic acid (AA) are essential for proper neurodevelopment in early life. These fatty acids are passed from mother to infant via the placenta, accreting into fetal tissues such as brain and adipose tissue. Placental transfer of LCPUFA is highest in the final trimester, but this transfer is abruptly severed with premature birth. As such, efforts have been made to supplement the post-natal feed of premature infants with LCPUFA to improve neurodevelopmental outcomes. This narrative review analyzes the current body of evidence pertinent to neurodevelopmental outcomes after LCPUFA supplementation in prematurely born infants, which was identified via the reference lists of systematic and narrative reviews and PubMed search engine results. This review finds that, while the evidence is weakened by heterogeneity, it may be seen that feed comprising 0.3% DHA and 0.6% AA is associated with more positive neurodevelopmental outcomes than LCPUFA-deplete feed. While no new RCTs have been performed since the most recent Cochrane meta-analysis in 2016, this narrative review provides a wider commentary; the wider effects of LCPUFA supplementation in prematurely born infants, the physiology of LCPUFA accretion into preterm tissues, and the physiological effects of LCPUFA that affect neurodevelopment. We also discuss the roles of maternal LCPUFA status as a modifiable factor affecting the risk of preterm birth and infant neurodevelopmental outcomes. To better understand the role of LCPUFAs in infant neurodevelopment, future study designs must consider absolute and relative availabilities of all LCPUFA species and incorporate the LCPUFA status of both mother and infant in pre- and postnatal periods. The N3 and N6 long-chain polyunsaturated fatty acid (LCPUFA) families comprise fatty acids with double carbon bonds at the N3 or N6 positions, respectively. The N3 family includes alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), while important N6 members include linoleic acid (LA) and arachidonic acid (AA). Humans cannot synthesise ALA and LA de novo, thus these LCPUFA are termed ‘dietarily essential’. Some conversion within the series of N3 or N6 fatty acids occurs endogenously, thus DHA and AA can be synthesized from their respective precursors. Synthesis occurs via enzymatic elongation and desaturation by 5- and 6-desaturase enzymes, but the efficiency of this process may be affected by genetic polymorphisms of the FADS gene . While iThis review aims to explore the current evidence surrounding LCPUFA supplementation to improve neurodevelopmental outcomes after preterm birth. We discuss the surrounding context of LCPUFA: how these fatty acids accrete into fetal tissues during gestation and after birth and how the physical nature of these LCPUFA molecules and their subsequent metabolism into bioactive mediators exert their wide-ranging effects upon infant brain development. This context provides a base from which we may view the effects of changes to infant LCPUFA status through the current standard of care or through interventional trials  and lipoxygenase (LOX) . IncreasHowever, the roles of AA and DHA within inflammation are not binary; some AA-derived metabolites regulate the process of inflammation resolution. The prostaglandin PGE2 promotes the initial inflammatory response but subsequently acts to inhibit LOX to reduce further production of pro-inflammatory prostaglandins . Some eiAlthough excessive inflammation is associated with negative outcomes , controlWhile the balance of N3:N6 fatty acids have the potential to disrupt the inflammatory process, there is not yet a broad base of evidence to understand how the amounts and timing of LCPUFA administration may influence outcomes after premature birth. Within the available evidence, an observational study of LCPUFA blood levels of prematurely born infants associated low levels of DHA with an increased risk of chronic lung disease, while low levels of AA in the blood were associated with an increased risk of sepsis . On the Premature birth itself may also be linked to inflammatory signaling, as illustrated by the actions of prostaglandins to precipitate preterm birth in sheep . In humaThe final trimester is associated with rapid growth of tissues, including adipose tissue, skeletal muscle, and brain tissue . BetweenAA transfer is high throughout pregnancy, with the biomagnification phenomenon maintaining fetal blood AA at levels twice those of the mother; at the beginning of the third trimester, the AA:DHA ratio is ~5.1 . The ratAccretion of LCPUFA continues in the postnatal period and throughout childhood up until the age of 18, but at a decreasing rate . MaternaWhile much of the LCPUFA transferred to the fetus in the final trimester contributes to brain growth and neurological development, by term, 90% of all maternally derived energy, including LCPUFA, is deposited into adipose tissue to result in seven-fold more DHA stored in adipose than in brain tissue , with thIt is estimated that the deposition of N3 and N6 fatty acids into adipose tissue outweighs that of brain and other neural tissues by ~46-fold, consuming 78% and 70% of all N6 and N3 PUFA transferred from mother to infant . While tThe importance of LCPUFA stored in adipose tissue is demonstrated by Cunnane et al., who compared the LCPUFA tissue content of infants fed either breastmilk or LCPUFA-deficient formula in the postnatal period . FormulaIn the absence of normal intrauterine LCPUFA supply, the deficits incurred by premature birth can be severe; an infant born at 35 weeks, despite being born at an appropriate weight for gestational age (AGA), will have half the LCPUFA content stored in their tissues of an AGA infant born at term . This ‘gIn summary, the final weeks of gestation are a key period for accretion of these fatty acids, with LCPUFA stores and adipose tissue mass effectively doubling. Premature birth disadvantages infants two-fold, by limiting DHA accretion directly into neural tissues as would happen in utero and by hindering the accumulation of adipose stores that the infant must rely on to continue optimal post-natal brain development.There is a heterogeneous population of studies assessing the effects of LCPUFA supplementation and neurodevelopmental outcomes of prematurely born infants. Two randomized controlled trials (RCTs) have studied neurodevelopment with regard to varying AA:DHA or N6:N3 ratios in premature infants. Alshweki et al.  studied The second trial to vary the AA:DHA ratio was the multicentre DINO trial . PretermStudies in Norway assessed the effects of LCPUFA-supplemented breastmilk for a median duration of 9 weeks to 141 premature infants weighing <1500 g and born at a mean GA of 28 weeks . BreastmIn addition to Alshweki et al., Clandinin et al. , and O’CO’Connor et al.  studied A further two studies evaluated the effects of 6 months of LCPUFA supplementation in small groups of infants. Van Wezel-Meijler et al.  randomizOn the other hand, Fang et al.  used staA collection of studies led by Fewtrell et al. measured neurodevelopmental outcomes after different durations of feeding with formulae of multiple LCPUFA compositions. In 2002, Fewtrell et al.  publisheSubsequently, the Fewtrell group (2004) studied a similar cohort of infants born at a mean GA of 31 weeks and mean birthweight ~1500 g, extending the duration of feed until 9 months CA . A diffeTwo older studies evaluated the sole provision of the N3 LCPUFA DHA and EPA. Carlson et al. (1996)  studied In a similar population of preterm infants <33 weeks GA (mean 29 weeks) and weighing <1400 g (mean 1103 g), Werkman et al.  providedThese studies insofar have excluded infants with comorbidities associated with premature birth. The UK based Dolphin  trial idFinally, the Preemie Tots  trial soA 2016 Cochrane systematic review found no statistically significant relationship between LCPUFA supplementation and neurodevelopment measured by BSID and indicated that the overall quality of evidence available was low ,72. LapiAlthough the studies above share few consistent concentrations of AA and DHA, the majority employ concentrations resembling the recommendations of the European Academy of Paediatrics and Child Health Foundation (2020) for formula to contain at least 0.3% but preferably 0.5% DHA, while AA should be at least equal to DHA content . Alshweki et al. showed that a formula containing 0.3% DHA and 0.6% AA (AA:DHA ratio 2:1) is at least better than LCPUFA-deficient formula in the absence of other LCPUFA (LA and/or ALA), demonstrating that these ratios were associated with neurodevelopmental scores most closely resembling those of breast-fed infants . ClandinIf 0.3% DHA and 0.6% AA appears to benefit infants, is there benefit to providing higher concentrations to mimic the physiological LCPUFA transfer rate of the placenta more closely? The concentrations of DHA and AA in standard feed are lower than the amounts of LCPUFA provided by the placenta in the final trimester, estimated at >6% of fatty acids for DHA and 14–20% of fatty acids for AA. The quantities of LCPUFA provided by standard formula feed is therefore around 10- to 50-fold less than the physiological expectation during the post-natal period after preterm birth . CurrentThus, there may be benefits to providing greater concentrations of LCPUFA than those given at present. DHA given at 1%, mimicking the ‘high end’ of breastmilk and still less than the transfer rate possible by the placenta, increases the brain DHA content of term neonatal baboons by 40% in comparison to 0% DHA feed . Human eAn interesting relationship between the degree of prematurity and benefit from supplementation is apparent, whereby infants born at greater immaturity may benefit more from LCPUFA supplementation. The DINO trial, providing 1% DHA and 0.6% AA, found improved neurodevelopment in the most premature or lowest birthweight infants but not in whole group analyses, including preterm infants of greater maturity . O’ConnoA further aspect to consider is the changing rate and ratio of placental LCPUFA transfer across gestation. Martinez et al. showed that in early gestation, transfer of AA is predominant, while DHA transfer is delayed until the final weeks of gestation ,80. ThisCurrent feeding regimes for preterm infants provide static concentrations of supplemental AA and DHA, but in pursuit of a greater depth of physiological mimicry, we may wish to vary formula AA and DHA concentrations with respect to infant CA to better simulate the dynamic functions of the placenta and demands of the infant. In this regard, it may be that prematurely born infants require several formulae during their postnatal period; an infant born at 25 weeks may require more AA than DHA, whilst an infant born after 33 weeks may require more DHA than AA. Formula provided after the premature infant’s ‘term’ date (corrected age) should resemble the LCPUFA composition of breastmilk for ongoing postnatal nutrition. Looking onwards, the LCPUFA composition of weaning foods and ongoing diet may be considered to be modifiable factors affecting LCPUFA assimilation and accumulation in childhood and later life. It is apparent that LCPUFA availability has differential effects upon the development of specific organs. Although postnatal supplementation of DHA and AA seem to be beneficial for neurodevelopment and retinal development in preterm infants ,32, recePremature birth brings many uncertainties and unexpected physiological challenges for the newborn, for which it is unprepared. Examples include exposure to air and increased oxygen tensions, infections, and varying types of brain and peripheral organ injury. LCPUFAs may be protective via their metabolism to eicosanoids and SPMs yet are also susceptible to peroxidation, which may propagate cellular damage ,82,83. WRegardless of the amount of supplemental DHA provided, we must also consider the effectiveness of the route chosen to provide DHA to prematurely born infants. Administration of high amounts of fatty acids such as DHA via the immaturely developed premature infant digestive system may be ineffective or problematic due to altered absorption. Additionally, intolerance of oral feeding and gastrointestinal complications such as spontaneous intestinal perforation and necrotizing enterocolitis are common with increasing prematurity. Parenteral lipid supplementation is therefore common in these infants but also does not fully mimic or replace normal fatty acid metabolism.Overall, supplementation of LCPUFA is greatly nuanced and has the potential to both benefit and harm the premature infant. Pragmatically, future research must also consider that measurement of fatty acid levels in blood might not correlate with the levels in specific tissues. It is evident that a greater understanding of changes in LCPUFA transfer rates with gestational maturity and the effects of LCPUFA availability upon specific tissue development is needed to optimize the growth and development of prematurely born infants. In addition to large differences in AA and DHA concentrations across the trials to date, there is large variation between the concentrations of LA and ALA provided in both control and intervention formulae. Historical formulae have included LA and ALA with an assumption of adequate endogenous conversion to AA and DHA, but in reality, this conversion is inadequate to meet infant demands. For instance, Cunnane et al. showed that infants fed DHA-deficient formula depleted their adipose stores to unmeasurable levels and had less brain DHA accumulation than infants fed DHA-repleted formula . Other aSimilarly, Bockmann et al. showed that feeding LA-rich feed to preterm infants alters the adipose LCPUFA composition to become dominated by LA and depleted of AA and DHA. These results show that, while dietary ALA and LA are inadequate to supply infant needs of DHA and AA, excessive LA is stored and persists in fetal adipose tissue . FurtherInstead, excess LA in infant diet and infant tissues has the potential to alter the LCPUFA composition of multiple tissues, as well as the overall N6:N3 ratio. This ratio is of significance due to metabolic competition at the level of the desaturase enzymes, whereby excesses of one series of LCPUFA may out-compete others for metabolism into downstream metabolites, contributing to a relative deficiency of these other LCPUFA despite adequate oral intake. It is known that dietary PUFA composition can alter the N6:N3 balance of tissues in both animals and humans. Hsieh et al. demonstrated competition between AA and DHA in infant baboons who were given feed containing varying N6:N3 ratios by providing a fixed percentage of AA and ‘standard’, ‘medium’, or ‘high’ percentages of DHA. The baboons with the highest DHA intake demonstrated a reciprocal decrease in brain AA content, representing competition between N3 and N6 fatty acids for placement within neuronal membranes . Infant Similarly, findings have been seen in human infants born >35 weeks GA provided total parenteral nutrition (TPN) high in LA for a maximum of 12 days after birth. In these infants, hepatic levels of LA increased three-fold, while levels of DHA halved, with a consequent dramatic reduction in the N3:N6 ratio . While tThe DIAMOND ,79 trialInfants in the DIAMOND trial that were fed formula containing DHA had improved visual acuity at 3 months and superior Bayley Scale of Infant Development (BSID-II) Motor Development Index (MDI) scores, emotional regulation, and language abilities at 18 months in comparison to infants that were fed the formula containing 0% DHA ,79. FurtThe DIAMOND studies generally found no benefit to greater concentrations of DHA in feed; the majority of benefits were found at a ratio of 2:1 or 1:1, with no significant benefit found at lower N3:N6 ratios or higher DHA concentrations. At greater intakes of 0.96% DHA, erythrocyte DHA increased with a reciprocal decrease in AA to reach a level below those of infants fed AA-deplete formula, confirming the findings of other studies demonstrating interaction and displacement between series of LCPUFA . Furthermore, there appear to be more complex synergistic effects between AA and DHA. The Mega Donna Mega trial analyzed DHA and AA serum concentrations with regard to risk of ROP development in premature infants and found that higher serum DHA only conferred reduced ROP risk when AA was above a threshold concentration; the same DHA concentration did not reduce ROP risk in infants with low serum AA .The cumulative storage of and interaction between dietary AA, DHA, and their parent LCPUFAs likely represent an important confounding factor that may affect our interpretation of all LCPUFA-supplementation studies that report neurocognitive outcomes at timepoints in later childhood. After feeding until 9 months, Fewtrell et al. found no neurocognitive benefits a  or 18 moIn summary, there is evidence for an interaction between AA, DHA, and other essential LCPUFA in animal models and both preterm and term humans. The DIAMOND study is the only evaluation of the effects of the N3:N6 balance and found neurodevelopmental benefits across multiple time points in childhood but is specific to infants born at term ,79. The maternal fatty acids that supply the infant in utero and in breastmilk are derived from the maternal diet and stores in adipose tissue ; the quaIn the 1980s, Clandinin et al. remarked that the N6 content of fetal adipose tissue was higher than previous studies, attributing this to changing maternal dietary habits to include more vegetable oils , intakesAside from diet, genetic polymorphisms of the FADS gene affecting expression of the fatty acid desaturase enzymes that convert LCPUFA precursors to AA and DHA may affect maternal LCPUFA synthesis and thus availability of LCPUFA to the infant. FADS gene polymorphisms may reduce the maternal ability to provide LCPUFA in breastmilk ,94 and cDietary LA also increases with decreasing socioeconomic status . One of Maternal LCPUFA status may also affect the trajectory of infant development with regard to gestation length and in-utero development. A 2018 paper found that low maternal blood EPA and DHA concentrations increased risk of preterm birth by 10-fold , and a rOn the other hand, results from large cohort studies that assessed the overall dietary LCPUFA intake during pregnancy have suggested that increases in the N6:N3 ratio are associated with developmental delay. Kim et al. estimated the N3 and N6 PUFA intakes of 960 mothers during pregnancy, and of their infants via breast milk and weaning feeds in the postnatal period, to assess the effects of N3 and N6 intakes upon neurodevelopment at 6 months as measured by BSID-II; greater LA:ALA and N6:N3 ratios were both associated with lower MDI and PDI scores in infants, while multiple logistic regression analysis showed that infants of mothers with the highest LA:ALA ratios had >2 times the risk of neurodevelopmental delay of infants whose mothers had the lowest LA:ALA ratios . SimilarAfter birth, increased maternal N6 LCPUFA status may directly affect infant tissue LCPUFA composition via the breastmilk, while weaning onto a N6-rich modern diet may contribute to ongoing competition between N6 and N3 fatty acids. The fatty acid composition of maternal milk and the wider food environment are unaccounted factors that may influence the results of trials assessing the long-term effects of DHA in the post-natal period. In summary, it is evident that the maternal dietary intake and storage of LCPUFA, in combination with genetic factors, can affect the availability of these LCPUFA to developing infants and affect the consequent fatty acid composition of infant tissues. Furthermore, a suboptimal maternal LCPUFA status may increase the risk of preterm birth, while a positive LCPUFA status may improve infant growth and thus reduce the risk of neonatal morbidity. Whether maternal LCPUFA status affects neurodevelopment in preterm infants is yet unknown and is largely unstudied, thus the authors welcome future studies incorporating data of maternal LCPUFA status into calculations of absolute and relative LCPUFA availability to the developing infant. LCPUFA transfer from mother to fetus occurs via the placenta throughout pregnancy, with particularly high rates of DHA transfer during the final trimester. LCPUFA accrete into brain tissue and are essential for neurodevelopment; however, the bulk of LCPUFA are stored in adipose tissue to provide vital endogenous LCPUFA stores in the postnatal period. Infants born before term do not receive this placental transfer of LCPUFA and hence can be severely LCPUFA-deficient in both brain and adipose tissue in comparison to term-born infants. The evidence supporting post-natal LCPUFA supplementation for prematurely born infants is weakened by heterogeneity within the populations studied, durations of feeding and the concentrations of AA, DHA, LA, and ALA provided. While firm conclusions cannot be drawn, it is apparent that providing DHA at 0.3% and AA at 0.6% provides greater benefit to the neurodevelopment of prematurely born infants than feeding LCPUFA-deficient formula. Future studies should consider the absolute and relative availability of AA and DHA and the timing of these fatty acids with respect to normal placental physiology. Furthermore, the availability of other LCPUFA such as LA and ALA within infant formula should be seen as factors interacting with the relative availability of AA and DHA within the infant. More widely, the role of maternal LCPUFA status is poorly understood, but may directly affect the risk of premature birth and infant neurodevelopment.


The pathogenesis of diabetic neuropathy is complex, and various pathogenic pathways have been proposed. A better understanding of the pathophysiology is warranted for developing novel therapeutic strategies. Here, we summarize recent evidence from experiments using animal models of type 1 and type 2 diabetes showing that low-grade intraneural inflammation is a facet of diabetic neuropathy. Our experimental data suggest that these mild inflammatory processes are a likely common terminal pathway in diabetic neuropathy associated with the degeneration of intraepidermal nerve fibers. In contrast to earlier reports claiming toxic effects of high-iron content, we found the opposite, i.e., nutritional iron deficiency caused low-grade inflammation and fiber degeneration while in normal or high non-heme iron nutrition no or only extremely mild inflammatory signs were identified in nerve tissue. Obesity and dyslipidemia also appear to trigger mild inflammation of peripheral nerves, associated with neuropathy even in the absence of overt diabetes mellitus. Our finding may be the experimental analog of recent observations identifying systemic proinflammatory activity in human sensorimotor diabetic neuropathy. In a rat model of type 1 diabetes, a mild neuropathy with inflammatory components could be induced by insulin treatment causing an abrupt reduction in HbA1c. This is in line with observations in patients with severe diabetes developing a small fiber neuropathy upon treatment-induced rapid HbA1c reduction. If the inflammatory pathogenesis could be further substantiated by data from human tissues and intervention studies, anti-inflammatory compounds with different modes of action may become candidates for the treatment or prevention of diabetic neuropathy. The prevalence of diabetes and prediabetes has continued to increase worldwide in recent years ,2,3,4,5.Small nerve fibers are considered to be most vulnerable structures for the development of DN since early damage was described even on patients with impaired glucose tolerance and without the expected overt chronic hyperglycemia .Despite intensive research in recent years, there is still no unifying concept of the pathophysiology of DN nor efficacious and safe specific treatments available.In this short review, we summarized the more recent aspects of two pathomechanisms based on our own experimental studies in diabetic disease models, namely inflammatory mechanisms and the modulation by manipulating systemic iron levels. These has been discussed against other recent findings and hypotheses. We conclude that re-examining these inflammatory mechanisms in human DN seems warranted.In some rare forms of diabetic polyneuropathies, such as radiculoneuropathy in a nerve plexus distribution or cranial neuropathies, an inflammatory component has already been substantiated by nerve biopsies in distinct cases ,13,14,15In our experimental studies, we could demonstrate that all animals with type 1 or type 2 experimental diabetes and neuropathy showed an increased inflammatory response, with mild infiltration and proliferation of macrophages and T cells as well as increased concentrations of soluble cytokines in the sciatic nerve ,17,18. Mob/ob mice, a model of type 2 diabetes. This was associated with loss of myelinated and unmyelinated nerve fibers and axonal damage, indicative of overt neuropathy ..21].ob/ob mice without hyperglycemia as a model of the metabolic syndrome or prediabetes. Even in the absence of chronic hyperglycemia, symptoms of inflammation were evident in peripheral nerves ..72].We concluded that autonomic nervous dysfunction may be positively influenced by an intervention leading to weight loss, even as early as during childhood, an observation that may translate to improved cardiovascular health and mortality later in life .Another important aspect is that physical activity in humans including aerobic, resistance training and especially combined exercise may improve inflammation-linked biomarkers and thus suggests an anti-inflammatory effect through these interventions ,74.The findings from experiments using animal models of type 1 and type 2 diabetes and using animal models for obesity and the metabolic syndrome lead us to suggest that low-grade intraneural inflammation may be a major facet of the common types of diabetic neuropathy. If the inflammatory phenomena observed in animal experiments could be confirmed as a pathogenic principle of DN in humans, anti-inflammatory therapies would become a candidate for anti-inflammatory therapeutic strategies. Based on the profound intraepidermal small fiber degeneration as shown in our animal models, minimally invasive skin biopsies are recommended in future prospective trials aimed at defining inflammatory mechanisms.Neurodegeneration along with inflammatory activity caused by iron deficiency has not yet been investigated systematically in humans. In practical terms, our experimental observations lead us to discourage the use of iron-reducing therapies in diabetes until more is known about the effects of iron intake in patients and until these human studies have been done.In a new animal model mimicking TIND in diabetes, the affliction of small nerve fibers could be reproduced and associated with inflammatory pathology, and this may be a consequence of an abrupt lowering of HbA1c. Still, the exact pathomechanism needs to be defined in future studies before novel treatment strategies can be proposed.