It is unclear whether vasopressors can be safely administered through a peripheral intravenous (PIV). Systematic review and meta-analysis methodology was used to examine the incidence of local anatomic adverse events associated with PIV vasopressor administration in patients of any age cared for in any acute care environment.MEDLINE, EMBASE, CINAHL, the Cochrane Central Register of controlled trials, and the Database of Abstracts of Reviews of Effects were searched without restriction from inception to October 2019. References of included studies and related reviews, as well as relevant conference proceedings were also searched. Studies were included if they were: (1) cohort, quasi-experimental, or randomized controlled trial study design; (2) conducted in humans of any age or clinical setting; and (3) reported on local anatomic adverse events associated with PIV vasopressor administration. Risk of bias was assessed using the Revised Cochrane risk-of-bias tool for randomized trials or the Joanna Briggs Institute checklist for prevalence studies where appropriate. Incidence estimates were pooled using random effects meta-analysis. Subgroup analyses were used to explore sources of heterogeneity.I2 = 93.7%). In children, meta-analysis from four studies and 388 patients demonstrated a pooled incidence proportion of adverse events as 3.3% . Subgroup analyses did not detect any statistically significant effects associated with stratification based on differences in clinical location, risk of bias or design between studies, PIV location and size, or vasopressor type or duration. Most studies had high or some concern for risk of bias.Twenty-three studies were included in the systematic review, of which 16 and 7 described adults and children, respectively. Meta-analysis from 11 adult studies including 16,055 patients demonstrated a pooled incidence proportion of adverse events associated with PIV vasopressor administration as 1.8% (95% CI 0.1–4.8%, The incidence of adverse events associated with PIV vasopressor administration is low. Additional research is required to examine the effects of PIV location and size, vasopressor type and dose, and patient characteristics on the safety of PIV vasopressor administration.The online version contains supplementary material available at 10.1186/s13054-021-03553-1. Intravenous vasopressors are commonly used to augment systemic blood pressure . CurrentA previous systematic review of predominantly case reports and case series found that among 263 patients who received vasopressors through a PIV there were 318 adverse events, of which 86 were extravasation with no injury and 179 were skin necrosis . Local tThis study was conducted and reported according to Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines  and regiThe search strategy was developed in consultation with a medical librarian (see acknowledgements) and peer reviewed using the Peer Reviewed of Electronic Search Strategies checklist . At the title and abstract level, if either reviewer  deemed a citation potentially relevant it progressed to more detailed review at full text screening . Disagreements were resolved by discussion or involvement of an additional reviewer (DJN). For articles not written in English, Google Translate was used  and if tStudies were included in the systematic review if they met the following eligibility criteria: (1) cohort, quasi-experimental, or randomized controlled trial design; (2) examined continuous vasopressor infusion administration through PIVs in humans ; and (3) investigated local anatomic adverse events associated with PIV vasopressor administration. Studies were subsequently included in the meta-analysis if they reported (1) data required to calculate the incidence of adverse events per patient  and (2) the PIVs were inserted and managed in a manner consistent with contemporary clinical practices.Data was extracted independently and in duplicate using a standardized form developed in Microsoft Excel. For studies where important data was missing, corresponding authors were contacted. Extracted data included study characteristics, participant characteristics and adverse events. The primary outcome was the incidence proportion per patient of local anatomic adverse events associated with PIV vasopressor administration. This incidence proportion was calculated by dividing the number of patients who experienced a local anatomical adverse event by the number of patients prescribed PIV vasopressors.Risk of bias was assessed independently and in duplicate by two reviewers  using the Revised Cochrane risk-of-bias tool for randomized trials (RoB 2)  or the JI2 statistic and Cochran’s Q test. Since proportion estimates were at or close to the border of permissible values (no or very few adverse events reported), the Freeman–Tukey double arcsine transformation was used for the pooled estimate [p < 0.05.The pooled incidence proportion of adverse events associated with PIV vasopressor administration was estimated using a random effects model and the ‘metaprop’ package  in Stataestimate . The exaestimate . Possiblestimate . Publican = 465, 46%), or did not employ the desired study design . Of the eight articles excluded from the meta-analysis, the most common deficiency related to not clearly reporting the number of patients that received vasopressors through a PIV (n = 5) [n = 2) [n = 1) [The search strategy identified 9814 citations, from which 7923 unique citations were screened for inclusion  –28. Othe [n = 2) , and out [n = 1) . Two rec [n = 1) .Fig. 1PRn = 18, 78%), but also included randomized control trials (RCTs) . Of the cohort studies, most were retrospective . Among the 16 adult studies, five (31%) described patients in emergency departments (ED), six (37%) described patients in ICUs, and the other five (31%) included patients from a mixture of clinical settings. Among children, clinical settings included the pediatric intensive care unit , neonatal intensive care unit , and mixture of ED and PICU .Of the 23 studies in the systematic review, 16 were in adults, and seven were in children (Table n = 5) of studies reporting an average duration between 12 and 24 h. In the six adult studies that reported the size of the PIV used to administer vasopressors, 88% of PIV vasopressor infusions were through PIVs that were 16–20 gauge (1283/1464), of which most were through a 20-gauge PIV . Seven adult studies commented on the anatomical location of PIV, of which four quantified how many infusions were recorded through each location. From these studies, 64% (446/692) of vasopressors were administered through a PIV in the arm proximal to the wrist and 27% (188/692) were administered in the hand or wrist.The characteristics of patients who received PIV vasopressors are presented in Additional file n = 7), followed by epinephrine (n = 4 studies), and norepinephrine (n = 3 studies). Most of the studies in children (n = 5) described patients receiving multiple vasopressors, with one study explicitly describing patients receiving multiple vasopressors simultaneously through the same PIV [n = 3 studies) reporting a duration under six hours. Gauge was reported in four studies with 24 gauge as most common PIV size.In the seven studies done in children, the average age ranged from 1 day to 9.3 years. Dopamine was the most commonly administered vasopressor and was administered to patients in all of the child studies (same PIV . Averagen = 2 patients) was the only long term complication [Adverse events associated with PIV vasopressor administration are described in Additional file lication  reportedOf the 19 studies that reported adverse events, seven reported the resolution of adverse events without treatment , four reported minor treatment (e.g. phentolamine injection and nitroglycerin paste application), and the other eight studies did not mention use of any treatment. In one adult study, three patients had PIV access challenges  which were resolved by obtaining a new PIV site in one patient and inserting a CVC in the other two patients . One stun = 16,055 patients) estimated the incidence proportion of adverse events associated with PIV vasopressor administration as 1.8%   estimated the incidence proportion of adverse events as 3.3%  .Random effects meta-analysis pooling data from 11 adult studies  of operative patients where event rates appeared to be lower to examine whether its inclusion was driving the main result for the adult studies [n = 1670 patients wherein location of care was primarily ICU and/or ED) was 2.1% (95% CI 1.2–3.1%) with a marked decrease in interstudy heterogeneity  compared to the main analysis  among adults and 3.3% (95% CI 0.0–10.1%) among children. In addition to the generally low incidence, 19 of the 23 studies reported either no adverse events or only mild local tissue reactions like infiltration or extravasation, with only one study  reportinOur results are consistent with prior literature examining the risks associated with PIV vasopressor administration. A prior systematic review that could not estimate incidence due to the inclusion of case series and case reports suggested adverse events appear associated with longer duration of infusion and distal PIV sites . GeneralWhile the incidence of adverse events associated with PIV vasopressor administration is low, it is difficult to make clinical recommendations without direct comparisons to current practice through CVCs. Designing a study to make those comparisons faces several challenges. First is defining the primary outcome of interest, wherein the adverse events associated with vasopressor administration are different for PIVs and CVCs. Medication extravasation, local tissue ischemia or sudden loss of intravenous access resulting in severe hypotension are events that would be more commonly associated with vasopressors administered through a PIV. Similarly, pneumothorax, arterial puncture, and catheter-related bloodstream infection are more commonly associated with CVCs. In an RCT done by Ricard et al., comparing all-purpose initial access with a PIV (not just for vasopressor administration) to a CVC in 263 patients admitted to ICUs in France, major complications were more common in those initially treated with a PIV . HoweverThough our study highlights the need for additional high-quality research, taken together with existing guidelines, our results help inform current clinical practice with regard to PIV vasopressor administration. The most recent Surviving Sepsis Campaign Guidelines for management of adults with sepsis do not make explicit recommendations regarding CVC versus PIV vasopressor administration, though it is highlighted that patients no longer require assessment of specialized data obtained from the CVC such as central venous oxygen saturation as part of their initial resuscitation . PediatrThe findings of this study should be interpreted in the context of its strengths and limitations. It was conducted by rigorously following methodological guidelines for meta-analyses, and to our knowledge, is the first meta-analysis to examine the safety of PIV vasopressor administration in children, and the largest in adults. However, most included studies were single-centre, retrospective cohort studies, inherently at risk of reporting bias, and were generally at high/some risk of bias. Subgroup and meta-regression analyses suggest a reduction in heterogeneity among those studies at lower risk of bias, however the adverse event estimate remained similar. In addition, there was significant interstudy heterogeneity. Subgroup and stratified analyses examining factors known to contribute to clinical heterogeneity such as location of clinical care, PIV gauge, and vasopressor infusion duration were identified as factors contributing to the observed statistical heterogeneity. Owing to non-uniform reporting of data within the included studies we were unable to comment on contributions of vasopressor concentration and/or dose, two factors predicted to be important determinants of the safety of PIV vasopressor administration. Sensitivity analysis excluding a large study of operative patients by Pancaro et al. wherein data was primarily obtained from an electronic database  and adverse event rates were reported to be lower , resulteThe incidence of adverse events associated with PIV vasopressor administration in adults and children is low. When adverse events did occur, they tended to be minor. Additional research is required to examine the effects of PIV location and size, vasopressor type, concentration, dose and duration, and patient characteristics on the safety of PIV vasopressor administration. However, in the meantime, these results suggest that with careful monitoring, administration of vasopressors through PIVs is safe.Additional file 1. Table S1: MEDLINE search strategy. Table S2: Conference abstracts searched. Table S3: Demographic and clinical characteristics of patients who received peripheral intravenous vasopressors. Table S4: Adverse event characteristics. Table S5: Revised Cochrane risk-of-bias tool for randomized trials (RoB 2). Table S6: JBI Critical Appraisal Checklist for Studies Reporting Prevalence Data. Fig. S1: Funnel plot of studies included in the meta-analysis. Fig. S2: Forest plot examining the incidence proportion of adverse events associated with peripheral intravenous vasopressor administration of adult studies stratified by location of care. Fig. S3: Forest plot examining the incidence proportion of adverse events associated with peripheral intravenous vasopressor administration of Adult studies stratified by study design. Fig. S4: Forest plot examining the incidence proportion of adverse events associated with peripheral intravenous vasopressor administration of adult studies stratified by the mean/median duration of infusion. Fig. S5: Forest plot examining the incidence proportion of adverse events associated with peripheral intravenous vasopressor administration of adult studies stratified by Intravenous gauge. Fig. S6: Forest plot examining the incidence proportion of adverse events associated with peripheral intravenous vasopressor administration of Adult studies stratified by anatomical location. Fig. S7: Forest plot examining the incidence proportion of adverse events associated with peripheral intravenous vasopressor administration of adult studies stratified by vasopressor type. Fig. S8: Forest plot examining the incidence proportion of adverse events associated with peripheral intravenous vasopressor administration of adult studies stratified by patient sex. Fig. S9: Sensitivity analysis of forest plot examining the incidence proportion of adverse events associated with peripheral intravenous vasopressor administration in adults.


P value thresholds (PT) with phenotypes. Altogether ten SNPs were associated with distinct phenotypes after correction for multiple testing (PEMP2 ≤ 0.05). The TLR7/TLR8 locus was associated with disease onset before and the SH2B3/ATXN2, ITGA4/UBE2E3 and IL2/IL21 loci after 7 years of age. The latter three loci were associated with a more severe small bowel mucosal damage and SH2B3/ATXN2 with type 1 diabetes. Patients at the highest wGRS39 tertiles had OR > 1.62 for having coeliac disease-related symptoms during childhood, a more severe small bowel mucosal damage, malabsorption and anaemia. PRS was associated only with dermatitis herpetiformis . Independent coeliac disease-susceptibility loci are associated with distinct phenotypes, suggesting that genetic factors play a role in determining the disease presentation. Moreover, the increased number of coeliac disease susceptibility SNPs might predispose to a more severe disease course.The phenotype of coeliac disease varies considerably for incompletely understood reasons. We investigated whether established coeliac disease susceptibility variants (SNPs) are individually or cumulatively associated with distinct phenotypes. We also tested whether a polygenic risk score (PRS) based on genome-wide associated (GWA) data could explain the phenotypic variation. The phenotypic association of 39 non-HLA coeliac disease SNPs was tested in 625 thoroughly phenotyped coeliac disease patients and 1817 controls. To assess their cumulative effects a weighted genetic risk score (wGRS39) was built, and stratified by tertiles. In our PRS model in cases, we took the summary statistics from the largest GWA study in coeliac disease and tested their association at eight  Food antigens do not generally cause a systemic immune response in healthy individuals, but rather lead to the induction of oral tolerance. However, in approximately 1–2% of individuals, the ingestion of dietary gluten results in the development of coeliac disease, an immune-mediated enteropathy . This muThe predisposition for coeliac disease runs in families, and relatives of coeliac patients are at an increased risk . The disCurrently, there’s only limited information about the connection of the non-HLA risk SNPs with different coeliac disease phenotypes. Therefore, the aim of this study was to investigate whether previously identified coeliac disease-susceptibility SNPs are associated with distinct disease phenotypes and to gain insight into possible biological pathways and processes underlying the identified genotype–phenotype associations. Moreover, the objective was to study whether GRS or PRS give clues to the factors contributing to the clinical heterogeneity of the disease.n = 1817) with information on gender and HLA-type were obtained from the FINRISK and Health 2000 population cohorts .Imputation was carried out with Beagle 4.1 In the post-imputation QC, the SNPs with good imputation quality metrics (INFO score ≥0.8) and MAF > 0.01 were included. The VCF files with the genotypes probabilities dosages were handled by using bcftools (r2 ≥ 0.8) with the phenotype-associated SNPs were identified using HaploReg v.4.1  in each male and gender strata  and high [3rd tertile] wGRS39 risk categories) to a reference . The associations were quantified by ORs with 95% CIs, significant at https://www.cog-genomics.org/plink/1.9), PLINK 2.0 (https://www.cog-genomics.org/plink/2.0/), PRSice v2 and RStudio .All data analysis, conversion of required input formats in the imputation analysis and graphs were performed using PLINK 1.90 . This SNP was associated with the age of coeliac disease diagnosis in girls  also in the case only analysis comparing coeliac disease patients with the given phenotype to those without (detailed results in Supplementary Table SH2B3/ATXN2), rs13010713 (at ITGA4/UBE2E3), rs13151961 (at IL2/IL21), rs11712165 (at CD80) and rs10936599 (at MYNN) showed an association. Except for rs10936599, the same variants were also associated with an intermediate HLA risk. Moreover, rs653178, rs13010713 and rs13151961 showed an association with the presence of gastrointestinal symptoms and with the presence of more severe mucosal damage (TVA/SVA) at diagnosis. In addition, rs653178 was also associated with extraintestinal manifestation as well as coeliac disease associated conditions in general and more specifically with AITD and T1D.Of the previously identified 94 coeliac disease-associated variants, 39 were typed to sufficient extent and thus were selected for the study. When comparing coeliac disease patients to controls, ten SNPs were associated with specific phenotypes Table . Rs59797In order to address the association of all the 94 coeliac disease associated SNPs, we carried out imputation. Out of the 2442 subjects in our study, 299 individuals (285 cases and 14 controls) were excluded due to heterozygosity and missingness. Of the 94 coeliac disease SNPs selected, five variants were missing  and one (rs12998748) was multiallelic in the Finnish reference panel, and thus dropped out of the analysis. All the remaining 88 coeliac disease variants were successfully imputed and tested with the phenotypes , revealed as associated  with distinct phenotypes  remained associated with the same phenotypes as in the original analysis. Seven imputed SNPs  had strong regulatory function on transcription with a RegulomeDB score ≤ 3a, indicating that the variant likely lies within a potential functional region. Of these rs13098911 was enriched with proxies with the lowest scores (1a and 1f). Out of the 148 extracted proxies to the other phenotype-associated SNPs , 42 had a putative functional role in gene regulation (RegulomeDB score ≤ 3a)  were enriched (FDR ≤ 0.002) in chemokine signalling and cytokine-cytokine receptor interaction KEGG pathways. These genes are likely to be involved in modulation of chemokine and cellular defence responses, cell chemotaxis second messenger-mediated signalling and divalent inorganic cation homoeostasis based on the GO associated terms (FDR ≤ 0.0001). Coeliac disease diagnosis above 7 years of age, gastrointestinal symptoms, coeliac disease-associated conditions, EI manifestations and intermediate HLA risk were the phenotypes connected to a set of trans-eQTL genes. Among KEGG pathways, they were most significantly enriched (FDR ≤ 0.01) in the B cell receptor signalling, Pertussis, NOD-like receptor signalling and in MicroRNAs in cancer. They were enriched (FDR ≤ 2.2E-09) for biological processes GO terms mostly involved in adaptive immune response, T cell activation, response to interferon-gamma, leucocyte cell-cell adhesion, cytokine secretion, regulation of leucocyte activation and response to molecule of bacterial origin    Fig. . Best fi12) Fig. .Fig. 1PoIn this study, by using genotyped variants in our cohort, we identified a genotype-phenotype association for ten SNPs previously associated with coeliac disease susceptibility. In addition, our results demonstrate that combining 39 coeliac disease-associated genotyped SNPs into wGRS39 was more informative than a PRS to assess the genetic risk for distinct phenotypes of coeliac disease.TLR7 and TLR8 genes was associated with a decreased risk of diagnosis before 7 years of age in girls and notably this association was detected also in the case only comparison. Our RegulomeDB analysis did not retrieve rs5979785 as having regulatory function of transcription although it has previously been reported to decrease the TLR8 expression in the blood [According to our results, rs5979785 located in the proximity of the he blood . As disthe blood , 34, addSH2B3/ATXN2 with an increased risk to several other phenotypes, including concomitant T1D. Our findings are thus in accordance with previous studies where the 12q24/SH2B3 locus has been associated with both coeliac disease and T1D [Our results also revealed an association of rs653178 at  and T1D , 36. In  and T1D , has beeIn addition to having a likely regulatory function on transcription and an eQTL effect of the expression of SH2B3 along with many other genes in our analysis, rs3184504 SNP has been also associated with a higher expression of SH2B3 in the intestinal mucosal of patients with active coeliac disease , islet acis-eQTL effects on a set of genes enriched in chemokine pathway. This finding most likely reflects the location of this variant in the chemokine gene cluster.In pursue of clarifying the molecular mechanisms exerted by the susceptibility SNPs on the phenotypes, we identified that only three of our phenotype-associated SNPs had significant eQTL effects on regulating gene expression. This would suggest that the SNPs included in our analysis are mostly proxies and either situated in the proximity of the causative variant or further away. In the KEGG pathway and GO biological process analysis the phenotypes-associated rs13098911 SNP was identified as the only variant having trans-eQTL genes identified largely identical pathways for most of our phenotypes. All these phenotypes were associated with rs653178 and its proxy rs3184504 revealed considerable effects on the expression of several distal genes. This pleiotropic effect of rs3184504 thus likely explains our finding of the pathway enrichment analysis of the trans-eQTL genes. Thus, further studies to infer the true phenotype-causal variants and the mechanisms that might mediate their effects on phenotypic variation are needed.The enrichment results of the Regarding our weighted 39 SNP-based risk score model, the high-risk tertile in wGSR39 was associated with a higher risk of having coeliac disease symptoms in childhood, more severe small bowel mucosal damage, malabsorption and the occurrence of anemia. These phenotypes can be considered as signs of a more severe disease course , 41, thuA major strength of the current study is the clinically well-characterised large cohort of coeliac patients. In addition, a strength is the careful phenotyping of the patients, allowing us to investigate the association of different genotypes with various phenotypes. Moreover, the imputation analysis allowed us to explore the phenotype association of further 49 previously coeliac disease SNPs not typed in our cohort. The use of the most comprehensive available imputation panel in Finnish population increased our opportunities for identifying new coeliac disease SNPs associated with the phenotypes. Our highly conservative procedures in the imputation analysis diminished the risk of false positive associations. It must be noted though that 47.8% of coeliac disease patients dropped out from the analysis. Moreover, for distinct phenotypes, we had a fairly low number of patients which affected the statistical power in some cases. Moreover, we included in our GRS study only the 39 genotyped SNPs from independent coeliac disease risk loci, and not all the 94 previously associated with coeliac disease, and it might be the case that other coeliac susceptibility SNPs have a modulatory role on the phenotypesWe conclude that independent coeliac disease-susceptibility loci are associated with distinct coeliac disease phenotypes, suggesting that distinct SNPs might play a role in modulating the disease presentation in a yet to determined mechanism. Moreover, while PRS seems not to explain the variance in phenotypes, more severe coeliac disease phenotypes could possibly be contributed by higher amount of coeliac disease risk SNPs. Our GRS approach might thus be useful to identify patients at risk of developing a severe disease course unless identified and treated early. Further studies with a larger number of SNPs are called for in independent well-characterised patient cohorts to better understand how genetic variants contribute to the different coeliac disease phenotypes.Supplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Table 6Supplementary Table 7Supplementary Table 8Supplementary Table 9


Late infantile neuronal ceroid lipofuscinosis (CLN2 Batten disease) is a rare, progressive neurodegenerative disease of childhood. The natural history of motor and language regression is used to monitor the efficacy of CNS treatments. Less is known about CLN2 retinopathy. Our aim is to elaborate the nature, age of onset, and symmetry of CLN2 retinopathy using visual electrophysiology and ophthalmic imaging.We reviewed 22 patients with genetically confirmed CLN2 disease; seventeen showing classical and five atypical disease. Flash electroretinograms (ERGs), flash and pattern reversal visual evoked potentials (VEPs), recorded from awake children were collated. Available fundus images were graded, optical coherence tomography (OCT) central subfoveal thickness (CST) measured, and genotype, age, clinical vision assessment and motor language grades assembled.p 0.001 level, Wilcoxon ranked signed test. Cone ERGs show a functional deficit before CST thinning in classical disease. Optomap hyper fundus autofluorescence (FAF) at the fovea was noted in three patients with normal ERGs. The oldest patient showed an ovoid aggregate above the external limiting membrane at the fovea, which did not affect the PERG.ERGs show cone/rod system dysfunction preceded by localised macular ellipsoid zone disruption on OCT from 4.8 years. Electroencephalogram (EEG) time-locked spikes confounded both pattern 6/17 (35%) and flash VEPs 12/16 (75%). Paired right eye (RE) and left eye (LE) ERG amplitudes did not differ significantly for each flash stimulus at the ERG findings in CLN2 retinopathy show symmetrical cone-rod dysfunction, from 4y10m in this series, but a broad range of ages when ERG function is preserved. Batten disease is a collective description for all forms of neuronal ceroid lipofuscinosis (NCL) , 2. NCL Historically NCLs were grouped according to the age of onset into congenital, infantile (INCL), late infantile (LINCL), juvenile (JNCL) and adult-onset forms, or by eponymous names such as Santavuori-Haltia disease for INCL (onset 6–24 months) or Vogt–Spielmeyer–Sjogren disease for JNCL onset 5–15 year). Now NCLs are described by numerical genotype. The most common types are CLN1 (INCL), CLN2 (LINCL), CLN6 (variant LINCL) and CLN3 (JNCL) [5 year. NClassical CLN2 LINCL or Jansky–Bielschowsky disease results from the depleted expression of the lysosomal enzyme tripeptidyl peptidase 1 (TTP1) caused by mutations in the CLN2 gene on chromosome 11p15 . ChildreTPP1-null dogs [Clinical management is palliative, but promising treatments are being developed and evaluated . Enzyme ull dogs . The advThe natural history of rare diseases can be used to monitor the efficacy of treatment when control groups or randomisation studies are not feasible or ethical. Natural clinical history scales for CLN2 have been developed –23. The Our study is a retrospective case series of patients with genetically confirmed CLN2 at Great Ormond Street Hospital for Children (GOSH) that seeks to contribute functional ERG and visual evoked potential (VEP) measures, and structural imaging data to the natural history of CLN2 retinopathy.The records of patients biallelic for mutations associated with CLN2 were reviewed. Visual electrophysiology tests, flash skin ERG, pattern VEP and flash VEP were carried out in awake children using techniques developed at GOSH. In brief, skin electrodes are positioned on the cheek below the eye and referenced to the outer canthus. Flashes are presented 3/s using a handheld Grass (Gr) strobe at different settings to produce, in the dark, a predominantly rod driven ERG to dim blue Gr1 (3/s), a maximal flash a-wave to white Gr16 (1/20 s), a mixed rod-cone ERG to white Gr4 (3/s) and in the light, a cone ERG to white Gr4 (3/s) and a flicker ERG to Gr4 30/s). The skin ERGs produce similar shapes and have broad physiological equivalence to ISCEV ERGs, , 30. The/s. The sPattern and flash VEPs were recorded to ISCEV standards with both eyes open . A rangeOphthalmic imaging in alert children was attempted using OPTOS Optomap ultra-widefield red green and fundus autofluorescence (FAF) programmes, and/or spectral-domain optical coherence tomography (OCT) using the Heidelberg Spectralis OCT, Monaco and Flex full or fast scan programmes and/or hand-held Bioptigen Envisu. In addition, one child was imaged using the Bioptigen whilst under GA for an MRI. OCT images were graded using the Weill Cornell Ophthalmic Severity Score . The retThis study was registered with the Great Ormond Street Hospital for Children GOSH Clinical Governance and Audit office #2864.Twenty-two patients with CLN2, including three siblings pairs, were reviewed. Eighteen patients had at least one of the common mutations C509-1G>C or c.622C>T . Most frFlash ERG recordings were attained in 20/22 patients. Two patients declined flash testing, one on one occasion, because of concerns about photosensitive epilepsy. Testing was unsuccessful in one patient because of agitation. Nine of twenty recorded patients had normal flash ERGs at the last test, four had absent ERGs. The amplitudes and time to peaks of measurable ERGs are tabulated in Appendix To compare these ERG data with other studies that have used different recording methods the ERG amplitudes were graded by dividing the measured value by the fifth centile reference limit for each stimulus. These are plotted for each eye and each stimulus as a function of age in Fig. The ERG grades from all stimuli for each eye were averaged to provide a single ERG index for each patient and these are plotted for 20 patients as a function of age in Fig. Pattern reversal VEPs recordings took place with 19 patients, examples produced by the ISCEV standard large check width (50′) are shown in Fig. OCT reference images have been arrayed to highlight the progression of foveal retinal thinning to generalised retinal atrophy in Fig. p 0.0003 , correlates with the average ERG score that sums activity from the majority of cones in the more peripheral retina (6.5 million), , 38. ThiMaintenance of CST in some cases may relate to bilateral ovoid accumulations above the outer limiting membrane at the fovea, seen most clearly in the oldest patient. Interestingly, the Dachshund model of CLN2 disease is accompanied by an accumulation of autofluorescent storage material along the outer limiting membrane as well as in the ganglion cells . The accThe lack of a frank electronegative ERG waveform highlights the CLN2 retinopathy in these children is not a primary inner retinal dysfunction, , whilst patients with no detectable ERG and profound retinal dysfunction are aged 6.25–8.75 y (75–105 m) broadly aligning with the trajectory of visual loss over 3 y described by Steinfield . Our serThe lack of visual response from children with CLN2 reflects post-retinal neurodegeneration of the visual pathway and occipital cortex as well as retinal dysfunction. Visual pathway function is reflected in the VEP extracted from the electroencephalogram (EEG). In CLN2 the EEG is often the first test to detect irregular activity as slowing of background activity, and epileptiform abnormalities in posterior regions . In contThe flash VEP waveform shows wide inter-individual variability and the pattern reversal VEP waveform is preferred clinically because it has a relatively constant single positive peak throughout life. The reversal VEP is a strong index of macular pathway function. Pattern reversal VEPs in 2/18 patients showed a bifid waveform which is associated with a central scotoma, caused by maculopathy or macular pathway dysfunction such as optic atrophy. Although a majority of patients (16/18) showed evidence of PVEPs to check widths of 50′ or smaller at the first recording many  were atypically early with large amplitudes due to the intrusion of pattern-driven paroxysmal EEG activity. Pattern sensitive or pattern-driven paroxysmal responses can falsely augment and disguise the true PRVEP as seen in Fig. The symmetry of retinal disease progression is very important for future therapies if the fellow eye may act as a control. Kovacs et al.  reportedThe limitations of this case series are in common with retrospective studies but accentuated in studies of rare diseases when children are very challenging to examine. Data were acquired after the children had completed their fortnightly TPPI ERT infusions. There are missing data when compliance became insufficient for reliable data collection. It was physically difficult to sustain the best position with a desktop mounted system. An arm-mounted OCT was used with some success with the fast acquisition programme, but the large machinery can be nudged easily by the children. The Bioptigen is a smaller handheld option. Studies comparing axial CST measurements from the same instruments show excellent inter-observer agreement, but different instruments require a correction factor to translate data from one platform to another. For our study ×1.025 is suggested from model eye evaluations to translate from handheld Bioptigen Envisu™ to Heidelberg Spectralis™ . The morAs noted by others the clinical fundus is found often to be unremarkable in CLN2 retinopathy , 36. TheOur large case series of children with CLN2 demonstrate a wide phenotypic variability with age, but the classical disease retinopathy fits broadly within the CST OCT prediction interval suggested by Kovacs et al. . Those wStructurally, OCT data show Batten disease CLN2 retinopathy starts as a maculopathy and progresses to involve cones and rods.Clinical assessment of children with CLN2 is challenging and grading scales may help.Functionally, ERG data show CLN2 retinopathy is cone-rod dystrophy.Children with CLN2 retinopathy do not show the profoundly electronegative ERGs described in the canine CLN2 model or in human CLN1 and CLN3 phenotypes.ERG changes can precede central subfoveal thickness change in classical CLN2 disease.There is a phenotypic variation of CLN2 retinopathy in classical and non-classical diseases and the age when normal ERGs are recordable.Appendix 1 - Measured ERG valuesAppendix 2 - ERG data plotted wrt reference rangesAppendix 3 - Statistical tables


Manihot esculenta) identified peaks of transcriptionally engaged RNA polymerase at both the 5′ and 3′ end of genes, consistent with paused or slowly moving Polymerase. In addition, we identified divergent transcription at intergenic sites. A full genome search for bi-directional transcription using an algorithm for enhancer detection developed in mammals (dREG) identified many intergenic regulatory element (IRE) candidates. These sites showed distinct patterns of methylation and nucleotide conservation based on genomic evolutionary rate profiling (GERP). SNPs within these IRE candidates explained significantly more variation in fitness and root composition than SNPs in chromosomal segments randomly ascertained from the same intergenic distribution, strongly suggesting a functional importance of these sites. Maize GRO-Seq data showed RNA polymerase occupancy at IREs consistent with patterns in cassava. Furthermore, these IREs in maize significantly overlapped with sites previously identified on the basis of open chromatin, histone marks, and methylation, and were enriched for reported eQTL. Our results suggest that bidirectional transcription can identify intergenic genomic regions in plants that play an important role in transcription regulation and whose identification has the potential to aid crop improvement.Control of gene expression is fundamental at every level of cell function. Promoter-proximal pausing and divergent transcription at promoters and enhancers, which are prominent features in animals, have only been studied in a handful of research experiments in plants. PRO-Seq analysis in cassava ( Gene expression in plants is a highly regulated process controlling the production of coding and noncoding RNA molecules. Proper regulation of expression is central to the development and phenotypic plasticity. The dynamics of transcriptional regulation have been extensively studied in several model organisms, including humans, yeast, and fruit flies . These sNascent RNA sequencing techniques such as Global nuclear Run-On sequencing (GRO-seq) . PromoteEnhancers are key eukaryotic regulatory elements that control spatiotemporal gene expression and are especially important during development . While tOnly a few studies on a limited number of species (Manihot esculenta) and maize (Zea mays), and to reveal if promoter-proximal pausing and bidirectional transcription at intergenic regions are present in these species. To do so, we quantified nascent transcription in cassava and maize using PRO-seq and re-analyzed a maize GRO-seq dataset (The objective of this study was to characterize nascent transcription in cassava (2 and stored at −80°C. Most of the nuclei extraction protocol took place in a cold room (4°C) with all reagents on ice. A fraction of the nuclei preparations were stained with DAPI  and visualized under a fluorescence microscope to test for concentration and nuclei integrity.Cassava accession “Nase 3” (synonymous with “IITA-TMS-IBA30572” and “Migyera”) cuttings were grown in tubes containing enriched medium. Tubes were placed in growth chambers with 12 hours of light at 30°C for 6 weeks before tissue collection. Stem and leaves of approximately 25 gr were ground with liquid nitrogen to a fine powder using a mortar and pestle. The resulting powder was transferred to a coffee grinder containing cold 1X NIB buffer. We then used the CelLytica PN Plant nuclei isolation/extraction kit (Sigma-Aldrich) following the instructions for the “Highly pure preparation of Nuclei.” The resulting solutions were frozen in liquid NMaize inbred line B73 seeds were put into growth chambers. Shoots were collected 9 days after germination. Around 10 grams of plant tissue were ground with liquid nitrogen to a fine powder. Five grams of ground tissue were transferred into 50 ml fresh SEB extraction buffer , incubated on ice for 20 minutes, and then filtered through 2 layers of 100um nylon mesh. Triton X-100 was then added to a final concentration of 0.5% and incubated on ice for another 10 minutes. Then the lysate was centrifuged at 2000 rcf for 15 minutes at 4°C and the supernatant was recovered. The pellet was suspended in another 25 ml SEB extraction buffer and centrifuged again at 2000 rcf for 15 minutes. We added 10 ml nuclei storage buffer  in the pellet and centrifuged at 2000 rcf for 5 minutes at 4°C. This step was repeated using 1 ml nuclei storage buffer. Finally, the pellet was resuspended and stored in 105 µl nuclei storage buffer. The protocol was conducted in a cold room (4°C).The PRO-seq protocol was performed as described by http://hannonlab.cshl.edu/fastx_toolkit/), and the 3′ molecular barcode was removed. Reads were trimmed to a maximum length of 36 bp, and the reverse complement was calculated because the HiSeq apparatus starts sequencing from the 5′ end. All downstream alignments were performed using Bowtie2 . Low-quality alignments were filtered and only reads mapping once to the genome were considered for further analysis (Supplementary Table S1). Bedtools (https://github.com/ENCODE-DCC/kentUtils).The fastq files were scanned for any residual adapter sequence (5′-TGGAATTCTCGGGTGCCAAGG -3′) using fastx_clipper from the FASTX_toolkit ( Bowtie2 . BecauseBedtools  was usedhttps://github.com/corcra/bed-metric.git) . Normalized BigWig files representing the mapped reads were used to visualize each strand of the genome separately in the Integrative Genomics Viewer (IGV) (https://github.com/bdo311/metagene-maker). Since the cassava genome is not readily available to work with HOMER, feature annotations were created separately, and the HOMER config files were modified. We approximated the transcription start site (TSS) as the beginning of the 5′ UTR because the cassava genome annotation lacked precise TSS annotations. The same approach was used for Arabidopsis and Maize genomes.The percentage of the cassava genome transcribed was calculated using bedtools . A saturation curve, which calculates the number of unique positions covered as a function of read depth was obtained using the bed-metric scripts , jatropha (Jatropha curcas), castor bean (Ricinus communis), willow , flax (Linum usitatissimum), and poplar (Populus trichocarpa) as previously described by The GERP scores for conservation were calculated using different species from the Malpighiales clade, including rubber  of the Genetic Gain (GG) collection, which comprises 709 elite and historically important clones. Along with these, we analyzed 2302 clones developed as the cycle 1 of IITA’s Genomic Selection (GS) breeding program. In total, 3011 clones were used as the source for phenotypes. For further details on the populations used, see We analyzed data from the IITA cassava breeding program in Nigeria, including a fraction . The GBS markers were combined with the Cassava HapMap v2.0 variants from 241 deep-sequenced cassava accessions (Genotyping-by-sequencing (GBS) libraries were prepared as described previously (cessions . The impr2 > 0.9 and were closer than 100 kb to IRE markers). We also built 10 random sets of 9665 regions with the same average length and approximate distribution in the genome as these elements. As with the IREs, random sets were forced to be outside any annotated gene , and markers from the ROG in close physical distance and high LD were removed.Genomic partitioning analyses are imprecise in highly related populations because of high LD between partitions. We mitigated this problem by eliminating markers from the rest of the genome (ROG) in high LD with the 9665 cassava IREs  and ROG genomic partitions using the software LDAK5 following the ideas of Genomic relationship matrices (GRMs) were calculated for focal l ∼ Ng ∼ Nh ∼ N or ROG (RGRM) partitions. The incidence matrices loc.yrZ, and Z relate observations to the levels of trials and clones, respectively. Variance components were estimated using the “emmremlMultikernel” function implemented in the “EMMREML” R package . For clarity, the four main libraries used through this study will be referred to as PRO-cassava, PRO-maize, GRO-maize  in the cassava genome. We identified ∼2000 peaks in intergenic regions using Homer’s  of the NextGen Cassava Breeding Project ^3 = 7.51e-4.Finally, to test the functional relevance of the plant IREs, we estimated the percentage of the SNP heritability  IREs using dREG and again observed a clear pattern of bi-directional transcription . Our PROIt is unknown whether dREG can accurately predict plant enhancers. The traditional way to identify enhancer candidates is based on open chromatin sites as assessed by DNase-seq or ATAC-seq, together with histone modification marks. To test if dREG was able to tag previously reported enhancer candidates in maize, we analyzed the transcription pattern of 1495 (Supplementary File S3) enhancer candidates identified in a separate maize study . MoreoveTo further characterize the IRE identified by dREG and explore whether they house more phenotypically relevant variants, we compared our results to a list of previously identified expression Quantitative Trait Loci (eQTLs) in maize kernels , which is likely required for a kinase-regulated release of paused Pol II . Some of the transcriptional activity observed in the IREs might be driven by RNA polymerase dependent DNA methylation (RdDM). Thus, we expect the IRE regions to not only capture enhancers but also regulatory RNAs  and transposons. This would partially explain the high methylation profiles and low evolutionary conservation at the IRE sites.IRE regions were shown in cassava to have low levels of evolutionary conservation, a bi-directional pattern of transcription, and a specific DNA methylation profile. It is worth mentioning that PRO-seq cannot differentiate between Pol II and other polymerases  leads to a superset or subset of sites relative to the other. We clearly show, however, that bi-directionally transcribed regions in maize have the characteristic of active enhancers of affecting gene transcription.In mammals, there are various enhancer states enriched with different histone marks: poised or inactive enhancers, primed enhancers, and active enhancers . PreviouRecently, The ability of PRO-seq to detect functional intergenic regions has also been explored in rice. While building a fitness consequence map of the rice genome A. thaliana: they validated 10 of the 14 enhancer candidates tested using a reporter assay. None of the Arabidopsis GRO-seq data, however, displayed strong evidence of transcription in the regions identified by Transcription of genomic enhancers was first described in 1992  there haThe results reported herein cassava and maize suggest that plant transcriptional regulation may be more similar to that of mammals and other metazoans than previously thought. The lack of transcription previously observed in putative Arabidopsis enhancers may have been the result of different growth conditions, tissues, or even low read depth. Recent research, however, has shown that bidirectional transcription could be observed in 113 noncoding regions in Arabidopsis when using exosome mutants  outside of genes in species with larger genomes. This hypothesis has recently been supported in a study that analyzed CRES in 13 plant species, where distal CRES were most abundant in larger and more complex genomes  under accession GSE114758. All other data needed to evaluate the conclusions in the paper are present in the paper or the supplementary materials. Custom scripts for the NGS analysis, metagene plots, and genomic partitioning, among others, have been made publicly available through the GitHub repository:


The consequences of this modified intracellular signaling can manifest at the level of protein quality control, dysfunctional mitochondrial homeostasis, autophagy and lysosomal dysregulation, abnormal presence of stress granules, synaptic toxicity, neuroinflammation or maladaptive innate immune response.Neurodegenerative diseases are characterized by the progressive loss of specific subsets of neurons. The original trigger of neuronal death is still unknown, although aging is considered a risk factor for neurodegenerative diseases. Many of these disorders share an abnormal accumulation of misfolded peptides or proteins in the brain and spinal cord. These deposits of insoluble peptides or proteins could accumulate with time, and they could be more toxic when neurons are old. The accumulation of neurodegeneration-related peptides or proteins, such as AβCell Signaling and Neurodegeneration” of the International Journal of Molecular Sciences covers eight original articles and six reviews that provide new insights regarding different signaling pathways that can be involved in neurodegenerative diseases.The Special Issue “Lenzi et al. investigate the role of clusterin (a molecular chaperone) in the -synuclein aggregation process. They report that clusterin is a key protein of the biochemical response conducted by the cell in order to regulate α-synuclein expression. They further suggest that clusterin is involved in the dynamic α-synuclein aggregation process .2O2-induced activation of the STING pathway is protective against cell death. They reveal that STING maintains efficient autophagy flux and protecting against H2O2-induced cell death. This investigation points out an important role for the STING pathway in the underlying cellular mechanisms contributing to the pathogenesis of neurological disorders [Abdullah et al., studying the stimulator of interferon genes (STING) pathway in cell death induced by elevated stress, describe that HDash et al. create a gene profile of amyotropic lateral sclerosis (ALS) by analyzing the differentially expressed genes, the Kyoto Encyclopedia of Genes and Genomes pathways, the interactome and the transcription factor profiles that would identify altered molecular/functional signatures and their interactions at both transcriptional (mRNAs) and translational levels. The authors find that different genetic ALS forms are singular diseases rather than part of a common spectrum. This is crucial for individualized medicine approaches in ALS .Avchalumov et al. examine whether acute methamphetamine treatment requires dopamine receptor type 1 activation in order to alter high-frecuency stimulation (HFS)-induced LTP in the dorsal striatum. They reveal that aberrant dopamine receptor type 1 function in the dorsal striatum by methamphetamine produces synaptic depression in the dorsal striatum. Therefore, the inhibition of dopamine receptor type 1 function may correlate with normalizing synaptic plasticity in the dorsal striatum and reducing the reinforcing properties of methamphetamine .2+ load and calpain activity and provides neuroprotection against excitotoxicity in an ISR-independent manner [Ruiz et al. show that the transient integrated stress response induced by NMDA is followed by an ATF4-independent peIF2α dephosphorylation. However, Sephin1 strongly attenuates NMDA-induced excitotoxicity whereas it is ineffective against ER stress-produced neuronal death. The authors find that Sephin1 reduces NMDA-triggered cytosolic Cat manner .Tinelli et al. determine the level and function of circulating endothelial progenitor cells (cEPCs) in a cohort of Italian Moyamoya angiopathy patients in order to consider if cEPCs may be qualified as a potential pathogenic marker or just an epiphenomenon of Moyamoya angiopathy. This patholgy is a rare, chronic and disabling cerebrovascular disease with a prevalence of 0.086–10.5/100,000. They conclude that cEPCs level more than endothelial progenitor cell functionality seems to be a potential marker of Moyamoya angiopathy [Bayer et al. investigate whether DNA methyltransferase 1 (DNMT1), which acts on neuronal survival in the aged brain, affects critical aspects of the proteostasis network. They found that DNMT1 negatively impacts retrograde trafficking and autophagy, with both being involved in the clearance of aggregation-prone proteins by the aggresome–autophagy pathway .2+ entry (SOCE) component, in modulating SOCE in human neuroglioma cell line H4. The authors suggest that ORAI2 downregulation can become a potential tool to rescue defective SOCE in Alzheimer’s disease, while preventing plaque formation [Scremin et al. focus their investigation on the role of ORAI2, a key store-operated Caormation .Martínez Cué and Rueda provide an overview of the signaling pathways implicated in each of the main neuropathological aspects of Alzheimer’s disease in individuals with and without Down syndrome as well as the interrelation of these pathways. In the fourth decade of life, all individuals with Down Syndrome develop neuropathology identical to that found in sporadic Alzheimer’s disease, including the development of amyloid plaques and neurofibrillary tangles, loss of neurons and synapses, reduced neurogenesis, enhanced oxidative stress, mitochondrial dysfunction and neuroinflammation. Down syndrome can be considered a useful model to study AD etiopathology and to search for new therapeutic strategies .Arrazola et al. go deeper into the dysregulated/aberrant signaling pathways controlled by the small GTPaese of the Ras superfamily that culminate in neurodegeneration. The authors specifically focused on the two most studied families of the Ras superfamily: Ras and Rho. They summarize the latest findings of small GTPases of the Ras and Rho families in neurodegeneration in order to highlight these small proteins as potential therapeutic targets capable of slowing down different neurodegenerative diseases .Grant and DeMorrow, in this review, provide a detailed discussion about the findings of recent studies highlighting bile acid-mediated therapies and bile acid-mediated signaling and the roles they play in neurodegenerative and neurological diseases .Alcover-Sánchez et al. offer a thorough review of the tight relation between R-Ras 1 and R-Ras 2 GTPases and myelination processes and discuss its potential as novel elements of crosstalk between the pathways. They conclude that a better understanding of the crosstalk elements orchestrating myelination mechanisms is essential to identify new potential targets to mitigate neurodegeneration .Sanz and García-Gimeno extensively review the recent progress made in the field of “Reactive glia inflammatory signaling pathways and epilepsy”, focusing on the main glial signaling pathways involved in neuroinflammation, how they are affected in epileptic conditions, and the therapeutic opportunities they offer to prevent these disorders .Filipek and Leśniak provide a detailed discussion about “S100A6 and its brain ligands in neurodegenerative disorders”. The authors focus on the expression/localization of S100A6, CACYBP/SIP and SGT1 proteins in various brain structures and on their possible involvement in neurodegenerative diseases, such as Alzheimer’s disease, amyotrophic lateral sclerosis, epileptogenesis, Parkinson’s disease, Huntington’s disease, and others .


POLE) and immunohistochemistry (mismatch repair and p53 proteins) to assign patients with EC to one of four molecular subtypes: POLEmut, MMRd, p53abn and NSMP (no specific molecular profile). Unlike histopathological evaluation, the molecular subtyping of EC offers an objective and reproducible classification system that has been shown to have prognostic value and therapeutic implications. It is an exciting time in EC care where we have moved beyond treatment based on histomorphology alone, and molecular classification will now finally allow assessment of treatment efficacy within biologically similar tumours. It is now recommended that molecular classification should be considered for all ECs, and should be performed routinely in all high grade tumours. It is also recommended to incorporate molecular classification into standard pathology reporting and treatment decision-making algorithms. In this review, we will discuss how the molecular classification of EC can be used to guide both conventional and targeted therapy in this new molecular era.Following the discovery of the four molecular subtypes of endometrial cancer (EC) by The Cancer Genome Atlas (TCGA) in 2013, subsequent studies used surrogate markers to develop and validate a clinically relevant EC classification tool to recapitulate TCGA subtypes. Molecular classification combines focused sequencing ( Patients with advanced and recurrent EC represent a major therapeutic challenge, with 5-year overall survival rates of only 17% in patients with distant disease.2 Approximately 80% of women with early-stage EC have a favourable prognosis, with 5-year overall survival rates of 95%.3 The remaining 20% of women with early-stage disease have one or more high risk features associated with an increased risk of cancer-related death. A major diagnostic challenge is identifying which patients with early-stage EC have low-risk disease with a risk of recurrence <5%, and thus can be managed by surgery alone, in contrast to patients with high-risk disease who need adjuvant therapy.Globally, the incidence of endometrial cancer (EC) has increased significantly by 0.69% per year between 1990 and 2019,5 Stage assignment is generally considered more objective and consistent; however, pathology review of all patients eligible for the PORTEC-3 trial for high risk EC found not only significant disagreement in grade and histotype, but also in stage- and risk-group-defining criteria such as cervical stromal invasion, myometrial invasion and lymphovascular invasion (LVI).6 As such, a risk stratification where all three of the critical parameters are inconsistent leads to an imprecise estimation of the risk of recurrence and death, which subsequently results in both the over- and under-treatment of many women.For decades, the EC risk stratification used to guide treatment has been based largely on histological type, grade and stage. Both histotype and grade assignment have been shown to be poorly reproducible even amongst expert pathologists, with no agreement in histotype diagnosis in one-third of cases.7 A recent National Cancer Database review showed out of 19,594 EC patients who met the national guidelines criteria for adjuvant radiation, 47% did not receive radiation, which was associated with worse overall survival. Omission of adjuvant radiation was more common among African-American, Hispanic and Asian patients, and patients with lower household income, lack of health insurance and treatment at non-academic hospitals.8 This highlights the need for an objective EC classification system with fair and consistent delivery of care.What is also concerning is the significant variation in clinical practice that is currently observed in administering adjuvant therapy in EC.POLE ultra-mutated, microsatellite instability hypermutated, copy-number low and copy-number high.9 Following this discovery, two groups developed and validated a molecular classification tool using surrogate markers to recapitulate TCGA subtypes using more pragmatic, cost-effective and clinically applicable methods.1014 This classifier uses a combination of focused sequencing to identify mutations in DNA polymerase epsilon (POLE) and immunohistochemistry (IHC) for mismatch repair proteins and p53, yielding four molecular subtypes: POLEmut, mismatch repair deficient (MMRd), p53abn and NSMP . The molecular classification of EC has been shown to be highly reproducible among pathologists, with high concordance between the preoperative biopsy and final hysterectomy specimens, as well as high interlaboratory concordance.1517 The prognostic value of molecular classification has been demonstrated repeatedly, across unselected population-based series, clinical trials, and even narrowly defined age- or histotype-stratified subsets.1821 What has been most promising, however, is the recent data supporting the predictive implications of molecular subtype assignment. This new reproducible and objective classification system creates a much needed framework to approach treatment decisions in both conventional and targeted therapy. Given the new recommendations for integration of molecular parameters in to standard pathology reporting in the 2020 5th edition of the WHO Female Genital Tumours, and the integration of molecular classification into the 2020 ESGO/ESTRO/ESP risk stratification and treatment algorithms, it is imperative that clinicians are aware of the rationale for molecular subtype-specific care.22We are now in an exciting era in EC research and clinical care, moving to a subtype specific approach proven successful in other disease sites. In 2013, The Cancer Genome Atlas (TCGA) discovered four molecular subtypes of EC-based genomic architecture, with each group having distinct clinical outcomes; In this review, we will discuss how the molecular classification of EC can be used to help guide systemic treatment in this new molecular era.914 These tumours have loss of DNA mismatch repair, which results in a high mutational burden (‘hypermutated’), exceeding >10 mutations per megabase.9 Mismatch repair deficiency can be identified through microsatellite instability testing, or testing for the loss of expression of one or more mismatch repair proteins . Approximately 90% of MLH1/PMS2 loss is due to somatic MLH1 promotor hypermethylation, and germline mutations in one of the mismatch repair genes, termed Lynch syndrome, accounts for approximately 10% of MMRd ECs and 3% of all ECs.24 Post et al.24 also recently assessed the prognostic value of Lynch Syndrome within MMRd EC and found a trend towards improved recurrence free survival in patients with Lynch Syndrome compared with MLH1 hypermethylation.The MMRd/microsatellite unstable molecular subtype accounts for 25–30% of all ECs and they have an intermediate prognosis.POLEmut ECs, are known to be highly immunogenic tumours. Previous work has demonstrated cancers that have a high mutational burden have a substantially increased production of tumour mutated antigens (neoantigens), which correlates significantly with improved patient survival.25 The increased neoantigens results in a high abundance of tumour-infiltrating lymphocytes (TIL), in particular CD8+ cytotoxic T cells, with an upregulated T-cell mediated antitumour response.2527 Cancers can develop mechanisms of immune escape, largely through upregulation of inhibitory immune checkpoint receptors, such as programmed death-ligand 1 (PD-L1), which subsequently block activated T-cell-mediated tumour cell death.27 These immune checkpoint interactions between tumour cell PD-L1 receptors and programmed death protein 1 (PD-1) on T-cells can be blocked by the use of antibodies, thus making these tumours susceptible to reactivation of the immune response when treated with immune checkpoint blockade therapy.The ‘hypermutated’ MMRd and ‘ultramutated’ versus 79% in the chemoradiation group (p = 0.445).28 Adjuvant radiation on the other hand, may play a more important role in MMRd EC, compared with other EC molecular subtypes. Pre-clinical work has shown increased sensitivity to radiation in MSH2 deficient cell lines.29 In a review of 128 patients with stage Ib/II grade 3 endometrioid endometrial cancer, Reijnen et al. showed that adjuvant radiation was associated with improved disease specific survival in the MMRd group, but not in MMR-proficient cases.30 A more recent study compared adjuvant chemotherapy and radiation with chemotherapy alone in advanced MSI-high EC, and found an improved progression-free survival with the addition of radiation.31 This evidence suggesting MMRd EC may have an increased sensitivity to radiation needs to be validated in prospective studies.The role of adjuvant chemotherapy in MMRd EC has been questioned recently by the molecular analysis of PORTEC-3. This trial assessed chemotherapy used in addition to adjuvant radiation in high-risk EC. The molecular analysis found no benefit with the addition of chemotherapy in the MMRd group, with the 5-year overall survival 84% in the radiation only group 33 This subsequently led to the United States (US) Food and Drug Administration (FDA) approval in 2017 of pembrolizumab in unresectable or metastatic MSI-H/MMRd solid tumours. The ORR of pembrolizumab in the MMRd EC cohorts in these studies were a striking 53–57%. Several other single agent immune checkpoint inhibitors have since been studied in advanced or recurrent EC, including nivolumab (anti-PD-1), avelumab (anti-PD-L1), durvalumab (anti-PD-L1) and dostarlimab (anti-PD-1), with the ORR in MMRd cohorts being 25%, 27%, 43% and 42%, respectively.3437Pembrolizumab (anti-PD-1) was the first immune checkpoint inhibitor shown to have favourable objective response rates (ORR) in metastatic or recurrent MMRd colorectal and non-colorectal cancers.versus radiation plus an immune checkpoint inhibitor  program of clinical trials plans to randomise patients with stage II/III MMRd EC to radiation n = 108) showed an ORR of 64% in the MMRd group and 36% in the MMR-proficient group.38 This led to the FDA approval of pembrolizumab plus lenvatinib in MMR-proficient EC patients who have disease progression following prior systemic therapy in 2019. KEYNOTE-775 is a phase III trial of pembrolizumab plus lenvatinib compared with physician’s choice chemotherapy (doxorubicin or paclitaxel) in advanced or recurrent EC in patients with at least one prior line of platinum-based chemotherapy. The results were presented at the recent Society of Gynecologic Oncology (SGO) annual meeting 2021 with significant improvement in both progression-free survival (7.2 months versus 3.8 months) and overall survival (18.3 months versus 11.4 months) in the pembrolizumab plus lenvatinib arm compared with chemotherapy in all comers (both MMR-proficient and MMRd EC).39 What remains to be determined is how much benefit is gained by the addition of lenvatinib to pembrolizumab within MMRd tumours and whether the additional toxicity is warranted in this group. There is another recently completed phase III study assessing this pembrolizumab plus lenvatinib combination but in first-line treatment compared with first-line carboplatin and paclitaxel in patients with advanced or recurrent EC (LEAP-001/ENGOT-en9).KEYNOTE-146 is a phase IB/II study of pembrolizumab in combination with lenvatinib – a multikinase inhibitor that targets vascular endothelial growth factor receptors 1–3 in selected advanced solid tumours. The EC cohort  in recurrent EC with prior systemic therapy was presented at ASCO 2020. This combination resulted in an ORR of 25% and a clinic benefit of 69% in this heavily pre-treated group.42 This suggests molecular classification used alone to direct use of immune checkpoint blockade therapy may miss a significant number of EC patients who may benefit, and further research to identify biomarkers that can accurately predict response to immunotherapy is required.There are also currently three clinical trials ongoing assessing the efficacy of immune checkpoint inhibition combined with first-line carboplatin and paclitaxel chemotherapy in advanced or recurrent EC. This includes the AtTEnd trial (AtTEnd/ENGOT-en7), which is using atezolizumab, RUBY (ENGOT-EN6/NSGO-RUBY), which is using dostarlimab and NRG-GY018 using pembrolizumab. The results of all of these ongoing and upcoming trials will help clarify the optimal timing of administration of immunotherapy in MMRd EC that is, whether it be adjuvant use in high-risk patients, combined use with first line platinum-based chemotherapy, combination use with targeted therapy or reserved for patients who have failed prior systemic therapy. Another important question is which patients with other EC molecular subtypes in addition to MMRd may benefit from immunotherapy. Two studies have assessed the immune response in large cohorts of molecularly classified ECs, and both groups found heterogeneity of TIL across EC subtypes with a proportion of both p53abn and NSMP subtypes surprisingly immune-rich.91821 The ECs in this molecular subtype were characterised by TCGA as having a very high number of somatic copy number alterations, low mutation rate and ubiquitous TP53 mutations.9 These tumours are now identified by a more pragmatic method of mutant-pattern p53 IHC staining, which has been shown to be an excellent surrogate maker for TP53 mutational status as determined by sequencing in EC biopsies.43 A large majority of serous carcinomas are p53abn; however, p53abn EC is seen across all EC histological types. The proportion of p53abn tumours for each histotype was assessed recently using pooled data from several EC cohorts that have been classified molecularly as follows; serous carcinoma 93%, carcinosarcoma 85%, clear cell carcinoma 38%, grade 3 endometrioid EC 22% and grade 1–2 endometrioid EC 5%.91844p53abn EC represents the most aggressive and lethal molecular subtype, and although it accounts for only approximately 15% of all EC cases, it is responsible for 50–70% of EC mortality.versus 36% with radiation alone (p = 0.019).28 This significant benefit from the addition of chemotherapy in the p53abn group was not observed in the other molecular subtypes. The overall test for interaction between molecular subgroups and treatment arm did not reach significance; however, the PORTEC-3 clinical trial was not originally powered for analysis by molecular subgroup. This signal of benefit with conventional chemotherapy observed within p53abn EC is promising. Although historical EC studies have shown very mixed results in terms of adjuvant platinum-based chemotherapy response, the ability to easily stratify (through p53 IHC) and identify a subset of women who appear to benefit will enable improved design of future trials. Further, this is a subset of women in whom better treatment options and opportunities to improve survival statistics are desperately needed. The evidence that p53abn molecular subtype is found in up to 5% of grade 1 and 2 endometrioid endometrial carcinomas45 highlights why it is important that molecular features are integrated into pathology classification and stratification of care. Patients with stage I and grade 1–2 endometrioid EC without LVI would otherwise be considered low risk by historical EC risk stratification systems, and might be treated by surgery alone. Although further work is needed to better understand this subgroup of cancers, the concern is without p53 stratification they may be undertreated, representing a missed opportunity for curative adjuvant therapy.The importance of conventional platinum-based chemotherapy in p53abn EC was demonstrated with the retrospective molecular analysis of PORTEC-3 trial for high-risk EC. This showed that patients with p53abn EC had significantly improved outcomes when chemotherapy was used in addition to pelvic radiation, with 5-year recurrence-free survival of 59% with chemotherapy and radiation TP53 mutated EC and both high-grade serous tubo-ovarian cancer (HGSOC) and basal-like breast cancer.9 Defects in the homologous recombination repair pathway are common in HGSOC and basal-like breast cancers, found in 55% and 46% respectively.47 Homologous recombination is essential for the repair of double-stranded DNA breaks, and tumours with homologous recombination deficiency (HRD) are known to be more responsive to both platinum-based chemotherapy and poly (ADP-ribose) polymerase (PARP) inhibitors. The prevalence of HRD in p53abn EC is currently unknown. De Jonge et al. assessed HRD in a small group of ECs using functional RAD51assessment and found HRD in 46% of p53abn EC,48 although this may be an over representation in this highly selected group. Another study assessed HRD in 19 uterine serous carcinomas using OncoScan SNP arrays to calculate HRD scores and found 53% had an HRD phenotype.49 However, another group found only 15% of copy-number high EC from TCGA dataset had mutational signatures that were HRD related.50 Siedel et al. used the Myriad HRD assay on two EC cohorts and found a cut-off HRD score ⩾4 was associated with worse survival, and the median HRD scores for endometrioid EC, mixed serous and endometrioid EC, and serous EC were 3, 15.5 and 28.5 respectively.51 The worse survival outcomes for high HRD score EC differs from HGSOC and basal-like breast cancer, where HRD high tumours show improved outcomes in both tumour types. This same group also determined the HRD scores of 12 EC cell lines. The three cell lines with highest HRD score were all TP53 mutated and all showed statistically significant increased sensitivity to cisplatin, paclitaxel and olaparib, compared with the low HRD score cell lines.Despite this benefit from conventional chemotherapy, we know the survival outcomes for patients with p53abn EC are poor and we urgently need therapeutic advances to improve outcomes for these patients. TCGA demonstrated several molecular similarities between 52NRG-GY012 is a phase II, three-arm study in recurrent and metastatic EC assessing the PARP inhibitor olaparib in combination with the anti-angiogenic cediranib, compared with both agents used alone in recurrent, refractory or metastatic EC. The results, presented at the SGO annual meeting 2021, showed the combination of olaparib and cediranib demonstrated modest efficacy, but was not significantly different compared with cediranib alone. Single agent olaparib was not effective in this patient population and the molecular analysis including subtype distribution and HRD scores from this trial cohort are still pending.versus conventional chemoradiation in early and late stage serous or p53abn endometrial cancer). The TransPORTEC RAINBO program of clinical trials plans to assess adjuvant chemoradiation with or without a DNA damage response (DDR) targeting agent in p53abn EC  is another ongoing phase II study looking at olaparib combined with the immune checkpoint inhibitor durvalumab in recurrent or metastatic EC. The efficacy of PARP inhibitors used in combination with adjuvant first line chemotherapy in p53abn EC will also be assessed in CAN-STAMP  therapy. HER2 overexpression and/or amplification has important prognostic and therapeutic implications in breast and gastric cancer. HER2 overexpression and/or amplification is seen in EC, with variable reported frequencies. Unlike breast and gastric cancer, there is no standardised scoring system of HER2 in EC and, consequently, what is reported as ‘HER2 positive EC’ varies considerably between studies and makes it difficult to interpret the literature. Buza 55 there is now data demonstrating improved outcomes within serous ECs. The phase II trial from Fader et al.57 showed the addition of trastuzumab to carboplatin and paclitaxel chemotherapy for women with advanced or recurrent uterine serous carcinoma, that were HER2 positive (3+ IHC or 2+IHC with amplification confirmed on fluorescence in situ hybridisation) significantly improved both progression free survival (12.6 months versus 8.0 months) and overall survival (29.6 months versus 24.4 months) compared with chemotherapy alone. The largest benefit was seen in patients with stage III/IV disease, with a progression free survival of 17.9 months with chemotherapy plus trastuzumab versus 9.3 months with chemotherapy alone. Molecular stratification has not been performed on this series.Despite disappointing results in a previous clinical trial assessing single agent trastuzumab in previously treated, non-stratified ECs,in situ hybridisation). The correlation between p53abn and HER2 status was significantly stronger than between serous histology and HER2 status.58 The HER2 positive cases were seen across all p53abn histotypes; including serous, endometrioid, and clear cell carcinomas. Similar findings were also recently reported in a cohort of 238 p53abn ECs which underwent sequencing of >400 cancer related genes. ERBB2 alterations were found in 21% of cases, and the frequency of ERBB2 amplification did not differ between histological types.59 Both of these studies highlight the importance of molecular subtype directed clinical trials rather than histotype directed, to maximise the number of patients who may derive clinical benefit from anti-HER2 therapy.HER2 status was recently assessed retrospectively in PORTEC-3 cases that had undergone molecular classification. They found 25% of the p53abn molecular subtype were HER2 positive  was assessed in recurrent or persistent EC after at least one prior line of chemotherapy, with 13.5% of patients demonstrating a clinical response .61 The MITO END-2 trial compared carboplatin, paclitaxel and bevacizumab in 108 patients with advanced or recurrent EC, with chemotherapy alone. Bevacizumab combined with chemotherapy failed to demonstrate a significant improvement in progression-free survival; however, it did show a significant increase in 6-month disease control rate (70.4% versus 90.7%).62 GOG-86P was a recent randomised three-arm trial assessing the addition of either bevacizumab, temsirolimus or ixabepilone, with standard paclitaxel and carboplatin in advanced or recurrent EC. Progression free survival was not increased in any of the three experimental arms compared with historical controls. In a post hoc analysis, assessment of TP53 mutation status showed that women with TP53 mutant EC had both improved progression free- and overall survival when treated with bevacizumab and chemotherapy, whereas women with TP53 wild-type tumours showed no difference in outcomes.63 The authors concluded p53/TP53 could be used as a biomarker to help predict patients with EC more likely to respond to bevacizumab treatment.Anti-angiogenic agents have been successful in many cancer types but have generally shown disappointing results in EC to date.TP53 mutant cells that have impaired G1 checkpoint repair and therefore rely on the G2 pathway for DNA repair.64 A phase II study looking at the use of single agent Wee1 inhibitor adavosertib in patients with recurrent uterine serous carcinoma and at least one prior line of platinum-based chemotherapy was presented at the ASCO 2020 meeting. They found an objective response rate of 29.4% and a clinical benefit rate of 50%, which is higher than observed with monotherapy in other disease sites.65 ADAGIO (a study of adavosertib as treatment of uterine serous carcinoma) is a larger phase II study looking at single agent adavosertib in the same patient population.Wee1 is a protein kinase that regulates the G2 checkpoint and prevents entry into mitosis in response to DNA damage. This G2 checkpoint is important for POLE mutations, mismatch repair or p53 abnormalities. They are the most common molecular subtype, accounting for approximately half of all ECs, with an intermediate prognosis. They are arguably the most challenging molecular subtype, given the lack of predictive biomarkers that can identify patients within this large heterogeneous group that have a higher risk of disease recurrence, and may benefit from more aggressive therapy. One potential stratification biomarker is β-catenin (CTNNB1) mutation status. Mutations in exon 3 of CTNNB1 are present in 52% of NSMP ECs and studies have shown low-grade endometrioid ECs that have a CTNNB1 mutation are a more aggressive subset with a higher risk of disease recurrence.66 L1-cell adhesion molecule (L1CAM) overexpression has also been shown to be an independent prognostic marker for distant recurrence and overall survival within NSMP ECs.67 Immune profiling of 695 ECs that had been molecularly classified demonstrated 36% of NSMP tumours had high immune cell infiltrate, and intraepithelial CD8+ cell density was a significant predictor of recurrence, and thus can be used to refine prognostication in this molecular subtype. Whilst we await further molecular refinement within NSMP EC, grade, stage, LVI and histotype will continue to play a role in determining adjuvant therapy for this subtype.Deriving from ‘copy number low’ TCGA subtype, NSMP ECs are characterised by a low number of somatic copy number alterations, low mutational burden and high levels of oestrogen and progesterone receptor (ER/PR) expression. As implied by their name ‘no specific molecular profile’ they are defined by their lack of pathogenic 68 Interpreting the current hormonal therapy literature in EC is challenging due to the unselected EC cohorts included in historic trials, different response criteria used in different studies, different hormonal treatment regimes and different cut offs used to define ER/ PR positive EC.Despite being used for several decades in ER-/PR-positive EC, mostly in the recurrent/metastatic setting, the role of hormonal therapy in EC remains poorly defined. In contrast to breast cancer, hormonal therapy in ER/PR positive EC is effective only in a minority of women, with response rates of approximately 22% to first-line hormonal therapy in advanced disease.69 Studies such as this would be extremely valuable in EC and may explain why response rates to hormonal agents in ER/PR positive EC have been disappointingly low. Given the high levels of ER and/or PR expression seen in NSMP EC, the TransPORTEC RAINBO program of clinical trials plans to randomise NSMP EC to adjuvant chemoradiation versus radiation plus hormonal therapy  4 and 6 inhibitor palbociclib. Palbociclib has been shown to inhibit growth of ER-positive breast cancer cells, and palbociclib plus letrozole resulted in significantly longer progression-free survival compared with letrozole alone in ER-positive breast cancer.72 A phase II study of palbociclib combined with letrozole in patients with ER-positive advanced or recurrent EC was presented at ESMO 2020 . The palbociclib and letrozole combination resulted in a disease control rate of 64% compared with 38% in letrozole only, and a significantly improved progression-free survival (8.3 months versus 3 months).73Hormonal therapy combined with targeted therapeutic agents known to disrupt ER pathways appears to be an effective mechanism of increasing response rates. One example is the use of mTOR inhibitors. Mutations in the PI3K/Akt/mTOR pathway are frequently seen in EC, and specific to NSMP EC, 74Obesity and diabetes are common in women with EC, especially within patients with NSMP molecular subtype. The efficacy of the antidiabetic drug metformin was assessed in combination with paclitaxel and carboplatin in 469 patients with treatment naïve advanced or recurrent EC (GOG-0286B). Progression-free survival and overall survival were not increased significantly with the addition of metformin to chemotherapy; however, translational studies are ongoing to identify potential biomarkers that may predict response to metformin treatment.POLEmut EC is the least common molecular subtype, accounting for approximately 10% of all ECs. These ECs have pathogenic mutations in the exonuclease domain of DNA polymerase epsilon (POLE), a protein involved in DNA replication. This results in extremely high somatic mutation frequencies (‘ultramutated’), exceeding 100 mutations per megabase.9 Most POLEmut ECs are endometrioid and, despite many having high-risk features, such as high-grade and LVI, this molecular subtype has exceptionally favourable survival outcomes.1028 One proposed explanation for this excellent prognosis is the host response to tumour. The extreme tumour mutation burden in POLEmut ECs was shown to result in a striking CD8+ lymphocytic infiltrate, as well as marked upregulation of cytotoxic T-cell effector markers in a study of 60 POLE mutant tumours.75 Another possible explanation for favourable outcomes had been that POLEmut ECs are hypersensitive to adjuvant treatment. Van Gool et al. created POLE mutant mouse-derived embryonic stem cell lines and demonstrated POLE mutations did not exhibit increased sensitivity to radiation or commonly used chemotherapeutics compared with POLE-wildytpe cell lines.76 Further, a recent meta-analysis of all published cases of POLE mutations in EC showed that the excellent survival outcomes of stage I/II POLEmut EC was independent of adjuvant treatment received.77 The molecular analysis of PORTEC-3 – a trial comparing chemoradiation versus radiation only in high risk EC – again demonstrated the excellent prognosis of POLEmut EC, even within this high risk cohort, with 5-year overall survival of 100% with chemoradiation and 97% with radiation alone (one recurrence event).28POLEmut EC . Further, there is a trial under consideration with the NCI Community Oncology Research Program (NCORP) and the imminent TransPORTEC RAINBO Blue-POLE trial , there are no data to infer efficacy. For rare advanced or recurrent POLEmut ECs, consideration may be given to immune checkpoint blockade therapy given the ‘ultramutated’ phenotype, with high TIL and PD-1/PD-L1 expression levels.7882There is currently insufficient evidence to guide conventional adjuvant treatment of advanced stage (III–IV) POLEmut tumours, secondary TP53 mutations/p53 IHC abnormalities or less commonly, MMR protein loss can be found. Leon-Castillo et al. reported on ECs that have more than one molecular feature.83 They demonstrated that tumours with a pathogenic mutation in POLE that also have MMRd and/or mutations in TP53, have morphology, molecular profiles and clinical behaviour aligning with POLEmut EC. These findings suggest that these secondary mutations are a later event acquired during tumour progression that do not affect the clinical outcome. This can sometimes be suggested based on histopathological examination, for example, the presence of  subclonal mutant-like IHC expression of p53 (POLE mutation status to avoid overtreatment. With the 2020 ESGO/ESTRO/ESP guidelines for the management of patients with endometrial carcinoma, any stage p53abn EC with myometrial invasion is considered high risk and is recommended adjuvant chemotherapy with or without radiation. A stage I–II POLEmut EC however is considered low-risk and omission of adjuvant therapy should be considered.22 Clarification of pathogenicity of POLE mutation is also imperative, with pathogenic list of mutations considered ‘actionable’ for POLEmut assignment currently limited to 11 well characterised missense mutations.84 Assessing clinical outcomes in women with mutations limited to the list of 11 pathogenic POLE mutations versus other non-pathogenic mutations in the recently assembled meta-analysis cohorts demonstrated statistically significant differences in outcomes.77 Women with non-pathogenic POLE mutations should not be offered de-escalation of adjuvant therapy.Given the high mutational burden observed in n of p53 . This alMolecular classification of EC offers an objective and reproducible classification system that has important prognostic and therapeutic implications. Molecularly directed EC clinical trials must be a priority to enable assessment of treatment efficacy within biologically ‘like’ tumours and to enable improvements in outcomes in this disease site. As we move forward in this new era, further stratification within each molecular subtype will likely refine our application of systemic therapy in EC. It is now recommended that molecular classification should be considered in all ECs and be incorporated into management decisions.


The aim of this systematic review is to explore the current available knowledge about tendon disorders and orthobiologics derived by preclinical experiments to evaluate their role and efficacy in the different stages and conditions related to the tendon healing processes.The systematic review was performed according to the PRISMA guidelines. Different electronic databases  were searched for studies investigating orthobiologics (PRP and cell-based products from adipose tissue or bone marrow) in animal models or veterinary clinical trials for tendon pathologies . Data regarding the specific product used, the treatment site/pathology, the host and the model were collected. The results were classified into the following categories: histological, biomechanical, molecular and imaging.A large pool of preclinical studies on tendon disorders have been found on platelet-rich plasma (PRP), while data about stromal vascular fraction (SVF) and bone marrow concentrate (BMAC) are still limited and frequently focused on expanded cells, rather than orthobiologics prepared at the point of care.The effect of PRP is related to an acceleration of the healing process, without improvements in the final structure and properties of repaired tendon. Cell-based products have been reported to produce more durable results, but the level of evidence is currently insufficient to draw clear indications.The preclinical results about orthobiologics applications to tendon pathologies would support the rationale of their clinical use and encourage the performance of clinical trials aimed to confirm these data in human subjects.The online version contains supplementary material available at 10.1186/s40634-022-00468-w. Tendon injuries, especially those affecting Achilles, forearm extensor, patellar and rotator cuff tendons, represent a very common condition , 55, 112While orthobiologics have been used extensively for the treatment of bone and cartilage diseases, the application to tendon disorders of this large variety of products is under-investigated. The aim of this systematic review is to explore the current available knowledge about orthobiologics for the treatment of tendon disorders in in vivo preclinical studies to evaluate their efficacy in the different tendon conditions. Given the little knowledge available from the clinical trials in this field, the evidence provided by preclinical studies may allow for the identification of the most promising applications in each different condition, in order to guide further clinical research on a subset of specific products and strategies.This systematic review was prepared according to the PRISMA guidelines and Cochrane Collaboration methodology . Three eThe screening process was focused on identifying preclinical studies or veterinary clinical trials investigating the effects of blood-derived products, cell-based products derived from adipose tissue and bone marrow in tendon disorders. The following reports were excluded during the screening process: in vitro studies or clinical trials on human subjects; studies evaluating the effects of orthobiologics on other tissues; studies investigating other techniques; documents written in languages other than English. Study selection was performed by two independent researchers. Figure The search for articles reporting preclinical studies concerning blood-derived products and tendons retrieved 89 papers after title/abstract screening. Further 10 papers were identified by manual search within the references of full-texts included in the analysis. They were all evaluated by abstract screening, and 37 were excluded because considered non-inherent for various reasons or because they did not satisfy inclusion/exclusion criteria. Then, 62 studies were included in the qualitative analysis.The search for studies reporting the efficacy of cultured mesenchymal stem cells or bone marrow aspirate concentrates (BMAC) in tendon conditions retrieved 19 documents. Of these, 8 were excluded after title/abstract screening, resulting in 11 studies available for analysis.Seventy-eight studies were identified during database search after title/abstract screening and 5 more were found among references of full-texts included in the analysis. Sixty-two studies were excluded because considered non-inherent for various reasons or because they did not satisfy inclusion/exclusion criteria, resulting in 21 studies included in the analysis.Product specifications, treatment site (or specific pathology), type of host and model, and experimental results  were collected for all studies included in the qualitative analysis.Risk of bias was determined for all studies using SYRCLE’s tool for animal studies. Two independent investigators rated the risk of bias for each study as low, high or unclear, depending on specific items dedicated to the identification of selection, performance, detection, attrition and reporting biases .n = 28) or rabbit (n = 26) models. Three and 2 studies respectively used horse and sheep models, while only one study was conducted in mice. In addition, two veterinary trials involving dogs were selected. Thirty-three studies used models of tendon ruptures, 8 addressed spontaneous or experimentally-induced tendinopathy, 10 investigated the rotator cuff tears and 6 studies involved tendon-bone junctions. The effect of PRP alone was evaluated in 57 studies, while 9 reports on PRP in addition to other therapies (drugs or cells). In 6 cases, PRP was used in combination with scaffolds. Platelet-rich fibrin (PRF) and plasma rich in growth factors (PRGF) were evaluated in 4 and 1 studies, respectively. According to SYRCLE’s tool, 22 studies were at high risk of bias, 9 due to the lack of control group and 13 due to lack of randomization and/or blinding of caregivers and outcome assessment  or detrimental effect of the experimental products (n = 2)  Table . StudiesEnhancement of biomechanical properties of the tissue was obtained in 20 studies, while in 5 cases no improvements were observed compared to controls Table . ControvThe treatment with blood-derived products was able to enhance the gene expression and protein deposition of collagen type I, as reported by 8 different studies involving both tendon rupture models and collagenase-induced tendinopathy , 74, 119Imaging techniques and behavior assessments demonstrated overall positive results using blood-derived products for the treatment of tendon-bone injury, tendon ruptures and collagenase induced tendinopathy, with 7 studies reporting satisfactory results and 1 showing no improvement compared to controls (Table n = 4), followed by horse (n = 3), rat (n = 2) mouse (n = 1) and dog models (n = 1). Concerning the pathological models, 3 focused on tendon ruptures, 2 on tendinopathy,2 on rotator cuff tears and 3 studies addressed lesions of the enthesis. Six studies employed BMAC, comprising 1 study combining this product with a scaffold and another study combining it with PRP. Five studies used in vitro cultured bone marrow (BM)-MSCs, 4 of which in combination with scaffolds or fibrin glue. The analysis performed using SYRCLE’s Risk of Bias tool, identified 5 studies at high risk of bias, due to the lack of a control group (n = 2) or differences between groups at baseline (n = 3). Only 1 randomized study and 2 studies with blinded outcome assessors were identified among the selected studies , while contemporary reducing the expression of collagen type III, MMP-3, MMP-13 , 99. Then = 1) or behavior/functional (n = 2) outcomes of BM-derived products for tendon pathologies reported positive results , horses (n = 2), mice (n = 1) and sheep (n = 1). Eleven studies addressed models of tendon ruptures, 5 focused on spontaneous or experimentally-induced tendinopathy and 5 focused on lesions of the tendon-bone junctions. In 5 cases, the site of injury was the supraspinatus tendon . Cultured adipose derived mesenchymal stem cells (ASCs) were frequently used either alone (n = 8) or in combination with scaffolds/hydrogels (n = 6) or with other treatments . Four studies used freshly isolated stromal vascular fraction (SVF) alone (n = 2) or in combination with fibrin glue (n = 2). Risk of bias analysis performed using SYRCLE’s tool demonstrated high risk only in 3 studies, due to lack of randomization and blinding of outcome assessment. Notably the frequency of randomized studies is higher among reports investigating adipose-derived products compared to studies about blood- and bone marrow-derived products . Possible safety issues about the use of orthobiologics for tendon pathologies are related to the formation of fibrotic tissue (fibrocartilage) and ectopic bone. On the other hand, this possible side effect turns into an advantage when treating tendon-bone junction injuries, as confirmed by studies assessing enthesis repair.Additional file 1.