To describe the complications associated with the different gastrostomy techniques  performed in the last 26 years in a terciary hospital.n (%)].The comparison of complications between patients who underwent different techniques was performed with Fisher’s test.Retrospective observational study. Patients who underwent gastrostomy at the Virgen del Rocío University Hospital between 1995 and 2021 were included. For PEG, the PULL technique was performed until 2018 and subsequently the PUSH technique predominantly. For PRG, a pigtail catheter was used until 2003, a balloon catheter between 2003 and 2009, and a balloon catheter with gastropexy between 2015 and 2021. For SG, the conventional technique (CSG) was performed until 2009 and since then the laparoscopic assisted percutaneous gastrostomy (PLAG) technique. Descriptive analysis was performed obtaining the median and quartiles of the quantitative variables [P50 (P25-P75)] and the frequency for the qualitative variables [n = 1,070 . The three most frequent indications were head and neck tumors, neurological diseases and gastroesophageal tumors. The percentage of patients who had any complication was 48.9% (PEG-PULL), 23.7% (PEG-PUSH), 38.5% (pigtail PRG), 39.2% , 29.7% , 87.3% (CSG), and 41.26% (PLAG). 2 (0.18%) patients died from gastrostomy-related complications. 18(1.68%) presented with peritonitis and 5 (0.4%) presented with gastrocolic fistula. The rest of the complications were minor.Gastrostomy in any of its modalities is currently a safe procedure with a low rate of complications, most of which are minor. The description in 1981 of the percutaneous radiological gastrostomy (PRG) technique by Preshaw  and in 1Surgical techniques (SG) have also evolved. In addition to conventional direct techniques with manual ostoma preparation (Stamn and Janeway techniques), the percutaneous PUSH technique can be performed. All three techniques can currently be performed by laparotomy or laparoscopy. On the other hand, in our centre, and with the aim of reducing complications, we developed the percutaneous laparoscopic assisted gastrostomy (PLAG) technique, associated with a similar level of complications to PEG and PRG . The latCurrently, since percutaneous endoscopic and radiological techniques are associated with fewer complications than direct surgical techniques, their use is more widespread . SurgicaThe indications have also expanded and varied over time. Currently, the most frequent pathologies for which these techniques are indicated are head and neck cancer, neurological diseases, malignant and benign pathologies of the oesophagus and stomach, and maxillofacial pathologies .Although these procedures have a low morbidity, they are not free of complications , 10, incA large number of gastrostomies are performed at our tertiary hospital by teams with extensive experience in the three techniques. In addition, we have a nutrition unit, with specific consultations for the follow-up of these patients by a specialised nursing team and a medical team made up of specialists in endocrinology and nutrition, where a large volume of patients are seen. As technological improvements have become available, the number of complications in our series has declined , 11–14.The primary objective of our study is to describe the complications associated with the different gastrostomy techniques used in the last 25 years in our centre. The secondary objectives are: to compare the complications associated with the new techniques with those associated with the techniques previously used and currently in disuse in our centre, and to describe the indications for these techniques as well as their evolution over this period.2.2.1.A retrospective observational study was carried out including all adult patients who had undergone gastrostomy by endoscopic, radiological or surgical technique at the Hospital Universitario Virgen del Rocío and who were subsequently followed up at the hospital’s Nutrition Unit between 1995 and 2021.2.2.Data collection was carried out retrospectively using the clinical records of the Andalusian Health Service. Until around 2008–2009, medical records were collected on paper and subsequently began to be collected in Digital Medical Records. Written informed consent was obtained from all the patients before the procedure. All the required clinical and ethical guidelines of our center were followed.The study variables included were: sex, age, date of performance, indication for gastrostomy , type of gastrostomy, presence of complications, presence of major complications  after the procedure and gastrocolic fistula), presence of minor complications  and death due to gastrostomy complications. The follow-up period ranged from 3 to 24 months until the patient’s loss of follow-up in the Nutrition Unit.2.3.n (%)]. Fisher’s test was used to analyze the differences between patients who underwent different techniques and their complications.Statistical analysis was carried out using Statistical Package for Social Science (SPSS®) 25 version for Windows . Descriptive analysis was performed by obtaining the median and quartiles for quantitative variables [expressed as P50 (P25-P75)] and the frequency for qualitative variables [expressed as 2.4.The technique used in each patient was not randomised, but decided by a team specialised in Clinical Nutrition. SG was used in those cases in which PEG and PRG were not possible .PEG: the PULL endoscopic technique (Ponsky-Gauderer technique) was performed exclusively until 2018, when the PUSH technique (Sacks-Vine technique) was introduced. The latter was the predominant technique in 2019 and 2021.PRG: until September 2003, only catheters with internal fixation of the pigtail type (Cope type) were used, then simple balloon catheters were used until 2009 (for simplicity it will be referred to as PUSH technique without pexy) and finally the PUSH technique with internal fixation with balloon and additional anchoring system with pexy (for simplicity it will be referred to as PUSH technique with pexy) was used from 2009 onwards.SG: conventional direct surgical techniques  were performed until 2010, when they began to be performed using the PLAG technique (described by our group in 2016 ).Enteral nutrition was started 6 h after the procedure in the case of PEG and PRG techniques and 24 h in the case of SG, except in those cases where there was clinical suspicion of peritonitis. After admission, patients were assessed in Clinical Nutrition consultations at 1 month and every 3 months thereafter.3.A total of 1,070 patients were included. PEG was performed in 608 patients , PRG in 344 patients  and SG in 118 patients (55 CSG and 63 PLAG).The demographic characteristics of the patients (gender and age) are shown in The pathologies that led to the indication of gastrostomy in these patients are shown in p < 0.001), exudate (p < 0.001) and granuloma (p < 0.001), more frequent in the PEG PULL group and bleeding (p = 0.023), more frequent in the PEG PUSH group. All bleeding events were mild.Complications associated with the PEG performed are shown in The complications associated with the different PRG techniques performed in our centre between 1995 and 2021 are shown in S. aureus, K. aerogenes, and E. faecalis) secondary to the gastrostomy tube migration fistula. He was treated with intravenous antibiotherapy (cefazolin and piperacillin/tazobactam) and a PLAG gastrostomy was performed.Gastro-colic fistula has occurred in two cases (1%) with the PUSH type PRG technique with pexy. The first case was a 73-year-old man with a head and neck tumour who 10 months after the procedure presented with severe malnutrition and diarrhoea with similar characteristics to the enteral nutrition formula he was receiving. A CSG gastrostomy was performed to resolve the situation. The second was a 64-year-old male with a head and neck tumour who presented severe malnutrition, diarrhoea and polymicrobial bacteraemia (p < 0.001). In the former, the most frequent complication was irritation (81.8%) and in the latter, exudate . There were also statistical differences regarding exudate and burn (both were more frequent in the CSG group). A 23-year-old patient with a diagnosis of ALS presented with acute respiratory failure after the procedure in 2019. He required non-invasive mechanical ventilation and a 10-day ICU admission. Another 45-year-old patient with an oesophageal carcinoma died after balloon rupture and leakage of gastric contents leading to diffuse peritonitis with hepatic abscesses.Complications associated with the SG techniques used are shown in 4.The present study evaluates the rate of complications associated with the performance of 1,070 gastrostomies for enteral nutritional support in adult patients with different PEG, PRG and SG techniques employed over the last 25 years. The techniques were performed in a tertiary hospital by experienced staff and the patients were closely followed up in an experienced Nutrition Unit. In summary, the techniques that have been implemented over the years have decreased the percentage of associated complications, and these have been mostly minor, so they could be considered as safe techniques.The most frequent indication for PEG was neurological diseases followed by head and neck tumours, as described in other studies , 2. For Comparison of complications with other studies is complex due to the lack of uniformity in the definition of complications and the variants of the techniques themselves. Regarding PEG and PRG, our results are comparable to those described in the study by Galaski et al. . This stStill on complications associated with PEG and PRG, our data differ from those obtained in the study by Kohli et al., in which the total complications encountered were lower . This isAdditionally, prospective multicenter studies and meta-analyses have also been performed, such as that of Grant et al. , althougRegarding surgical techniques, there are few studies evaluating the new laparoscopic techniques and their long-term complications, but the PLAG technique has been shown to be safe , 10. In It should be noted that our results show that the appearance of new techniques  has led to a decrease in complications, as previously described in the scientific literature both by our group , 15, 16 These safety data are consistent with the results of the meta-analysis by Strijobs et al.  in whichIt is worth mentioning that caution should be exercised with regard to the higher rate of bleeding (even if slight) with the recently implanted PEG PUSH technique. Precisely in the Endoscopic management of enteral tubes in adult patients guidelines of the European Society of Gastrointestinal Endoscopy (ESGE), it is considered a relative contraindication recent gastrointestinal (GI) bleeding due to peptic ulcer disease . In addiThe main limitation of this study, in addition to its observational nature and retrospective data collection, is the heterogeneity of the patients included in terms of the techniques performed and the types and calibres of tube used. On the other hand, it would have been interesting to take into account some variables that were not collected and that may affect the number of complications: use of prophylactic antibiotic therapy , use ofThe inclusion in the study of patients with different pathologies and indications may represent another bias and sub-analyses should be performed in the future in these groups, since complications may vary among them and the indication for one technique or another may depend on this, and the choice of technique should be made in the most individualised manner possible as recommended in guidelines . Finally5.We present a series of patients who underwent gastrostomy by endoscopic, radiological or surgical technique in a tertiary hospital over the last 26 years. The indication for gastrostomy has varied over this period. Gastrostomy in any of its modalities is currently a safe procedure with a low rate of complications, most of which are minor. However, patients should be carefully selected, the modality of gastrostomy should be discussed in a multidisciplinary way, experienced endoscopists, radiologists, and surgeons should be available, and any major complication should be analysed from a risk management perspective. Further studies, especially randomised clinical trials that analyse complications based on each indication, will allow us in the future to individualise the choice of the most appropriate technique for each patient.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by CEI de los Hospitales Universitarios Virgen Macarena – Virgen del Rocío de Sevilla. The patients/participants provided their written informed consent to participate in this study.AP-G and PG-L: conceptualization. SG and RV: methodology. PG-L: software, resources, supervision. PS-A, JP-C, and PG-L: validation. AP-G: formal analysis, writing—original draft preparation, visualization. AP-G, IG-N, DT-D, PG-D, PR-R, and AM-O: investigation. SG, RV, and AP-G: data curation. AP-G, JP-C, MS, SM-C, JB-G, FG-F, and PG-L: writing—review and editing. ÁI-L, VN, and PG-L: project administration. ÁI-L and PG-L: funding acquisition. All authors contributed to the article and approved the submitted version.


The development of microbiome-targeted strategies is limited by individual differences in gut microbiome composition and metabolic responses to interventions. In vitro models that can replicate this variation allow us to conduct pre-clinical studies and assess efficacy. This study describes the exposure of 16 individual fecal microbiota samples to 5 different fibers using an in vitro system for the anaerobic cultivation of bacteria. The individual microbiota differed in composition and metabolite profiles (short-chain fatty acids and branched-chain fatty acids) after incubation with the fibers. Furthermore, microbiota composition after fiber incubation was significantly different between subjects with good intestinal health and subjects with Inflammatory Bowel Disease (IBD). α-diversity was differently affected by dietary fibers; for example, exposure to psyllium resulted in increased diversity in the healthy group and in decreased diversity in the IBD group. Instead, the functional metabolic profile did not differ between the two groups. Finally, the combination of all fibers, tested on the microbiota from IBD subjects, resulted in stronger overall effects on both microbiota composition and metabolite production compared to the single fibers. These results confirm that incubation with dietary fiber results in different compositional and functional effects on individual microbiota and that in vitro models represent successful tools for studying individual fiber effects. A healthy diet is crucial to maintain, promote, and restore health. Although some dietary patterns are known to positively influence health and reduce the risk of non-communicable diseases , a one-sFibers are dietary components that are non-digestible by human digestive enzymes and associated with positive intestinal and systemic health effects. Higher fiber intake is linked to lower body weight and decreased risk for certain conditions, including obesity, diabetes, cardiovascular, and gastrointestinal diseases . To a grEach person has a unique gut microbiota, potentially resulting in different metabolic responses and health outcomes to the same food ingredient . Indeed,This study describes the exposure of 16 individual fecal microbiota samples to 5 different fibers using an in vitro system for the anaerobic cultivation of bacteria . After fTo collect the sample, the subjects used the FecesCatcher, a specimen collection device consisting of biodegradable paper to be placed under the toilet seat (fecesvanger.nl). Fecal material was collected with a sterile plastic spoon and placed in a tube that was, in turn, placed in an anaerobic jar  with an AnaeroGen Sachet (Thermo Fisher Diagnostics GMBH). The jar was kept at 4 °C until delivery at the laboratory (within 24 h from collection). There, the material was introduced in an anaerobic chamber, diluted 1:3 with phosphate-buffered saline, and homogenized. Finally, 20% glycerol was added before storing the material at −80 °C.® 260 resistant starch, product code 22000B00, CAS number 9005-25-8, Ingredion, Westchester, IL, USA), psyllium , cellulose , and pectin .Five fibers with different composition, structure, and solubility were selected: β-glucan , resistant starch . Additionally, all fibers were combined in equal proportions to create a fiber mix that was tested on fecal material from IBD donors. The fiber mix was tested at two concentrations: 4 mg/mL, for comparison with the single fibers, and 12 mg/mL, representative of a high-fiber intervention.9 CFU/mL. The microbiota was cultured in SIEM with pH adjusted to 5.8. All compounds were tested in triplicate. After 24 h of anaerobic fermentation, the incubation material was sampled for DNA isolation and metabolite analysis.The fecal material was incubated anaerobically in the i-screen  system . First, Following incubation, samples were collected, and DNA was isolated as described by Ladirat et al.  with minChanges in the microbiota composition were analyzed by using 16S rDNA amplicon sequencing. The V4 hypervariable region was targeted. A total of 100 pg of DNA was amplified as described by Kozich et al. , with thSequence pre-processing, analysis, and classification were performed using modules implemented in the Mothur software platform . Chimerig, 5 min). Clear supernatant was filter-sterilized (0.45 µm). A mixture of formic acid (20%), methanol, and 2-ethyl butyric acid  was added. A 3 µL sample with a split ratio of 75.0 was injected on a GC-column  in a gas chromatograph .SCFA covering acetate, propionate, and n-butyrate and branched chain fatty acids (BCFA) covering iso-butyrate and iso-valerate were analyzed as described by Jouany , with moAll data analysis was performed using R version 4.1.2 . All figUnivariate regression on the microbiome count data was in all cases performed using linear mixed models from the dream package, version 1.24.0, with the variancePartition extension ,22. ThisMultivariate analysis and ordinations were performed using the vegan package, version 2.5-7 . This pap-values for the terms included in the model. A total of 103 permutations were used for all reported results. Count data were normalized using the Wisconsin double standardization after square-root transformation in the case of PERMANOVA, RDA, and MDS ordinations. In the case of the distance analysis, PERMANOVA, and MDS, the Bray–Curtis distance measure was used.The multivariate models fitted by PERMANOVA were tested via permutation analysis in order to produce Type III  The 16S data were filtered to include only those taxa that contribute to the first 95% of all counts in the data. This step was not performed for the alpha-diversity analysis. The filtering procedure consisted of the steps denoted below.Given the count table of taxa Let the row count normalized column sums of our matrix The cumulative sum of the column sums normalized for column count is then denoted as Any taxa where Statistical analysis of the SCFAs and α-diversity measures was performed using the lme4 and lmerTest packages, with the emmeans package for post hoc analysis ,21,22. Ap < 0.001 for overall fiber effect) but not for the IBD microbiota (p = 0.0501). Linear model analyses indicate a significant group-treatment effect (p < 0.001), meaning that the effect of fiber on microbiota diversity was significantly different between the healthy and the IBD groups. p < 0.001), and it was significantly increased after treatment with psyllium (p = 0.015) and resistant starch (p = 0.022); in the IBD group, α-diversity was significantly decreased after treatment with psyllium compared to the untreated control (p = 0.016).The overall effect of fiber on the α-diversity was significant for the healthy microbiota  and in the IBD group . Furthermore, ANOVA revealed a significant subject–treatment interaction , meaning that the effect of fiber on microbiota composition was significantly different across individuals . This is illustrated in After 24 h of fiber incubation, the microbiota composition was significantly different from the untreated control, both in the healthy group . Specifically, different bacterial genera were affected by fiber treatment in the healthy versus the IBD microbiota. p < 0.001) affected by the fibers in the healthy and IBD microbiota, compared to the untreated control. Lachnospira was strongly increased in both groups after treatment with pectin. Of all tested fibers, pectin affected the highest number of bacterial taxa in both the healthy and the IBD microbiota; the second largest effect was shown by β-glucan on the IBD microbiota.PERMANOVA revealed that, after incubation with fibers, microbiota composition was significantly different between the healthy and the IBD group . The overall effect of fiber, both in the healthy and IBD group, was significant on all metabolites measured: acetate, propionate, n-butyrate, iso-valerate, iso-butyrate, valerate, and 2-methylbutyrate. The largest effect of fiber was observed on acetate . This effect was observed for all metabolites except acetate. This means that the individual microbiota had different metabolite profiles after exposure to fiber; however, their acetate profile was similar. Interestingly, an opposite trend was observed for the group–treatment interaction: the effect of fiber on metabolite levels was not significantly different between healthy and IBD subjects, except for acetate (p < 0.005). This means that the effect of fiber on acetate levels may be mediated by the microbiota composition of the person.Linear mixed-effect models indicate that fiber’s effects on metabolite levels are significantly different across individual microbiota  illustrates the effect of different fibers on individual bacterial genera in the microbiota . The pinThis study described the exposure of 16 individual fecal microbiota samples to 5 different fibers in vitro. Effects on microbial composition and functional profiles were determined, and we explored whether these effects differed among individuals and between two groups with different intestinal health.All fibers tested in this study influenced the microbiota, albeit with individual variations in effect size. However, the differences in microbiota composition between individual study subjects were larger than the differences induced by fiber treatments. The α-diversity was significantly decreased after treatment with pectin, but only in the healthy group. Treatment with psyllium resulted in a significant increase in α-diversity in the healthy group and a significant decrease in α-diversity in the IBD group. High diversity and richness of the gut microbiota are generally considered biomarkers of intestinal health, but there is also evidence that diets rich in fiber, while inducing positive metabolic responses, also reduce overall microbial diversity by promoting the growth of specific bacteria ,25.Fibers can produce specific effects on the microbiota depending on their structural properties, solubility, and fermentability . In theiLachnospira was significantly increased after exposure to pectin in both the healthy and the IBD groups. Bifidobacterium and Faecalibacterium, two bacterial genera that are considered beneficial for gut health because of their capacity to ferment dietary fiber [Bifidobacterium and Faecalibacterium were only increased in the IBD group. Furthermore, we observed that fiber mixes promote bigger shifts in microbiota composition compared to single fibers: this is easily explained because fiber mixes contain a variety of different molecules.In this study, pectin had an especially considerable effect, causing significant shifts in many bacterial groups. Interestingly, these microbiome changes were often related to the intestinal health status of the microbiota donor. For example, ry fiber  and to pry fiber , were siThe mechanisms that modulate the health effects of fibers include the production of specific metabolites, such as short-chain fatty acids (SCFAs). Aside from shifts in microbiota composition, it is, therefore, important to consider the functional effects of fiber interventions. The two can go along hand in hand, and individual differences in microbiome composition can result in different functional responses to dietary fiber. Indeed, the microbiota of different enterotypes has been shown to produce different levels and ratios of SCFA after in vitro fiber fermentation . The micThe small sample size represents a limitation in this study, creating a risk of bias and the presence of outliers. The healthy group included five subjects, most likely not enough to represent the diversity of a healthy population. Especially in microbiome studies, considering the complexity of gut bacterial populations and the high interindividual diversity, larger sample sizes are needed to detect significant effects and differences between subjects . Still, Another limitation of this study was the lack of baseline information that prevented us from studying microbial responses, i.e., differences between microbiota composition and function before and after fiber fermentation. To overcome this issue, we compared taxonomic and SCFA patterns in the fiber-treated samples to the untreated control samples to infer fiber effects. Future studies should include baseline data to determine whether microbiome profiles can be used as predictors of responsiveness or non-responsiveness to fiber interventions .This study described the in vitro exposure of individual fecal microbiota to different fibers and showed that in vitro models could effectively detect compositional and functional differences between individual microbiota. The effects of dietary fibers observed in this study were significantly different among individual microbiota but also between the healthy and IBD groups. Furthermore, the combination of all fibers resulted in stronger effects on microbiota composition and metabolite production compared to single fibers. It seems in vitro models are well suited to study fibers in early screening stages and can be applied to test hypotheses about the specific effects of novel ingredients or combinations thereof. Future studies should evaluate to what extent observations from in vitro models, such as the i-screen applied here, translate to in vivo situations.


The occurrence of ischemic stroke (IS) is associated with nonalcoholic fatty liver disease (NAFLD). The cancer burden of NAFLD complicated by IS also warrants attention. This study aimed to identify candidate immune biomarkers linked to NAFLD and IS and analyze their association with cancer.via weighted gene coexpression network analysis (WGCNA). Subsequently, utilizing machine learning  and immune cell infiltration analysis, immune-related candidate biomarkers for NAFLD with IS were determined. Simultaneously, a nomogram was established, the diagnostic efficacy was assessed, and the role of candidate biomarkers in cancer was ascertained through pan-cancer analyses.Two of each of the NAFLD and IS datasets were downloaded, differentially expressed genes (DEGs) were identified, and module genes were screened RRS1 was strongly associated with the prognosis, immune cell infiltration, microsatellite instability (MSI), and tumor mutation burden (TMB).In this study, 117 and 98 DEGs were identified from the combined NAFLD and IS datasets, respectively, and 279 genes were obtained from the most significant modules of NAFLD. NAFLD module genes and IS DEGs were intersected to obtain nine genes, which were enriched in the inflammatory response and immune regulation. After overlapping the results of the three machine learning algorithms, six candidate genes were obtained, based on which a nomogram was constructed. The calibration curve demonstrated good accuracy, and the candidate genes had high diagnostic values. The genes were found to be related to the immune dysregulation of stroke, and PTGS2, FCGR1A, MMP9, VNN3, S100A12, and RRS1) of NAFLD and IS were identified, and a nomogram for diagnosing NAFLD with IS was established. RRS1 may serve as a candidate gene for predicting the prognosis of patients with cancer who have NAFLD complicated by IS, which could aid in their diagnosis and treatment.Six common candidate immune-related genes ( It is aRecent studies have shown that NAFLD may be linked to a higher risk of ischemic stroke (IS) . IS, oneAccording to reports, the onset and progression of NAFLD involve crosstalk or their temporal involvement among immune cells such as innate-like T cells, neutrophils, monocytes, B cells, and Dendritic cells . In addihttp://www.ncbi.nlm.nih.gov/geo) database. The “Limma” package was used to identify the differentially expressed genes (DEGs) in NAFLD and IS. Subsequently, weighted gene coexpression network analysis (WGCNA) was applied to determine the critical module in NAFLD. To discern the candidate immune-related biomarkers for NAFLD with IS, functional enrichment analysis, protein-protein interaction (PPI) network creation, application of machine-learning algorithms , evaluation of nomogram and receiver operating characteristic (ROC) curve, and analysis of immune cell infiltration were performed. Furthermore, pan-cancer analysis was conducted to assess the functions of biomarkers in cancer.In this study, two NAFLD and IS datasets were downloaded from the Gene Expression Omnibus | >0.6 as the screening criterion.The 4 original datasets were first subjected to background correction and then normalized by using the “affy” package in the R software. Subsequently, probe-level data were converted to their respective gene expression values. When a gene corresponds to multiple probes, the gene expression value is replaced with the average expression value. Following the integration of the datasets, the batch effect was eliminated by using the combat function of the “SVA” package in R, as there was a notable batch difference across datasets from different platforms. Finally, in order to identify DEGs between the disease and control groups, the “Limma” package in R software was used with the false discovery rate (FDR) of <0.05 and |log2.3The weighted co-expression network for the expression matrix of the NAFLD dataset was created using the “WGCNA” package in the R software. For further analysis, the genes with a median absolute deviation (MAD) >25% were selected. To ensure data integrity, the “goodSamplesGenes” function was applied. To determine and validate the optimal soft threshold (β) and construct a scale-free network, the “pickSoftThreshold” was used. Next, the adjacency matrix was turned into a topological overlap matrix (TOM), and the gene modules were constructed using hierarchical clustering and the dynamic pruning-tree algorithm. The gene significance (GS) and module significance (MS) were calculated by using phenotype and module data in order to examine the relationship between modules or genes and the clinical features.2.4http://string-db.org/) for Protein–Protein Interaction (PPI) network construction. The intersection genes of the most significant module genes of NAFLD and the DEGs of IS were imported into the String database, and then the species was selected as “Homo sapiens”, and finally it was considered significant when it was greater than the minimum interaction score of 0.4.The “clusterProfiler” package in the R software, the functional enrichment analysis of the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed, and P < 0.05 was considered to indicate statistical significance. GO and KEGG analyses were performed in accordance with the intersection genes of the most significant module genes and DEGs of NAFLD, and the intersection genes of the most significant module genes of NAFLD and DEGs of IS, respectively. In addition, we also utilize the String database  was computed to assess the prediction accuracy of NAFLD with IS.2.7Using the ssGSEA algorithm of the “GSVA” package in R software, the immune infiltration of IS was quantified. Spearman’s correlations were computed to investigate the correlation between immune-infiltrating cells and the intersection of genes of machine-learning algorithms.2.8The “forestplot” package in R software was used to run univariate Cox regression analyses, after which the p-value, HR, and 95% CI values were computed. Subsequently, the data was visualized through a forest plot.2.9TIMER, xCELL, MCPCOUNTER, CIBERSORT, EPIC, and QUANTISEQ algorithms were used for further exploring the correlation between candidate gene expression and immune infiltration in all TCGA tumors. Then, we looked at the relationship between candidate genes and the expression of genes relevant to immunological checkpoints in diverse cancers. Moreover, we examined the relationship between MSI or TMB in different cancers and the expression of candidate genes.2.10https://www.r-project.org/) was utilized for all statistical analyses and graph generation. The Student t-test was utilized to compare group differences. The predictive performance of the candidate genes utilized to build the predictive model was evaluated using the ROC curve. P<0.05 was considered to indicate statistical significance.R software  to fit the gene expression associated with a scale-free network  were recognized , MMP9 , PTGS2 , RRS1 , S100A12 , and VNN3  . Therefore, the immune-related gene RRS1 was selected for further pan-cancer analysis. In the TCGA database, RRS1 was highly expressed in 18 cancer types, including CHOL, BRCA, and BLCA, with a statistical significance , disease-free survival (DFS), disease-specific survival (DSS), and overall survival (OS). RRS1 expression was substantially linked to LIHC and LUAD in OS analysis and acted as a risk factor in both LIHC and LUAD  for NAFLD and IS were identified via comprehensive bioinformatic analysis and machine learning algorithms.NAFLD, a disease closely associated with metabolic dysfunction, has become the most common cause of chronic liver disease worldwide . NAFLD, Atherosclerosis, which can cause blood flow restriction and potential plaque rupture risk, is one of the causes of IS , 19. PTGPrevious studies have demonstrated that inflammatory response and immune regulation are involved in the development of IS , 36. WitFurthermore, a relationship was observed between the levels of various immune cells and the expressions of candidate genes. MMP-9 is one of the widely studied members of matrix metalloproteinases (MMPs) . In inflRRS1 (the gene with the greatest prediction performance of NAFLD with IS) was further investigated in pan-cancer. In TCGA and GTEx databases, the differential expression analysis of RRS1 in 28 tumor types, including BRCA, LIHC, and STAD, showed statistical significance. Moreover, the findings of this research signified that RRS1 is a prognostic risk factor in various cancers. These results indicate that RRS1 can promote the development of tumors and may be a potential marker for poor prognosis. RRS1 can promote the development of HCC by enhancing ribosome biogenesis and attenuating RPL11-MDM2-P53 signaling  associated with NAFLD and IS were identified using bioinformatics methods and machine learning algorithms, and a diagnostic nomogram of NAFLD patients with IS was established. Meanwhile, we pointed out that dysregulation of RRS1 may be a high risk factor for cancer in patients with NAFLD complicated by IS. Further research on RRS1 is expected to aid in the diagnosis and treatment of this type of cancer patient.Six common immune-related genes (The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/HB and BZ contributed to data analysis and manuscript drafting. JL contributed to data analysis and revised the manuscript. JH participated in data collection and figure editing. DS and LL conceived the study and revised the manuscript. All authors contributed to the article and approved the submitted version.


Since the COVID-19 pandemic, working environments and private lives have changed dramatically. Digital technologies and media have become more and more important and have found their way into nearly all private and work environments. Communication situations have been largely relocated to virtual spaces. One of these scenarios is digital job interviews. Job interviews are usually—also in the non-digital world—perceived as stressful and associated with biological stress responses. We here present and evaluate a newly developed laboratory stressor that is based on a digital job interview-scenario.N = 45 healthy people participated in the study . Salivary alpha-amylase (sAA) and cortisol were assessed as measures for biological stress responses. Furthermore, perceived stress was rated at the time points of the saliva samplings. The job interviews lasted between 20 and 25 min. All materials, including instructions for the experimenter  and the data set used for statistical analysis, as well as a multimodal data set, which includes further measures, are publicly available.Typical subjective and biological stress-response patterns were found, with peak sAA and perceived stress levels observed immediately after the job interviews and peak cortisol concentrations 5 min afterwards. Female participants experienced the scenario as more stressful than male participants. Cortisol peaks were higher for participants who experienced the situation as a threat in comparison to participants who experienced it as a challenge. Associations between the strength of the stress response with further person characteristics and psychological variables such as BMI, age, coping styles, and personality were not found.Overall, our method is well-suited to induce biological and perceived stress, mostly independent of person characteristics and psychological variables. The setting is naturalistic and easily implementable in standardized laboratory settings. Stress is an ubiquitous phenomenon, which is accompanied by the subjective feeling of being stressed as well as a variety of biological stress responses. The biological stress response is complex and involves several stress systems. In acute stress situations, the two most important ones are the activation of the sympathetic nervous system (SNS), which is a part of the autonomous nervous system and the activation of the hypothalamic–pituitary–adrenal (HPA) axis . Our objective was 3-fold: First, we developed the setting and provided a detailed description of the materials. Second, we conducted an evaluation study to investigate whether our scenario, indeed, induces stress . Third, we tested for potential associations between the strength of the stress responses and person characteristics .N = 52 healthy people, who were non-smokers, participated in the study. Data from n = 7 participants had to be excluded from statistical analysis . The final sample size was N = 45 . The study has been approved by the local Ethics Committee of the Medical Faculty of the Friedrich-Alexander-Universität Erlangen-Nürnberg (protocol number: 21-408-S).Overall, 0) was taken. After this, participants were equipped with an eye tracker and a finger-clip pulse sensor . Then, the participants were asked about their dream job, which they would apply for at the job interview, and they were seated in front of a computer.The study consisted of two parts . Prior t1) was taken. The actual job interview started afterward on the computer screen in front of the participants and lasted about 20–25 min . The interviewer sat in another room and was hidden from the participant until the job interview started. The job interview was conducted via Zoom. Immediately after the job interview, the next saliva sample (s2) was taken. After this, participants were interviewed about their experiences and emotions during the job interview (Interviews preceding the job interviews). Further saliva samples were taken 5 (s3), 20 (s4), and 35 (s5) min after s2. The time course of the general procedure is shown in The preparation phase lasted about 15 min, and participants were instructed to prepare for the job interview and to take notes if necessary. After the end of the preparation phase, the next saliva sample  realistic and (2) associated with acute stress responses  for most participants. As described in Introduction (1), we decided to use a digital job interview-scenario based on a setting, which has proven to be perceived as stressful and associated with negative emotions in a pilot study Introduction of the applicant Interruption of the introduction Questions regarding the employer/company Job change explanation Expectations of the applicant Advertising of the applicant Hypothetical situation Applicant's future vision Practical task Basic knowledge questions Subject-specific questions Questions regarding the applicant's outfit Ideas regarding salary and working hours , participants rated whether they perceived the job interview rather as a threat or as a challenge and whether they experienced stress during the job interview. The rating as threat vs. challenge was used for the following analyses.Immediately after the job interview :Coping has been assessed using the German 24-item version of the Coping Inventory for Stressful Situations , were videotaped, and movements were recorded using a Microsoft Kinect 2. Furthermore, they were equipped with a finger-clip sensor (iom) on the non-dominant hand. This data is reported in Heimerl et al.  and standard deviations (SD) were calculated. Prior to further analysis, sAA and cortisol concentrations, as well as age and BMI, were transformed using the natural logarithm (ln) to achieve a normal distribution. Participants with values that were more than three standard deviations away from the participant's mean were treated as outliers and excluded from further analysis.For statistical analysis, IBM SPSS Statistics (version 29 for Windows) was used. For descriptive statistics, means (0-s5). If necessary, sphericity violations  were calculated for sAA, cortisol, and perceived stress ratings with the within-subjects factor ‘time' which reflected the 6 measurement time points  with sex or threat vs. challenge as a between-subjects factor. Similar analyses were conducted for female participants who reported the use of contraceptives and for participants with the same vs. a different sex than the interviewer.1 were calculated and tested for significance.To test whether the stress responses were associated with ln(age), ln(BMI), number of previous job interviews, time of day, coping, perceived stress during the last month, personality, trait anxiety or depression, Pearson's correlations between these variables and the difference between the maximum sAA, cortisol, and perceived stress level and the value at sAn adjusted alpha level of 0.05/3 = 0.017 was used for all analyses because three main outcome variables  were evaluated.https://osf.io/5bdyf/. Furthermore, an annotated multimodal data set, which has been created during the study and which contains further modalities , is available upon request from http://hcai.eu/fordigitstress.All materials used in the study, a comprehensive overview of the job interview-scenario and the data set which has been used for the analyses within this article, are freely available as The characteristics of the final sample, including the psychological variables, are reported in F = 6.32, p < 0.001, Post hoc t-tests indicated that sAA levels were significantly higher at s2  than before the job interview at s1 , s3 (t(42.94) = 3.70, p < 0.001, Cohen's d = 1.74), and s4  and s4  and s4 [t(43) = 2.64, p = 0.006, Cohen's d = 1.38; 1, it should be noted that the interviewer was not known at this time point.Finally, it was investigated whether the stress responses differed between participants who had the same sex as the interviewer  was expected regarding the fast time course of SNS responses. However, peak cortisol levels already occurred 5 min later, which was earlier than expected based on our experience with further stressors and which are usually reported . However, as described in the Introduction (1), the acute stress response is complex, and a variety of stress measures is conceivable , and future research can build on it.Overall, our digital job interview-scenario is very well-suited to induce biological and perceived stress responses, mostly independent of person characteristics and psychological variables such as personality traits and coping styles. Therefore, it can be applied to a variety of different people. All materials, including the specific questions that were used during the job interviews as well as the rating scales, are freely available (https://osf.io/5bdyf/.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: Open Science Framework: The studies involving human participants were reviewed and approved by Ethics Committee of the Medical Faculty of the Friedrich-Alexander-Universität Erlangen-Nürnberg . The participants provided their written informed consent to participate in this study.LB and AH designed and conducted the study and supervised data collection. LB analyzed the data and wrote the manuscript. AH and EA provided critical feedback on the manuscript. LB and EA received funding for this manuscript. All authors read and approved the final version of the manuscript.


We systematically identified the prevalence of triplex infections (combined human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV)) in pregnancy. I2 was used as the statistical measure of heterogeneity. The checklist was created by Hoy and colleagues. The study protocol was registered on PROSPERO, under the registration number CRD42020202583.  To gather information on the frequency of triplex infections, we searched the databases of PubMed, CINAHL, and Google Scholar. Without regard to language, we utilized search terms that covered HIV, HBV, HCV, and pregnancy. Pregnant women with triplex infections of HIV, HBV, and HCV were included in studies that also examined the prevalence of triplex infections. Review Manager 5.4.1 was employed to conduct the meta-analysis. Critical appraisal and bias tool risk data were provided as percentages with 95% confidence intervals (95% CIs), and P < 0.001) in general obstetric population. Moreover, there was a significant difference in the pooled prevalence between studies published between 2001 and 2010 and between 2011 and 2021 )) and participants recruited in the period between 2001 and 2011 and between 2012 and 2021 )), respectively.  Eight studies involving 5314 women were included. We identified one ongoing study. Pooled prevalence of triplex infections was 0.03% (95% CI: 0.02–0.04%) according to meta-analysis. Subgroup analysis demonstrated a significantly high prevalence of 0.08%  in HIV-positive population than 0.00% (95% CI:−0.00-0.00; 1451 women;  The combined prevalence of prenatal triplex infections was 0.03%, with rates notably higher among the group of pregnant women who were HIV-positive and during the recruitment period that took place before 2012. This prevalence still necessitates screening for these infections as necessary. Human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) triplex infections in pregnant women continue to be serious public health issues , 2. ThisAlthough HIV and AIDS were once thought to be terminal illnesses, highly active antiretroviral therapy (HAART) has transformed these conditions into a manageable chronic infection by lowering the rates of mother-to-child (MTCT) HIV transmission. Since hepatitis viruses are known to have adverse effects on people with HIV, triplex infections may negate the positive effects of HAART . As a reAlthough there may be fewer pregnant women who have triplex infections, the combination of HIV, HBV, and HCV is an unacceptable coexistence  and coulTo the best of the authors' knowledge, there has not yet been published data on the prevalence of triplex infections  among expectant women. Thus, the purpose of the study is to estimate the pooled prevalence of triplex HIV, HBV, and HCV infections among pregnant women.The study protocol was registered on PROSPERO, under the registration number CRD42020202583.In the current study, there has been a thorough review and meta-analysis. The revised Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA-P) standards were followed in the reporting of this review.Nigeria is a nation in West Africa. It is divided into five geopolitical regions: the north-central area, where the capital city Abuja is located, the south-east, south-south, south-west, and north-east. In 2018, there were more than 200 million people living in Nigeria.The study population was defined as pregnant women with triplex infections of HIV, hepatitis B, and hepatitis C viruses.(1) Study Design. Cross-sectional, case-control, or cohort studies.(2) Participants. Pregnant women with triplex infections of HIV, hepatitis B, and hepatitis C viruses residing in Nigeria.(3) Studies of Interest. Studies reporting the seroprevalence of HIV-HBV-HCV triplex infections ), and/or HCV infection markers  in pregnancy or enough data to compute this estimate in pregnancy even though the prevalence data are missing, i.e., if the prevalence data are missing but there is sufficient data to calculate the prevalence, the study was included.(4) Outcome Measurement. Results were measured by looking at the presence of anti-HIV antibodies I and II, HBsAg, and/or infectivity (HBe antigen), as well as anti-HCV.(5) Types of Publication. Published and unpublished data.Studies carried out among pregnant Nigerian women, populations living outside of Nigeria, and nonpregnant populations were all eliminated. Studies in subgroups of participants chosen based on the presence of any other viral hepatitis other than HIV-HBV-HCV triplex infection, such as case series (20 participants) of the hepatitis D (delta) virus (HDV), were also excluded. Studies reporting only mono-infection , or only dual infections in pregnancy, were also excluded, as were reviews, letters, commentaries, and editorials. In addition, studies lacking primary data and/or explicit method description after two unsuccessful requests addressed to the corresponding author were also excluded. The study was also disqualified if the abstract lacked conclusion and the full text was unavailable. The most thorough and recent version was utilized for duplicate publications (research published in more than one report).∗, triple infection, pregnant women, and Nigeria. For the full search strategy, see The new PRISMA-P  standard was used when conducting this review . A thoroReference lists of eligible articles and relevant reviews were manually searched to identify additional studies.The pooled prevalence rates of HIV-HBV-HCV triplex infection among pregnant women in Nigeria are the primary outcome of the study.For research comparing participants from the HIV-positive group to the general prenatal population, studies from 2001 to 2010 to studies from 2011 to 2021, and studies from 2001 to 2011 to studies from 2012 to 2021, subgroup analyses were conducted. Only pregnant women with HIV-positive status were recruited or studied in the HIV-positive study population by the authors of the primary studies. However, in the general obstetric population , the authors of the primary research recruited or studied pregnant women without taking into account whether they are HIV-positive or not.The software EndNote version X9 was used to import the retrieved articles and check them for duplication. Two researchers (GE and OL) independently reviewed each record's titles and abstracts to determine whether they should be included in the review. Two researchers (HU and GA) acquired the complete texts of papers deemed potentially eligible and evaluated them for eligibility. Two investigators mutually agreed to keep studies for inclusion. Any differences of opinion were settled by a third researcher (PF).A pretested data extraction form created in Microsoft Excel was used by two independent reviewers (CO and RE), with a third reviewer (IM) resolving any differences. First author, year of publication, period of participant recruitment, region of recruitment, site, setting, timing of data collection, study design, eligibility requirements, sample size, mean or median age, diagnostic criteria, number of samples tested for HIV, HCV, and/or HBV, number of participants with HIV I and II, HBsAg, HBeAg, HCVAb, and HCV detectable viral load, and prevalence rate were among the pertinent information that the reviewers independently extracted.HIV I and II, HBsAg, or HCVAb for HIV, HBV, and HCV, respectively, were used to define infection. HIV I and II for HIV, HBsAg, and/or HBeAg for HBV, and both HCVAb and detectable viral load for HCV were considered indicators of infection. On the other hand, in order to estimate the percentage of pregnant women who can pass the virus onto their unborn child, we reported the prevalence of HIV I and II, HBeAg, and HCV detectable viral load, respectively, among HIV, HBV, and HCV positive women. We made at least two separate attempts to get in touch with the study's corresponding author personally when pertinent data were not readily available.Using a modified version of the risk of bias instrument for prevalence studies, which was created by Hoy and colleagues , the metEach chosen study received a special identification. The overall score was classed as follows, with a total score ranging from 0 to 9: 0–3: “low risk,” 4–6: “moderate risk,” and 7–9: “high risk” of bias. Better quality was indicated by a lower score.p value for the heterogeneity test was >0.10. The random effects model was applied in the other cases. We conducted subgroup and meta-regression analyses to look into potential sources of heterogeneity when significant heterogeneity (I2 > 75%) was found. P values less than 0.05 were regarded as statistically significant for all other tests other than the heterogeneity test.The Nordic Cochrane Center, Copenhagen, Denmark, provided Review Manager 5.4.1 software for use in all statistical calculations. For the entire study as well as for the various subgroups, the findings were presented as pooled prevalence with 95% confidence intervals (CI). The frequency of HIV, HBV, and HCV positive laboratory results was used to determine prevalence. The prevalence of triplex infection in the obstetric population was pooled, and the percentage  was utilized as the effect size. The pooled effect was then calculated using the generic inverse variance approach, because the effect of a single rate and its standard error are similar to the rate difference (RD) at this time . The Q tAdditional hand searches also produced 7 citations, bringing the total number of results from the search approach to 843. Sixteen citations were chosen by the reviewers for full-text analysis after titles and abstracts were scrutinized. According to our selection criteria , 16–23, The eight included studies' –23 primaIn addition, the majority of studies  involved pregnant women in the HIV-positive obstetric population , 19–21, I2 = 95%, p < 0.001 showed that there was significant heterogeneity among the studies. The random effects model was used as a result. According to the findings of our meta-analysis, the combined prevalence of triplex infection in the eight studies that were included was 0.03%   .p < 0.001; I2 = 98.0%) when we eliminated one study at a time, but it remained the same overall  and 0.03%  respectively . The subgroup analysis did increase the heterogeneity, indicating that the HIV-positive study population is the source of heterogeneity (I2 = 0.0% vs 99.0%).Considering pregnant women who are HIV-positive may have different prevalence rates to the general obstetric population, we performed subgroup analyses for HIV-positive population and for the other general obstetric populations. The subgroup analyses revealed that the prevalence of triplex infection in the general obstetric population and the HIV-positive population were 0.0%  and 0.02%  in the studies published between 2001 and 2010 and in the studies published between 2011 and 2021 . The fact that the heterogeneity was not reduced by the subgroup analysis (I2 = 98.0% vs. 99.0%) shows that the year of publication is not the cause of the heterogeneity.A subgroup analysis of studies published between 2001 and 2010 and those published between 2011 and 2021 revealed that the prevalence of triplex infection was, respectively, 0.14%  and 0.00% , respectively, in the studies whose participants were recruited between the years of 2001 and 2011 and in the studies whose participants were recruited between the years of 2012 and 2021. Significant differences were found in the tests for subgroup differences . The heterogeneity was increased by the subgroup analysis, which showed that the participants' recruitment period between 2001 and 2011 (I2 = 99.0% vs 0.0%).The majority of studies (7/8) were rated as having low bias risk. One study was at a moderate risk of bias. The domains on which studies most frequently did poorly were the use of an acceptable case definition in the study (since one was not used in the study) and the use of inappropriate numerators and denominators for the study's parameter of interest . Each included study's specific quality and bias risk were detailed in Because there were less than 10 included research studies, we were unable to evaluate the included studies' publication bias. Due to a lack of statistical power, the available tests have a very significant risk of bias when less than 10 studies are included .The World Health Organization's (WHO) global goal of eradicating HIV and viral hepatitis infection by 2030 was put in jeopardy because pregnant women who are triple infected with HIV, HBV, and HCV are at a high risk of passing these viruses from mother to child. This is because people who have three coinfections are more likely to present with lower CD4+ counts, which increases the likelihood that their host immunity will be weakened, which can have more negative effects on pregnancy. According to the results of this systematic review and meta-analysis, the prevalence of triplex coinfections with HIV, HBV, and HCV during pregnancy was 0.03% overall, 0.08% in the community of HIV obstetrics, and 0% in the general obstetric population.However, the prevalence in the nonobstetric population was lower than in earlier systematic reviews and meta-analyses reported in Africa , Iran 3, China , and intThe decreased prevalence of triplex infection in the current study may be attributable to the study's unique demographic, which of course includes pregnant women. It could also be a result of the passage of time, as more successful methods for reducing mother-to-child transmission of HIV and HBV have been adopted. One of these methods is the elimination of mother-to-child transmission services. The latter could therefore be explained by ongoing efforts to lower mother-to-child transmission and potential HBV vaccine initiatives. Nigeria started its hepatitis B vaccination program in 2004; however, there have not been many program reviews , 36.However, a change in the vaccination policy in Nigeria in February 2015 restricted hepatitis B-birth dose, vaccine administration to only within 24 hours of birth, and Expanded Programme for Immunization (EPI) tools were revised accordingly , 36. DesThe management of these triplex viruses during pregnancy also depends on the status of HBV and HCV . A diagnIn addition, the WHO advises starting highly active antiretroviral therapy (HAART) in HIV patients who are also coinfected with HBV or HCV regardless of their CD4 T lymphocyte count; however, the regimen to use depends on which of these viral infections is present because failing to do so could put the patient at higher risk for hepatotoxicity . ImproviIn addition to the participant subgroup analysis based on HIV positivity status, the recruiting period-based subgroup analysis is also more important. This is due to the fact that participants were recruited for four of the eight research studies between 2006 and 2011. Given that all three of the documented cases of triplex infection occurred during recruitment periods that began before 2012, this obviously has an impact on the findings of the analysis and the prevalence. This may also be the cause of the recently reported moderate-to-low rates of mother-to-child transmission of HIV and HBV in Nigeria .I2 > 75%) for the main result. A random-effects model was applied to the meta-analysis in order to lessen this effect.Following a thorough evaluation of the studies' quality that were a part of this systematic review, we found significant methodological issues with the included research, particularly with regard to the diagnostic criteria, and they were given a “moderate” risk of bias rating. Although the included studies were considered to be of high to moderate quality, their individual study designs and definitions of the outcomes of interest had biases. There was significant heterogeneity (This work has a huge impact on subsequent research. Because none of the included studies had their primary objectives focused on figuring out the prevalence of triplex infections in pregnancy, prospective studies must be created and carried out to assess the prevalence of triplex infections directly and objectively. Only one active study was found , and earThe seroconversion or seroclearance of triplex infections in Nigeria, however, was not examined in this investigation . The smaThe combined prevalence of HIV, HBV, and HCV triplex coinfections during pregnancy was 0.03%, with a frequency that was noticeably greater in the obstetric population that tested positive for HIV and during the recruiting period that took place before 2012. This prevalence nevertheless necessitates screening for these illnesses and, where necessary, treating and/or immunizing the sick or exposed newborns. To completely eradicate these triplex infections in the obstetric community, health policymakers must continue to implement practical measures. To increase effectiveness and build on current successes in the eradication of triplex infection, it is necessary to increase political will and support, expand treatments based on scientific evidence, and make better use of financing sources.It is also necessary to do larger, more meticulous prospective investigations using PCR technology for diagnosis. Due to the reported burden of triplex infection among the HIV-positive obstetric population in our meta-analysis, we also advise that antenatal viral screening tests be prioritized to cover HIV, HBV, and HCV infections.


Stereotypes have traditionally been considered as “mental pictures” of a particular social group. The current research aims to draw the structure of gender stereotypes and metastereotype schemes as complex systems of stereotypical features. Therefore, we analyze gender stereotypes as networks of interconnected characteristics.N = 750), participants listed the common female and male features to build the structure of the gender stereotypes. Participants also listed the common features of how members of one gender think they are viewed by people of the other gender to build the structure of gender metastereotypes.Through an online survey . Thus, focusing on central nodes can be critical to understanding and changing the structure of gender stereotypes. The research about gender stereotypes has been a longstanding storyline in social science and has inspired much research in gender theories Eagly, . Social Stereotypes have been traditionally considered “mental pictures” of a specific social group , a parallel process could be happening from interpretation to attributed characteristics (top-down), which would be guided by schemes . There were 585 participants that indicated that Spanish was their native language (30 were not and 135 missing language information). There were no exclusions, so we include all the participants in the analyses. Participants' sociodemographic features are available in We conducted an online study by sending an email to the university community of a city in southern Spain. There were 750 respondents to our study , who ranged from 18 to 64 years old  you know—i.e., relatives, friends, work or university colleagues, women (men) who appear on television, on social networks, in books…—What are the features that all those women (man) have in common?” Participants were asked to write ten open-ended answers. We suggested answering for at least five features.Afterward, we asked them about their gender, and according to their answer, we asked them about the metastereotype,Given that participants provided open-ended answers, we obtained a high diversity of words referring to similar features. Thus, three researchers manually performed the lemmatization of the data corpus, which consisted in keeping the base form of each word (lemma) by removing the inflectional ending or word derivations participants used . This strategy is commonly used in linguistic processing to reduce the variability of words that capture the same meaning. Although this process might lead us to lose sight of some nuances, this is a practical procedure to simplify large amounts of information that can be redundant, which allows us to reach a more substantive interpretation. Furthermore, this process was performed manually, allowing researchers to detect potential ambiguities and select the most appropriate lemma according to the context.https://osf.io/cmf6a/).Because our original data corpus was mostly composed of single words in Spanish, after the lemmatization, we automatically translated each word into English. Then, two researchers verified the translation's accuracy by reviewing whether those words fit the original meaning properly. This stage allowed a new check on the quality of lemmatization and appropriate use of words. The raw and coded material is available in the online We used the co-occurrence matrix of features the participants mentioned to conduct the network analyses. The network analysis strategy allowed us to account for the relationship of all the features simultaneously to reveal patterns of association. Networks are made up of two components: nodes and edges. Nodes correspond to participants' responses , and edges reflect the associations between nodes . Thus, we had as many nodes as named features regardless of how many times these features were mentioned, and edges reflect the co-occurrence of every pair of features the same participant mentioned. To illustrate the procedure, imagine that a participant A answered three features of female stereotypes: intelligent, resilience, and hardworking  and the pattern of association between them . The network analysis was supported by Gephi 0.9.2 software , loving–intelligent (18), intelligent–hardworking (17), fighter–hardworking (17), and fighter-intelligent (15). As notably seen in intelligent (215hardworking (149), and fighters (139). These results suggest that these features had more relevance in this community, given they were the most connected , patience (35), and proud (34). Finally, the third community (colored by blue) represents 5.39% of the network, and their nodes with the highest degree are chatty (84), long hair (71), and hips (38).The female stereotype that men showed was composed of a network that included 853 nodes, with 4,398 co-occurrences collapsed in 3,474 edges. Communality analyses showed three large clusters of features. The strongest co-occurrences were intelligent-strong (92), hardworking–intelligent (86), fighter–strong (66), strong–hardworking (66), and fighter-hardworking (64). As seen in intelligent (352), strong (312), and hardworking (299). Similarly to the female stereotype projected by men, there are additional communities but with lower representation. The second community (colored by green) represents 10.79% of the network, and their nodes with the highest degree are long hair (67), thin (35), and bosom (34). Finally, the third community (colored by blue) represents 8.68% of the network and their nodes with the highest degree are unsafe (84), feminist (71), and dependent (38).The female stereotype that women provided (in-group stereotype) was composed of a network that included 853 nodes, with 13,600 co-occurrences collapsed in 7,711 edges. The strongest co-occurrences were intelligent, strong, hardworking, and fighter. The exception was that one of the main links in the women stereotypes projected by men was intelligent-loving, suggesting an important nuance in the meaning of the term intelligent when men attributed it to women. The weight this community has in the network suggests that the female stereotype is mainly, although not wholly, homogeneous. We should note that although most central features in this community might be considered agentic features, there are also other features more related to communion . The results also showed that there were additional small communities that characterized the female stereotype. Both women and men conveyed their female stereotype with physical features—i.e., long hair, thin, bosom, and hips.In short, the female in-group stereotype was similar to the stereotypes that men have about women. These results suggest that the female stereotype (established by both women and men) is composed mainly by one community characterized by positive valued features linked among them such as unsafe and dependent). It is noteworthy that in this community, the feature of feminist also appeared. However, this feature should be interpreted into their network context, not only by their direct meaning. The label feminist has been traditionally stigmatized and used by some people as a negative feature to describe women , strong–intelligent (40), strong–hardworking (36), nice–intelligent (32), and nice-hardworking (29). As seen in strong (320), intelligent (305), and hardworking (259). There are additional communities but with lower representation. The second community (colored by green) represents 13.67% of the network, and their nodes with the highest degree are high (102), beard (58), and brunettes (45). Finally, the third community (colored by blue) represents 10.95% of the network, and their nodes with the highest degree are simple (96), tough (75), and rough (54).The male stereotype provided by women included 995 nodes with 11,794 co-occurrences collapsed in 8,378 edges. The strongest co-occurrences were intelligent-hardworking (13), strong–intelligent (13), likable–intelligent (9), nice–intelligent (9), and strong-hardworking (9). As seen in intelligent (141), hardworking (131), and nice (120). Interestingly, there is a second extensive community. This second community (colored by green) represents 13.67% of the network, and their nodes with the highest degree are strong (149), aggressive (89), and hardheaded (64), showing a relatively strong interconnection between those features: strong-aggressive (4), strong-hardheaded (3), and strong-hardheaded (2). Finally, the third community (colored by blue) represents 7.14% of the network, and their nodes with the highest degree are cheerful (56), chatty (45), and beard (44).The male in-group stereotype included 995 nodes, with 4,145 co-occurrences collapsed in 3,582 edges. The strongest co-occurrences were strong, intelligent, and hardworking, which we could define as a traditional male stereotype .Finally, a similarity between the male stereotype projected by both women and men, and shared with the female stereotype, is that a small part of the network is based on physical features , weak–sensitive (28), intelligent–hardworking (27), friendly–intelligent (25), and sensitive-intelligent (24). As seen in sensitive (238), weak (226), and motherly (172). The nodes with the highest degree of the second community are intelligent (288), hardworking (222), and pretty (200). Finally, the third community (colored by blue) represents 5.22% of the network, and their nodes with the highest degree are made-up (27), long hair (26), and friendship (22).The female metastereotype included 632 nodes, with 9,742 co-occurrences collapsed in 6,851 edges. The strongest co-occurrences were sensitive, weak, and motherly. By contrast, the other community is characterized by the features of intelligent and hardworking, which seem to reflect the modern female stereotype. However, there is a crucial nuance in this last community given the role of pretty, which qualify importantly the meaning of intelligent and hardworking. It must be emphasized that these two communities are similar to women's in-group stereotypes except for their relative size. Whereas, women's in-group stereotype is mainly representative of modern female stereotypes, considering the traditional one is only a small part of the network; when women think of how men stereotype them, both traditional and modern communities take on a similar relevance. These results suggest that most women tend to in-group stereotype as modern women , although a few of them keep the traditional stereotype . However, when looking at their beliefs about how men see them, women show more disagreement in the features mentioned. There is a similar number of women who think men see them as modern and traditional. Nevertheless, this does not appear to be the case. In other words, the female stereotype (both projected by men and women) is more homogeneous than the female metastereotype. Both women and men think about the female stereotype mainly as a modern stereotype. We did not find evidence that the female stereotypes projected by men reflected traditional features. Moreover, a part of the female metastereotype is represented by physical features—i.e., long hair, thin, bosom, hips—similar to the female stereotype.These results suggest that the structure is heterogeneous because it is represented mainly by two communities. One community seems to reflect the traditional female stereotype which is characterized by the features of chauvinist-strong (8), strong–intelligent (8), strong–brave (8), strong–aggressive (6), and protective-hardworking (5). As seen in hardworking (94), intelligent (78), and friendly (70). The second community (colored by green) represents 23.54% of the network, and their nodes with the highest degree are strong (135), chauvinist (120), and insensitive (71). There is a third community (colored by blue) which represents 13.83% of the network, and their nodes with the highest degree are violent (49), heavy (35), and basic (33). Finally, the fourth community (colored by dark gray) represents 9.71% of the network, and their nodes with the highest degree are beautiful (53), athletes (31), and educated (25).The male metastereotype included 412 nodes, with 3,115 co-occurrences collapsed in 2,756 edges. The strongest co-occurrences were intelligent and hardworking) and traditional negative male stereotype . Therefore, the male metastereotype was quite similar to the male in-group stereotype but different from the male stereotype women project, which was homogeneous with a main community of the traditional positive male stereotype . The main network analysis results are summarized in The male metastereotype's structure was heterogeneous, given that it was mainly built from two main communities. These communities were quite similar to those found for the male in-group stereotype: traditional positive male stereotype , and another community characterized by features such as strong, aggressive, and hardheaded, which could be interpreted as a traditional negative male stereotype.The aim of this research was to map the structure of gender stereotypes and metastereotypes as complex systems Additionally, according to our results, women and men show a heterogeneous metastereotype. Two central communities characterize both female and male metastereotypes. The female metastereotype is characterized by what could be defined as a modern female stereotype as well as a traditional female stereotype. The male metastereotype, on the contrary, is characterized by what could be defined as a traditional positive male stereotype as well as a traditional negative male stereotype. We highlight that both women and men project a metastereotype differently than the gender stereotype that the opposite gender projects. Therefore, it seems that both women and men project a distorted metastereotype in comparison with the opposite gender views, but for a different reason. Women think that men's beliefs about women are different from the beliefs that women have about themselves, which seems not to be the case because both women and men seem to share a similar female stereotype. However, men think that women's beliefs about men are the same that men have about themselves, which seems not to be the case because women and men showed different male stereotypes. It is worth highlighting this point because of the mismatch between metastereotype and how out-group stereotype in-group might condition the intergroup relationships  will allow people to understand some of the underpinnings of their behavior and hopefully engage strategies to change it for the better.Identifying the structure of the current gender stereotypes is important for practice. Our results showed that there is a contradictory view of how each gender thinks the other gender sees them. Women are still thinking that men see them in traditional way as weak and sensitive people, and men think women see them as mainly competent but also tough. However, neither case corresponds to how people perceive other persons of the opposite gender. We are therefore dealing with a possible source of conflict between groups. People's misperception of how the other gender sees their gender can negatively impact—directly or indirectly—the behavior toward the out-groups  and others without direct translation . Therefore, the final terminology might not be a crystal-clear mirror of the original language used. Indeed, even using the same language, the features used for depicting gender stereotypes can be shaped by the cultural context and diverse connotations linked to similar words. Future research could conduct a similar procedure in other languages and cultural contexts to examine to what extent the structure of gender stereotypes is shared across different contexts.Fourth, this research was conducted in the Spanish language and translated into English. There are linguistic terms in the Spanish language with two or more potential translations  that might drive the differences found in their structures in comparison with stereotypes. Future studies should address how these motivational components could affect or be affected by the structure of metastereotypes and their implications . Moreover, gender stereotypes and metastereotypes are broad categories that could be divided into subcategories . Previous research has found that there are differences between subgroups of stereotypes. For instance, DeWall et al.  found thhttps://osf.io/cmf6a/).In sum, our findings could be extended and refined in several ways. Thus, one of our contributions to this field is to apply text and network analyses to map gender stereotypes from a complex dynamic perspective in such a way that it can be easily followed by other researchers using open-source tools .The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: Open Science Framework (The studies involving human participants were reviewed and approved by Ethical Clearance ID: 1696/CEIH/2020. The patients/participants provided their written informed consent to participate in this study.ÁS-R and EM-B contributed to the conception and design of the study and organized the database. ÁS-R and EG-S performed the analyses of the data. ÁS-R wrote the first draft of the manuscript. EM-B and EG-S wrote sections of the manuscript. All the authors contributed to manuscript revisions, read, and approved the submitted version.


Nerve Growth Factor (NGF) and Brain derived Neurotrophic Factor (BDNF) mature/precursor imbalance in tears and serum is suggested as a risk factor and symptomatology aggravation in ophthalmology and neuropsychiatric disturbances. Cognitive and mood alterations are reported by patients with Graves’ Orbitopathy (GO), indicating neurotrophin alterations might be involved. To address this question, the expression levels of NGF and BDNF and their precursors in serum and tears of GO patients were analyzed and correlated with the ophthalmological and psycho-cognitive symptoms. Hamilton Rating Scale for Anxiety (HAM-A) and Depression (HAM-D), Temperament and Character Inventory (TCI), and Cambridge Neuropsychological Test Automated Battery (CANTAB) test were used as a score. NGF and BDNF levels were measured using ELISA and Western Blot and statistically analyzed for psychiatric/ocular variable trend association. GO patients show memorization time and level of distraction increase, together with high irritability and impulsiveness. HAM-A and CANTAB variables association, and some TCI dimensions are also found. NGF and BDNF expression correlates with ophthalmological symptoms only in tears, while mature/precursor NGF and BDNF correlate with the specific psycho-cognitive variables both in tears and serum. Our study is the first to show that changes in NGF and BDNF processing in tears and serum might profile ocular and cognitive alterations in patients. The Nerve Growth Factor (NGF) and Brain-Derived Neurotrophic Factor (BDNF) are expressed in human organs, including eyes, and a detectable amount of these neurotrophins (NTs) are measurable in human fluids, such as blood ,2,3, salIn serum, such as in tissues, mature and precursor NGF and BDNF forms have been detected, and their imbalance is suggested as a risk factor for the aggravation of clinical symptoms, including cognitive and mood alterations, in diverse pathological conditions ,7. The eThese questions have been addressed by this pilot study on a small group of patients affected by Graves’ Orbitopathy (GO), a typical ocular disorder affecting more than 50% of patients with the autoimmune GD .GO is characterized by dry eye disease of varying severity associated with signs and symptoms of periorbital soft tissues inflammation such as palpebral and/or conjunctival chemosis and hyperemia, proptosis, lid retraction, and extraocular muscles oedema followed by progressive diplopia . HoweverBecause of these clinical features, GO patients appear as a suitable group of study for investigating the trend of NTs expression in tears and serum in relation with the ocular and psycho-cognitive profile.In the present study, the evaluation of expression levels of NGF/proNGF and BDNF/proBDNF were performed by Western Blot (WB) analysis following protocols previously standardized in our laboratories to detect the mature and precursor NTs in ocular tissues and brains ,19. OphtThe results reported confirm the presence of proNGF and proBDNF in human serum and tears, and for the first time demonstrate the correlation between serum/tear NGF and BDNF, and their association with the patients’ ophthalmological and psycho-cognitive profile. The biological and clinical significance of these data are discussed, suggesting further multidisciplinary research involving cognitive behavioral and linked neurophysiological correlations.p = 0.026), but only one of them resulted high anxious (HAM-A > 34).All the subjects included in the study did not manifest pathological signs of depression as evaluate by Hamilton scale (HAM-D). Only two GO patients present low levels of depression (HAMD: 8–17), but no changes were found between the groups . A significant increase in anxiety (HAM-A score) was manifested by GO patients , and the medial tentative (PAL-MT p = 0.004), the SWM Between Errors Total (SWM-BE p = 0.036), and in RVP-A-Prime (p = 0.0054) (The cognitive assessment shows significant changes in Paired Associates Learning (PAL) measures in GO patients, such as the total errors (PAL-TE  0.0054) .p = 0.013) might justify this finding.The increased time in memorization, as well as the higher levels of distraction indicate a deficit in attention in GO patients. The significant correlation between the RVP-A and the anxiety rating found in GO patients , and Cooperativeness  (p = 0.011) and irritability . A correlation between HAM-A and TCI dimensions score in GO patients was also found  . In addiso found , indicatp < 0.05), while no changes in BDNF are observable  = 1.35).The data obtained by ELISA show that the NGF concentration is reduced in GO patients compared with HC  = 17.08, p < 0.001), but no difference in the NGF expression  = 3.26, p = 0.08) were found in patients with GO. The ratio NGF/proNGF is consequently increased in GO patients  = 16.53, p = 0.05)  A,B.p = 0.014) while no changes in proNGF expression levels were found between the two groups.Within the GO group, the higher levels of NGF are detectable in group II , and a little increase in proBDNF  = 4.05, p < 0.05) in serum of GO patients with respect to HC. The BDNF/proBDNF ratio resulted significantly reduced in GO patients (p = 0.01) (p = 0.009) and proBDNF  were both increased compared with the group II .The comparison analysis of the data obtained by WB analysis shows a 4-fold decrease in BDNF levels  = 13.92,  = 0.01) C,D. TakiIn our group of GO patients, no significant correlation between serum neurotrophins and T3 and T4 levels, as well as TRAb, were found.p < 0.05) and no variance of NGF  = 1.75 p = 0.54) expression levels in the tears of GO patients. A significant decrease in the NGF/proNGF ratio  = 37.53, p = 0.001) was thus found in GO patients’ tears (p < 0.05), while no changes in mature form were found  = 0.266, p = 0.82). The BDNF/proBDNF ratio in GO was significantly decreased with respect to HC  = 25.46, p = 0.01)  C,D.p < 0.001) and proBDNF  were found in group II compared with the group I . An increase in the NGF and proNGF levels, although it does not reach the significant levels, were found in the group II compared group I.Within the GO groups, decreased levels of tears BDNF , and CAS , but the NGF/proNGF is significantly negatively correlated with CAS only   and CAS , but no correlation between BDNF/proBDNF and CAS  was found  A. A negaas found B.p = 0.023; Spearman’s rho = −0.371 p = 0.192) levels, but not with serum BDNF/proBDNF levels .Patient’s CAS score was also positively correlated with serum NGF/proNGF .In line with the comparative analysis reported in paragraph 2.1, which demonstrated a selective deficit in GO in Paired Associates Learning (PAL) and Rapid Visual Information Processing (RVP), the regression analysis shows a significant correlation between the scores of these parameters and the NGF and BDNF expression in tears and serum , indicating the accuracy and latency increase parallel with the increase in BDNF (or decline of proBDNF). A negative correlation was found between the RVP - ML scores and the m/proBDNF levels in both serum and tear  .A far as the TCI is concerned, the correlation analysis between the TCI score and NT levels in GO patients shows differences between the serum and tears for both NTs, as reported in Only a few traits were found to correlate with both NT ratio in serum and tears. Specifically, NS1 and RD2 traits correlated with BDNF ratio , while NS2  and T traits  correlated with NGF ratio, as indicated in bold in The NGF and BDNF serum and tear profile was investigated in HC and GO patients by measuring the levels of NTs using commercial kits which recognize human NGF and BDNF, and WB analysis to detect the mature and precursor forms. The obtained results extend the previous observations that tear and serum NGF concentrations are altered in Graves’ patients ,21, showThe parallel measure of serum NGF and BDNF expression by ELISA and WB allow us to identify a correspondence between the individual NT concentration measured by the commercial immunoassay kits and the mature/proNT ratio. We found that ELISA BDNF and NGF concentrations are decreased and unchanged in GO patients, respectively, while the WB analysis shows a reduction in proNGF, but no NGF, and an increase in proBDNF in the presence of a low expression of BDNF. As observed by Malerba et al.  and suppMoreover, our WB analysis shows that NGF and BDNF, and their precursors, are expressed in the tears. Like in serum, and to what has been found previously in other tissues , the proIn this context, we found an increase in proNGF and proBDNF, but not changes in mature forms in GO tears compared with HC. Since the levels of NGF and BDNF in tears depend mainly on their synthesis by lacrimal grands , it is lThe clinical values of NT changes in tears in GO ophthalmological manifestations, which include conjunctival inflammation and eye dryness, is also in accordance with the evidences that the local production of NTs in eyes and their release in tears are crucial for the maintenance of healthy ocular surfaces, and exert a neuroprotective and anti-inflammatory role in ocular pathologies and dry eye .However, it cannot even be ruled out that NT imbalance in tears might have been already present in the preclinical phase and/or have a role in the manifestation of other symptoms reported in GO, such as memory impairment and mood disturbances, and psychosocial functions .Changes in NGF and BDNF levels in serum have been detected in patients with neurodegenerative and psychiatric diseases, anxiety, mood disorders, and stress ,19,27 buChiefly, we found that impairment of episodic memory in GO, as indicated by increased PAL scores, is associated with the tear and serum NGF and BDNF m/pro expression in a negative (low NGF levels) and positive manner (high BDNF levels), respectively, while only m/pro BDNF correlated with RVP.Because of the novelty of our data, the significance of these correlations can be only speculated. For example, it is interesting that all PAL tasks correlated with tear NGF and BDNF, while only some tasks correlated with serum. One possible explanation might arise from the evidence that visual memory is coupled with increased eye movements and blinking ,33, and The specific correlation between m/pro BDNF in both serum and tears with RVP might corroborate the involvement of the eye–brain pathway. Indeed, BDNF is known to be directly regulated by the visual stimulation ,39,40,41In this study, we found that in GO patients, the RVP score correlated with the HAM-A value indicating that the attention impairment is associated with anxiety. The correlation between anxiety and impulsivity, as well as irritability, further support the idea that mood status and increased stress sensitivity and/or reactivity might characterize GO patients. It is therefore possible that the correlation between m/pro BDNF and the RVP we found in the serum and tears of GO patients might be also representative of the emotional dimension of disease, and therefore impact on their ability to maintain attention.This observational study was addressed to analyze the daily NGF and BDNF profiles in the tears and serum of healthy women and in patients with GO. This study was performed at the Department of Sense Organs of Sapienza University of Rome.Ten healthy subjects (HC) and Twenty patients with GO were enrolled by an ophthalmologist with the following inclusion criteria: age older than 18 years, laboratory diagnosis of GD based on the finding of undetectable serum TSH and high blood thyroid hormone associated with the presence of circulating TSH receptors antibodies (TRAb), and signs and symptoms of GO according with EUGOGO guidelines . ClinicaTen healthy subjects, age matched, attending the outpatients’ service of the Eye Clinic of University of Rome “Sapienza”, were recruited as a control group by an ophthalmologist and were reported to not have signs and symptoms of GD and GO, to not take regular medications or drugs, to not have history of alcohol/drug addiction, to not be heavy alcohol drinkers , and to not have drank alcoholic beverages in the past 42 h. Women were included in the first week of their post-menstrual period. All the subjects were not family related, did not report any personal or familiar neurological or psychiatric diseases, or allergic, infective, nor inflammatory disorders. All GO patients and healthy controls were referred to psychiatrists for valuations, as described below.All subjects included in the study underwent a psychiatric interview (AI), and were tested for scoring their emotional and cognitive profile.Patients’ mood conditions were assessed using the Hamilton Rating Scale for Anxiety (HAM-A) and Depression (HAM-D) . The HAMThe HAM-D contains 17 items (HDRS17) pertaining to symptoms of depression experienced over the past week. A score of 0–7 is generally accepted to be within the normal range , while a score of 20 or higher indicates at least moderate severity.http://www.camcog.com) were administered to all participants by trained individuals (AQ). The CANTAB is a computer-administered, nonverbal  set of tasks, developed to examine specific components of cognition, particularly those associated with frontal and medial temporal regions of the brain [To assess the cognitive function, tasks from the Cambridge Neuropsychological Test automated Battery  developed by C. Robert Cloninger , based oTears and blood were collected at 10:30 a.m. on the same day of the psychiatric interview. Tear samples were collected in both eyes from all patients by imbibition of a sterile Sharp-tip Microsponges  placed at the inferior conjunctival fornix and removed after 30 s. Tears from the eyes of each patient were pooled, and after centrifugation at 13,000 rpm for 3 min, were stored at −20 °C until processing for WB analysis.Approximately 10 mL of blood was drawn from the subject’s antecubital vein and left at room temperature until clot forming and clear serum was obtained by centrifugation. All samples were aliquoted and stored at –70 °C until use.NGF and BDNF concentrations in serum and saliva were measured by using human NGF and BDNF immunoassay . All the assays were performed in triplicate, following the manufacturer’s instruction and using the recommended buffers, diluents, and substrates. The optical density of the color reaction was read using a microtiter plate reader  set for 450 nm. The intra- and inter-assay coefficients of variation were below 7%. The NT concentration (expressed as pg/mL) in each sample was calculated according to a standard curve.The expression levels of mature precursor forms of NGF and BDNF were analyzed by WB in tears and serum samples. Briefly, equal protein amounts were loaded on a 10% SDS-PAGE gel and electrophoretically transferred to nitrocellulose membranes later blocked in low-fat milk (10% in Tris buffer and 0.1% Tween-20) and washed three times for 10 min each at RT in TBS-T. Samples were exposed to the primary anti-NGF  and anti-proBDNF  antibodies for 1 h at room temperature (RT) on a shaker.The blots were washed and incubated for 1 h with anti-rabbit or anti-mouse horseradish peroxidase conjugated antibody  at RT on a shaker. Immunoblot analyses were performed using a chemiluminescence detection kit (ECL) as the chromophore  and exposed to Hyperfilm . The integrate density (ID) of the bands was recorded and quantified by the public domain image Fiji  Image processing and analysis program. Data are expressed as arbitrary units (a.u.).p ≤ 0.05. The correlations analysis between the levels of NTs in serum and tears, as well as between the NTs and the psychiatric and ocular variables, were determined by both Pearson’s and Spearman correlation, as suggested by the Rovetta’s study [p ≤ 0.05.Statistical analysis was performed by using the free open-source program for statistical analysis JASP  by the University of Amsterdam . Based o’s study . Again, In conclusion, the present study is one of the first to explore the relationship between the NGF and BDNF expression in serum and tears of humans, and to show that altered ratio of mature and precursor NTs in these fluids correspond to the ophthalmological symptoms (proptosis and CAS), and the psycho-cognitive profile of GO patients. These observations enlarge the information about NT involvement in ocular diseases, as well as neuropsychiatric disturbances, and open a new insight into the validation of tears and serum NTs as clinical and therapeutical biomarkers.