Aggress Behav, 43(1), pp. 74–84) overcomes this weakness by identifying four types of cyber‐aggression . However, the CATQ only considers aggression in cyberspace. We extended the CATQ to the face‐to‐face context by developing a corresponding Face‐to‐Face Aggression Typology Questionnaire (FATQ). The aim of this study was to investigate factorial and convergent validity and metric measurement invariance between four‐factorial cyber and face‐to‐face aggression. In total, 587 students from six Austrian universities filled out the CATQ, the FATQ, and additional scales during regular university lectures to examine convergent validity. Confirmatory factor analysis supported the four‐factor structure of both questionnaires, after excluding inconclusive items from the impulsive‐aversive/rage subscale of the FATQ. These items were also removed from the CATQ to obtain two symmetric questionnaires. Metric measurement invariance between the CATQ and the FATQ was confirmed. Convergent validity was largely observed. Our results support an extended four‐factor model of aggression. Having two parallel questionnaires, the FATQ and CATQ, enables future studies to investigate commonalities and differences in underlying drivers of aggressive behavior in the cyber and face‐to‐face contexts.Aggressive behavior in the face‐to‐face and cyber contexts is driven by underlying aggression . Common theories of aggression distinguish between reactive  and proactive  aggression. However, according to the quadripartite violence typology, this distinction conflates aspects of motivational valence with self‐regulatory processes. The Cyber‐Aggression Typology Questionnaire (CATQ; Runions et al., 2017,  However, reasons to engage in aggressive behavior are manifold . As with the CATQ, we aimed to confirm the four‐factor structure of the FATQ.Third, we tested the metric measurement invariance of the CATQ and the FATQ, which enables a comparison of factor variance and factor covariance across both instruments. That is, we assumed that corresponding items with matched item content referring to the cyber and the face‐to‐face context would have the same factor loadings.Fourth and finally, we tested the convergent validity of the CATQ and the FATQ by investigating the associations between both instruments and the dichotomous measure of aggression  but conducted in a controlled manner (like proactive aggression). We expected cyber and face‐to‐face reward aggression to be positively associated with proactive aggression. This is due to the deliberate and reward‐related nature of both conceptualizations. Finally, we hypothesized positive associations between cyber and face‐to‐face recreation aggression and both reactive and proactive aggression. This was assumed because recreation aggression is reward‐related (like proactive aggression) yet impulsive (like reactive aggression).We expected the following associations between the four factors of cyber and face‐to‐faceaggression and the behavioral inhibition and activation systems  from six Austrian universities answered online questionnaires via their smartphones during regular class time. A trained research assistant was present at all times. Participants gave informed consent and participation was voluntary. To avoid any systematic order effects, the order of the CATQ and the FATQ were randomly counterbalanced across participants.A total of 587 university students  for rage aggression, .92/.84 (cyberspace/face‐to‐face) for revenge aggression, .94/.85 (cyberspace/face‐to‐face) for reward aggression, and .92/.88 (cyberspace/face‐to‐face) for recreation aggression.Moreover, as previously discussed, we adapted all CATQ items to the face‐to‐face context, resulting in the FATQ. Thus, we considered two context‐specific questionnaires assessing four‐factorial cyber‐aggression as well as four‐factorial face‐to‐faceaggression. The items for the cyber context were presented after the following introduction: “The questions below are related to your behavior in cyberspace chats, social media, forums, blogs, etc.)”, while the items for the face‐to‐face context were presented after the following introduction: “The following questions are exclusively related to your behavior in direct, personal contact (“face‐to‐face”)”. Answers to all items were given on a four‐point response scale  showed an acceptable model fit for the measurement model for the RPQ removing one item from the reactive aggression scale (χ2(200) = 388.74, Comparative Fit Index [CFI] = .943, Tucker‐Lewis Index [TLI] = 0.934, root mean square error of approximation [RMSEA] = 0.040 and standardized root mean square residual [SRMR] = 0.079).We measured the dichotomous conceptualization of reactive and proactive aggression with the RPQ  = 737.22, CFI = .954, TLI = .947, RMSEA = .077 and SRMR = .079).To assess the personality dimensions of BIS and BAS sensitivity, we used the 20‐item short version of the ARES‐K  and a measurement model with factor loadings constrained to be equal between the CATQ and the FATQ factors  was estimated. To compare the configural and the metric invariance model, changes in CFI and RMSEA were examined to evaluate the equality of factor loadings. It has been suggested that a change in CFI of more than .01 and a change in RMSEA of more than .015  = 886.68, CFI = .952, TLI = 0.947, RMSEA = 0.049, and SRMR = 0.068). Measurement items and standardized factor loadings are shown in Table S3.Results of the CFA showed a very good model fit for the rage, revenge, reward, and recreation aggression scales (see Table S2). Accordingly, the four‐factor measurement model exhibited a very good model fit (3.2.2χ2(45) = 414.73, CFI = .936, RMSEA = .118, and SRMR = .077). Subsequently, standardized factor loadings and modification indices were inspected to identify items with poor psychometric properties. Inspecting the item contents revealed that both Item 6 (“I overreact before I have a chance to think about the consequences when someone says something mean face‐to‐face”) and Item 10  contained information about additional cognitive processes . Similarly, Item 7  and Item 11  also contained information about additional cognitive processes . However, inspecting the content of Item 4  indicated that spreading mean rumours might be not an appropriate example of impulsively‐driven behavior in the face‐to‐face context, despite being common in cyberspace. As a result, Items 4, 6, 7, 10, and 11 were removed from the measurement model. We discuss the incompatibility of these items when transferred from the cyber to the face‐to‐face context more detailed in Section χ2(242) = 712.97, CFI = .944, TLI = 0.936, RMSEA = .058, and SRMR = .078). Measurement items and standardized factor loadings are provided in Table S4.Results of the CFA showed a very good model fit for the revenge, reward, and recreation aggression scales (see Table S2). However, the measurement model for the rage aggression scale did not exhibit an acceptable model fit (χ25 = 414.7χ2(242) = 506.38, CFI = .971, TLI = .967, RMSEA = .043 and SRMR = .058).To ensure comparability between the CATQ and the FATQ, Items 4, 6, 7, 10, and 11 were also removed from the cyberspace version of the questionnaire resulting in a modified version of the CATQ (mCATQ). The model fit for the reduced four‐factor measurement model exhibited a very good model fit (3.3χ2(252) = 1800.15, CFI = .948, TLI = .943, RMSEA = .036 and SRMR = 0.074) and the metric invariance model (χ2(232) = 1856.57, CFI = .946, TLI = .941, RMSEA = .037 and SRMR = .080). Moreover, there was no meaningful decrease in model fit between the configural and metric invariance models (see Table S5). Thus, it can be assumed that factor loadings are invariant across the mCATQ and the FATQ.The results of the CFA for the measurement model comprising four factors of the FATQ and four factors of the mCATQ revealed a good model fit for the configural invariance model  differs across contexts . To examine, whether such comparisons could be validly done, metric measurement invariance between the FATQ and the modified CATQ was checked. Our results support this potential application. It can be assumed that factor loadings are invariant.Regarding convergent validity, we were able to confirm all hypothesized relationships between all four dimensions of cyber and face‐to‐face aggression and the two dimensions of the RPQ and thus to replicate Runions et al.'s  findingsAll other results were not in line with our hypotheses. Similar to Runions et al. , we founIn line with Runions et al.'s  results,In contrast to the positive association between cyber reward aggression and drive reported by Runions et al. , we obseIn line with Runions et al. , we founFinally, we observed a negative association between cyber recreation aggression and gratification and a nonrelationship between face‐to‐face recreation aggression and gratification, although positive relations were proposed. As recreation aggression refers to impulsive thrill‐seeking, it could be considered an intrapersonal motive, which might in turn make it independent of external influences such as reactions by others. Instead, recreation‐related aggressive behavior might predominantly relate to immediate pleasant arousal, resulting from the aggressive behavior per se. In contrast to our previous assumptions, achieving joy upon achieving an external goal  might not be decisive for this connection.In summary, we found limited results with respect to CATQ and FATQ's convergent validity vis‐a‐vis the ARES‐K. Although Runions et al.  also fou4.1Our study is based on a variant sample of students from several universities, studying different subjects. However, additional research with school students is needed. In our sample, the severity of four‐factorial aggression in both the face‐to‐face and cyber contexts was low. Future studies should investigate samples based on a more representative population. University students only represent a specific subsample of young people . The focus on younger students is expected to increase overall face‐to‐face and cyber‐aggression, as it would be more in line with the population at greatest risk for face‐to‐face and cyber‐aggression  with different motivational patterns within and between contexts to be identified and provided with tailor‐made interventions.The authors declare that there are no conflict of interests.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.


To elucidate the correlation between expression of thrombomodulin (TM) mRNA from 83 benign soft tissue tumors or soft tissue sarcomas (STS) and clinicopathological parameters and to analyze the outcome of high‐grade STS patients after 10 years.Total RNA was extracted from 83 primary soft tissue tumors . TM mRNA normalized to glyceraldehyde‐3‐phosphate dehydrogenase was measured with real‐time quantitative polymerase chain reaction and compared to various clinicopathological parameters. The log‐rank test and Cox proportional hazard analysis were used to evaluate recurrence‐free survival, metastasis‐free survival, and overall survival.n = 57) and low‐grade (n = 11) tumors. Following analysis of high‐grade STS at the 10‐year follow‐up, 21 patients had experienced a recurrence, 22 patients had experienced metastasis, and 23 patients had died of disease (DOD). TM levels were significantly higher in patients with metastasis or DOD patients. Receiver operating characteristic analysis for identifying 5‐year and 10‐year DOD determined the threshold for best sensitivity and specificity as 0.283. We divided patients into those with high (<0.283) and low (≤0.283) TM mRNA levels. Based on Kaplan–Meier analysis, a significant difference between the two groups was seen for recurrence‐free survival  and metastasis‐free survival . Furthermore, the high TM group showed significantly worse prognosis than the low TM group . Thus, high levels of TM mRNA are associated with highly recurrent and metastatic potential and lead to poor prognosis. In multivariate Cox proportional hazard analysis, only high TM showed a significant difference in metastasis‐free survival  and overall survival .Thrombomodulin mRNA levels were not significantly different between benign tumors and STS. In STS, TM mRNA levels were not significantly different between histologically high‐grade (High levels of TM mRNA may be a significant predictor of recurrence, metastasis, and a poor outcome in STS patients after 10 years. TM is a candidate molecular marker and may be clinically useful for devising a therapeutic treatment strategy by prediction of prognosis.  TM plays an essential role as a receptor for thrombin. Thrombin‐containing complexes activate the anticoagulant pathway of protein C. Activated protein C proteolytically inactivates coagulation factors Va and VIIIaThrombomodulin , a transmembrane glycoprotein with 557 amino acids, comprises an NH2+‐dependent cell–cell adhesionThrombomodulin is expressed on the cell surface of endothelial cells, monocytes, dendritic cells, neutrophils, platelets, megakaryocytes, smooth muscle cells, keratinocytes, meningeal cells, synovial lining cells, and mesothelial cellsThe purpose of this study is: (i) to elucidate the correlation between expression of TM mRNA from 83 benign soft tissue tumors or STS and clinicopathological parameters; and (ii) to predict the outcome of high‐grade STS patients after 10 years.Criteria for inclusion were: patients with primary soft tissue tumors who visited Mie University Hospital between 1982 and 2006 and who underwent complete tumor resection with a wide margin during the initial surgery. Criteria for exclusion were: patients with local recurrence or distant metastasis or who were referred for additional resection after inadequate resection in a previous hospital. A total of 83 patients were included in this study. The histopathological diagnosis was made by independent pathologists and classified according to the World Health Organization classification. Written, informed consent was obtained from each patient. For patients under the age of 19 years, informed consent was obtained from their parents or legal guardian. This study was approved by the Ethics Committee of the Mie University Graduate School of Medicine . All procedures performed in studies involving human participants were in accordance with the ethical standards of the Ethics Committee of Mie University and with the Helsinki Declaration.All tumor tissue samples were obtained from patients who underwent surgical resection or an open biopsy at the Department of Orthopedic Surgery, Mie University Graduate School of Medicine. The tissue specimens were immediately collected and frozen in liquid nitrogen (for RNA extraction) or fixed for 24 h in 10% buffered formalin solution and embedded in paraffin .Total RNA was extracted from each sample using ISOGEN , according to the manufacturer's instructions and then reverse‐transcribed into cDNA using the First Strand cDNA Synthesis Kit . The TaqMan Gene Expression Master Mix and the TaqMan Gene Expression Assay  were used to quantitatively analyze the expression of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and TM. GAPDH was used as an endogenous housekeeping gene for normalization. Real‐time quantitative polymerase chain reaction amplifications were performed using an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). Standard curves were generated using cDNA samples from HeLa cells. The relative expression levels of each target gene were indicated by calculating the ratio to the expression levels in the HeLa cells. All assays were performed in triplicate.U‐test for quantitative data. To evaluate the threshold for detecting mortality due to disease, receiver operating characteristic (ROC) curves were generated. ROC curves were created by plotting the sensitivity on the y‐axis and the false positive rate (1 − specificity) on the x‐axis. To measure the effectiveness of the TM level as a marker for mortality due to disease, the area under the curve (AUC) was assessed. Local recurrence‐free survival (RFS) was defined as the time from the initial treatment to the date of clinically documented local recurrence. Metastasis‐free survival (MFS) was defined as the time from the initial treatment to the date of clinically documented distant metastasis. Overall survival (OS) was defined as the time from the initial treatment to the date of mortality attributed to the neoplasm. Kaplan–Meier survival plots and log‐rank tests were used to assess the differences in time to local recurrence, distant metastasis, or OS. To adjust for the imbalance in prognostic factors among patients, Cox proportional hazard analysis was used. P < 0.05 was considered statistically significant.Clinicopathological analysis was performed to compare the TM mRNA levels to various clinical parameters using the Mann–Whitney P = 0.00488) (Table P = 0.0248). In addition, in STS, TM mRNA levels were not significantly different between histologically high‐grade (n = 57) and low‐grade (n = 11) tumors . The average age was 50.1 years (range: 0–85 years). Histopathological diagnosis was as follows: 15 benign tumors, including 5 schwannomas, 3 lipomas, 3 neurofibromas, and 4 others; 68 STS, including 21 liposarcomas, 17 undifferentiated pleomorphic sarcomas (UPS), 9 synovial sarcomas, 7 malignant peripheral nerve sheath tumors, 4 clear cell sarcomas, and 10 others. The 57 high‐grade STS included 17 UPS, 11 liposarcomas, 9 synovial sarcomas, 7 malignant peripheral nerve sheath tumors, 4 clear cell sarcomas, and 9 others. In the benign soft tissue tumors and STS, TM mRNA levels were not significantly different between patients according to sex and malignancy. Patients over 55 years of age showed significantly higher TM values than those under 55 years . At the 5‐year observation time point, 18 patients had experienced a recurrence, 19 patients had experienced metastasis, and 18 patients had died of disease (DOD). Recurrence, metastasis, and DOD of the top three pathological types were shown. TM mRNA levels were significantly higher in the patients with metastasis or DOD patients than in patients without metastasis or patients who were alive. After an additional 5 years, three more patients experienced a recurrence, three more patients experienced metastasis, and five more patients had DOD. Similar to the 5‐year observation time point, mRNA levels were significantly higher in the patients with metastasis or DOD patients at the 10‐year observation time point  0.676–0.922, 10 years: AUC 0.765, 95% CI 0.632–0.897). With a threshold of 0.283, the sensitivity and specificity for identifying 5‐year DOD were 88.9% and 71.8%, respectively, and for identifying 10‐year DOD were 78.3% and 73.5%, respectively. To divide patients into two groups for further analysis, a threshold of 0.283 was adopted, and low (≤0.283) and high (0.283<) TM mRNA groups were analyzed.P = 0.041, metastasis: OR 9.80, 95% CI: 2.06–46.0, h = 0.0040, DOD: OR 9.34, 95% CI: 2.05–42.7, P = 0.0038) . In MFS, high TM and tumor size showed a significant difference . In addition, to elucidate the association with multiple factors, multivariate Cox proportional hazard analysis was performed in MFS. Tumor size was not significantly different (P = 0.2961). High TM was the only independent metastatic factor . In OS, similar to MFS, not only high TM but also tumor size showed a significant association with the survival rate . In OS, additional multivariate Cox proportional hazard analysis with high TM and tumor size showed a significant difference only for high TM  : 2.95, 95% CI 1.21–7.19, In experimental studies, TM protein has the potential to prevent metastasis, because of its anti‐coagulant activityIn the current study, we found no difference in the level of TM mRNA between benign tumors and STS. In STS, no significant difference was seen between histological low‐grade and high‐grade tumors Table . HoweverThrombomodulin expression in STS cells is uncommon. Immunohistological staining for TM has been used as a marker of malignant mesotheliomaTranslational medicine efficiently facilitates medical advances from the basic sciences to the clinical situationThis retrospective study has some limitations. The number of patients was small. Soft tissue tumors, including sarcomas, have many subtypes, and the incidence rate of each is low; because of the small number of patients, we could not statistically analyze the tumors by subtype. Many studies have analyzed STS as a whole group rather than by each histological classificationThe association between TM and STS is newly recognized. We successfully demonstrated the relationship between TM mRNA in STS tissues and recurrence, metastasis, and poor outcomes. We believe that the small step of this translational study will help increase the understanding of the mechanism of soft tissue tumor malignancy and accelerate drug development.


RELA fusion. When a clear cell neoplasm is intraoperatively diagnosed, intracytoplasmic dot‐like inclusions by cytology are a useful cytopathological feature of ependymoma.Pediatric supratentorial ependymomas often have a clear cell morphology and reveal a  RELA fusion. When a clear cell neoplasm is intraoperatively diagnosed, intracytoplasmic dot‐like inclusions by cytology are a useful cytopathological feature of ependymoma.Pediatric supratentorial ependymomas often have a clear cell morphology and reveal a  We believe that intracytoplasmic dot‐like inclusions in neoplastic cells, in addition to perivascular pseudorosettes, might be a useful finding for ependymomas. Furthermore, we should recognize the presence of RELA fusion‐positive ST‐EPNs as a poor prognostic variant of ependymomas.Ependymomas are major primary glial neoplasms that mainly affect children and young adults, accounting for 2%‐9% of all neuroepithelial tumors.2An 8‐year‐old girl received symptomatic treatment for headache and vomiting in a local pediatric clinic. As her symptoms did not improve, she underwent brain computed tomography (CT) and magnetic resonance imaging (MRI) at a local hospital 6 days after onset. Brain CT and MRI revealed a mass with a cyst at the right temporal lobe. Therefore, subtotal tumor resection was performed 10 days after onset, and the resected brain tumor was diagnosed as anaplastic ependymoma. After the operation, postoperative radiation therapy  was performed at our hospital, following which the patient was observed at the outpatient ward of the hospital. However, 8 years after the operation, the right temporal tumor recurred and was resected again. As postoperative neuroradiological analysis revealed complete resection of the tumor, she received no postoperative therapy but was followed up at the outpatient ward of the hospital. We observed no tumor recurrence 1 month after the second operation.33.1A cytopathological specimen was obtained during the second operation via tumor tissue squash‐and‐imprint and stained using the standard Papanicolaou procedure and hematoxylin and eosin (HE) staining Figure . Cellula3.2In frozen section diagnosis, the tumor specimen had a high degree of cellular neoplasm with perivascular pseudorosettes formed by neoplastic cells elongating their processes into intermingled vessels Figure . Those nC11orf95 exon 3 and RELA exon 2, the most commonly identified fusion pattern. Therefore, integrated diagnosis of this pediatric supratentorial neoplasm was determined as “ependymoma, RELA fusion‐positive” according to the revised 2016 World Health Organization (WHO) classification of central nervous system (CNS) tumors.Among those pathological findings, the final diagnosis of the recurrent brain tumor was supratentorial anaplastic ependymoma with clear cell morphology, expressing L1CAM. RNA sequencing from the tumor tissue revealed a fusion of 4C11orf95‐RELA fusion, in particular, are defined as distinct variants of ependymoma, named “ependymoma, RELA fusion‐positive,” in the revised 2016 WHO classification of CNS tumors.RELA fusion‐positive ependymomas usually have anaplasia, including high cellularity, nuclear atypia, high mitotic activity, microvascular proliferation, and/or tumor necrosis.RELA fusion in ST‐EPNs.RELA fusion‐positive ST‐EPN affecting this 16‐year‐old girl recurred 8 years after the first operation even though postoperative radiation therapy had been administered. In addition, its histopathology was anaplastic ependymoma with clear cell morphology, resembling CC‐EPN. Furthermore, the neoplasm revealed L1CAM immunoreactivity. These clinicopathological and immunohistochemical features are compatible with a diagnosis of “ependymoma, RELA fusion‐positive.” Recently, genetic alterations of ST‐EPNs have been reported.RELA fusion,RELA fusion‐positive ependymoma.The ependymoma, in this case, was morphologically characterized by clear cell features. CC‐EPN is a rare histological variant of ependymoma, accounting for 6% of ependymomas.The major points of the differential diagnoses of CC‐EPN, OD, CN, and ccRCC are summarized in Table C11orf95‐RELA fusion.Immunohistochemically, CC‐EPN shows GFAP immunoreactivityRELA fusion. It is difficult to diagnose a RELA fusion‐positive ependymoma by intraoperative‐frozen pathological diagnosis. However, using the patient's age, tumor location, and several cytopathological findings including the intracytoplasmic dot‐like inclusions of neoplastic cells, we could suggest a diagnosis of ependymoma with a possible C11orf95‐RELA fusion: the poor prognostic variant of ependymoma.In conclusion, there are several histologic types of brain tumors showing clear cell morphology. Among them, some show biologically aggressive behavior and require suitable postoperative chemoradiation therapies. One of those tumors is ST‐EPN with None declared.Conceptualization: Taku Homma. Patient's care: Reina Mizuno, Tomonari Suzuki, Eita Uchida, Jun‐ichi Adachi. Pathological investigation: Taku Homma, Yu Miyama, Masanori Yasuda. Writing – original draft: Taku Homma. Writing – review and editing: Taku Homma.Appropriate consent has been obtained.


To assess the risk of three autoimmune diseases ‐ autoimmune thyroiditis (AIT), Guillain‐Barré syndrome (GBS), and inflammatory bowel disease (IBD) ‐ in females following AS04‐HPV‐16/18 vaccination.This meta‐analysis included data from 18 randomized controlled trials, one cluster‐randomized trial, two large observational retrospective cohort studies, and one case‐control study. Following vaccination, a risk window of 2 years was defined for AIT and IBD and 42 days for GBS. Odds ratios (ORs) were estimated using three methods: meta‐analysis inverse‐variance with continuity correction , pooled estimate, and beta‐binomial regression.In all studies apart from the case‐control study, 154 398 exposed and 1 504 322 non‐exposed subjects were included, among whom there were 141 and 1972 cases of (autoimmune) thyroiditis; 2 and 2 cases of GBS; and 43 and 401 cases of IBD, respectively. In the case‐control study, there were 97 cases of AIT and 13 of GBS; matched with 802 and 130 controls, respectively. The primary analysis OR estimates were 1.46 , 11.14 (2.00‐61.92), and 1.11 (0.75‐1.66) for (autoimmune) thyroiditis, GBS, and IBD, respectively.This meta‐analysis did not show an increased risk of IBD following vaccination with AS04‐HPV‐16/18. The 1.5‐fold increased risk of (autoimmune) thyroiditis does not allow us to conclude about a causal association. For GBS, the very low number of cases and wide 95% CIs negate any firm conclusion. There have been hypothesized concerns that vaccine adjuvants could lead to autoimmune conditions.This meta‐analysis examined cases of (autoimmune) thyroiditis, Guillain‐Barré syndrome, and inflammatory bowel disease following AS04‐HPV‐16/18 vaccination using all available data from clinical and post‐marketing observational studies.This analysis did not show a risk of inflammatory bowel disease.There was a 1.5‐fold increased risk of (autoimmune) thyroiditis, but no conclusion about a causal association could be drawn.The very low number of cases of Guillain‐Barré syndrome negated any firm conclusion.1Cervarix; GSK)Gardasil; Merck Sharp & Dohme Limited),Gardasil 9; Merck Sharp & Dohme Limited).O‐desacyl‐4′‐monophosphoryl lipid A (50 μg MPL) adsorbed on aluminium hydroxide (500 μg Al3+)Three human papillomavirus (HPV) vaccines are currently available: AS04‐adjuvanted HPV‐16/18 (AS04‐HPV‐16/18) vaccine  and post‐marketing observational studies that were identified in GSK internal repository of studies sponsored and supported by the Company, and information from Regulatory Authorities. In a complementary, systematic literature review searching for all studies published till end 2015. Details of the search strategy, the database consulted and number of references found and selected are described in Eighteen individually RCTs 9, 10A cluster‐randomized trial, in which communities of subjects received different vaccination schedules.Two large observational, retrospective cohort studies: a United Kingdom (UK) database cohort studyA French case‐control study,The following studies were included:2.2Subject‐level data were extracted from all studies except the French cohort studyThe following data were extracted for each study: numbers of subjects exposed and non‐exposed to AS04‐HPV‐16/18; mean ages; countries of enrollment; length of follow‐up; and numbers of cases of (autoimmune) thyroiditis, GBS, and IBD during the risk period (defined below).2.3Clinical definitions of AIT, GBS, and IBD events varied across studies, as detailed in In the French cohort study,2.4The post‐vaccination risk periods were determined based on the onset of the disease (acute or insidious) and possible or known pathologic mechanisms.As the clinical courses of AIT and IBD are generally insidious, 2 years between vaccination and disease onset was selected. For trials with longer follow‐up periods, only cases that occurred during the 2 years following first vaccination were included. For the French cohort study,For GBS, a shorter risk period (42 days following each vaccination) was considered for the main analysis, based on its anticipated acute onset. This period is also recommended by the Brighton Collaboration GBS Working Group,2.5To harmonize results across studies, odds ratios (ORs) and 95% confidence intervals (CIs) were calculated from the numbers of cases and total numbers of subjects for the combined clinical studies ; the cluMeta‐analysis of rare events is challenging due to the inclusion of studies with no event in one or both arms .In the inverse‐variance method , a continuity correction ,3.2There were an estimated 140.6 cases of (autoimmune) thyroiditis among 152 225 exposed subjects (92/100 000) and 1971.5 cases among 1 453 729 non‐exposed subjects (136/100 000). The OR using the inverse‐variance method with continuity correction (primary method) was 1.46 (95% CI 1.22‐1.76), the beta‐binomial regression method without the case‐control studyResults using the original analysis of the French cohort study3.3The only GBS cases were from the French cohort study,The primary method OR was 11.14  and 401.4 cases among 1 504 322 non‐exposed subjects (27/100 000). The primary method OR was 1.11 (95% CI 0.75‐1.66); the other methods gave similar estimates  that had been identified as safety signals in observational studies of AS04‐HPV‐16/18 or HPV‐6/11/16/18 vaccines.The AIT primary analysis showed a slightly increased risk (OR = 1.46) of (autoimmune) thyroiditis following AS04‐HPV‐16/18 vaccination. This is likely a slight overestimation of risk given that this was heavily influenced by the large French cohort study,The other analysis methods showed ambiguous results for AIT due to differences in their weighting of data from different studies. The beta‐binomial model estimate was similar to that from the primary analysis, but the pooled analysis estimate provided an OR of 0.68. This simple pooled estimate is biased because there was a much higher incidence of (autoimmune) thyroiditis in the French cohort studyThe GBS results were driven by two cases among exposed individuals in the French cohort study,The IBD primary analysis did not show an increased risk following AS04‐HPV‐16/18 vaccination. This is in line with previous AS04‐HPV‐16/18 pooled analyses.Our results are also supported by a recent systematic review and meta‐analysis that examined the risk of various autoimmune disorders after vaccination with any HPV vaccine.4.1This study highlights the potential value, as well as the limitations of, meta‐analysis as a tool to investigate safety signals related to rare outcomes, which is challenging due to the inclusion of studies with no events in one or both arms.i2 index  ‐ appeared to be very low, although the CIs were very broad, so the apparent lack of heterogeneity should be interpreted with caution.There was also heterogeneity between studies, in terms of study design, coding of medical events , case asRegarding study design, the AS04‐HPV‐16/18 exposed and non‐exposed arms of the RCTs should have been balanced by randomization, but there could have been multiple unknown confounders between arms in the observational studies, which it is impossible to adjust for. Also, subjects in both arms could have received additional vaccines, with or without adjuvants, further complicating interpretation of the results. Another difference between the RCTs and observational cohort studies is the level of medical surveillance. In the RCTs, vaccinated and control subjects were followed up according to protocol‐defined scheduled visits. However, in the two observational cohort studies,The case‐control studyCase definitions and ascertainment methods varied between studies. In the RCTs, all cases were fully medically validated, whereas in the French cohort study,Unfortunately, individual subject data (including time‐to‐event data) for the case‐control studyIdeally, disease onset would be from the date of first symptoms, but this is not necessarily known, especially in retrospective database studies. Using diagnosis dates, some autoimmune cases that were diagnosed after vaccination may have had their first symptoms prior to vaccination. Conversely, some cases with first symptoms within 2 years or 42 days following vaccination might have been diagnosed after these windows and therefore not have been included.Overall, our analysis illustrates that a meta‐analysis can be powerful tool, but its strength is related to the quality of the input data.5This meta‐analysis ‐ including approximately 150 000 AS04‐HPV‐16/18‐exposed and 1 500 000 non‐exposed subjects ‐ did not indicate an increased risk of IBD. The results of the analysis showed a 1.5‐fold increased risk of (autoimmune) thyroiditis, but based on existing epidemiological and mechanistic evidence, there is insufficient evidence to conclude a causal association with vaccination. No conclusion regarding the risk of GBS can be drawn as they were driven by two cases among exposed individuals, the times‐to‐onset of which were unknown. Although the GBS OR estimates were high, the number of cases was low and the 95% CIs were wide.Considering the current results and ongoing surveillance of AS04‐HPV‐16/18 vaccination including other available post‐marketing data and pooled analyses of clinical trial data,Given the overall safety data and the demonstrated high and sustained efficacy of AS04‐HPV‐16/18 against HPV‐16/18 infection and cervical lesions,The authors state that no ethical approval was needed.A.G., C.W., D.R., F.T.D.S., S.C., S.W. are employees of the GSK group of companies. A.G., D.R., F.S., F.T.D.S., S.W. hold shares in the GSK group of companies as part of their employee remuneration. F.S. was an employee of the GSK group of companies at the time the study was performed and is currently an employee of Janssen, Pharmaceutical Companies of Johnson & Johnson. F.S. owns shares in the GSK group of companies.D.R., C.W. and F.T.D.S. were involved in the design of the study, collection of the data, and interpretation of the results. D.R. and S.C. were involved in the statistical analysis. A.G. and S.C. were involved in the interpretation of the results. F.S. was involved in the design of the study and interpretation of the results.Data S1. Supporting Information.Click here for additional data file.Data S2. Supporting Information.Click here for additional data file.Data S3. Supporting Information.Click here for additional data file.Data S4. Supporting Information.Click here for additional data file.Data S5. Supporting Information.Click here for additional data file.Data S6. Supporting Information.Click here for additional data file.Data S7. Supporting Information.Click here for additional data file.Data S8. Supporting Information.Click here for additional data file.


The objective of this study was to examine clinical characteristics, cognitive decline, and predictors for time to dementia in prodromal dementia with Lewy bodies with mild cognitive impairment (MCI‐LB) compared with prodromal Alzheimer's disease (MCI‐AD).1–42 ratio). Longitudinal outcome measures included cognitive slopes  and time to dementia.We included 73 MCI‐LB patients  and 124 MCI‐AD patients  from the Amsterdam Dementia Cohort. Follow‐up was available for 61 MCI‐LB patients and all MCI‐AD patients (3 ± 2 years). We evaluated dementia with Lewy bodies core features, neuropsychiatric symptoms, caregiver burden (Zarit caregiver burden interview), MRI, apolipoprotein genotype, and cerebrospinal fluid biomarkers (tau/AβParkinsonism was the most frequently present core feature in MCI‐LB (69%). MCI‐LB patients more often had neuropsychiatric symptoms and scored higher on ZARIT when compared with the MCI‐AD patients. Linear mixed models showed that at baseline, MCI‐LB patients performed worse on nonmemory cognitive domains, whereas memory performance was worse in MCI‐AD patients. Over time, MCI‐LB patients declined faster on attention, whereas MCI‐AD patients declined faster on the Mini Mental State Examination and memory. Cox proportional hazards regressions showed that in the MCI‐LB patients, lower attention  and more posterior cortical atrophy  predicted shorter time to dementia. In the MCI‐AD patients, worse performance on memory  and executive functions  were independently associated with time to Alzheimer's dementia.Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.MCI‐LB patients have distinct neuropsychiatric and cognitive profiles with prominent decline in attention when compared with MCI‐AD patients. Our results highlight the importance of early diagnosis because symptoms already have an impact in the prodromal stages. © 2020 The Authors.  Dementia with Lewy bodies (DLB) is the second most common form of neurodegenerative dementia in the elderly.Some cross‐sectional studies suggest that the cognitive profile of MCI‐LB differs from MCI‐AD, with less pronounced memory impairment,123I‐FP‐CIT single‐photon emission computed tomography (SPECT) imaging, and EEG, can have prognostic value in the MCI stage.Only a few longitudinal studies on MCI‐LB have been described. One study found that nonamnestic MCI patients most likely develop DLB, whereas amnestic MCI patients likely develop AD.These former studies in MCI‐LB are limited by small sample sizes and/or cross‐sectional designs, therefore prognosis and disease course remain speculative at best. Here, we examined clinical characteristics, cognitive decline, and predictors of time to progression in MCI‐LB compared with MCI‐AD.We included 73 MCI‐LB patients and 124 MCI‐AD patients from the Amsterdam Dementia Cohort. All patients visited the memory clinic and underwent a 1‐day standardized diagnostic work‐up that included a semistructured medical history interview, informant‐based history, neurological and medical examinations, neuropsychological assessment, brain MRI, standard laboratory work‐up, and lumbar puncture.123I‐FP‐CIT SPECT (dopamine transporter (DAT)–SPECT; n = 6, 8%), and/or clinical diagnosis of probable DLB during follow‐up . DAT–SPECT imaging was available in 47 MCI‐LB patients, of which 46 patients (98%) showed abnormal tracer uptake. MRI, cerebrospinal fluid (CSF) AD biomarkers (tau/Aβ1–42 ratio) and apolipoprotein (APOE)‐e4 status was available in most MCI‐LB patients . Follow‐up neuropsychological assessment was available for 61 MCI‐LB patients (84%), and the average follow‐up time was 3.0 ± 2.0 years.MCI is diagnosed according to the current diagnostic criteria, that is, concern reflecting changes in cognition, objective impairment in cognition, preservation of independence of functional abilities, and not demented.1‐42 < 813 pg/mL, tau >375pg/mL, p‐tau >52pg/mL, all abnormal). Patients had a last clinical diagnosis of MCI or probable AD at follow‐up. We selected patients who had CSF, MRI, APOE‐ε4 status, and at least a 1 year neuropsychological follow‐up available (average follow‐up time 3.0 ± 2.2 years). MCI‐AD patients were matched on age range with MCI‐LB patients. We used selection criteria for MCI‐LB and MCI‐AD that optimized diagnostic accuracy for both diseases. This resulted in selection criteria based on clinical features for MCI‐LB and selection criteria based on biomarkers for MCI‐AD.Inclusion criteria for MCI‐AD were MCI with abnormal AD CSF biomarkers .The Disability Assessment for Dementia is an informant‐rated questionnaire measuring the competence in instrumental activities in daily living (IADL), with scores ranging from 0% to 100%, and higher scores indicating higher competence.z scores using the baseline data of an independent group of cognitively healthy subjects . We created domain scores for attention, memory, executive function, language, and visuospatial functioning. Progression to dementia was defined as having impairment in 2 or more cognitive domains. Impairment in a cognitive domain was defined as having a domain score of z score < −2. All patients that progressed to dementia also fulfilled the clinical diagnosis for dementia, including interference in daily living.Cognition was assessed with a standardized test battery.1‐42 and total tau concentrations were determined using Innotest (enzyme‐linked immunosorbent assay (ELISA); Innotest, Fujirebio, Gent, Belgium) or Elecsys Aβ1–42, tau, and p‐tau (181P) CSF assays  run on the cobas e601 analyzer (Roche Diagnostics). Elecsys values of Aβ1–42 and tau were converted to Innotest values by using previously described formulas.1–42 > 0.52 to define concomitant AD pathology.CSF was obtained via lumbar puncture.MRI scanning was performed according to standardized protocol.The APOE‐ε4 genotype was determined using the LightCycler ApoE mutation Detection Kit (Roche Diagnostics) after DNA isolation from 10 mL ethylenediamine tetraacetic acid (EDTA) vacutainer tubes. Based on the APOE‐ε4 genotype, patients were classified as APOE‐ε4 carriers (heterozygous and homozygous) or noncarriers.Orthostatic hypotension was defined as a 20 mmHg drop in systolic blood pressure or a 10 mmHG drop in diastolic blood pressure between supine and standing positions.t tests, Mann‐Whitney U tests, and χ1–42 ratio, and MRI markers as putative predictors. Subsequently, we constructed a multivariate model (model 2) with the significant predictors of the univariate models using backward modeling. Cox proportional hazards analyses were performed separately for the MCI‐LB and MCI‐AD patients to provide a comparison of predictors. For visualization, we constructed Kaplan‐Meier curves of the significant predictors in MCI‐LB. We dichotomized the variables using predefined cut‐offs. When no cut‐offs were available, we used the split‐half approach to dichotomize the variables using the median value as a cut‐off point.Analyses were performed using Statistical Package for the Social Sciences  and R . To assess group differences at baseline, P < 0.001). Age, years of education, and disease duration did not differ between the MCI‐LB and MCI‐AD groups. No differences were found on IADL dependency. The caregiver‐rated NPI indicated more NPS in the MCI‐LB patients, in particular apathy, which was more frequently noted in the MCI‐LB patients compared with the MCI‐AD patients (74% vs. 46%). As expected, based on our selection criteria, hallucinations and sleep disturbances were more frequently reported by caregivers of MCI‐LB patients . There were no differences in MRI characteristics between groups. The MCI‐LB patients were less often APOE‐ε4 carriers when compared with the MCI‐AD patients. Of the MCI‐LB patients, 35% had CSF profiles compatible with AD at baseline, whereas by definition, all MCI‐AD patients had abnormal CSF biomarker profiles.Orthostatic hypotension was present in 43% of the MCI‐LB patients compared with 26% of the MCI‐AD patients  progressed to dementia during follow‐up after an average of 2.7 ± 1.7 years. In the MCI‐AD group, 85 of 124 patients (69%) progressed to dementia at follow‐up after 2.6 ± 1.7 years. The progression rate and time to dementia did not differ between the MCI‐LB and MCI‐AD patients.1–42 ratio, or APOE‐ε4. Subsequently, in the multivariate model, PCA and attention remained independent predictors of progression to dementia  were reported in roughly half of the MCI‐LB patients. Second, we found that the MCI‐LB patients had more NPS, in particular apathy and depressive symptoms, and that they had distinct cognitive profiles with prominent declines in attention when compared with the MCI‐AD patients. Finally, lower attentional function and more posterior cortical atrophy at first visit were independent predictors of time to dementia in MCI‐LB, whereas in MCI‐AD lower memory and executive functions were predictive of time to dementia.Consistent with previous studies in established DLB, we found more noncognitive symptoms and higher caregiver burden in MCI‐LB compared with the MCI‐AD.The cognitive profile in MCI‐LB differs from (MCI‐)AD, with less severe memory impairment and more executive and visuospatial impairment.The progression rate and time to dementia did not differ between MCI‐LB and MCI‐AD. One other study investigated the progression to dementia in MCI‐LB and MCI‐AD and found similar progression rates and time to dementia.In addition, cortical atrophy in MCI‐LB was associated with swifter progression to dementia. Specifically, higher GCA scores and higher PCA scores were more commonly seen in patients who progressed to DLB, and they were determinants of progression to dementia. The prognostic value of these atrophy scales seems specific for MCI‐LB patients, as we did not find any prognostic value of GCA or PCA for MCI‐AD patients. Recent studies showed that GCA and PCA can occur in DLB patients, independent of amyloid pathology, but are likely related to tau pathology and α‐synuclein pathology.Core clinical features and CSF AD biomarkers did not predispose for rapid progression to dementia. Neither the separate core clinical features nor the amount of core features that were present at first visit predicted time to dementia. In addition, although concomitant AD pathology has previously been related to worse prognosis in the dementia phase, CSF AD biomarkers did not predict time to dementia in MCI‐LB.There are several strengths in the present study, of which most notably the large and well‐defined cohort of MCI‐LB patients and the longitudinal design. Possible limitations entail the retrospective nature of the study. Missing values of core clinical features were rated retrospectively based on information in the medical charts. With this method, there is some risk of information and recall bias. Also, the retrospective design limits us to the use of rating core clinical features as being present or not, and the severity of symptoms is left out. Future prospective studies should use standardized methods to assess symptom severity. Second, we defined the moment of progression to dementia based on decline on cognitive tests, and functional decline was left out. It is important to note that our patients were followed up in a clinical setting and that clinicians diagnosed the patients with dementia as well, therefore the patients also fulfilled the criterion for interference in daily living based on clinical decision making. Yet the moment of progression was determined by cognitive scores, which could potentially introduce bias. For future studies, it would be interesting to look at progression in a broader sense by taking into account decline in IADL functioning or progression of NPS. Third, not all patients had sufficient follow‐up available, and therefore not all patients had progressed to dementia. It is possible that patients who are still in the MCI phase progress to a different type of dementia than expected based on our selection criteria. To accurately estimate prognosis, longer follow‐up duration is necessary.MCI‐LB could be insufficient in capturing patients in these earliest stages of DLB because it still focuses on cognitive impairment. A diagnosis of prodromal DLB should change to a classification that is not as much focused on cognitive staging. For the sake of both adequate patient management as well as shortening the time of insecurity for patients and their relatives, early diagnosis is mandatory. There is an urgent need for specific biomarkers, and a new set of diagnostic criteria should be considered in which cognitive impairment is not necessarily leading. Analogs could be found in diseases as Huntington's disease or progressive supranuclear palsy in which a certain constellation of symptoms can lead to a high probability diagnosis.Our findings underline the importance of timely diagnosis. Our results show that impairment in MCI‐LB is not restricted to cognition, but there is broad symptomatology that affects patients as well as caregivers. Some of these noncognitive symptoms occur even prior to the onset of cognitive symptoms. The term In summary, MCI‐LB patients have distinct symptoms, cognitive trajectories, and predictors of progression when compared with MCI‐AD patients. Further studies should elucidate whether early treatment has an impact on disease progression.(1) Research project: A. Conception, B. Organization, C. Execution; (2) Statistical Analysis: A. Design, B. Execution, C. Review and Critique; (3) Manuscript: A. Writing of the first draft, B. Review and Critique.M.v.d.B.: 1A, 1B, 1C, 2A, 2B, 2C, 3AI.v.S.: 1A, 1B, 1C, 2C, 3BJ.v.d.Z.: 2C, 3BF.B.: 2C, 3BC.T.: 2C, 3BW.v.d.F.: 1C, 2C, 3BA.L.: 1C, 2C, 3BDr. Barkhof is supported by the NIHR biomedical research centre at UCLH. Research of Dr. Teunissen is supported by the European Commission , Health Holland, the Dutch Research Council (ZonMW), The WestonBrain Institute, Alzheimer Netherlands. Dr. Teunissen has a collaboration contract with ADx Neurosciences, performedcontract research or received grants from Probiodrug, AC Immune, Biogen‐Esai,CogRx, Toyama, Janssen prevention center, Boehringer, AxonNeurosciences,Fujirebio, EIP farma, PeopleBio, Roche. Research programs of Dr. van der Flier have been funded by ZonMW, NWO,EU‐JPND, Alzheimer Nederland, CardioVascular Onderzoek Nederland, Health~Holland, Topsector Life Sciences & Health, stichting Dioraphte, Gieskes‐Strijbisfonds, stichting Equilibrio, Biogen MA Inc, Life‐MI, AVID, Combinostics. Dr. van der Flier holds the Pasman chair. Dr. van der Flier has performed contract research for Biogen MA Inc. All funding is paid to her insitution. Dr. Lemstra has received funding from stichting Dioraphte and ZonMW Memorabel (project#733050509).The remaining authors report no actual or potential financial disclosures.Supplementary Table 1 Estimated baseline cognition and change over timeClick here for additional data file.Supplementary Figure 1 Number of core features present in MCI‐LB (n = 73).Click here for additional data file.


The objective was to study the effect of hydroxychloride trace minerals (HTM) on growth performance, carcass quality and gut microbiota of broiler chickens in comparison to sulphate trace minerals (STM). In total 1440 male Ross 308 day-old chicks were divided into 12 replicate pens with 30 birds each per treatment. Four different treatments were tested according to a 2 × 2 factorial study design, where the animals received a three phase diet containing either inorganic Zn from sulphates or Zn from HTM in high (80 mg/kg Zn) or low Zn dosage (20 mg/kg Zn). In all treatments 15 mg/kg Cu was added from the same mineral source as the Zn. Body weight and feed intake were measured on day 0, 10, 27 and 34, while carcass and breast meat yields were measured at the end of the study (day 34). In addition, high-throughput sequencing analysis was performed in digesta samples from ileum and cecum to study the gut microbiome (day 34).P < 0.05) body weight of broiler chickens fed HTM, regardless of Zn level, on day 27, while on day 34 this effect remained as a tendency (P = 0.0542). In the overall study period, birds fed HTM had a higher (P < 0.05) average daily gain and average daily feed intake when compared to birds fed STM. The mineral source did not affect the carcass characteristics, however, feeding 80 mg/kg Zn resulted in a significantly higher (P = 0.0171) breast meat yield, regardless of source. High-throughput sequencing analysis of the microbiota revealed a higher microbial diversity in the ileum and cecum of HTM fed birds compared to STM fed birds. Taxonomical differences were mainly found in the cecum, specifically between the group fed high and low Zn levels from HTM. This correlated with the mineral contents observed in the cecal digesta. Comparing both groups fed 80 mg/kg Zn, the HTM group had more Streptococcaceae, Streptococcus, Clostridia, Weissella and Leuconostocaceae compared to the STM group.The results showed an improved (HTM improved growth performance of broiler chickens; and the source and level of Zn modulated the gut microbiota communities in broilers differentially. Zinc is an essential trace mineral for broiler chickens. Zinc has been proven to play a role in immunity, enzymatic processes and eventually in growth , 2. Ther2/bird with wood shavings litter. Each group was randomly allocated to one of four treatments with 12 replicate pens each. The four treatments were applied according to a 2 × 2 factorial study design, in which two mineral sources of zinc (STM and HTM) were tested at two different zinc levels (high: 80 mg/kg and low: 20 mg/kg), whereas to all diets 15 mg/kg Cu was added from the same mineral source as the Zn. Sulphate trace minerals source were added to the feed in the form of Zn sulphate , and Cu sulphate (copper(II) sulphate pentahydrate, Sigma-Aldrich, Saint Louis, MO, USA). Hydroxychloride trace minerals were added at the same Zn  and Cu  levels as for the sulphate minerals. The composition of the diets is shown in Table In total, 1440 newly hatched male Ross 308 broiler chicks were housed at the Poultry Research Facility of Trouw Nutrition, Casarrubios del Monte, Spain. The birds were randomly allocated into groups of 30 animals per floor pen (3 m × 1 m) with 6 and 18 h of light/dark periods. Each pen provided 0.10 mAt the end of the experiment, 6 animals per pen were selected based on average body weight and euthanized by cervical dislocation according to the guidelines of the Ethics Committee of Poultry Research Centre of Trouw Nutrition for the humane care and use of animals in research. Animal procedures were verified by the External Ethical Committee of “Hospital General Universitario de Ciudad Real”, and approved by the Junta de Castilla-La Mancha Animal Welfare department as compliable with the RD 53/2013 of the 1st of February which establishes basic rules applied to protect animals used for research, and other scientific matters like teaching. The present study was approved by the Trouw Nutrition Animal Care Committee and followed recommendations of the Castilla-La Mancha Animal Welfare Department , in compliance with the European Union guidelines for the care and use of animals in research . From the six birds selected per pen, five birds were used for carcass and breast meat yields and the remaining one was sampled for both the microbiota and mineral content. Samples were collected aseptically by opening the abdominal cavity with a sterile blade, the intestinal content (digesta) from the ileum and cecum was placed into a sterile tube until further analysis. All samples were immediately snap frozen on dry ice and then transferred to a − 80 °C freezer.Body weight and feed intake was measured per pen at the start of the experiment at day 0 and at the end of each feeding phase at day 10, 27 and 34. The body weight and feed intake measures were used to calculate the average daily gain, the average daily feed intake and the feed conversion ratio (feed:gain). On day 34, the carcasses were weighted and both breast muscles were excised and weighted by qualified personnel. These weights were used to calculate carcass and breast yield, as the percentage of the live weight or the carcass weight, respectively.Zinc and copper content in the diets and in 1 ileal and 1 cecal digesta samples  per pen, resulting in 12 ileal and 12 cecal samples per treatment were analysed according to ISO (ANA-00281) using inductively coupled plasma-mass spectroscopy (ICP-MS)  with an autosampler coupled , Omaha, NE, USA). The chemicals used were nitric acid (67–69%) and hydrochloric acid (34–35%) of ultra pure grade for trace mineral analysis .From the ileal and cecal digesta 200 mg of samples were lysated with MagNA Lyser  prior to DNA extraction. DNA extraction was performed with PowerMicrobiome™ RNA isolation kit  following the manufacturer’s instructions with some modifications, such as omitting the β-mercaptoethanol and DNase I steps. The concentration of the extracted prokaryotic DNA in each sample was calculated by qPCR with 926F  and 1027Raw fastq files were imported, demultiplexed and processed using QIIME 2 (version 2018.6) . Paired-P < 0.05.The pen was the experimental unit for all parameters. Growth performance data were analysed according to the 2 × 2 factorial design using the MIXED procedure in SAS software . The model included source and level and feeding phase as fixed effects. The different time points were included as repeated measures. The carcass and breast meat yield were subjected to the GLIMMIX procedure in SAS. Post hoc analysis was done using Tukey. R in QIIME2 (version 2018.6) was used to analyse alpha diversity with Kruskal-Wallis and Spearman for correlations, beta diversity with PERMANOVA and ANOSIM with a sequencing depth of 234,000 and 175,000 sequences for ileum and cecum, respectively. Taxonomy data was analysed with LEfSE [P = 0.0229) higher body weight than birds fed STM, regardless of mineral levels. This continued until day 34 as a strong trend (P = 0.0542) towards an increased body weight in birds fed HTM compared to birds fed STM. Average daily gain (ADG) and average daily feed intake (ADFI) followed the same patterns as body weight throughout the study. In this case, between 10 and 27 days a tendency was observed towards an increased ADG (P = 0.0738) and ADFI (P = 0.0846) for birds fed HTM compared to birds fed STM. However, this tendency resulted in a significantly improved ADG (P = 0.0130) and ADFI (P = 0.0125) over the entire period for HTM fed birds compared to STM fed birds, regardless of mineral level. As a result, the feed conversion ratio (FCR) was not significantly different among treatments.Growth performance results are shown in Table P = 0.0171), while no significant differences were found between the sources or any source×level interaction.The effect of the treatments on carcass and breast meat yield were measured at the end of the study Table . No signP = 0.004). The Zn content in the ileum was proportional to the dietary Zn level, where birds fed a high dietary Zn presented significantly higher Zn than in birds fed low Zn (P < 0.0001). In the cecum, the Cu level remained similar across the treatments, while Zn content depended significantly on the supplemented levels (P < 0.0001). Surprisingly, there was a significant source×level interaction where birds fed 80 and 20 mg/kg Zn from HTM showed the highest and the lowest cecum values, respectively (P = 0.0472).The mineral content in the digesta from ileum and cecum is shown in Table plateau . Rarefactions curves of observed OTUs showed a P = 0.020) and PERMANOVA (P = 0.014) of unweighted unifrac and a trend in jaccard (P = 0.080), between microbial communities in the ileum of birds fed 80 mg/kg Zn from HTM to birds fed 20 mg/kg from HTM. The STM fed birds showed intermediate values not differing from any of the other treatments tested. The number of observed OTUs of alpha diversity was significantly higher in the group fed 80 mg/kg Zn from HTM (50 ± 14.1) than 20 mg/kg Zn from HTM (39 ± 8.3) (P = 0.049), as well as the phylogenetic distances, whereas both STM groups were in-between (Table P = 0.093) (data not shown). The taxonomy at phyla level (data not shown) showed Firmicutes as main phyla (99.8%), followed by Actinobacteria (0.15%), Proteobacteria (0.08%) and Patescibacteria (0.01%). Actinobacteria phyla was significantly higher in group fed 80 mg/kg Zn from HTM (0.34%) in comparison with the group fed 20 mg/kg Zn from HTM (0.03%) (P = 0.043). At genus level, the main organism present in the ileum was Lactobacillus (> 88%)  representing 100 OTUs. Beta diversity ordination represented by redundancy analysis  and Streptococcus (P = 0.042), in comparison to the diet containing 20 mg/kg Zn from STM, which in turn presented higher Lactobacillus than 80 mg/kg Zn from HTM (P = 0.010). The group fed 80 mg/kg Zn from HTM also presented higher Bifidobacterium (P = 0.018), Blautia (P = 0.034), Streptococcus (P = 0.043), Weissella (P = 0.039), Eubacterium hallii group (P = 0.033), Ruminococcus torques g (P = 0.043) than the group fed 20 mg/kg Zn from HTM (Table Lactobacillus (20 mg/kg) and in Streptococcus and Enterococcus (80 mg/kg), mainly because of the high influence of the group fed 80 mg/kg Zn from HTM. No significant difference was found by the overall source effect, regardless of Zn level.Differences in the remaining genera in the ileum are shown in Table TM Table . Dosage P = 0.019 and P = 0.027, respectively), promoted by significant differences between both groups fed 20 mg/kg Zn . Feeding 20 mg/kg Zn from HTM also presented marked differences in the ANOSIM and PERMANOVA of unweighted unifrac distances in comparison with feeding 80 mg/kg Zn from HTM  or from STM . The alpha diversity confirmed the differences found in the ordination of the bacterial communities (see Table P = 0.066), with the group fed 80 mg/kg Zn from STM having the lowest diversity in comparison with the group fed 20 mg/kg Zn from HTM (P = 0.049). The observed species in the cecum tended to be higher in birds fed 20 mg/kg Zn from HTM compared to the birds fed 80 mg/kg Zn from STM (P = 0.067). The evenness showed a trend towards an increased (P = 0.099) richness in the HTM groups compared to the STM groups, regardless of Zn level. Taking the phylogenetic distances into account, the Faith_PD parameter did show a trend towards a different community in the groups fed 20 mg/kg Zn compared to the groups fed 80 mg/kg Zn (P = 0.070), and specifically between 20 mg/kg Zn from HTM and 80 mg/kg Zn from STM (P = 0.024) (Table P = 0.0322), evenness (P = 0.046) or observed species  in the cecum. Also the ADFI and ADG performance indexes tended to be positively correlated to the richness or evenness of the microbiota  (data not shown).In the cecum, 17,250,171 sequences were recovered  representing 644 OTUs. Beta diversity ordination represented by redundancy analysis , Actinobacteria (9.5%), Bacteroidetes (3.1%), and in less extend Proteobacteria (0.06%), Tenericutes (0.05%) and Cyanobacteria (0.01%). On family level, the main taxa present in the cecum were the Lachnospiraceae, Ruminococcaceae, Lactobacillaceae and Bifidobacteriaceae.Lactobacillus, Faecalibacterium, unknown genus from Lachnospiraceae family, Bifidobacterium, several Ruminococcus groups, Butyricoccus, Blautia and Alistipes , Eubacterium brachy group, Pygmaiobacter, Peptococcaceae_other, whereas the group fed 80 mg/kg Zn from HTM showed significantly higher relative amounts of vadin BB60 group (Table Anaerotruncus level, Tyzzerella 3, Family XIII UCG-001, and Brachybacterium, and the group fed 80 mg/kg Zn from STM the highest CHKCI002 (Table Anaerostipes, Pygmaiobacter, unknown genus from Peptococcaceae family, Christensenellaceae R-7 group and family XIII in comparison with 80 mg/kg when analysed using the LEfSe approach [Streptococcus and Enteroccocaceae family, and STM groups of family vadin BB60 group and unknown genus from Christensenellaceae family.At genus level, the main organisms present in the cecum were es Table . Being 8approach . This reThe current study tested the effect of STM compared to HTM on the growth performance, carcass characteristics and gut microbiota of broiler chickens. In this comparison two different Zn levels from STM were compared to the same levels of Zn from HTM. In all diets the same level of 15 mg/kg added Cu was used in the same source as the Zn. Due to the combined use of Zn and Cu of each source, the observed effects can not directly be linked to either Zn or Cu. Vieira  describeThis study showed a beneficial effect of HTM on growth performance, specifically on body weight and average daily gain. The breast meat yield was not influenced by mineral source, however only by Zn level with higher Zn resulting in a higher breast meat yield. This is in contrast to the differences reported by Olukosi et al. , who desLactobacillus. This is in line with the study of Munyaka et al. [Lactobacillus to be the most abundant species in the ileum of birds fed high fiber diets. In the cecum the main species on family level were Lachnospiraceae, Ruminococcaceae, Lactobacillaceae and Bifidobacteriaceae. This is in line with Munyaka et al. [Generally, the main organism present in the ileum was a et al. , who alsa et al. , who obsa et al. .The current study shows indications for an improved richness and evenness in the diversity of cecal microbiota in birds fed HTM. In the literature, Zn is described to be important for microbiota diversity, since chronic Zn deficiency is related to a reduced species richness and diversity in the cecum . MoreoveBlautia, a fiber degrading organism, in ileal digesta of birds fed 80 mg/kg Zn from HTM may support this hypothesis. It is unknown whether the other different species observed in the current study may be responsible for the difference in growth performance, however the positive correlation of carcass yield and ADFI trend with Shannon and evenness indexes could support this fact.A difference in microbiota may also be related to the growth performance of broiler chickens. Stanley et al.  showed aStreptococcus species have been found in the cecum of broiler chickens [Weissella, which was increased in the ileum of birds fed 80 mg/kg Zn from HTM, it is known that Zn has a promoting effect on it. Vahjen et al. [Weissella in the ileal digesta of pigs fed high levels of ZnO, showing that high Zn may increase the presence of this bacteria in the intestinal content.Change in specific species of the microbiota may not always lead to a clear conclusion. For example, Clostridia are commonly found in the cecum of chickens, as shown both in laying hens and broilers , 30. Simchickens , and depn et al.  observedInterestingly, most of the differences were observed between the groups fed 80 or 20 mg/kg Zn from HTM, whereas the STM groups reached values in between. This might suggest that HTM is able to modulate the gut microbiota in both sections of the intestine studied.Streptococcus and two unidentified genus from vadin BB60 family and less CHKCI002 from Eggerthellaceae family, than the STM group. Therefore, results from this study show that HTM can clearly modulate the microbiota, increase bacterial diversity in the GIT and potentially increase the growth performance in chickens.Birds fed diets containing Cu and Zn from HTM performed better than birds fed minerals from STM. In the overall study, the ADG and ADFI were significantly higher in the HTM fed birds. Also a tendency towards an increased body weight was observed in HTM fed birds. This did not result in differences in carcass yield and breast meat yield. The microbial diversity in the ileum and cecum were higher in the HTM fed birds compared to the STM fed birds. Main differences in the bacterial composition were found in the cecum, especially between the group fed 80 mg/kg Zn from HTM and 20 mg/kg Zn from HTM. These were the two groups with the highest contrast in Zn levels in the cecum. Further, the main taxonomical differences found in the cecum were in between the two groups fed 80 mg/kg Zn, where the HTM group had more


Common types of musculoskeletal conditions include pain in the neck and shoulder areas. This study seeks to identify the genetic variants associated with neck or shoulder pain based on a genome-wide association approach using 203 309 subjects from the UK Biobank cohort and look for replication evidence from the Generation Scotland: Scottish Family Health Study (GS:SFHS) and TwinsUK.A genome-wide association study was performed adjusting for age, sex, BMI and nine population principal components. Significant and independent genetic variants were then sent to GS:SFHS and TwinsUK for replication.P = 1.66 × 10−11. The second most significant locus was located in the FOXP2 gene in chromosome 7 with P = 2.38 × 10−10 for rs34291892. The third locus was located in the LINC01572 gene in chromosome 16 with P = 4.50 × 10−8 for rs62053992. In the replication stage, among four significant and independent genetic variants, rs2049604 in the FOXP2 gene and rs62053992 in the LINC01572 gene were weakly replicated in GS:SFHS .We identified three genetic loci that were associated with neck or shoulder pain in the UK Biobank samples. The most significant locus was in an intergenic region in chromosome 17, rs12453010, having We have identified three loci associated with neck or shoulder pain in the UK Biobank cohort, two of which were weakly supported in a replication cohort. Further evidence is needed to confirm their roles in neck or shoulder pain. Musculoskeletal pain in the neck and shoulder areas is a major health problem for adults of working age as well as for elderly populations . Neck anEpidemiological studies have suggested that the prevalence of neck pain is 5–8% and 13% for shoulder pain . The GloGenetic studies have identified genes associated with neck or shoulder pain. Twin studies have shown that there is a genetic role in neck pain , though This study seeks to identify the genetic variants associated with neck or shoulder pain based on a GWAS approach in a cohort of 203 309 subjects from the UK Biobank cohort and to test significant results for replication in the Generation Scotland: Scottish Family Health Study (GS:SFHS) and TwinsUK. Similar approaches have been used to examine back pain, knee pain and headaches in the UK Biobank cohort .P < 0.001) between cases and controls. Three genetic loci including four significant and independent SNPs reached a GWAS significance of P < 5 × 10−8 (P = 1.66 × 10−10). The second locus was found in the FOXP2 gene located in chromosome 7, and the most significant SNP from this locus was rs34291892 (P = 2.38 × 10−10). The third locus was the LINC01572 gene located in chromosome 16, and the most significant SNP in this locus was rs62053992 (P = 4.50 × 10−10). The SNP heritability  from genome-wide complex trait analysis (GCTA) was 0.11 ± 0.017.In the UK Biobank, 775 252 responses to all options were received for the specific pain question. Of the 501 708 participants in the study, 123 061 participants reported having experienced activity limiting pain in the neck or shoulder in the previous month. 213 408 participants chose the ‘None of the above’ option which meant that they did not have activity limiting pain anywhere in the previous month. To create a homogeneous dataset, we first removed samples according to their ancestry information. In addition, those who were related to one or more others in the cohort  and those who failed quality control were also removed. The final number of those included in the case group after the above exclusions was 53 994 . 149 312  individuals were included in the control group. After single-nucleotide polymorphism (SNP) quality control, there were 9 304 965 SNPs available for GWAS analysis. Clinical characteristics of the case and control groups were compiled . Age, se5 × 10−8 , Table 2forkhead box protein P2 (FOXP2) gene and rs62053992 in the Long Intergenic Non-Protein Coding RNA 1572 (LINC01572) gene were replicated weakly  (P > 0.05). The clinical characteristics of the GS:SFHS and TwinsUK cohorts are summarized in These four significant and independent SNPs were tested for replication in GS:SFHS and TwinsUK. Among 20 032 subjects in the GS:SFHS subjects, 19 598 had complete relevant information. The whole-genome fastGWA results did not find any SNPs with GWAS significance associated with neck or shoulder pain. . Among tctively) . Of the Meta-analysis of the four significant and independent hits, combining UK Biobank, GS:SFHS and Twins UK found that the significance of their associations was increased .FOXP2 (P = 1.62 × 10−11), which is located in chromosome 7.In gene-based association analysis by MAGMA, 26 genes were found to be associated with neck or shoulder pain, all of which are represented in (P < 4.7 × 10−6 (0.05/10651)).In gene-set analysis conducted by MAGMA, 10651 gene-sets were analyzed using the default competitive test model. None of these gene-sets met genome-wide significance P = 9.53 × 10−5). Only expression in brain and pituitary tissue reached significant values of P < 1.67 × 10−3 (0.05/30). Tissue expression analysis of 53 specific tissue types by GTEx found expression in the nucleus accumbens of the basal ganglia to be the most significant (P = 3.55 × 10−5). In addition, the top 6 significant associations were all from brain tissues.Tissue expression analysis was conducted by GTEx, and the relationship between tissue specific gene expression and genetic associations was tested by using the average gene expression in each tissue type as a covariate. Two analyses were carried out, one investigating 30 general tissue types  and the P = 3.41 × 10−30), followed by insomnia . It was also found that the age at which they have their first child had the most significant and negative genetic correlations with neck or shoulder pain , followed by college completion . All the phenotypes with significant genetic correlation with neck or shoulder pain are shown in The genetic correlation analysis using the LD hub (v1.9.0) showed that symptoms of depression had the largest significant and positive genetic correlation with neck or shoulder pain . They are the FOXP2 gene in chromosome 7, the LINC01582 gene in chromosome 16 and an intergenic area in chromosome 17. In the replication stage, the FOXP2 and LINC01582 loci were weakly supported by the GS:SFHS cohort but not by TwinsUK.We have performed a GWAS on neck or shoulder pain using the UK Biobank resource and found three loci that have reached genome-wide significance . The nearest protein coding gene is CA10, which encodes a protein belonging to the carbonic anhydrase family and is responsible for catalyzing the hydration of carbon dioxide. It is also thought to contribute to central nervous system development particularly in the brain (syndrome . Howeverhe brain .N = 3982) was too small to replicate the loci for such a heterogeneous phenotype as neck or shoulder pain. In particular, for the 4 SNPs chosen for replication, the power to achieve a P value < 0.05 in the given TwinsUK sample ranged between 11.6 and 15.8% , greater than knee pain (0.08), multisite chronic pain (0.10), and less than hip pain (0.12), stomach or abdominal pain (0.14), headache (0.21), facial pain 0.24), and pain all over the body (0.31) ,45. Furt, and paiThe genetic correlation analysis results were perhaps to be expected. Like knee pain and back pain, which have been shown to be positively correlated with depression and neuroticism , neck orThe primary limitation of this study is that different  case and control definitions were used in the discovery and replication cohorts. This was a consequence of the pre-determined phenotypic information that was present in the relevant cohorts. We defined neck or shoulder pain cases and controls based on the responses by UK Biobank participants to a specific pain question. This question focused on neck or shoulder pain occurrence during the previous month that was sufficient to cause interference with activity. The severity, frequency, and exact location of the neck or shoulder pain were not documented. Hence, our phenotyping should be considered as broadly defined. In the GS:SFHS and Twins UK cohorts, the disease status of participants was also self-reported: while cases were those who reported neck or shoulder pain over the previous 3 months; controls included those who reported neck or shoulder pain for less than 3 months, or pain of any duration in other body sites, as well as those reporting no pain. However, in the UK Biobank, controls were defined as pain free for the previous month. Differences between  case and control definitions could have a negative impact on the power of the replication study and the replication results while this impact is hard to evaluate. There could also be some cases who report neck or shoulder pain as a result of underlying causes such as cancer and osteoarthritis in the neck and shoulder areas. Their impact on the results would be very limited due to the small numbers. In addition, as the cases and controls in the UK Biobank were self-reported, there could be some bias, for example from social desirability, recall period, sampling approach, or selective recall . FurtherP < 5 × 10−8) associated with neck or shoulder pain in the UK Biobank dataset using a GWAS approach. Two of these loci were replicated weakly in the GS:SFHS cohort. Identification of these loci now provides a foundation for future work into understanding genetic roles and etiology in neck or shoulder pain.In summary, we have identified 3 loci of genome-wide significance (https://www.ukbiobank.ac.uk/) is a project facilitating research into health and disease and involves over 500 000 participants aged between 40 and 69 years old at recruitment. Participants completed a detailed questionnaire which examined lifestyle, demographic factors and clinical history. Participants also underwent clinical measures and baseline body measurements such as height and weight. Biological samples including urine, saliva, and blood were also provided. The National Research Ethics Service granted ethical approval to the UK Biobank (reference 11/NW/0382). The genetic information of 500 000 participants was released to approved researchers in March 2018. The corresponding author of this paper was granted access to the genetic information under UK Biobank application number 4844. Detailed quality control information pertaining to these genotypes was described by Bycroft et al  is a multi-institutional, family-based cohort involving over 20 000 volunteer participants aged between 18–98 years old at recruitment, who provided blood samples from which DNA was extracted. Similarly, participants also completed questionnaires to provide detailed phenotypic and sociodemographic information. Clinical and biochemical measurements were also collected for the purpose of research. Permission was obtained for linkage of research data to routine health data in the form of electronic health records ,49. EthiReplication cohort 2—The TwinsUK cohort is a UK nationwide registry of volunteer same sex twins. It has recruited 14 274 registered twins aged between 16 and 98 years. Collection of data and biological materials commenced in 1992 and is ongoing. During study participation, participants regularly complete health and lifestyle questionnaires and visit collaborating clinics and hospitals for clinical evaluation. Ethical approval was provided by the Research Ethics Committee at Guy’s and St. Thomas’ NHS Foundation Trust. TwinsUK has the genetic information relating to 6921 participants. Detailed quality control information pertaining to the genetic information was described by Moayyeri et al .Discovery cohort—UK Biobank: Participants were offered a pain-related questionnaire, which included the question: ‘in the last month have you experienced any of the following that interfered with your usual activities?’. The options were: 1. Headache; 2. Facial pain; 3. Neck or shoulder pain; 4. Back pain; 5. Stomach or abdominal pain; 6. Hip pain; 7. Knee pain; 8. Pain all over the body; 9. None of the above; 10. Prefer not to say. Participants could select more than one option. (UK Biobank Questionnaire field ID: 6159).In this study, cases were defined as participants who reported having activity limiting pain in the neck or shoulder in the past month (option 3), regardless of whether they reported pain in other regions. The controls were defined as participants who chose the ‘None of the above’ option.Replication cohort 1—GS:SFHS: Participants were first asked ‘Are you currently troubled by pain or discomfort, either all the time or on and off?’. If yes was selected, then the participants were asked ‘Have you had this pain or discomfort for more than 3 months?’. If yes was selected once again, then they were asked ‘Where is this pain or discomfort?’ with options of ‘Back pain’, ‘Neck or shoulder pain’, ‘Headache, facial or dental pain’, ‘Stomach ache or abdominal pain’, ‘Pain in your arms, hands, hips, legs or feet’, ‘Chest pain’, and ‘Other pain’. If a participant selected the ‘Neck or shoulder pain’, then he/she was defined as a case. All other subjects were defined as controls.Replication cohort 2: TwinsUK: participants were asked ‘In the past three months, have you had pain in your neck or shoulders?’ Those who answered ‘Yes’ were defined as cases. Those who answered ‘No’ were defined as controls. Those with missing answers were not included in the study.https://jmarchini.org/bgenie/) was used as the main GWAS software. SNPs with imputation INFO scores < 0.1, minor allele frequency (MAF) < 0.5% were removed, as well as SNPs that failed Hardy-Weinberg tests P < 1.0 × 10−6.Discovery cohort—UK Biobank: BGENIE  <0.6 with any other significant SNPs.BGENIE was used to perform association studies using linear association tests, adjusting for age, sex, BMI, 9 population principle components, genotyping arrays, and assessment centers. Chi-square testing was used to compare gender difference between cases and controls. T-tests were used to compare age and BMI between case and control groups using IBM SPSS 22 . As is standard in GWAS, SNP associations were considered significant when tability . Only sihttps://cnsgenomics.com/software/gcta/#fastGWA). SNPs with INFO scores < 0.3, or MAF <1% were removed, as well as SNPs that failed Hardy-Weinberg tests P < 1.0 × 10−6. FastGWA was used to perform association studies using a mixed-effects linear model adjusting for age, sex, BMI and nine population principal components. Relatedness was adjusted for via a genetic kinship matrix.Replication cohort 1—GS:SFHS: GCTA fastGWA1.92.4 was the main software used for replication (https://github.com/genetics-statistics/GEMMA). A mixed-effects linear model adjusting for age, sex, BMI and relatedness via a genetic kinship matrix was used.Replication cohort 2—TwinsUK: GEMMA v 0.98.1 was used for replication (https://genomics.ut.ee/en/tools/gwama).Meta-analysis of the significant and independent SNPs combining the UK Biobank, GS:SFHS and TwinsUK was performed using GWAMA 2.2.2  provided the results of tissue expression analysis.MAGMA v1.06 (integrated in FUMA) was used to perform gene-based association analysis and gene-set analysis, both of which were generated from GWAS summary statistics . For genhttp://ldsc.broadinstitute.org/ldhub/). Any P value less than 2.1 × 10−4 (0.05/234) was considered statistically significant by Bonferroni adjusted testing.Genetic correlation analysis was also performed to identify genetic correlation between neck or shoulder pain and 234 complex traits based on the online tool LD hub v1.9.0 (Supplementary_Figure_S1_ddaa058Click here for additional data file.Supplementary_Table_S1_ddaa058Click here for additional data file.Supplementary_Table_S2_ddaa058Click here for additional data file.


Potential differences between homosexual and heterosexual men have been studied on a diverse set of social and biological traits. Regarding acoustic features of speech, researchers have hypothesized a feminization of such characteristics in homosexual men, but previous investigations have so far produced mixed results. Moreover, most studies have been conducted with English-speaking populations, which calls for further cross-linguistic examinations. Lastly, no studies investigated so far the potential role of testosterone in the association between sexual orientation and speech acoustic features. To fill these gaps, we explored potential differences in acoustic features of speech between homosexual and heterosexual native French men and investigated whether the former showed a trend toward feminization by comparing theirs to that of heterosexual native French women. Lastly, we examined whether testosterone levels mediated the association between speech acoustic features and sexual orientation. We studied four sexually dimorphic acoustic features relevant for the qualification of feminine versus masculine voices: the fundamental frequency, its modulation, and two understudied acoustic features of speech, the harmonics-to-noise ratio  and the jitter . Results showed that homosexual men displayed significantly higher pitch modulation patterns and less breathy voices compared to heterosexual men, with values shifted toward those of heterosexual women. Lastly, testosterone levels did not influence any of the investigated acoustic features. Combined with the literature conducted in other languages, our findings bring new support for the feminization hypothesis and suggest that the feminization of some acoustic features could be shared across languages. The gender atypicality hypothesis suggests that gender atypical traits in homosexuals could be used as cues to indicate sexual orientation. Differences between heterosexual and homosexual individuals have thus been studied on a diverse set of traits such as face  and the jitter. Indeed, both components are sexually dimorphic as women exhibit significantly higher values of HNR  and lower values of jitter  than men . We investigated the effect of sexual orientation on four sexually dimorphic acoustic parameters  and examined whether homosexual men’s vocal characteristics showed a feminization by comparing theirs with that of heterosexual women. Lastly, we examined the potential role of testosterone in the association between speech acoustic features and sexual orientation.The French National Commission of Informatics and Liberties approved all protocols used in this study (CNIL number 1261003). Participants were recruited by means of flyers handed out as well as advertisements posted on public and private locations in the city of Montpellier, France. In order to recruit as much as possible homosexual males, we contacted the local LGBTQ community to help advertise the study as well as directly advertising it in known local gay bars. All participants gave a written consent prior to the study and were given a financial compensation for their participation. In total, 150 women and 180 men participated in the study. All participants completed a questionnaire assessing their sexual orientation , their nationality, age, relationship status (single vs. in relationship), socioeconomic status (level of education and monthly income) as well as country of birth of their parents and grandparents.Recordings took place in a quiet room in our laboratory at the University of Montpellier. All recordings took place between 2:00 p.m. and 5:00 p.m. Each participant heard the French version of the story “The North Wind and the Sun” from the International Phonetic Association and were asked to tell the story back to the research assistant. The rationale for using semi-spontaneous speech is that it is more ecologically valid than sustained vowels or read speech while controlling for semantic content, as the latter produce very different acoustic speech characteristics that do not represent how an individual vocally behaves in social interactions Laan, . Speech M ± SD = 26.18 ± 5.41 years), 58 homosexual men (26.38 ± 5.06 years), and 54 heterosexual women (24.85 ± 4.34 years).Because origin . Pitch floors were set to 75 Hz with a ceiling of 300 Hz for both heterosexual and homosexual men and 85–400 Hz for heterosexual women. All other settings were kept as default. For each participant, we extracted four acoustic parameters: mean fundamental frequency , its variations , the jitter (%), and the HNR (in dB).In total, we analyzed these 160 speech samples with the Praat© software  and a non-periodic component  comprising a segment of voiced speech  were measured in saliva samples (pg/ml). This noninvasive technique has been previously validated and yields T-levels that are highly correlated with serum levels Ellison, . At the In order to examine the potential influence of T-levels and sexual orientation on men’s speech, we performed four linear models, one for each acoustic parameter studied. Each acoustic parameter was used as a response variable. To investigate the effects of sexual orientation and test the hypothesis of feminization on these vocal features, we used an explanatory variable called “SexOr” that considers both sex and sexual orientation with three modalities: heterosexual men, homosexual men, and heterosexual women. T-level was added as another explanatory variable. To investigate whether T-level mediates the association between acoustic features and sexual orientation, we also added the interaction between T-level and “SexOr.” When the interaction was not statistically significant, we removed it from the linear models. Age, monthly income, level of education, and relationship status were added as confounding variables. All continuous variables  were standardized. Then, to assess if homosexual men displayed vocal features with values shifted toward those of heterosexual women, post hoc analyses (Tukey HSD tests) were performed to compare which category  differ from one another. Thresholds of significance were corrected for the number of models and post hoc comparisons using the Bonferroni method.In order to assess the overall difference on speech acoustic features between heterosexual and homosexual men and to examine whether homosexual men’s vocal features are shifted toward those of women, we conducted a linear discriminant analysis (LDA). LDA attempts to model whether a set of variables  is effective in predicting category membership . In other words, we used a LDA to find the linear combinations of the four acoustic features that gives the more accurate separation between the three categories of participants. Then, individuals’ coordinates were computed from the two linear discriminant functions. Those coordinates were used to produce a continuous axis of vocal femininity and masculinity to determine where homosexual men were positioned within this axis. The coordinates of the three groups were then entered in linear models followed by post hoc comparisons (Tukey HSD tests) to assess the overall difference in acoustic speech features.All statistical analyses were performed under the R software (version 3.1.2).Descriptive statistics of all acoustic parameters and T-levels are shown in Table F = 2.31, p = .10), F0-SD  = 0.07, p = .93), jitter  = 0.24, p = .78), and HNR  = 0.22, p = .79). These interactions were thus subsequently removed from the linear models. “SexOr” showed a significant effect on mean F0  = 225.07, p < .001, Table F= 95.94, p < .001, Table F = 13.59, p < .001, Table F = 55.64, p < .001, Table p < .01). In addition, homosexual men displayed a significantly higher F0-SD and HNR than heterosexual men (respectively t(157) = − 4.48, p < .001; t(157) = − 2.97, p < .01), with values shifted toward those of heterosexual women (Tables t(157) = 0.51, p = .86; t(157) = 1.79, p = .17). Age had a significant positive effect on F0-SD and jitter  = 7.82, p < .01; F = 10.82, p < .01, Tables p > .05, Tables The interactions between T-level and “SexOr” did not have a significant effect on mean F0  = 0.08, p = .77), the overall acoustic difference between the three groups was assessed using the coordinates of the first function. Post hoc comparisons revealed significant differences between all three groups: heterosexual and homosexual men  = − 3.69, p < .001), heterosexual men and heterosexual women  = − 33.78, p < .001), and homosexual men and heterosexual women  = − 33.65, p < .001). Homosexual men showed a total of 10.65% differences in overall speech acoustic features compared to heterosexual men, slightly but significantly shifting toward those of heterosexual women . Secondly, our findings showed that French homosexual men displayed a more modulated and less breathy voice than French heterosexual men, thus supporting and extending previous studies conducted mostly with English speakers. Our results for the LDA showed that French homosexual men attested a slight but significant vocal feminization when considering speech acoustic features altogether (up to 10.65%), which support the feminization hypothesis.  Lastly, testosterone levels did not mediate the association between vocal patterns and sexual orientation.Consistent with previous findings in English-speaking populations, no significant differences were observed in mean F0 between French-speaking heterosexual and homosexual men Gaudio, . The resTo our knowledge, this is the first study to report an association between men’s vocal breathiness and sexual orientation. Interestingly, vocal breathiness has been suggested to be an important component of vocal femininity in female voices , our results indicated that some vocal traits differed between heterosexual and homosexual men  with values shifted toward heterosexual women’s vocal characteristics. Combined with the literature conducted in other languages, our findings bring new support for the feminization hypothesis (at least for some acoustic features) and suggest that the feminization of some acoustic features could be shared across languages. Further studies are needed to test whether intonation and vocal breathiness are perceptually salient to distinguish homosexual and heterosexual men, and whether overall differences are due to biological and/or sociolinguistic reasons.


Dear editor,We read with great interest the brief report by Xu and colleagues  about th2 < 93% despite supplemental oxygen (rather than PFR) as an indication to trial PP [2/FiO2 (and removing the need for PEEP) may allow COVID-19-related ARDS to be diagnosed in patients on HFNC or face-mask oxygen without the need for NIV or blood gases. This may have advantages in healthcare facilities with more limited resources. We suggest considering awake PP in patients with SpO2 > 94% requiring either 0.3–0.6 FiO2 (with HFNC or NIV) or an oxygen flow rate of 2–10 L/min . These oxygen requirements correspond to an SpO2/FiO2 range of 140–315 which approximates a PFR 100–300 mmHg, indicating mild to moderate ARDS [First, which criteria should we use to identify appropriate candidates for awake PP in COVID-19? Xu et al. used PFR < 300 mmHg as their only criteria. Other studies have not used blood gas analysis. A study of 50 COVID-19 patients treated with awake PP used SpOSecond, what is the optimal duration of awake PP? In the report by Xu et al. , the tar2 may fall temporarily and a judgement must be made whether to continue. A sustained PFR < 100 mmHg or SFR < 140 mmHg after PP may indicate need for intubation [Finally, how should we monitor for failure of awake PP and avoid delay in intubation? Xu et al.  do not dtubation .In summary, a protocol for an early recognition and better monitoring of awake PP in this population is proposed in Fig. a, Weihua LuaQiancheng XuaDepartment of Critical Care Medicine, The First Affiliated Hospital of Wannan Medical College , No.2, West road of Zheshan, Jinghu District, Wuhu, Anhui, 241000, ChinaDear editor,Critical Care [We would like to thank Dr. Bower for their insightful comments on our paper recently published in cal Care . Studiescal Care . Prone p2 is greatly affected by FiO2. Therefore, PP may be delayed if SpO2 and RR are used as criteria. In our study, a P/F ratio ≤ 300 mmHg was used as the only criterion, whereas SpO2 and respiratory rate were used as reference indicators. We also agree with Dr. Bower’s view that this approach may require additional resources.In clinical cases, many patients with respiratory failure/hypoxaemia do not present symptoms of dyspnoea (silent hypoxaemia), especially elderly patients , and the2 as the target of oxygen therapy rather than the P/F ratio, which reflects the degree of lung injury. There was a patient with a P/F ratio ≤ 100 mmHg who avoided intubation in our study. Siemieniuk et al. [2 range of 90–94% seems reasonable for most patients. A previous programme proposed a COVID-19 SpO2 maintenance target in non-pregnant adult patients ≥ 90% and 92–95% for pregnant patients [2 less than or equal to 90% at an FiO2 of 100% for at least 5 min or SpO2 > 90% but with high respiratory load, respiratory acidosis, haemodynamic instability, multiple organ dysfunction, and mental disorders.For the timing of conversion to invasive ventilation, since the therapy is applied to avoid organ damage secondary to hypoxaemia, we focused on SpOk et al.  recommenThe specific duration of the PP should be determined with consideration of the local medical resources and the patient’s condition. Each of our treatment units consisted of a nurse, a respiratory therapist, and a psychotherapist, so they could closely monitor conditions that can prolong the prone position. However, there were difficulties in healthcare facilities without sufficient staff.Finally, comprehensive treatment of COVID-19 may be more important. Our treatments could include convalescent plasma, tocilizumab, and anticoagulant therapy to reduce the duration of virus replication and respiratory support. Therefore, early awake PP can effectively prevent hypoxaemia in patients with severe COVID-19.