The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function. The full extent of the genetic basis for hearing impairment is unknown. Here, as part of the International Mouse Phenotyping Consortium, the authors perform a hearing loss screen in 3006 mouse knockout strains and identify 52 new candidate genes for genetic hearing loss. To date, around two-thirds of the genes for non-syndromic hearing loss loci are known4, and, while the causative genes for several of the well-known hearing loss syndromes have been identified5, the vast majority of genes underlying syndromes with hearing loss are unknown. As such, we are far from having a complete understanding of the genetics underlying hearing function. Importantly, we do not know the extent of the mammalian gene set that impacts auditory function. Mouse genetics has played an important role in our understanding of the development and functioning of the mammalian auditory system7. Utilising forward-genetic screens, many hearing loss mouse mutants have been identified9, and characterisation of these has enabled genes critical for hearing function to be elucidated. Allied with gene-driven approaches, mouse mutants continue to help identify the molecular mechanisms and physiological bases of human hearing impairment. Moreover, the availability of a disease model that allows for the study of pathological changes occurring within the cochlea is invaluable for understanding disease onset and progression.Hearing impairment is the most common sensory deficit in the human population. According to figures from the World Health Organisation, there are currently around 360 million people worldwide living with mild to profound hearing loss. Approximately half of these cases have a genetic basis, with the hearing loss either occurring as an isolated condition  or presenting with additional phenotypes . Around 150 non-syndromic loci have been identified in humans (hereditaryhearingloss.org) and over 400 genetic syndromes are known that include hearing losswww.mousephenotype.org)11. Until recently, IMPC has generated mouse mutants using principally the International Knockout Mouse Consortium (IKMC) tm1b null allele . Homozygous viable mutants, or heterozygotes in the case of homozygous lethal lines, enter an extensive adult phenotyping pipeline that includes a range of phenotyping tests generating broad-based phenotype information across diverse physiological systems, including hearing10. To date, the IMPC has generated over 5800 genotype confirmed mutant strains and of these, 3006 have been subject to Auditory Brainstem Response (ABR) testing to determine their hearing thresholds.The International Mouse Phenotyping Consortium (IMPC) aims to generate and phenotype a null mutant for every gene in the mouse genome .In order to identify genes required for hearing function, the consortium uses an ABR test in the adult pipeline at week 14 that assesses hearing at five frequencies—6, 12, 18, 24 and 30 kHz. The consortium aimed to analyse a minimum of four mutant mice for each gene and, in most cases, both mutant males and females were analysed. Data from each IMPC centre is uploaded to the IMPC Data Coordination Centre, where it is subject to data validation and quality control (QC) procedures prior to statistical analysis and public access . Thus we adopted a different method to undertake a more robust analysis of the ABR data set.To date for ABR data, a mixed model approach has been employed in IMPC, using sex and genotype as fixed parameters and date-of-procedure as a random term . The genotype parameter is tested for its significant effect on the model, and this 13. Within this function, a reference range is created by ordering all of the baseline data points and creating a 98% reference range. This is achieved by taking the 1% percentile and 99% percentile data values, between which 98% of all values lie. The reference range is a non-parametric technique that makes no assumptions about the data distribution. The number of observations within and without this range is counted for the baseline and the mutant data values. A two-way contingency table is thus created. A Fisher’s exact test is then used to compare the baseline values against the mutant value, producing a p-value that reflects whether the two sets of counts are from the same data set. For our analysis we employed all the available IMPC ABR mutant data, and C57BL/6N wild-type data. For each genotype, the mutant and matched wild-type data were used to create reference ranges, build contingency tables, and calculate p-values.The PhenStat package includes a ‘reference range plus’ functionp-values provide an initial indicator of possible biological significance. As such, the ABR phenotyping data for each of these 328 lines underwent expert-led manual curation to assess phenodeviance and identify a robust data set of candidate hearing loss genes with a low false discovery rate.Using this approach, the available ABR data from 3006 lines yielded 328 candidate hearing loss genes  were also removed from the list. Employing these criteria led to 261 strains being removed from further analyses. Strains that showed highly concordant elevated thresholds at either, low frequency (6 kHz), high frequency (30 kHz) or across two or more frequencies, were deemed to be true hearing-impaired mutants (65 strains). In addition, strains with hearing thresholds that were discordant between sex, but concordant within sex were also deemed to be true hearing-impaired mutants (2 strains). Using these criteria, our analyses generated a list of 67 mutant strains that we deemed to be robust auditory pheno-deviants ; only the lower frequencies affected—Low-frequency hearing loss (10 genes); if three or more frequency thresholds are ≥45 dB above the centre median—severe hearing loss 25 genes); and, if <3 frequency thresholds are ≥45 dB above the centre median—mild hearing loss (19 genes)  allows us to calculate a false omission rate for loci that may have been missed by our stringent manual curation of the set of 261 genes for which there was initial evidence of an auditory phenotype. Overall, the false omission rate for known loci is 3/261 or around 1%. Extrapolating to novel loci, based on the ratio of novel to known loci (52/15) within the set of 67 candidate hearing loss genes, we surmise that around 10  additional loci would be undiscovered from the set of 261 genes. This provides an estimate of 13/261 for the false omission rate.Elmod1, Myo7a and Ush1c, which are known hearing loss genes. The respective allelic mutants roundabout, shaker-1 and deaf circler have previously been reported to exhibit vestibular dysfunction16. However, aside from the CSD test, there is no obvious pattern to the prevalence of additional phenotypes observed in the mutants tested. Of the 60 mutants that display additional phenotypes: 44 have 1-to-10 additional phenodeviant parameters; 10 have 11-to-20 additional phenodeviant parameters; 4 have 21-to-30 additional phenodeviant parameters; and 2 have >50 additional phenodeviant parameters detected. In contrast, seven of the mutants do not show any significant phenodeviance for any other phenotype parameter tested.The screen we have undertaken is effectively unbiased for both the developmental and physiological origin of the auditory phenotype, as well as for syndromic features that might be associated with the observed hearing loss. Without further investigation of individual mutants, we are unable to distinguish between hearing loss due to a deficit at the level of the central auditory pathway, a sensorineural origin in the inner ear, or due to conductive hearing loss due to middle ear problems, such as otitis media. However, we can take advantage of the comprehensive phenotype assessment that each mutant undergoes in the IMPC phenotyping pipeline, and are able to assess the prevalence of additional phenotypes in the hearing loss mutants. In Supplementary Table The products of the 52 novel candidate hearing loss genes encompass a very wide range of functions, from structural proteins to transcription factors, which reflects the complexity and cell type diversity of the auditory system. We assessed for potential interactions between the encoded proteins of the 52 genes with known human hereditary hearing loss dominant (DFNA), recessive (DFNB) and X-linked (DFNX) gene products. The predicted network interaction map shows that a significant number of known genes (65) formed a densely connected hub that incorporated 11 IMPC novel genes Fig. . HoweverSpns217, Nptn19, Slc4a1020—that emerged subsequent to identification in the IMPC screen. For example, the identification via the IMPC catalogue of Neuroplastin (Nptn) as a novel hearing loss gene has recently been extended in a report that demonstrates a role for this protein in synaptogenesis in inner hair cells in the organ of Corti in the mouse18. Neuroplastin, along with Embigin (Emb) and Basigin (Bsg), form a small family of neural cell adhesion molecules, belonging to the Ig superfamily. It is noteworthy that we report here Emb as a candidate hearing loss gene demonstrating high-frequency hearing loss, indicating that this family of neural CAMs merits further investigation as to their wider role in the auditory system.For three genes, recent reports from the Consortium confirm the hearing loss in mutant mice—We have assessed 3006 mouse mutants for hearing using an ABR test, and identified a large number of candidate hearing loss genes. After automated statistical analysis of the data set followed by manual curation, we uncovered a set of 67 mutants with hearing impairment. Of the 67 genes, 15 were known hearing loss loci, while the vast majority, 52, were novel candidate hearing loss genes that had not previously been associated with hearing loss.Atp2b1) or to play a role in some aspect of hair cell function in the cochlea 22. However, for the majority of candidate loci, it will be important to further explore expression and function in the auditory apparatus. In particular, an ABR screen is relatively unbiased and will reveal auditory phenotypes that arise due to conductive hearing impairment as well as sensorineural hearing loss, or possibly a combination of both. We cannot rule out that some of the candidate hearing loss genes impact on middle ear function, or cause otitis media. This class of hearing loss genes is unlikely to be represented by mutations with severe hearing loss or high-frequency hearing loss, and would be more likely to be present in the class of genes with low-frequency or mild hearing loss. The mouse has proved an excellent model for the identification of genes involved in chronic otitis media25 and it will be interesting to determine if the set of candidate hearing loss genes uncovered here further extends our knowledge of the genetic pathways involved in this form of hearing loss.Each of the 52 candidate hearing loss genes will merit further investigation. Some of the candidate genes are known to be expressed in the inner ear . There are over 400 genetic syndromes that include a hearing loss component2. However, most of the genes underlying syndromic hearing loss are unknown5. Our analysis thus reveals a very extensive and unexplored genetic landscape of genes required for auditory function and uncovers a large set of novel candidate hearing loss genes. We should emphasise that 450 genes represents a minimum set, as our screen may have failed to uncover several classes of genes, which contribute to hearing in either mouse or human. First, we discuss above our estimate of the false omission rate arising from our stringent manual curation of the set of 328 candidate genes. Moreover, we cannot rule out that there are hearing loss genes, in particular those with modest threshold effects, which our ABR test failed to identify. Also our analysis of the ABR data from 3006 genes did not include a waveform analysis that may have revealed further mutants. Second, we are unable to assess the contribution to auditory function of embryonic lethal, developmental mutations, which on different genetic backgrounds or in different genetic contexts may be homozygous viable and manifest a hearing loss phenotype not detectable in heterozygotes. Third, the hearing loss screen will not identify age-related hearing loss (presbycusis) genes, which again are likely to add substantially to the total number of hearing loss loci20. Finally, the data from a parallel screen of around 1000 genes reports similar findings to IMPC27.In our screen for auditory-related genes, we have explored around 15% of the mouse genome. Thus, we calculate that at a minimum the mammalian genome carries around 450 genes required for hearing function. In humans, around 150 non-syndromic hearing loss  loci have been mapped, with 103 underlying genes identified  and Knockout Mouse Project (KOMP) resourcehttp://www.mousephenotype.org) has a webpage dedicated to each gene with a link to the allele created. Targeted alleles were validated by a combination of short-range PCR, quantitative PCR and non-radioactive Southern blot30. These included allele-specific assays undertaken at the level of the ES cell and of the resulting mouse. The CRISPR-engineered alleles were validated by PCR amplification of the targeted locus and Sanger sequencing to confirm introduction of the required editing event. All QC data, including for CRISPR-engineered alleles and the guides employed, and Sanger sequencing of the engineered allele, are centrally deposited in the iMits  database. This centrally held IMPC database tracks allele generation and enables all mouse clinics to assess data of any generated strain. These data are freely available on request. In addition, when mice or germplasms are distributed from an IMPC repository, the full allele-specific genotyping details are communicated.Each mutant line underwent allele validation at the centre where the mouse model was produced. The consortium website . In addition, pertinent metadata parameters for each IMPC centre are also available from the portal including, cage-type, caging density, bedding and enrichment details, feed constituents, lighting regimes, temperature and humidity of animal holding rooms, and full strain nomenclature.Housing and husbandry data are captured for each IMPC Centre as described in Karp et al.https://www.mousephenotype.org/impress), defines the experimental design, detailed procedural information, the data that is to be collected, age of the mice, significant metadata parameters and data QC for IMPC pipeline tests. Unlike most experiments, we cannot randomly allocate animals to experiment groups, rather we rely on Mendelian inheritance to provide the randomisation method. However, at different institutes a variety of approaches are taken to minimise bias such as order effects including: alternate animal order; cage casual randomisation; and casual randomisation within a cage. There were no consistent approaches to blinding for ABR data collection and annotation across the institutes within IMPC. These issues are discussed with regard to the ARRIVE guidelines at http://www.mousephenotype.org/about-impc/arrive-guidelines.We have used data collected from the IMPC high-throughput phenotyping pipeline, which is based on a pipeline design where a mouse is characterised by a series of standardised and validated set of tests underpinned by standard operating procedures (SOPs). The IMPReSS  database . Hearing was assessed at five frequencies—6, 12, 18, 24 and 30 kHz. Tone pips were presented from 0 to 85 dB sound pressure level (SPL) in 5 dB intervals and were 5 ms in duration with a 1 ms rise/fall, presented 256 times at 42.6/sec. If hearing loss was suspected for a particular mutant line , stimulus presentation levels were extended to 95 dB SPL. Tone stimuli were presented in decreasing frequency order for a particular sound level and from low to high stimulus level. A click-evoked ABR is optional, and some centres, but not all, also acquired click data, but we have not included this in our analysis. The ABR test is conducted open field with hearing thresholds determined for one ear. Importantly, each centre records significant metadata parameters including: equipment manufacturer; equipment model; recording environment; anaesthetic agent; and anaesthetic dose. In addition, the Combined SHIRPA and Dysmorphology test [IMPC_CSD_003], carried out at 9 weeks, includes a click box test that provides a crude assessment of hearing9. The click box, which emits a high-frequency (~20 kHz) tone stimulus at 90 dB SPL, was held 30 cm above the mouse. When the click box is activated, a hearing mouse will elicit a ‘Preyer’ reflex (the pinnae flick backwards). A lack or reduction of Preyer reflex indicates possible hearing impairment.The IMPC phenotyping pipeline included an ABR test31 and is available from the IMPC portal (http://www.mousephenotype.org/about-impc/arrive-guidelines). As a high-throughput project, the target sample size of four animals per knockout strain is relatively low. The IMPC determined to set the lowest sample size that would consume the least amount of resources while achieving the goal of detecting auditory threshold abnormalities. At times, viability issues or the difficulty in administering the ABR test might further limit the number of animals. As such, when data are visualised on the IMPC portal, the number of animals phenotyped is listed.At each IMPC Centre, ABR phenotyping data from each mutant strain, and age-matched wild-type mice of equivalent genetic backgrounds, were collected at 14 weeks of age. When possible, cohorts of at least four mutant mice were tested, with a preference for two male and two female mice. If no homozygotes were obtained from 28 or more offspring of heterozygote intercrosses, the strain was scored non-viable. Similarly, if <13% of the pups resulting from intercrossing were homozygous, the line was scored as being subviable. In such circumstances, heterozygote mice were used in the phenotyping pipelines. The random allocation of mice to experimental group (wild-type vs. knockout) was driven by Mendelian inheritance. The individual mouse was considered the experimental unit within the studies. Further detailed experimental design information  for each phenotyping Centre, or the blinding strategy implemented is captured with a standardised ontology as detailed in Karp et al.The data generated in each IMPC centre are captured centrally by the Data Coordination Centre (DCC), where a team of data wranglers perform QC. The QC process involves data wranglers checking all data both manually and with automated methods. QC issues are raised through a QC web interface, where IMPC centres can respond to confirm it is an issue or alternatively that the data are correct. Each QC issue is then tracked with the corresponding data points until it has been corrected by the data contributing centre. Once the data have passed QC, it is released to the Core Data Archive (CDA) at the European Bioinformatics Institute, through a regular release schedule. The data set analysed here consists largely of data from the IMPC data release 5.0 with a smaller set of pre-QC data from the DCC. For the purpose of this article, the ABR data in this smaller set were QC’d and manually curated.Wild-type data sets were assembled for a trait by selecting wild-type mice that were generated by the same IMPC Centre. The wild-type mice are analysed with the same genetic background, sex, pipeline and metadata parameters .http://www.mousephenotype.org/impress/) were analysed by comparing the knockout strain with the appropriate wild-type control data. The default statistical analysis employed for the IMPC annotation pipeline is a mixed model approach, or a Mann–Whitney inference test may be employed as an alternative. However, for analysis of ABR data neither approach is suitable when there is lack of variation within the data set  or due to the step nature of the data, respectively. Therefore for this analysis, we used an enhanced version of PhenStat version 2.6.013 that includes a reference range plus approach, which allows identification of hearing impairment even when there are few data points or a lack of variation within the data set. The ‘testDataset’ function within range plus approach was used with a 98% reference range, produced from the wildtypes for each sex. Per knockout line, range plus approach classifies phenotype values as normal or low  or high  for each sex. A Fisher’s exact test is used to compare the proportions of classification, independent of sex, between wild-type and knockout  and the lowest p-value returned. This approach was used due to the small sample size of the experiment. A critical p-value (p ≤ 0.01) was used to generate a list of putative auditory pheno-deviant knockout lines. This list was used for further manual curation by domain experts.ABR data from IMPC release v5.0 along with additional pre-QC data deposited up until 6 September 2016 was used for the detection of putative pheno-deviants. In total, data from 3006 mutant strains were available for analysis. Each knockout strain was examined in three ways: only males, only females and both sexes combined. A minimum of 20 wild-type data points and 3 mutant data points was required for the analysis to proceed. The parameters assigned for annotation in the ABR procedure, stored in IMPReSS  at a combined medium confidence setting of ≥0.434.A single data set consisting of 103 known human hereditary hearing loss genes  and 52 IMPC novel hearing loss genes was used for functional assessment. We used g:Profiler (version r1665_e85_eg32) to find enrichment within the gene ontology, categories include; molecular function, cellular component and biological processwww.mousephenotype.org. An API in the form of RESTful interfaces is available for accessing post-QC data from the Core Data Archive (http://www.mousephenotype.org/data/documentation/data-access), and pre-QC data are also available for manual download (via Phenoview) or on request (http://www.mousephenotype.org/contact-us).Data from this study are available from IMPC at Supplementary InformationSupplementary Datapeer review file


Pan troglodytes verus) and sooty mangabeys (Cercocebus atys atys). In both species, rather than increasing their own access to preferred partners, bystanders intervened mainly when an alliance between groomers could have a negative impact on them: when the lower-ranking groomer was close to the bystander in rank, when either groomer was an affiliation partner whose services they could lose, or the groomers were not yet strongly affiliated with each other. Thus, bystanders in both species appear to monitor grooming interactions and intervene based on their own dominance rank and social relationships, as well as triadic awareness of the relationship between groomers. While the motivation to intervene did not differ between species, mangabeys appeared to be more constrained by dominance rank than chimpanzees.Grooming interactions benefit groomers, but may have negative consequences for bystanders. Grooming limits bystanders' grooming access and ensuing alliances could threaten the bystander's hierarchy rank or their previous investment in the groomers. To gain a competitive advantage, bystanders could intervene into a grooming bout to increase their own grooming access or to prevent the negative impact of others' grooming. We tested the impact of dominance rank and social relationships on grooming intervention likelihood and outcome in two sympatric primate species, Western chimpanzees ( They arIn primates, individuals have been shown to bias their grooming towards higher-ranking grooming (HRG) partners ,37,39–44The triadic nature of competitive events is lost when aggregating the distribution of multiple grooming bouts over time, impeding our understanding of grooming competition. While grooming has mainly positive effects for the groomers, it does not take place in a social vacuum. Individuals in many animal species monitor group members' mating behaviour –55, poliGrooming between two individuals can negatively affect bystanders in at least three ways. Owing to time constraints, grooming between two individuals may influence their availability to others, but also how the groomers will allocate other services like feeding tolerance or support during aggressive interactions. Thus, groomers may restrict bystanders in their attempt to maximize their own benefits ,45. Secoany behaviour of a bystander directed at one or both groomers that could change the ongoing grooming bout. Interventions into affiliative interactions have been shown to effectively reduce subsequent affiliations [Because of these three reasons, bystanders are predicted to not passively observe, but rather to actively alter the outcome of grooming interactions in their favour. Importantly, countering the negative impacts of threatening alliance formation or affiliation partner defection requires bystanders to keep track of and evaluate grooming between other group members and to then act upon dominance rank and social relationship information. To test how bystanders influence grooming interactions, we use direct observations of grooming interventions, i.e. liations . In primliations ,43,71,72liations ,67–69. Oliations ,67,69,72liations ,64), oneliations . In the Cercocebus atys atys) and Western chimpanzees (Pan troglodytes verus), in the Taï National Park, Côte d'Ivoire, as part of a project investigating the link between social complexity and cognitive abilities in these two species. Both have large multi-male, multi-female social groups [We studied two sympatrically living species of non-human primates, sooty mangabeys , and rel groups ,82,83. Al groups . Thus, il groups . While sWe predict that species differences in the impact of rank on resource monopolization will influence grooming interventions, with chimpanzee interventions being less defined by the ranks of groomers and bystanders. In chimpanzees, the difference in attractiveness of low- and high-ranking individuals should be less pronounced than in mangabeys, where high-ranking individuals can offer stronger benefits in return for grooming. Thus, we predict that mangabeys should intervene more to gain access to high-ranking individuals than chimpanzees. Priority of access in mangabeys, but not chimpanzees, should shape intervention patterns by preventing low-ranking individuals from intervening, also reducing their success rate if they try.We investigate when bystanders intervene into grooming bouts, which of the groomers they target (i.e. which groomer they attempted to groom themselves), what determines intervention success, and how intervention patterns compare in the two primate species. Our goal was to determine whether interventions function to gain access to attractive grooming partners (pro intervention ), or if 2.2.1.Data on grooming interventions were collected in Taï National Park, Côte d'Ivoire  from 201We included two chimpanzee communities to get an indication of whether effects are group-specific. Data were collected following the same protocol in both species, using customized CyberTracker data collection software (CyberTracker Conservation 2013). We recorded dyadic and polyadic social interactions within focal observations. In addition, we recorded all occurrences of grooming interactions in the party  that werInterventions were defined as any behaviour of a bystander directed at one or both of the groomers that could potentially change the course of a grooming bout. We included grooming interactions and interventions  into the dataset if all three individuals involved were focal individuals during this study  and if all of the following were available: identity of groomers, duration of the bout, identity of all bystanders present that could potentially intervene, the amount of time they were present, type of intervention , and the outcome of the intervention. We considered consecutive dyadic grooming interactions as part of the same bout if they involved at least one of the same groomers and started within 5 min of the end of the last grooming interaction. We treated polyadic grooming bouts as multiple dyadic interactions. If an individual intervened multiple times into the same grooming dyad during the same bout, we only considered the first attempt, but multiple bystanders could intervene in the same grooming dyad.2.2.et al. [Hierarchy ranks of all communities were calculated dynamically, using a modification of the Elo rating method ,89 as pret al. . We usedet al. ) in chimet al. . For sooet al. ) to estak, the modification by Foerster et al. uses maximum-likelihood estimation to optimize the k and allow individuals to enter the hierarchy with different start values. This reduces the need for a burn-in phase where ranks are relatively uncertain, does not assume rank changes where none exist, and reduces the need for a priori decisions by the researcher. However, it shows some problematic properties when facing sparse datasets, as we see for female chimpanzees in the communities studied here. We, therefore, added a further modification to the script provided by Foerster et al. [k and start values of the Elo rating by optimizing only the winning likelihood, which might underestimate the size of the k in sparse datasets and, therefore, miss rank changes, we additionally optimized the number of correct classifications  by varying the number of iterations of the optimization algorithm and selecting the solution that maximizes the number of correct classifications . Assuming that the chosen interaction type is overwhelmingly unidirectional , this m2.3.To account for the fact that affiliative relationships between individuals might change over time, we adopted a method similar to Elo ratings, the dynamic dyadic sociality index (DDSI ), to cal2.4.We fitted multiple models to test the impact of dominance ranks and affiliative relationships on grooming interventions . All models were generalized linear mixed model (GLMM) with binomial error structure and logit link function , implemen = 550) differed from non-intervening bystanders (n = 20 656). Owing to model complexity, we fitted two separate models.Model 1 tested which factors influenced the likelihood that a bystander would intervene into a grooming bout. For this, we analysed every combination of each dyadic grooming interaction  with each bystander for this interaction, comparing whether intervening  and with whom they had the lower DDSI score , on intervention likelihood. To test for the impact of affiliative social relationships in both species, we included the three-way interaction between the relationship score of bystander and PG, the relationship score of bystander and the NPG, and group identity as test predictor. To test for group differences in the impact of the groomers' affiliative relationship, we entered a two-way interaction between group identity and the DDSI score of the groomers as a test predictor. We controlled for the rank values of all three individuals. We included the log-transformed presence time of each bystander during the grooming bout as an offset term into models 1.1 and 1.2 to account for differences in the opportunity to intervene in grooming bouts of different durations , and forn = 456), thereby excluding interventions that did not lead to grooming by the intervener. To be able to control for non-independence of data and retain the random effects structure, but still test the impact of the fixed effects on individual choice, we used a ‘repeated measures’ design [χ2, parameter estimates, standard errors, z-values and p-values across the 1000 models as the result. Test predictors for this model were: the three-way interaction of the dominance rank of the target with that of the intervener and group identity, to test whether individuals in either species targeted higher-ranking or closely ranked groomers; and the two-way interaction of group identity with the DDSI of the intervener with the target, to test whether individuals in either species targeted the groomer they were more affiliated to. We controlled for grooming direction at the time of intervention.In model 2, we analysed whether intervening individuals preferably target HRGs or their preferred partners. We used only interventions in which the target (the first grooming partner after the intervention) could be determined  or subsequently received grooming from or remained as the sole grooming partner of either groomer (joining). It was not successful if the two groomers continued grooming, or if the intervener joined but was ignored by the groomers and left without receiving grooming. Of the 462 interventions with a known outcome and target, 271 were successful. Owing to model complexity, we divided the model into models 3.1 and model 3.2. In model 3.1, we included the three-way interaction of the intervener rank, group identity and the rank of the non-target, to test whether the impact of rank relations on intervention success differed between the two species. We also included the three-way interaction between the two groomer ranks and group identity, to test whether it is easier to intervene into grooming between individuals with higher rank distance. In model 3.2, we tested whether interveners in either species succeeded more if they were more affiliated with either groomer, or if the two groomers were affiliated less with each other, by including two-way interactions between the relationship scores of intervener and each groomer with group identity, and the interaction between the relationship score of the groomers with group identity.z-standardized to a mean of zero and a standard deviation of one [We included the three identities of bystander and groomers  and the three dyad combinations between them (513 dyads) into models as random effects, to account for individual and dyadic differences in intervention behaviour and the fact that not all individuals and dyads were equally likely to be observed grooming . We trien of one . We testn of one  and compn of one . Variancn of one  were dern of one  applied 3.3.1.χ215 = 30.23, p = 0.011; electronic supplementary material, table S2a). None of the three-way interactions were significant. After they were removed, the interaction between group identity and bystander rank was significant , while south showed the opposite effect, with LRGs having a higher likelihood to intervene . East showed no effect . The interaction between bystander rank and the rank of the LRG was significant in all three groups . High-ranking bystanders intervened more if the LRG was itself high-ranking. Low-ranking bystanders intervened more when the LRG was also low-ranking  and the LRG  were male.The full null model comparison for model 1.1 showed a significant impact of the test predictors . After removing non-significant interaction terms, it became clear that the intervention likelihood in all communities was higher with higher relationship scores between bystander and both the PG  and the NPG . Additionally, in all groups there was a lower likelihood to intervene when relationship scores between the groomers were high  or the PG  or NPG  was male.The full null model comparison for model 1.2, focusing on affiliation strengths, revealed that the test predictors had a significant impact on the response , with mangabey interveners of high rank being significantly more likely to succeed, which was not observed in either chimpanzee community , and male interveners were more successful than females in both species .There was a species difference in the intervention outcomes : mangabeχ29 = 5.58, p = 0.782; electronic supplementary material, table S6), indicating that bond variables did not determined how successful the intervention was.Model 3.2 was non-significant  whether a grooming intervention occurred, (ii) who the target of the intervention was, and (iii) intervention success. We found that rank and affiliative relationships affected grooming intervention behaviour in both sooty mangabeys and chimpanzees. We argue that, while they differed in who could intervene, the motivation to intervene was similar in all three groups and across both species. Interventions seemed to occur mainly when bystanders could gain from impeding grooming of that dyad, indicating that both species alter grooming interactions between others to their advantage.While it is hard to identify species differences when comparing only three communities, we nonetheless found some persistent differences distinguishing the mangabey group from both chimpanzee communities. The lack of polyadic grooming in mangabeys limited who could intervene and how. Aggression in the intervention context was rare (around 4% of interventions) and of low intensity in both species, so most interventions initially involved one individual approaching a grooming dyad. In the mangabeys rather than the chimpanzees, bystanders higher in rank than at least one groomer were more likely to approach and one or both groomers would react by abandoning the grooming bout, as previously reported . Also, hThe factors motivating bystanders to intervene seem to be similar in mangabeys and chimpanzees. Interventions were not more likely in any group if the HRG was high in rank or close in rank to the bystander, as would be predicted if interventions were a way to gain access to attractive grooming partners. However, individuals in all groups were more likely to intervene if the lower-ranking (but not the higher-ranking) groomer was close to them in rank. This would fit a scenario where bystanders intervene to impede alliance formation of competitors who are close to them in rank, as the HRG could provide coalitionary support as a return for the grooming ,64. The In both species, bystanders intervened more when they had a strong affiliative relationship with one or both groomers, which could be evidence for an attraction to affiliation partners . HoweverMangabey and chimpanzee bystanders intervened more when the relationship score between the two groomers was low. The relationship score between groomers had no obvious effect on intervention success, indicating that the lower intervention rate was not a result of affiliated groomers being harder to separate. Interventions might impede the formation of new alliances rather than restrict existing stable bonds, as reported for ravens . BystandWe provide quantitative evidence that grooming interventions could function to reduce the negative impact for the bystander of others' grooming in two primate species. Bystanders used grooming interventions towards dyads whose grooming could threaten their rank position or relationships, particularly dyads of close-ranked competitors, own affiliation partners and emerging alliances. While similar patterns have been found in other species ,68, this


GnRH) mRNA with the lack of LH surge and elevation of prolactin (PRL). TCS mice had lower kisspeptin immunoreactivity and kiss1 mRNA in anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC). Moreover, the estrogen (E2)-enhanced AVPV-kisspeptin expression was reduced in TCS mice. In addition, the serum thyroid hormones (triiodothyronine (T3) and thyroxine (T4)) in TCS mice were reduced with increases in levels of thyroid stimulating hormone (TSH) and thyroid releasing hormone (TRH). In TCS mice, the treatment with Levothyroxine (L-T4) corrected the increases in PRL, TSH and TRH; the administration of L-T4 or type-2 dopamine receptors agonist quinpirole inhibiting PRL release could rescue the decline of kisspeptin expression in AVPV and ARC; the treatment with L-T4, quinpirole or the GPR45 agonist kisspeptin-10 recovered the levels of serum LH and FSH and progesterone, and GnRH mRNA. Furthermore, TCS mice treated with L-T4 or quinpirole resumed regular estrous cycling, follicular development and ovulation. Together, these results indicate that exposing adult female mice to TCS (≥10 mg/kg) reduces thyroid hormones causing hyperprolactinemia that then suppresses hypothalamic kisspeptin expression, leading to deficits in reproductive endocrine and function.Triclosan (TCS), a broad-spectrum antimicrobial agent, is widely used in clinical settings and various personal care products. The aim of this study was to evaluate the influence of TCS on reproductive endocrine and function. Here, we show that the exposure of adult female mice to 10 or 100 mg/kg/day TCS caused prolongation of diestrus, and decreases in antral follicles and corpora lutea within 2 weeks. TCS mice showed decreases in the levels of serum luteinizing hormone (LH), follicle-stimulating hormone (FSH) and progesterone, and gonadotrophin-releasing hormone ( Triclosan  is a synthetic antibacterial compound largely utilized in personal and household products such as toothpastes, shampoos, cosmetics, antibacterial soaps, deodorants, kitchen utensils, bedding and clothing  axis. The pulse release of gonadotrophin-releasing hormone (GnRH)/luteinizing hormone (LH) and generation of preovulatory surge-like LH release (LH-surge) are altered by the feedback action of estradiol  for 50 days. Our results suggest that in adult female mice, TCS exposure (≥10 mg/kg) through reducing thyroid hormones causes hyperprolactinemia that then suppresses hypothalamic kisspeptin synthesis, thereby disrupting the reproductive endocrine and ovarian function.ad libitum. Their body weight was measured every day. All efforts were made to minimize animal suffering. Every early morning (09:00 h), estrous cyclicity was examined using vaginal cytology  at 12 weeks of age (30 ± 2 g) were housed in stainless steel cages with wood bedding to minimize additional exposure to endocrine disrupting chemicals  in Animal Research Center of Nanjing Medical University. They received food and water TCS (>99% purity) was purchased from Sigma-Aldrich . TCS was dissolved in dimethyl sulfoxide (DMSO), and then diluted with corn oil . After 3–4 regular estrous cycles were determined, the mice were given the oral intake of TCS at doses of 1, 10 and 100 mg/kg per day at 08:00 h. A recent study  within 12 h after the administration of TCS were collected and stored at −80°C until measurement. The TCS concentrations of urinary (1 ml/mouse) were measured using an established method  injection chloral hydrate (400 mg/kg). Both ovaries were dissected and fixed in Bouin’s fluid. The samples were dehydrated through a graded series of alcohol, cleared in xylene, and then embedded in paraffin wax. After the sections (5 μm) were deparaffined and rehydrated, the sections were stained with hematoxylin and eosin (HE). The classification of follicular stages was made following the morphological criteria as described previously  cells in AVPV (AVPV-kisspeptin+ cells) and ARC (ARC-kisspeptin+ cells) were observed by conventional light microscope  with a 40× objective.Mice were anesthetized with chloral hydrate  and perfused transcardially with 4% paraformaldehyde. Brains were transferred gradually into 15% and 30% sucrose until they settled. Sections (40 μm thick) through the AVPV (Bregma +0.50 to +0.02 mm) the ARC area . The measurement of T3, T4, thyroid stimulating hormone (TSH), thyrotropin-releasing hormone (TRH) and PRL had no limit for any estrous cyclicity (group II). The serum sample (5 μl) was needed for each assay  using commercial enzyme-linked immunosorbent assay (ELISA) kits . The measurement of each sample was repeated 2 times to obtain an average value. The sensitivities were 47.1 pg/ml for T3, 1.4 ng/ml for T4, 19.3 pg/ml for TSH, 0.18 μIU/ml for TRH, 2.0 pg/ml for E2, 0.2 ng/ml for P4, 0.2 ng/ml for LH, 0.4 ng/ml for FSH and 0.4 ng/ml for PRL, respectively. The intra- and inter-assay coefficients of variation were 4.5% and 7.2% for T3, 4.3% and 7.5% for T4, 3.2% and 9.5% for TSH, 5.6% and 7.2% for TRH, 6.0% and 5.8% for E2, 5.8% and 8.4% for P4, 5.5% and 8.9% for LH, 4.3% and 10.3% for FSH, 4.7% and 4.9% for PRL. For determination of the LH surge, the repetitive blood sampling was undertaken at 1600, 1700 and 1800 h, respectively, on the day of proestrus (group III). The mice were anesthetized with ketamine (80 mg/kg) and xylazine (4 mg/kg) and a needle was inserted into a caudal vein at 1400 h. The mice were gently restrained in a cardboard tube, and the blood sample (~50 μl per time) was collected without anesthesia using heparinized syringes. After each blood collection, an equivalent volume of heparinized saline  was injected. Serum (~15 μl) was stored at −80°C for subsequent ELISA of LH.Orbital blood (~300 μl) was obtained under anesthetized conditions with chloral hydrate  at 1600–1700 h. Serum (~100 μl) was separated by centrifugation at 4°C and stored at −80°C until assay. The levels of serum estradiol (E2), progesterone (P4), LH and follicle-stimulating hormone (FSH) were measured on the day of diestrus . There was no difference in GAPDH expression among the groups. The relative expression of genes was determined using the 2−∆∆ct method with normalization to GAPDH expression. On the basis of melting curve analyses, there were no primer dimers or secondary products formed.The POA area  dissolved in 0.9% saline was subcutaneously (s.c.) injected at dose of 20 μg/kg/day /metastin ] (Sigma-Aldrich Corp) was dissolved in DMSO, and then was diluted by 0.9% saline to a final concentration of 0.5% DMSO. For repeated intracerebroventricular (i.c.v.) injection of kisspeptin-10, the mice were anesthetized with chloral hydrate  and then placed into a stereotaxic instrument . A small hole (2 mm diameter) was drilled in the skull using a dental drill. A guide cannula  was implanted into the right lateral ventricle  and anchored to the skull with three stainless steel screws and dental cement. On day 3 after surgery, the dummy cannula was removed from the guide cannula, and replaced by infusion cannulas (30 gauge) connected by polyethylene tubing  with a stepper-motorized micro-syringe . The kisspeptin-10  under the anesthetized conditions with intramuscular injections of ketamine (80 mg/kg) and xylazine (4 mg/kg) at day 43 of TCS exposure. After surgery, the OVX mice received a subcutaneous implant of a silastic tubing  that was filled with 20 μg/ml of E2 in olive oil. The E2-treatment for 5 days produced a physiological level of serum E2 (20.71 ± 6.25 pg/ml) in adult female mice. On day 6 after implanting silastic tubing, the mice were given the injection (s.c.) of E2 at doses of 100 μg/kg for two consecutive days to produce a preovulatory high level of E2 (314.38 ± 66.91 pg/ml) that could exert a positive feedback action in AVPV-kisspeptin neurons  = 1.129, P > 0.05; Figure Female mice (12 weeks old) were treated with the oral gavage of TCS at 1, 10 and 100 mg/kg/day for 50 days, hereafter referred to as 1-TCS, 10-TCS, or 100-TCS mice  = 18.31, P < 0.01; Figure F = 24.17, P < 0.01; Figure P < 0.01) and 90% of 100-TCS mice (P < 0.01) had a persistent diestrus (lasting more than 5 days) starting from the 2nd week of TCS exposure.The estrous cycle was monitored using the vaginal smear test. As shown in Figure P > 0.05, n = 20). To examine the possible influence of TCS on follicle development and ovulation, we counted the number of antral follicles and corpora lutea at diestrus using morphological criteria. As shown in Figure F = 5.675, P < 0.05; Figure F = 3.371, P < 0.05; Figure P < 0.05) and corpora lutea (P < 0.05).Ovary weights of 1-TCS mice (7.48 ± 1.55 mg), 10-TCS mice (7.69 ± 2.18 mg) and 100-TCS-mice (7.92 ± 1.44 mg) were not significantly different than control mice  = 3.349, P < 0.05; Table F = 2.889, P < 0.05), P4  = 4.035, P < 0.05) and PRL  = 3.376, P < 0.05) and GnRH mRNA  = 9.422, P < 0.01), were all affected by TCS exposure. Specifically, 10-TCS mice and 100-TCS mice showed a modest but significant decrease in the levels of LH (P < 0.05 and P < 0.01), FSH (P < 0.05), P4 (P < 0.05) and GnRH mRNA (P < 0.01) compared to controls, which were associated with an obvious increase in the level of PRL (P < 0.05). Although the TCS exposure had a tendency to decrease the serum E2 level, this difference did not reach statistical significance  = 2.083, P > 0.05). A surge-like LH release (LH surge) was observed between 1600 and 1700 in proestrus control mice and 1-TCS mice, but not in 10-TCS mice and 100-TCS mice  = 11.512, P < 0.01; Figure kiss1 mRNA at diestrus  = 3.614, P < 0.05; Figure P < 0.01, n = 10). In comparison with OVX control mice, the level of AVPV-kiss1 mRNA in OVX 10-TCS mice was reduced by approximately 40% . Although the high dose of E2 could elevate the level of AVPV-kiss1 mRNA in OVX 10-TCS mice , their level of AVPV-kiss1 mRNA still was less than half of E2-treated control mice .To further investigate the targets of TCS-reduced HPG axis and LH surge production, we examined kisspeptin expression in AVPV of proestrus mice and ARC of diestrus mice. In proestrus control mice, the kisspeptin+ cells in AVPV (AVPV-kisspeptin+) were located along the third ventricle Figure . In compF = 5.170, P < 0.01) and T4  = 15.267, P < 0.01), TSH  = 3.919, P < 0.05) and TRH  = 3.882, P < 0.05) were each affected by TCS exposure. While T3 and T4 were substantially reduced in 10-TCS (P < 0.05) and 100-TCS (P < 0.01) mice, TSH (P < 0.05) and TRH (P < 0.05) were increased. Interestingly, the administration of L-T4 at the dose of 20 μg/kg for 20 days starting from day 30 of 10-TCS exposure  and PRL .To further explore the underlying mechanisms of TCS-enhanced secretion of PRL, we measured serum total thyroid hormones (T3 and T4), TSH and TRH Figure . As showkiss1 mRNA at proestrus  and FSH .Thyroid hormone replacement in TCS mice corrected their hyperprolactinemia, next experiments were designed to examine whether the hyperprolactinemia causes the suppression of kisspeptin expression and hypothalamic-pituitary-reproductive endocrine. The results showed that the treatment of 10-TCS mice with L-T4 for 20 days was able to correct the decrease in the level of AVPV-P < 0.05, n = 10; Table P > 0.05, n = 10) and TRH . Similarly, the administration of quinpirole for 20 days in 10-TCS mice restored the levels of AVPV-kiss1 mRNA , ARC-kiss1 mRNA  and GnRH mRNA , as well as serum LH .Until now, there have been no commercially available the PRL receptor antagonists  and FSH  without altering the increased levels of TSH  and TRH , or PRL .To demonstrate that TCS-induced hyperprolactinemia through reducing kisspeptin expression suppresses the reproductive endocrine, the 10-TCS mice were given the injection (i.c.v.) of the GPR45 agonist kisspeptin-10 (Kp-10) for 7 days  reduces thyroid hormones, causing hyperprolactinemia which then suppresses hypothalamic kisspeptin neurons, ultimately disrupting reproductive endocrine and function.The results in the present study provides via reduced negative feedback of the hypothalamus-pituitary axis, which promotes the release of both TSH and PRL. The present data suggest that TCS-induced hypothyroidism presents the same scenario, as the elevated levels of serum PRL, TSH and TRH in 10-TCS mice were corrected by L-T4 administration. Hypothyroidism can also induce elevation in vasoactive intestinal peptide, which is capable of increasing PRL secretion  to reduce the estradiol metabolism and clearance  survey of 2003 and 2004 (range: 2.4–3, 790 μg/L; Calafat et al., X-YC, XH, and J-WX performed the experiment. X-YC and W-TZ analyzed the experimental data and wrote the manuscript. LC and JZ designed the studies and revised the manuscript. All authors approved the final version for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There are no published data regarding the sensitivity and specificity of the unaided APHABu. Therefore, we investigated these parameters for detecting hearing loss of at least 25 dB at any frequency between 0.5 and 8.0 kHz. We used the APHABu to determine hearing loss in 245 subjects aged 50 years and older without any reported disease of the ears. Due to incomplete answering of the APHAB form, 55 subjects have been excluded. We also measured the pure-tone thresholds by air conduction for all octave frequencies between 0.5 and 8 kHz. Receiver operating characteristic (ROC) curves and the Youden Index were used to determine the diagnostic value of the APHABu, particularly sensitivity and specificity, in three different ways: (1) separately for ease of communication (ECu), background noise (BNu), and hearing with reverberation (RVu) subscales; (2) with the mean value of ECu, BNu, and RVu; and (3) with a logistic regression model. The area under the ROC curve was lower for BN only (0.83) and nearly equal for all other methods (0.87–0.89). Depending on how we performed the analyses, the sensitivity of the APHABu was 0.70–0.84 , 0.76 , or 0.85 (logistic regression model). The specificity was 0.79–0.95. The use of single APHABu subscales for determining the sensitivity and specificity of the APHABu due to confusing results. In comparison, the use of the mean value of ECu, BNu, and RVu and the use of the logistic regression model due to equal values in the ROC curves but a higher sensitivity in the logistic regression model. Therefore, we would recommend the last method for determining the sensitivity and specificity of the APHABu.Subjective hearing loss in hearing-impaired patients can be assessed by inventory questionnaires. The abbreviated profile of hearing aid benefit (APHAB) measures subjective hearing loss in four typical hearing situations . It is used to fit hearing aids in patients with statutory insurance in Germany. In addition, the unaided APHAB (APHAB Grades of hearing loss are objectively measured by pure-tone and speech audiometry while self-reporting questionnaires can be used to measure subjective hearing impairment. Recently, the abbreviated profile of hearing aid benefit (APHAB), developed by Cox and Alexander , 2, has The APHAB comprises 24 single questions divided into four subscales that measure hearing loss in everyday hearing situations. The ease of communication (EC) scale examines basic hearing situations without ambient noise in a quiet environment, the background noise (BN) scale examines hearing situations with background noise, the reverberation (RV) scale investigates hearing situations in large spaces with echoes, and the aversiveness (AV) scale measures the perception of loud sound events.u) in the EC and RV subscales. In contrast, they did not find any association between individual hearing loss typified to standard audiograms introduced by [u scores in any subscale [u scores and hearing loss at all octave frequencies between 0.5 and 8 kHz. Moreover, they investigated all of the subscales in 5% steps and 5 dB level steps were investigated [u scores for the EC and RV subscales, but not for the BN subscale. As in speech audiometry, this is probably due to the ability of individuals to learn how to compensate for BN. Hearing with RV is rarer than problems with BN; thus, there are fewer opportunities for individuals to experience RV and develop ways to compensate for it. Moreover, hearing problems in normal, low-noise conditions (EC) generally affect only individuals with severe hearing loss, with, again, fewer opportunities to learn how to compensate. The AV subscale score does appear to be negatively associated with hearing loss [u values correlated to increasing dB values of hearing loss [Löhler et al.  recentlyduced by  and APHAsubscale . Moreovesubscale . Finallysubscale  investigstigated . Löhler ing loss , 10. Theing loss , 9. In cing loss .u, BNu, and RVu subscales; (2) calculation of the mean of the ECu, BNu, and RVu subscales; and (3) a logistic regression model for the ECu, BNu, and RVu subscales. Due to the mentioned effect of the opposite character of the AV subscale (measuring the aversiveness of loud situations), it is not rational to include the AV subscale for determining the sensitivity and specificity of the APHABu. The high variance of individual compensating effects will limit the use of BN subscale for the detection of sensitivity and specificity as well. Therefore, it may be of benefit to focus on the EC and RV subscales within our investigation.To the best of our knowledge, no published studies have addressed the sensitivity and specificity of the APHAB for detecting a defined hearing loss. This lack of data is typical of most inventories . BecauseWith the values of specificity and sensitivity, the last missing main characteristic of the APHAB will be described. Together with the known attributes , 9–11 thu to 245 subjects aged 50 years and older who had no actual or reported disease of the ears or hearing impairment. Thus, we used the APHAB as a primary diagnostic tool for hearing loss. In addition, we measured and recorded the pure-tone thresholds of the participants at all octave frequencies between 0.5 and 8.0 kHz by air conduction. The database did not include a record of patients who had a difference in hearing loss of >60 dB in comparisons of air conduction for both ears at frequencies at 0.5, 1.0, and 2.0 kHz, based on the three-frequency table [In Germany, an APHAB database has been established several years ago . Betweency table , 9–11. Wu to detect a hearing loss of 25 dB in at least one of the octave frequencies between 0.5 and 8.0 kHz in any ear. Using 5 dB steps in pure-tone thresholds, this will lead to the same results as using a cutoff value for hearing loss of more than 20 dB. Logistic regression was used to construct the ROC curves. We determined the diagnostic value of the APHABu by the area under the ROC curve (AUC) with 95% confidence intervals. The threshold of the APHABu was identified with the Youden Index [We used receiver operating characteristic (ROC) curves , 15 to een Index  and calcu by means of the following three methods:u, BNu, and RVu individually.We determined the optimal cutoff values for detecting hearing loss by considering ECWe determined the optimal cutoff value using the arithmetic mean value of the unaided EC, BN, and RV subscales: p denotes the probability of hearing loss in any frequency ranging from 0.5 to 8.0 kHz:We determined the probability (cutoff) for the unaided EC and RV subscales with a logistic transforming regression analysis mode using a logistic regression model with random effects . The folIn accordance with the aim of our study, we analyzed all of the data to determine the sensitivity and specificity of the APHABu scores were the independent variables. Gender and site of hearing loss were additional independent variables. The choice of a random effect model serves two purposes. One, we are able to model variability between patients and second we are able to take the paired data structure into account (left vs. right ear). Calculations were performed with SAS software version 9.4, PROC GLIMMIX  while one hundred and twelve were women . Forty-three of the participants (17.6%) had normal thresholds (maximum hearing loss of 20 dB in one or more octave frequencies between 0.5 and 8.0 kHz). Fifty-five subjects (22.4%) did not answer all of the APHAB questions; this left 190 full data sets for analysis in this study. APHABu, BNu, and RVu scores and the ability of the average score of these subscales , as demonstrated in Table u  leads to nearly similar cutoff values for ECu and RVu, and, by comparison, the cutoff score for BNu was even higher (Table u (0.10) and RVu (0.12) than the BNu value (0.23) by the single use of the subscales. As has been reported, BN subscale scores are not associated with individual hearing loss [u values of the normal hearing group in EC and RV (Table We found that neither the side of the ear nor gender influenced the APHABing aids , 9, 10. er Table . This maing loss . Maybe, d RVu 0.1 than theRV Table , which uu scores. In fact, including 8.0 kHz might be very strict and not used in MAT [It might be surprising that a hearing loss of 25 dB has an influence on APHABd in MAT , and incd in MAT , 10. In d in MAT . In contd in MAT .u is being used as a screening inventory or as a primary audiological diagnostic method. Use of the logistic regression model to determine the diagnostic value of the APHABu may be justified by the weighted influence of the RV subscale. Recent investigations have found that the likelihood of individual compensatory effects is highest for BN and lowest for RV and that the influence of the EC subscale is limited to cases with increased hearing loss [u adds to the knowledge of this widely used inventory in Germany. We suggest that future studies investigate the values of these parameters separately for each frequency. Together with the recently published percentile distribution curves and box plots of the unaided and aided APHAB and the resulting benefit [u scores, pure-tone thresholds, and speech-audiometric results, it is well possible to interpret an individual hearing loss.At present, some rather difficult methods in conjunction with the APHAB to measure the quality of hearing aid fitting in patients with statutory insurance are used in Germany , 4. Theying loss , 10. In  benefit  and toge


To investigate the expression of cathepsins B, D, and G, in relation to the cancer stem cell (CSC) subpopulations, we have previously characterized within isocitrate dehydogenase (IDH)-wildtype glioblastoma (IDHWGB).in situ hybridization (CISH) were conducted to investigate the transcriptional activation of genes encoding for cathepsins B, D, and G. Data obtained from cell counting of DAB IHC-stained slides and from NanoString analysis were subjected to statistical analyses to determine significance.3,3-Diaminobezidine (DAB) immunohistochemical (IHC) staining for cathepsins B, D, and G, was performed on 4μm-thick formalin-fixed paraffin-embedded IDHWGB samples obtained from six patients. Two representative DHWGB samples from the original cohort of patients were selected for immunofluorescent (IF) IHC staining, to identify the localization of the cathepsins in relation to the CSC subpopulations. NanoString gene expression analysis and colorimetric + and SALL4+ CSC subpopulations. NanoString gene analysis and CISH confirmed the abundant transcript expression of these cathepsins. The transcriptional and translational expressions of cathepsin G were minimal and were confined to cells within the microvasculature.Cathepsin B and cathepsin D were detected in IDHWGB by DAB IHC staining. IF IHC staining demonstrated the expression of both cathepsin B and cathepsin D by the OCT4This study demonstrated the expression of cathepsin B and cathepsin D but not cathepsin G within the CSC subpopulations of IDHWGB at both the transcriptional and translational level. Cathepsin G was expressed at low levels and was not localized to the CSC population of IDHWGB. The novel finding of cathepsin B and cathepsin D in IDHWGB suggests the presence of bypass loops for the renin-angiotensin system, which may facilitate the production of angiotensin peptides. Elucidating the precise role of these cathepsins may lead to better understanding and more effective treatment of this aggressive tumor. Glioblastoma multiforme (GBM) is an aggressive primary brain cancer, associated with a 5-year survival rate of 2% , 2. DespRecent WHO guidelines for classification of GBM attempt to capture the complex molecular and histological nature of GBM, by separating tumors into two otherwise morphologically indistinguishable categories , 8, baseCancer stem cells (CSCs), a small population of cells within cancer that possess phenotypic characteristics of embryonic stem cells (ESCs), acquired either from the accumulation of mutations in normal ESCs or progenitor cells, are attributed to the poor prognosis associated with GBM –12. CSCs+ subpopulation represents the most primitive CSCs within GBM, which subsequently differentiate to form SOX2+/SALL4+ progenitor cells  . The claThe RAS is highly complex involving many other signaling pathways and enzymes such as cathepsins B, D, and G –32 Figu. We haveThe expression of cathepsin B has previously been identified in GBM tumor cells , specifiin situ hybridization (CISH).We hypothesized that cathepsins B, D, and G are expressed by the CSC subpopulations within IDH-negative GBM. In this study, we investigated the presence of these isozymes at the translational level using immunohistochemical (IHC) staining and at the transcriptional level using NanoString gene analysis and colorimetric Six IDH-wildtype glioblastoma (IDHWGB) tissue samples from three male and three female patients aged 42–81  years were sourced from the Gillies McIndoe Research Institute Tissue Bank for this study, which was approved by the Central Regional Health and Disability Ethics Committee (ref. no 15CEN28). Written informed consent was obtained from patients included in this study.Hematoxylin and eosin (H&E) staining was performed on 4μm-thick formalin-fixed paraffin-embedded sections of GBM samples from six patients, to confirm the diagnosis of IDHWGB by an anatomical pathologist (HDB). 3,3-Diaminobenzidine (DAB) and immunofluorescent (IF) IHC staining were then performed, as previously described , 23, on To further characterize the expression of cathepsins B, D, and G, two representative samples of IDHWGB from the original cohort of six patients included in DAB IHC staining were selected for IF IHC staining. Dual IF IHC staining was performed using identical primary antibodies and concentrations as for DAB IHC staining, in conjunction with primary antibodies for tryptase , OCT4  and SALL4 . An appropriate fluorescent secondary antibody of Vectafluor Excel anti-mouse  or Alexa Fluor anti-rabbit 594  was used for detection.DAB IHC-stained slides were mounted in Surgipath Micromount mounting medium . IF IHC-stained slides were mounted in Vectashield HardSet anti-fade mounting medium and counter-stained with 4′6-diamino-2-phenylinodole .Positive controls were demonstrated on human placenta for cathepsin B, human breast tissue for cathepsin D, and mouse bone marrow for cathepsin G. Negative controls for DAB IHC staining were performed on sections of IDHWGB using a matched isotype control for both mouse  and rabbit  primary antibodies, to determine the specificity of the amplification cascade. Negative controls for IF IHC staining were performed using a section of GBM tissue with the combined use of primary isotype mouse  and rabbit  antibodies.DAB IHC-stained slides were viewed and the images were captured using the Olympus BX53 light microscope fitted with an Olympus DP21 digital camera and processed with the CellSens 2.0 Software . IF IHC-stained slides were visualized and imaged using the Olympus FV1200 biological confocal laser-scanning microscope and processed with CellSens Dimension 1.11 software using 2D deconvolution algorithm (Olympus).RNA extracted separately from ~20 mg of snap-frozen IDHWGB tissues obtained from six patients was used for NanoString nCounter™ Gene Expression Assay , as previously described . Probes n = 3) selected from the original cohort of six patients were used for mRNA CISH staining, using the Leica Bond Rx autostainer and detected using the ViewRNA red stain kit . Probes for cathepsin B (NM_001908), cathepsin D (NM_001909), and cathepsin G (NM_001911) were obtained from Affymetrix. Human placenta, human breast tissue, and mouse bone marrow were used as positive controls for cathepsin B, cathepsin D, and cathepsin G, respectively. Negative controls were exhibited on sections of GBM tissue using a probe for Bacillus .About 4 μm-thick formalin-fixed paraffin-embedded sections of IDHWGB tissues  and cathepsin G (p = 0.010); and level of expression of cathepsin D was more abundant than that of cathepsin G (p = 0.002).NanoString gene expression analysis was used to investigate the presence of mRNA transcripts for cathepsins B, D, and G with all expression values normalized against the housekeeping gene PGK1. Presence of mRNA coding for cathepsin B and cathepsin D was detected in all six IDHWGB samples, while the corresponding expression of cathepsin G was present in only four of the six samples Figure . Statistin situ hybridization demonstrated the presence of mRNA transcripts coding for cathepsin B and cathepsin D within the tumor cells , while both were much greater than cathepsin G   was significantly greater than the level for cathepsin D  Figure .The function of the RAS extends beyond its role in regulating cardiovascular homeostasis. The existence of a paracrine RAS within the local tissue that functions independently or synergistically with the endocrine RAS to maintain systemic and cellular homeostasis has been proposed . Dysregu+ CSC subpopulation being the most primitive, which we hypothesize proliferate and differentiate to give rise to the SOX2+/SALL4+ progenitor cells .TI and ST formulated the study hypothesis. TI, AW, and ST designed the study. SK, AW, HB, ST, and TI interpreted the DAB and IF IHC and the CISH data. SK, AW, ST, and TI interpreted the NanoString gene expression analysis data. SK performed cell counting on DAB IHC-stained slides. RM conducted statistical analysis and interpreted the results. SK, AW, ST, and TI drafted the manuscript. All authors commented on and approved the manuscript.TI and ST are inventors of the PCT patent application (No. PCT/NZ2015/050108) Cancer Diagnosis and Therapy and Cancer Therapeutic (62/452479). The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Multi-channel electroencephalography of cortical evoked activity was measured repeatedly over time at: 1) acute (0.5 ± 0.7 weeks); 2) early chronic (1.1 ± 0.2 months); and 3) chronic (5.8 ± 3.4 months) cochlear implant stimulation. Results indicated consistent responses from the normal right ear with marked changes in activity from the implanted left ear. Atypical distribution of peak amplitude activity from the implanted ear at acute stimulation marked abnormal lateralization of activity to the ipsilateral left auditory cortex and recruitment of extra-temporal areas including left frontal cortex. These abnormalities resolved with chronic implant use and contralateral aural preference emerged in both auditory cortices. These findings indicate that early implantation in young children with single sided deafness can rapidly restore bilateral auditory input to the cortex needed to improve binaural hearing.Early treatment of single sided deafness in children has been recommended to protect from neurodevelopmental preference for the better hearing ear and from social and educational deficits. A fairly homogeneous group of five young children (≤3.6 years of age) with normal right sided hearing who received a cochlear implant to treat deafness in their left ears were studied. Etiology of deafness was largely cytomegalovirus ( In the present study, we examined whether cochlear implantation of the deaf ear in a fairly homogeneous group of five young children with normal or near-normal hearing in the other ear can restore expected organization of the auditory cortices.There are significant consequences of single sided deafness (SSD) in childhood on auditory development and function6, and has known developmental and educational consequences8. These effects relate, in part, to poor spatial hearing10; indeed, unilateral listening in childhood reorganizes cortical areas involved in spatial awareness and attention12. Children with bilateral deafness who used one cochlear implant (CI) for some time prior to bilateral implantation are a unique group of single sided listeners who experience neurodevelopmental preference for the first/better hearing ear both in the brainstem15 and cortex16. Similar findings are reported from kittens born with unilateral deafness18 or experimentally induced unilateral/asymmetric hearing in young animals21. Such auditory asymmetries have consequences for processing binaural timing and level cues22 which are integral for locating and distinguishing one sound amongst many23. Importantly, bilateral cochlear implantation without delay protects from development of the “aural preference syndrome”16. Thus, to avoid problems of single sided listening and preserve opportunities for binaural hearing, it has been recommended that hearing loss  be treated by providing the most appropriate device in each ear as soon as possible3.The prevalence of childhood unilateral hearing loss is estimated to be 0.06 to 3.0%4. In the case of SSD where an auditory nerve is present, a CI is arguably the most appropriate device to stimulate the impaired ear26 but this is not the present standard of care. Potential for success is suggested by benefits of electrical stimulation from a CI in one ear and amplified acoustic input through a hearing aid contralaterally  in children with asymmetric hearing30 and from adults with SSD who were implanted to treat disruptive tinnitus in the deaf ear26. Benefits for listening to speech in noise are realized over listening with the unimplanted ear alone and increase as the duration of deafness decreases25. Early studies of implantation in children with SSD show early signs of benefit34 and one case report in an older child suggests the potential for longitudinal changes in crossmodal plasticity35. To understand the functional outcomes and define an optimal period for implantation in SSD, it is essential to address whether expected function in bilateral auditory pathways can be restored during early important developmental periods.Despite research-based recommendations, treatment in children with unilateral hearing loss has been inconsistentIn the present study, plasticity of the neural input to auditory cortices was measured to assess whether expected representation can be restored by providing electrical stimulation from a CI in one ear with normal hearing in the other ear in early development. Results in a group of young children (≤3.6 years) who were deaf in their left ears from infancy demonstrate marked and rapid uptake of input from the newly implanted ear, restoring symmetric representation of both ears in the auditory brain.37, accounting for an additional 5.4 ± 1.7 hours/day that the CI was worn. Daily CI use in the 4 children with complete data did not vary with CI experience (χ2(1) = 0.3, p = 0.61). This time was mostly spent in environments with moderate sound levels (50–70 dB A)  = 18.6, p < 0.001) at all three time points  = 0.5, p = 0.62; level × time: F = 0.2, p = 1.0)  was confirmed by datalogs available from the CI speech processor Fig. . One chi.0) Fig. , consistF = 0.3, p = 0.74; ear: F = 0.3, p = 0.58; time × ear: F = 0.2, p = 0.80; N2: time: F = 1.9, p = 0.18; ear: F = 0.2, p = 0.67; time × ear: F = 0.6, p = 0.54) or latencies  = 3.1, p = 0.07; ear: F = 2.5, p = 0.13; time × ear: F = 0.2, p = 0.84; N2: time: F = 2.9, p = 0.08; ear: F = 2.0, p = 0.17; time × ear: F = 2.2, p = 0.14) over time for either the hearing or CI ear. Opposite polarities of P1  and N2  are largely consistent from the normal hearing ear in topographical plots over time  were identified in the mean global field power responses  = 0.0, p = 0.85; time: F = 1.5, p = 0.25; P = 0.34; ear × time × coordinate: F = 0.8, p = 0.45). Peak dipole moments did not significantly change with CI stimulation in either auditory cortex for either the normal right ear (Left Cortex: χ2(1) = 0.09, p = 0.49; Right Cortex: χ2(1) = 0.007, p = 0.93) or left CI ear (Left Cortex: χ2(1) = 0.87, p = 0.35; Right Cortex: χ2(1) = 0.48, p = 0.49).Peak dipoles were measured from the voxels with the highest pseudo-Z in left and right auditory cortices  = 7.6, p = 0.006). Lateralization from the normal right ear was initially variable for the group , bilateral (n = 2), and abnormal ipsilateral (n = 1)) but consistent for each child over time (χ2(1) = 0.003, p = 0.96). Cortical lateralization from both ears, plotted for each child at initial CI use (two early time points) and after chronic CI stimulation . After chronic CI use, a distribution of expected contralateral aural preference had been established in both auditory cortices.The aural preference of each auditory cortex  was variable at acute stimulation Fig.  with abn35 and may reflect recruitment of the arousal and attention network38 for early stage cortical processing of sound. Thus, the naiveté of the ear coupled with the atypical input delivered by the CI induced heightened cortical reactions at the initial test. With chronic CI use, responses normalized with a marked reduction in extra-temporal activity.Cortical recovery from SSD occurred rapidly in a small but relatively homogenous group of young children (≤ 3.6 years old) who consistently wore their CI for several hours daily. Unexpected cortical responses to acute CI stimulation were characterized by abnormal distribution over the surface of the head , corresponding to high activity both within the defined temporal auditory areas and beyond in areas including the left frontal cortex. The extra-temporal activity identified in these five young children is consistent with a recent case study17. Similar results occurred with unilateral implant use in children with bilateral deafness16; importantly, those abnormalities often did not resolve despite several years of bilateral use and were associated with asymmetric speech perception16. By contrast, repeated measures in the present cohort of young children reveal remarkable developmental plasticity within a 6 month period likely attributable to both the relatively early stage of cortical development, during which synaptogenesis may still be possible39, and the relatively short duration of unilateral deprivation to cells expecting binaural input40.Auditory immaturity of the deaf ear resulted in asymmetric input to both auditory cortices at early stages of CI use. Consistent activity levels (dipoles) in auditory cortices were evoked over time but intra-subject measures indicated a shift with CI use from abnormal toward expected contralateral cortical lateralization from each ear and to expected contralateral aural preference in each auditory cortex. Thus, excitatory inputs from the deaf ear are preserved but initially reduced in number and/or strength41. This is particularly important in light of the high incidence of cCMV and EVA as etiologies of SSD9 which come with a risk of progressive loss of hearing in the normal ear43. Benefits of implantation to spatial hearing have been reported in older children with SSD31 but could be improved with better integration of bimodal input and preservation of binaural cues than presently possible47. Binaural disruptions by spectral cues coming from the pinna (outer ear) on only one side also need to be resolved. Importantly, the present findings demonstrate that bilateral pathways are available for these future efforts to promote binaural hearing when young children with SSD are provided with cochlear implants.Behavioral data in young children at such an early stage of device use were not possible to obtain but findings from children with longer term bilateral implant experience suggest that protection of bilateral pathways will promote symmetric speech perceptionIn conclusion, cochlear implantation in young children with SSD effectively treats unilateral deafness by promoting bilateral auditory development. This gives an unparalleled opportunity to advance opportunities to promote binaural hearing in children deprived of this important spatial information.48. The children included in the present study were the first 5 with early onset single-sided deafness to undergo cochlear implantation in our program. Parental written informed consent was obtained for all participants according to study protocol #100000294 approved by the Hospital for Sick Children Research Ethics Board. Four of five children were diagnosed with congenital cytomegalovirus (cCMV) based on presence of CMV DNVA detected by PCR of the neonatal dried bloodspot (n = 3) or cCMV associated white matter changes on MRI (n = 1). MRI revealed an enlarged vestibular aqueduct (EVA) on the left side for the other child (S2). Four children were referred to our clinic once unilateral deafness was detected through neonatal hearing screening and upon parental concern in one child with cCMV (S3). Candidacy for cochlear implantation was determined by the multidisciplinary CI team based on protocols established in children with bilateral hearing loss50. Implantation of the left ear occurred at 1.1 years of age in one child (S5) and between 3.3 and 3.6 years (3.4 ± 0.1 years) in the other four children.Five children  with normal or near-normal hearing in their right ears  and severe to profound deafness in their left ears  participated in the present study. Hearing thresholds were obtained 1.9 ± 0.9 months (range: 1.0–2.9 months) prior to implantation at age 2.8 ± 1.0 years (range: 1.0–3.4 years old) using visual reinforcement  or play  audiometry with insert earphones. The decision to implant children with single-sided deafness (SSD) in our program has been a multi-stage process involving families and the multi-disciplinary cochlear implant team. A more detailed accounting of the factors involved in our population of children presenting with SSD has recently been reported36); datalog data was only available at one time (chronic stimulation) for S1. Because it is typically difficult to obtain or measure behavioral changes to speech during early stages of cochlear implant use in young children31, auditory function and plasticity were monitored using electrophysiology.Daily use of the CI was confirmed by datalogs available from the CI speech processor. Complete datalog data were available for four of the five children  beamforming method51. Briefly, the linearly constrained minimum variance type beamformer suppressed 97% of the CI artefact corresponding to the largest four singular vector values between −80 to 10 ms51 before localizing activity evoked by the implanted ear. Age-dependent head geometry and tissue conductivities were accounted for when calculating lead potentials for 63,307 3 × 3 × 3 mm voxels using a boundary element model mesh that was constructed from age-appropriate Montreal Neurologic Institute (MNI) head model templates generated using the Template-O-matic toolbox52. Activity in each hemisphere was evaluated by supressing the other hemisphere53. Peak activity in both the left (X ≤ −55 mm) and right (X ≥ 55 mm) auditory cortical areas  were analysed. Maximum dipole moment (nAm) and latency were extracted for all voxels, and the voxel with the largest signal-to-noise ratio (pseudo-Z54) above a statistical baseline threshold of noise (one-tailed omnibus-noise T-test55) in both auditory cortical areas was chosen. Consistency of coordinates and peak dipole moments and latencies were verified in the top 10 voxels with highest pseudo-Z values in these defined regions.EEG measures were recorded at three time points: 1) acute stimulation (0.5 ± 0.7 weeks of implant use); 2) early chronic stimulation (1.1 ± 0.2 months of implant use), and 3) chronic stimulation (5.8 ± 3.4 months of implant use). Recording was missed at the second time point (early chronic stimulation) for one child (S1) due to scheduling conflicts. Stimuli were 36 ms trains of acoustic clicks (100 µs) delivered at 250 Hz via an insert earphone to normal hearing ears or electric biphasic pulses (57 µs pulse-width) delivered at 250 Hz via an L34 processor to an apical electrode (#20) of the CI. These trains of stimuli were presented at 1 Hz. Levels were confirmed by maximum auditory brainstem response (ABR) wave V/eV amplitude to ensure similar activation of both ears at the upper part of the dynamic range (loud but comfortable)58), linear mixed effects regression59 with random intercept and slope for each child was conducted with the lme4 package60 to evaluate individual changes in daily CI use and source cortical activity with log-transformed duration of CI use while controlling for repeated values from the same child. Significance of the regression was determined using a likelihood ratio test. Repeated measured ANOVA was used to analyze average daily CI use across environments with different level ranges in dB A.Group surface activity was analyzed using repeated measures ANOVA. Given the progressive increase in follow up intervals, duration of CI use at testing was log-transformed. This log transformation permitted linear regression while preserving the effective non-linear relationship. As frequently used in biomedical sciences (e.g.,The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.


The vestibular implant seems feasible as a clinically useful device in the near future. However, hearing preservation during intralabyrinthine implantation remains a challenge. It should be preserved to be able to treat patients with bilateral vestibulopathy and  intact hearing. This case study investigated the feasibility of hearing preservation during the acute phase after electrode insertion in the semicircular canals.A 40-year-old woman with normal hearing underwent a translabyrinthine approach for a vestibular schwannoma Koos Grade IV. Hearing was monitored using auditory brainstem response audiometry (ABR). ABR signals were recorded synchronously to video recordings of the surgery. Following the principles of soft surgery, a conventional dummy electrode was inserted in the lateral semicircular canal for several minutes and subsequently removed. The same procedure was then applied for the posterior canal. Finally, the labyrinthectomy was completed, and the schwannoma was removed.Surgery was performed without complications. No leakage of endolymph and no significant reduction of ABR response were observed during insertion and after removal of the electrodes from the semicircular canals, indicting no damage to the peripheral auditory function. The ABR response significantly changed when the semicircular canals were completely opened during the labyrinthectomy. This was indicated by a change in the morphology and latency of peak V of the ABR signal.Electrode insertion in the semicircular canals is possible without acutely damaging the peripheral auditory function measured with ABR, as shown in this proof-of-principle clinical investigation. Bilateral vestibulopathy is the disease resulting in a reduced or absent function of the vestibular organs, the vestibular nerves, or a combination of both . It can This implant, in a concept analogous to the cochlear implant, captures motion and processes this information into an electrical stimulus that is delivered by electrodes to the vestibular nerves . HoweverAt this moment, two surgical strategies have been described: the intralabyrinthine approach  and the A 40-year-old woman who had to undergo a planned partial resection followed by radiosurgery of a vestibular schwannoma Koos Grade IV on her right side was selected. She still had normal hearing, with a Pure Tone Average tone threshold  of 10 dB on the right side and 5 dB on the left side  and posterior semicircular canal (PSCC) were bluelined, and the peripheral auditory function was monitored. Second, the LSCC was opened first, without disrupting the membranous labyrinth, and the peripheral auditory function was monitored again. Third, a conventional dummy electrode  was inserted in the LSCC approximately up to the cupula, and the peripheral auditory function was monitored. Following the principles of soft tissue surgery, the insertion was performed extra slowly to avoid strong hydraulic forces that could be transmitted throughout the inner ear. The electrode was inserted until a resistance was met, and from insertion depth it was deduced that the tip of the electrode was adjacent to the ampulla Figure . This siThe synchronized ABR video recordings of the surgery were analyzed in consensus by three authors . Regarding surgery, special attention was paid to the observation of damage to the membranous labyrinth and leakage of endolymph.All surgical steps of the study procedure and removal of the vestibular schwannoma were performed without complications. When opening the LSCC and PSCC, the membranous labyrinth was not disrupted at the point of fenestration. After insertion of the electrode and taking it out again, damage to the membranous labyrinth was observed in the lateral as well as the PSCC. However, no leakage of endolymph was observed, until the semicircular canals were opened completely as a part of the translabyrinthine surgery of the schwannoma.Auditory brainstem response audiometry results showed a stable morphology and a consistent peak V during the first 11 min after opening the LSCC. That is, the functional status of the cochleovestibular pathway appeared to be stable even after the LSCC was opened and after the electrode was inserted and removed. After opening the PSCC, peak V was still clearly present but it was delayed approximately 0.5 ms compared to baseline. ABR morphology, however, remained normal, and peak V remained present during opening of the PSCC and during insertion and removal of the electrode. Even after completely opening all three semicircular canals, ABR results appeared stable, with still a clearly visible peak V. However, after opening the vestibulum, ABR no longer showed any identifiable peaks, implying that functional hearing was clearly distorted. A largely delayed peak V seemed to return at a later moment, but morphology of the ABR signal and thus functional hearing still appeared to be compromised at this stage of surgery Figure . The posThe objective of this case study was to evaluate whether hearing can be preserved during the acute phase after electrode insertion in semicircular canals. It was shown that intralabyrinthine electrode insertion is possible without inducing significant damage to the peripheral auditory function measured by ABR, in the first half hour after opening the labyrinth and subsequent electrode insertion and removal. This is congruent with the experience in other intralabyrinthine surgeries such as semicircular canal plugging for intractable benign paroxysmal positional vertigo or superior canal dehiscence syndrome, in which hearing is often preserved , 29.Major difference between electrodes in this study and “plugs” in previous studies is the fact that electrodes do not immediately close the whole semicircular canal and that they might penetrate the membranous labyrinth. This is important, since leakage of endolymph and perilymph is generally believed to induce loss of cochlear and vestibular function , 30. In However, in another research group, previous vestibular implantations with electrodes designed to be selectively inserted in the perilymphatic space of the semicircular canals, resulted in hearing loss in all four Meniere’s patients following the implantations. Taken this into account, probably multiple factors could influence hearing during intralabyrinthine surgery. First, regarding the leakage of endolymph, patients with a hydrops (such as Meniere’s patients) or a gusher could be more susceptible to hearing loss , 31. WheMost of the abovementioned factors remain still partially speculative. Therefore, it should still be investigated which factors contribute to which extent to the occurrence of hearing loss during the acute phase of intralabyrinthine surgery. Until then, it could be advised to use “soft surgical” skills like preservation of the membranous labyrinth as long as possible, slow insertion of the electrodes, and avoiding suction directly on the perilymph or membranous labyrinth. Furthermore, it could be considered to open the labyrinth more far away from the ampulla and to not yet select patients with a suspicion of endolymphatic hydrops or gusher. In these cases, an extralabyrinthine approach might at this point be the safest procedure, since the labyrinth is not opened. Ideally, vestibular implantation surgery should be standardized. This would imply a safe surgical procedure and an optimal electrode design that complement each other, while both taking into account anatomical variabilities. At this moment, no optimal surgical procedure and electrode design are present yet , 21, 24,Regarding monitoring of hearing, the use of other (complementary) techniques could be considered like electrocochleography, or directly recorded cochlear nerve action potentials, in order to improve specificity. This, since ABR measurements are highly sensitive in detecting auditory damage, but specificity is poor .Previous unsuccessful attempts for hearing preservation during vestibular implant  might haFinally, after a protocol for hearing preservation in the acute stage of intralabyrinthine insertion is established, the long-term effects should still be investigated. For example, delayed neo-osteogenesis, reactive fibrous tissue formation around the array, and delayed inner ear toxicity  might all appear and could lead to delayed hearing loss .Due to obvious ethical limitations, the time for the study procedure was limited. This resulted in two main limitations. First, not all semicircular canals were inserted with an electrode. At this moment, intralabyrinthine vestibular implants in humans consist of three electrode branches that are inserted in each semicircular canal , 21. FutElectrode insertion in the semicircular canals is possible without acutely damaging the peripheral auditory function as measured with ABR and shown in this proof-of-principle clinical investigation.The procedures in this investigation were in accordance with the legislation and ethical standards on human experimentation in the Netherlands and in accordance with the Declaration of Helsinki (amended version 2013). Approval was obtained from the ethical committee of Maastricht University Medical Center (NL54761.068.15/METC162006). All procedures were performed at Maastricht University Medical Center.All authors contributed extensively to the work presented in this paper. JT, RS, and YT were the surgeons in this case. EG analyzed the ABR measurements. RB wrote the manuscript. FL created and edited the figures of the manuscript. FL, NG, JT, EG, J-PG, HK, MH, YT, JO, AP-F, and RS reviewed the manuscript and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.