Families of children affected with atopic dermatitis (AD) often report fear and anxiety regarding treatment with topical corticosteroids (TCS), which may lead to reduced compliance. The objective of our study was to measure, through a standardized questionnaire, fear of TCS in families of pediatric patients with AD and to identify items associated with fear.Families of pediatric patients with AD were enrolled in 9 Italian centers of pediatric dermatology. Enrolled parents were invited to fill in a questionnaire including questions on sociodemographic and clinical characteristics and 3 sets of questions on corticosteroid phobia . Determinants of the level of general fear were investigated through multivariable analysis.P = 0.011); believing that TCS may be dangerous independently from the specific side effect (P < 0.001). Moreover, TCS fear was associated with fear of applying too much cream (P = 0.001).A total of 300 outpatients with AD were enrolled. Most parents (80%) had a high instruction level. Eighty-one percent reported to have a certain amount of fear of TCS. At the multivariable analysis, fear of TCS was associated with the following items: believing that TCS treatment advantages do not overweight disadvantages  contains supplementary material, which is available to authorized users. Atopic dermatitis (AD) is a common chronic, pruritic, inflammatory skin disease, mostly affecting children, with heterogeneous lifetime prevalence figures, ranging worldwide from 8 to 18% –4. TakinWith the present multicenter study, we investigated prevalence and determinants of steroid phobia in a large group of Italian families of pediatric patients affected by AD.This is a multicenter, cross-sectional study conducted in the dermatology units and general pediatric units of 9 pediatric centers located in 9 Italian cities . The included centers represent some of the main Italian centers for pediatric dermatology.From September 2013 to September 2014, families of pediatric patients with AD consulting for the first time at one of the above mentioned centers were consecutively enrolled. Inclusion criteria were age <18 years and a diagnosis of AD. Exclusion criteria were presence of contraindications to the use of TCS  and presence of other forms of dermatitis.Prior to enrolment, one caregiver was asked to sign an informed consent to the study.The study was approved by the Bambino Gesù Children’s Hospital Ethical Committee (coordinator center) and by the Ethical Committees of all the other involved hospitals.At enrolment, a self-filling questionnaire was given to one of the caregivers attending the patient.The questionnaire included items on sociodemographic characteristics, previously identified atopic conditions, symptom duration, reason for the outpatient visit, clinical characteristics and SCORAD index at visit, quality of life assessment (DLQI), treatment prescriptions, previous therapeutic education.Finally, the questionnaire included a standardized set of questions on fear of corticosteroids , dividedIn the first section, fear of TCS was investigated, based on a 10-point visual analogue scale (VAS).In the second section, specific fears and beliefs were investigated through 21 items. The level of agreement to each item was measured through a 4-point Lickert-scale.In the third section, frequency of parents’ behaviours regarding TCS treatment was investigated through 10 items based on a 4-point Lickert-scale.AD was diagnosed according to Hanifin e Rajka criteria . SeveritWe estimated a sample size of 302, based on an expected prevalence of TCS phobia of 80%, with a ±5% precision level and a 97% confidence interval.The VAS scale used for expressing fear of corticosteroids was translated into categories as follows: 0–1: Not at all; 2–3: A little; 4–6: Moderately; 7–8: A lot; 9–10: Very much.The ordinal qualitative variables  of the second and third section of the questionnaire were converted into quantitative, defined as a score ranging from 0 to 3 .Additionally, we classified each item of the second and third part of the questionnaire into one category among efficacy, safety, compliance and quality of life. We then created 4 new “synthetic” variables, named after these categories. The value of the new variables was the sum of the scores of the items in each category, normalized to 100.Proportions were calculated excluding missing values. Results of the descriptive analysis are reported as mean and SD or median and range as appropriate.We performed a univariate analysis through simple linear regression to investigate factors associated with TCS fear, among the following: sociodemographic variables ; comorbidities ; clinical data including age at AD diagnosis, AD duration, AD evolution, AD treatment , SCORAD, median weekly pruritus, DQLI; having received therapeutic education; each item of the second and third section of the questionnaire and each of the synthetic variables .p < 0.05) at the univariate analysis. The first model included only the single items of the questionnaire . The second model included the synthetic variables. Normality was verified with Shapiro-Wilk test. Results were considered statistically significant with p < 0.05. Statistical analysis was performed using Stata 11.After checking for multicollinearity, multiple linear regression was used to create two multivariable models. Both models included fear of TCS as the dependent variable, and, as independent variables, those significantly associated with fear of TCS  were females.A large proportion of respondent caregivers had a high education level (80% had a high school or university degree).Twenty-seven percent of the included patients had comorbidity, mainly asthma or allergic disorders .Regarding AD, in most cases, the disease had been diagnosed during the first year of life (median age at AD diagnosis: 5 months), and the median AD duration at enrolment was 1.8 years. Most patients had a moderate-severe AD (61.7%). The mean pruritus score was 5 (SD: 2).A total of 287 patients were taking a treatment for AD at enrolment. A large proportion of patients were on topical steroids (82.2%), while 84% were on moisturizers. Concerning other therapies, 56 patients were (19.5%) on systemic steroids, 31 (10.8%) on topical immunosuppressants and 10 (3.5%) on systemic immunosuppressants. Almost 30% of patients were treated with an elimination diet. Two thirds of respondents had received therapeutic education, either continuous or occasional.Mean DLQI score was 3.Overall, a high proportion of patients reported having fears regarding TCS treatment: at the VAS scale, “No fear at all” was reported by 18.8% of the enrolled patients only . Responses to the other questionnaire items are reported in (Table P = 0.011); believing that TCS may be dangerous independently from the specific side effect (P < 0.001); moreover, TCS fear was associated with fear of applying too much cream (P = 0.001). Results of univariate and multivariate analyses are reported in Additional file The multivariable analysis identified few items significantly associated with fear of TCS treatment: believing that TCS treatment advantages do not overweigh disadvantages P = 0.01; believWhen subgrouping the items into categories, items concerning safety were more associated with fear compared with items concerning efficacy, compliance or quality of life.In the present study, we show that, in Italy, fear of TCS is very common among families of pediatric patients affected with AD.We investigated corticophobia in an exclusively pediatric population. Only few previous studies on TCS phobia have been conducted on a population of children –14.According to our results, fear of TCS is not motivated by specific reasons: most respondents reported that, according to them, TCS are dangerous independently from the specific side effects, and the treatment disadvantages are not balanced by its advantages. In previous studies, the predominant concerns about TCS were cutaneous side-effects and failure to thrive , 15, 16.Old generation TCS had a high systemic absorption rate, especially in children , 18. TheOur study also shows that fear of TCS is related to the fear of applying too much cream. Thus, corticophobia may trigger a vicious circle leading to treatment failure: a reduction of compliance may cause prolonged and frequent relapses, which reduce trust in the treatment, thus further decreasing compliance.Several studies highlighted the important role of the patient-doctor relationship in order to reduce fear of TCS , 14–16. Interestingly, 82% of our population was under TCS treatment, demonstrating that, despite fear, the parents were compliant to the prescription of their caregivers. Nevertheless, it is not possible to prove whether the use of TCS was adequate.A large proportion of participants (two thirds) had received a therapeutic education for the management of atopic dermatitis. However, therapeutic education did not seem to affect fear of TCS among participants. In Italy, a therapeutic patient education program has been introduced in the last 5–10 years, in the third-level centers participating in the study. This education program aims at informing the families of pediatric patients about the disease course and at empowering them towards an autonomous therapeutic management of the disease . The theThe present study has several limitations. The main limitation concerns selection bias. The patients were enrolled from amongst those presenting to the pediatric dermatology outpatient clinics of the above mentioned hospitals. Nevertheless, this is a multicenter study involving hospitals that are located in different Italian regions, therefore the sample should be sufficiently representative of the Italian population of pediatric patients with AD. Moreover, only a very limited number of families refused to answer to the questionnaire, nevertheless we cannot exclude that patients whose parents refused to answer to the questionnaire differed in terms of age and sex from those that agreed to enrolment. Another limitation concerns the score used to assess TCS phobia. We adopted the score proposed by Aubert-Wastiaux . The samAD is a chronic disorder and its treatment, mainly based on topical agents, is a long-term “therapeutic management”, rather than a common therapy. Awareness of AD treatments’ side effects is crucial, nevertheless, truths and misconceptions regarding this issue should be precisely addressed in order to ensure compliance. A specific educational program should follow a holistic approach and establish a strong relationship between the patient’s family and caregivers. In fact, the results of our study prove the need of implementing and focusing the therapeutic education on the use of TCS and to spread it to all professionals involved in the management of AD. It is mandatory that caregivers dedicate time to inform parents about the safety of the new generation products, whose main advantage is a clearly improved risk/benefit ratio.


A number of assessment tools exist to evaluate the impact of hearing loss, with littleconsensus among researchers as to either preference or psychometric adequacy. The itemcontent of hearing loss assessment tools should seek to capture the impact of hearing losson everyday life, but to date no one has synthesized the range of hearing loss complaintsfrom the perspectives of the person with hearing loss and their communication partner. Thecurrent review aims to synthesize the evidence on person with hearing loss- andcommunication partner-reported complaints of hearing loss. Searches were conducted in CosConference Papers Index, the Cumulative Index to Nursing and Allied Health Literature,Excerpta Medica Database, PubMed, Web of Science, and Google Scholar to identifypublications from May 1982 to August 2015. A manual search of four relevant journalsupdated the search to May 2017. Of the 9,516 titles identified, 78 records  met inclusion criteria and were taken through to data collection.Data were analyzed using meta-ethnography to form domains representing the person withhearing loss- and communication partner-reported complaints of hearing loss as reported inresearch. Domains and subdomains mutual to both perspectives are related to “Auditory”, “Social” , and “Self” . Our framework contributes fundamental new knowledge and a unique resource thatenables researchers and clinicians to consider the broader impacts of hearing loss. Ourfindings can also be used to guide questions during diagnostic assessment and to evaluateexisting measures of hearing loss. Hearing loss affects about 300 million adults worldwide , and thefull range of patient-reported domains relating to the everydayimpact of hearing loss. For example, of the nine questionnaires developed to measure theimpact of hearing loss on day-to-day life, only five explicitly involved patient involvementusing qualitative methods, namely the Communication Profile for the Hearing Impaired  or methodsused to collect potential content information. However, without this knowledge, we cannot besure that examining questionnaire items or subscales in isolation will give the Impaired ,PerformImpaired , SatisfaImpaired , the SpeImpaired , and theImpaired . The remIn 2001, the World Health Assembly endorsed the ICF for use as an international standardfor describing and measuring health and disability . It offecommunication partners is here taken as referring to “those withwhom the person with hearing impairment communicates with on a regular basis … their spouse,siblings, children, friends, relatives, colleagues, and carers”  the personal impact ofhearing loss from the perspective of the PHI  and (b) the impact hearing loss from the perspective ofcommunication partners (“DoHL-CP” framework). These frameworks consist of a hierarchicalframework with supradomain, domain, and subdomain groupings, all using inductive(data-driven) methods. A “domain” refers to a broad area of life that is negatively affectedby hearing loss . A “subdomain”refers to a distinct aspect of life that is affected by hearing loss such as a particularsituation or scenario . A “supra-domain” aims to broadlycategorize the domains . Subsequently, we then sought to (a) identifysimilarities and differences in the evidence collected from people with hearing loss andfrom communication partner(s), (b) compare the DoHL-P with the ICF Core Set for hearingloss, and (c) investigate whether any domains or subdomains vary as a function of hearingloss severity (data permitting).To date, there has been no comprehensive synthesis of what patients We followed the search strategy, data collection and synthesis methods, and the qualityassessment as laid out in a predefined protocol . Note thWe searched for studies that have reported what adults with hearing loss andcommunication partners report as problematic in everyday life. To be eligible forinclusion in the review, studies must have recruited adults (men and/or women) ≥ 18 yearsold who had been diagnosed with mild-to-profound hearing loss as the primary condition ofinterest or communication partner(s) who could be of any age or hearing status.Participants were required to use oral communication as their primary mode ofcommunication, but there was no restriction to those people using hearing aids or otherassistive listening devices. Any studies that investigated the perspective ofprofessionals only regarding the impact of hearing loss were excluded because it was notin the scope of our research question.We included intervention studies where data in these studies were taken at the initialassessment, as well as non-intervention studies. There was no restriction on the type ofstudy design. Resource and language limitations within the team led us to limit studies tothose published in the English language. The search was limited to publications on orafter May 1, 1982, because the HHIE questionnaire  was publEarand Hearing, International Journal of Audiology,Audiology, and Journal of theAmerican Academy of Audiology) from August 2015 to April 2017. This finalmanual search was conducted on May 3, 2017.To support an exhaustive literature search, published articles were identified throughnumerous electronic databases: Cos Conference Papers Index, the Cumulative Index toNursing and Allied Health Literature, Excerpta Medica Database, PubMed (includingMEDLINE), and Web of Science. Google Scholar was also searched page by page until itcontained no relevant articles. All electronic searches were conducted on August 31, 2015.Finally, to ensure that the review was up-to-date, we conducted a manual search of the topfour journals in which eligible studies had been sourced . Studies that were evidently irrelevant to the eligibility criteria ofthe systematic review based on the title were excluded. Next, the abstracts of studiesthat passed the title screen were independently screened in an unblinded standardizedmanner by two reviewers (DAH and VV). The reviewers screened the abstracts according tothe eligibility criteria of the review, such as the objectives, methods, and language ofthe study. Disagreements between reviewers were resolved through discussion. The full textof studies that met the eligibility criteria based on the abstract or where there wasuncertainty were obtained for review. The full text of studies were then independentlyscreened by the same two reviewers according to the eligibility criteria. Those that metinclusion of the review were retained for data collection.Data collection was conducted using a prespecified electronic data collection form. Tominimize observer bias, guidance material was created prior to the data collection process(see Supplementary file A), and then the data collection form and guidance were bothpiloted and revised across three iterations by VV and DAH. VV carried out data collectionand consulted with DAH and MAA to resolve any uncertainties.For each included study, we recorded the researcher performing data collection, studyauthors, title, year of publication, type of publication , and country of origin. For the study characteristics, werecorded the study design, whether or not hearing loss was the primary condition ofinterest, the wording of questions , sample size, andtheoretical framework reported by authors (if any). For the data items relating toparticipant characteristics, we recorded their mean age, gender, setting , and hearing status .For the complaints reported by both the PHI and their communication partners, we recordedthe measure used to obtain each hearing loss complaint or domain , the domain as described in the text, author examples orparticipant quotes describing their complaints, and perspective (referring to self or toother). For studies using closed-set questionnaires to assess the impact of hearing loss,we extracted data only for those subscales or questionnaire items that had beenhighlighted by the study findings or conclusions as reflecting experienced complaints. For intervention studies, data pertaining to our research question wasonly extracted at the initial assessment and therefore we did not extract informationabout effectiveness of treatments. Given that our primary research question was toidentify what are the reported complaints in everyday life experienced by adults withhearing loss as well as their communication partners, our data collection carefullyconsidered those complaints, examples, and quotes given by each party in terms of howhearing loss affected them personally. The terminology used by study authors, in the formof reported examples or quotes, was important to help us understand each authors’epistemological frame and hence to interpret their concept of each domain.Reported complaints, examples, and quotes associated with hearing loss typically referredto the negative functional impact on hearing ability or other psychosocial consequences ofhearing loss, but examples were wide ranging. The aim of the data synthesis was toidentify and group together similar data characteristics across studies into domains, andso data synthesis used a meta-ethnographic approach . Meta-etThe specific research question that data synthesis aimed to address was to collect andsynthesize generic and hearing-specific complaints in everyday life that are reported bypeople with hearing loss and also by communication partners.The scope of the synthesis was to focus on what studies had reported the personalimpact of hearing loss on individuals with hearing loss and the personal impact ofhearing loss on communication partners. Studies included in the data synthesis wereassumed to be of acceptable quality in terms of methods and reliability of results.Familiarization of the studies was first conducted in the study screening stage of theabstracts and full text of the studies. Data pertaining to the research question wasextracted for data synthesis during the data collection process.The first step of synthesizing the data required searching for and grouping the domaindata under descriptive labels that contained recurring keywords, such as “stigma” and“withdrawal.” All of the extracted complaints and domains were printed onto card foranalysis by the research team. To clearly identify which complaints were comparable, theprinted cards were sorted into groups, then the researchers looked through the cards forcommon and recurring themes. The extracted qualitative data were synthesized at domainlevel in the first instance. Preliminary domain groupings emerged from the given words,phrases, and sentences taken directly from the full texts (without any abstraction).Complaints that appeared to be nonspecific or contained limited information  weretemporarily placed in a “miscellaneous” group and carried forward to further review.Individual data items that could not be consolidated into the final domain groupingframework are reported as Supplementary file (available in Supplementary file B).The preliminary groupings were then thoroughly reviewed by the research team. Thedescriptive labels used to name each grouping were also reviewed based on the reviseddomain keywords. Suggestions were shared among all three authors leading to aharmonization of the domain classification .Auditory, Social, Self) at a higher level of abstraction , Cumulative Index to Nursing and Allied HealthLiterature , Excerpta Medica Database , PubMed , Web ofScience , and Google Scholar (n=25). The flow of studies through the review process is illustrated in The electronic search identified 12,096 studies in total: Cos Conference Papers Index(n=49). Fewer studies investigated its effects on thecommunication partner (n=11), but there were another 18publications exploring both perspectives as part of the same study. The age ofparticipants ranged from 18 to 92. Sixty-four studies reflected the views of both men andwomen; two publications recruited men only  and Domains of hearing loss – Communication partner (DoHL-CP). Allcommunication partner participants in studies that investigated this perspective werespouses or partners of the PHI. Included studies typically reported a mixture of domainsand illustrative examples, either in terms of individual questionnaire items orparticipant quotes. For this reason, we considered the data items supported in the primarydata synthesis to be equivalent, irrespective of the study design, or analysismethodology. The four additional studies identified in the updated manual search weresubjected to the same data collection. The extracted complaints of hearing loss wereconsidered with our frameworks to decipher if any new information or complaints werereported in these studies. However, these studies did not identify any new domains orsubdomains that were not already in our frameworks.The primary objective of this review was to collect and synthesize the complaints withhearing loss for people with hearing loss- and communication partner-reported complaintsof hearing loss. In the following section, the main findings are presented separately forthe two domain grouping perspectives (people with hearing loss and communication partner).Using the domain groupings, two frameworks were developed, termed n. Note that the numberof complaints only marks how often the complaint appears in the surveyed literature; itdoes not determine the importance of a complaint, either to an individual or everyone.Our classification scheme comprises of three overarching supra-domains. “Auditory” refersto domains relating to perception of sound and speech. “Social” refers to domains thatrepresent the impact of hearing loss on activities with friends and family, as well asattitudes to hearing loss. “Self” refers to domains relating to self-perception andpersonality. Each supra-domain encapsulated constituent domains and subdomains. “Auditory”had 4 domains and 35 subdomains . “Socialn=8complaints) such as fire alarms. Specific problematic situations reported includehearing the telephone ring (n=7 complaints),television/radio sounds (n=5 complaints), and the doorbell(n=4 complaints). While these complaints were notdirectly reported in the context of hearing in the DoHL-CP, communication partners didobserve the increase in the volume of the television and constantly having to answer thetelephone.This domain corresponds to the passive function of hearing, the ability to accesssound, but not always in the context of listening to speech. The seven subdomains withinthis domain describe difficulties in hearing various types of sounds. The most commonlyreported complaint was hearing warning sounds (n=17complaints).For people with hearing loss, the most commonly reported problem of the 12 subdomainswas listening to speech in noisy environments (n=3 complaints) “ … a hearing aid would be wonderful if they could solve theproblem … “ ; for example, “Originally I was told that I would lose my job if I needed touse interpreters”  as well as having to tell the PHI the phone is ringing (n=2 complaints):having to answer the telephone seemed to be that’s a source of annoyance becauseit’s never for me … and I have to take the call and then I have to go through the Ohyeah, I’m well thanks, How are you? This is due to the complaints relating to the telephone in the patientdomains, whereby a PHI avoided having to answer the phone due to difficulty hearing thetelephone ring, as well as listening to conversation while on the phone. Anotherreported subdomain, having to act as an interpreter (n=3complaints), refers to communication partners having to speak on behalf of the PHI,particularly in social situations, “ … by the time you’ve tried telling him what theyhave said, they’ve moved on … “ (n=2 complaints) which more generallycaptures the accommodations by communication partners to the PHI. One explicitlyreported strategy, “speech production by partner” (n=4complaints), represents the communication partner having to raise their voice in orderto facilitate effective communication with the PHI.This domain refers to any additional responsibilities or roles the communicationpartner has had to take on as a result of the limitations hearing loss has imposed ontheir communication partner that prevents them from carrying out certain tasks. The fivesubdomains within this domain describe roles relating to listening and communication inthe home and social settings. The most commonly reported task reported by communicationpartners was having to answer the telephone  to the extent that several examples reportedindividuals with hearing loss avoiding taking phone calls (n=7 complaints) across the 29 studies that questionedpartners. The next frequently reported difficulty for people with hearing loss waslistening to speech in noisy environments (n=17complaints). Listening to the television and radio was also common, resulting in havingto raise the volume (n=14 complaints). There was a directequivalent for communication partners, which was that of having to listen to thetelevision or radio louder than what they would normally prefer (n= 9 complaints).This domain refers to difficulties experienced when hearing is purposefully engaged,implying some degree of attentional effort, especially but not always in the context oflistening to speech. The 13 subdomains see  within tne calls . Thecorn=13 and n=12 complaints, respectively). Nextconcerned asking people to repeat things (n=11complaints). The result of that for communication partners was having to repeat to theirpartner which was mentioned seven times (n=7 complaints).The second most frequently reported challenge for partners was conversing on aone-to-one basis (n=5 complaints). Examples and quotesindicate that this subdomain predominantly refers to conversations with the hearingimpaired spouse: “When you’ve spent forty years able to converse easily and then onegoes deaf it’s very difficult to adjust … “ . For communication partners, speaking problems also relatedto an increase in volume of their own voice when talking to their hearing-impairedpartner (n=3 complaints). For example, “I have to raise myvoice” . There was no overlap in the four subdomainsacross the DoHL-P and DoHL-CP. Quotes given by people with hearing loss made referenceto the inability to fully engage in a social event or social gathering, and physicallyremoving themselves from the situation due to experienced difficulties. For example, “Ifind myself avoiding company because conversation is too much effort” . For example, “ … he’s not participating in the actual conversation andthere’s just all this noise going on around him he just switches off” . For example, “My hearingloss makes me feel isolated from other people” (n=8 complaints). Forexample, “We don’t go along to our senior’s group anymore … isolated at parties”. Furthermore, they reported feeling isolated as acouple at social events (n=3 complaints).The domain “sense of isolation” encompassed feelings of separation and exclusion fromothers, especially in relationships or during social gatherings. Again, there was nooverlap in the three subdomains across frameworks. For people with hearing loss, theperception of isolation was very much in the context of themselves in certain socialsituations . Spouses or partners were oftenidentified as the primary motivator for seeking an audiological appointment usually as aresult of the strain on communication: “My wife threatened me with divorce” . For example,“My partner’s hearing difficulties has an effect on our intimate relationship” . For example, “there is no use indiscussing the problem with him…it does not work, it always ends up in a conflict”; “just can’t hearwhat they’re saying to me … it’s just awful” (n=14 complaints) of the difficulties hearing loss imposed on several aspectsof life such as at the compensation for the social dependence of the impaired spouse orhaving to undertake additional responsibilities. Another reported subdomain was theburden of adjustment to hearing loss (n=4 complaints)experienced by communication partners: for example, “I feel that it’s actually the otherpeople who are with him who suffer more than him because I think they’ve got to adapttheir living style rather than him” . The emotionalconsequences of having to raise the volume of the television or radio was frequentlyreported (n=6 complaints); for example, “What I find iswhen it gets up too high, it aggravates me. I don’t get any pleasure out of it … I’m nothaving a happy time” . This corresponds to the earlierresult that in the Auditory: Listening domain, loudness of the television or radio wasfrequently reported as a problem.Several emotional responses to hearing loss, and to the secondary problems caused byhearing loss were reported across both frameworks . In totat awful” , p. 279.n=13 complaints; partners: n=12 complaints) and across both frameworks. People with hearingloss frequently reported exerting greater effort in order to listen and follow aconversation (n=8 complaints), and consequently reportedfeelings of fatigue (n=6 complaints). For example, “I fellasleep when I was at the meeting and after they said to me I know how you fell asleep,because you couldn’t concentrate the whole period” : for example,“ … there’s no connection, you can’t hear. Well, it actually becomes part of yourself-esteem as well” (n=6 complaints); “You feel incomplete … mutilated” or hearing aids (n=7 complaints). For example,“having been diagnosed, I feel I have labelled myself” (n=11complaints). For example, “When I became conscious of it the hearing loss I kept tryingto deny [it]” (n=7 complaints). For example, “Lots of times it is useful if you tellsomebody something and they say, ‘OK.’ Rather than no response” . In particular, this was made in reference to aging or asign of “getting old” .Communication partners mentioned only the stigma of hearing loss (n=3complaints) or unwilling to accept their partner’s hearing difficulties were due tohearing loss “ … as a spouse you actually perpetuate. I suppose you deny it yourself asa well as a spouse. You say ‘Oh well, maybe it isn’t as bad as that, maybe I’m justimpatient … ‘“ .Communication partners also reported being in denial  their data that was coded as “component not covered”within the ICF , then maWe also investigated the pattern of reported complaints in relation to severity ofhearing loss, where hearing status was adequately specified. Only 27 studies reported thehearing status of participants, and even in these studies, there was considerablevariation in the way hearing loss was reported . Where possible, wegraded severity into three categories using UK audiometric descriptors based onComplaints relating to communication and speech comprehension were the most commonlyreported across the included studies, as well as emotional subdomains. The data extractedin the present review showed considerable overlap across hearing loss severity and thedomains extracted. The social impact of hearing loss, particularly social withdrawal wasalso represented across both (mild-moderate) hearing loss severities. The classificationof hearing loss severity, however, was based on pure-tone averages only. Informationregarding the onset of hearing loss and duration of hearing aid use are both importantpieces of information for examining the lived experiences of hearing loss as a function ofthe time that an individual has to adapt to their hearing loss. Neither of theseparameters was consistently reported.only enter our frameworks if they have been mentioned by a patient orcommunication partner and then reported in a paper. That is, should a potential topic notbe covered, or a patient forgets to say something, then potentially important data couldbe missed. In general, this cannot be ruled out, and so some measure of its potentialextent can be gauged by formally assessing the quality of the studies. Such appraisalsgive a general overview of the quality of reporting of the included studies and isconsidered an important component of reviews .Since we used an entirely data-driven approach, subdomains can The application of quality criteria to qualitative research is widely debated . This isWe chose the Critical Appraisal Skills Programme  to determine the assessment. This isThe remaining 44 studies  were subjected to a second quality appraisalas defined in the protocol . StudiesThe primary objective of our review was to collect and synthesize generic andhearing-specific complaints in everyday life that are reported by people with hearing lossand their communication partners. After extensive searching, we found 78 eligible studies.Information presented across these studies broadly encapsulated auditory and nonauditorycomplaints due to hearing loss. These complaints were extracted from the studies thenorganized as two hierarchical frameworks, DoHL-P and DoHL-CP, each comprising the same threesupra-domains .Across both frameworks, there were 14 domains and 79 subdomains representing the impact ofhearing loss. There was considerable overlap in some of these reported across bothframeworks. Many of the subdomains within “Auditory: communicating” were experienced by bothindividuals with hearing loss and communication partners. For example, individuals withhearing loss reported difficulties participating in conversation in several differentsituations, which in turn had impacted on the communication partner. Another subdomain inthe DoHL-P was difficulty hearing the telephone ring and listening to speech on thetelephone. This was represented conversely in the DoHL-CP within “Self: role ofcommunication partner” domain as “having to answer the telephone” and “having to say thatphone is ringing.” This domain overall comprises the accommodations or additional rolescommunication partners have had to undertake as a result of their partner’s hearing loss. Itrepresents the ways in which hearing loss has multifaceted effects on those close to a PHI,rather than purely at an individual level of someone experiencing hearing difficulties.Across domains that were common to both frameworks, there was some variability within thesubdomains: For example, the subdomains within “Social: relationships” of the DoHL-P fromthat of the DoHL-CP. For people with hearing loss, this was not acknowledged apart from theeffects of hearing loss on relationships with communication partners and family members ingeneral. Within the domain “Social: social life,” communication partners reported that as aresult of the negative experiences encountered by people with hearing loss, they experienceda reduced enjoyment of social activities and attending social events alone. This was alsorepresented in the “sense of isolation” subdomains where communication partners reportedfeeling isolated as a couple at social events and a reduction in attending events as acouple. This ties in with the PHI domains of the subdomain of social withdrawal.There were considerably more subdomains reported in the “Self: emotions” domain bypartners. Communication partners reported the burden and stress of having to adjust to theirpartner’s hearing loss as well as the emotional consequences hearing loss imposed on therelationship with their partner. Communication partners reported feelings of guilt and upsetin relation to the way they reacted to hearing loss and their lack of understanding of thePHI’s difficulties. Furthermore, communication partners reported far more subdomains withinthe “Self: effort and fatigue” domain. Effort in this context was particularly associatedwith having to accommodate and additional responsibilities and strategies undertaken toadapt to hearing loss. It is noteworthy that fatigue in relation to communication is presentin both perspectives rather than the person experiencing hearing loss alone.The “Auditory: communicating” domain contained the greatest overlap between patients andcommunication partners. This was particularly true for subdomains relating to conversationin different situations such as in noise or among a group of talkers. There was also someoverlap in the subdomains within “emotions” such as the frustration, anger, and the upset ofcoping and living with hearing loss across patients and communication partners. In addition,self-image was a subdomain present in both frameworks. For communication partners, this wasin relation to the image of themselves as a couple in society following hearing loss, aswell as the image of the individual with hearing loss alone. Feelings of fatigue, inrelation to listening and participating in conversation, were also reported by individualswith hearing loss and communication partners which again the shared problems hearing losshas on communication which involves interaction with other people.Perhaps unsurprisingly, most of the subdomains in the “Auditory” supra-domain were uniqueto people with hearing loss, particularly those associated with listening and hearingsounds. In addition, the occupational impact of hearing loss on those experiencing hearingdifficulties is only present in DoHL-P. These subdomains did not appear in DoHL-CP.We presume that the overlap across the two frameworks reflects different manifestations ofthe same domain: that is, how a particular difficulty of someone with hearing loss can havea secondary impact on their communication partner. Audiological assessments in clinicemphasize the hearing status of an individual. Many clinical measures do not adequatelycapture the psychosocial consequences of hearing loss from both perspectives, and they arenot typically aimed at communication partners. Considering the challenges encountered bycommunication partners as a result of hearing loss may contribute a more complete clinicalprofile of a patient undergoing audiological assessment and help optimize rehabilitationoutcomes. Future research should thus explore ways in which these domains can be measured inthe laboratory in order to integrate a holistic approach to clinical assessment of hearingloss: the DoHL-P and DoHL-CP can be used to guide the development of such measures.We found in the survey that 19 different questionnaires were used. We mapped theirindividual items onto the domains and subdomains of the DoHL-P; see Supplementary file F. Theresulting distribution is quite uneven. About 20% to 30% of the possible cells within theAuditory: hearing, Auditory: listening, and Self: emotions domains are represented by items,but only about 15% of the Auditory: communication and Self: identity domains, and just 5% orless of Auditory: Speaking, Self: Effort & Fatigue, and Self: Stigma. Some subdomains were entirelyunrepresented. No single questionnaire asks about every domain: the closest is theHearing-Dependent Daily Activities Scale  with sevTaken together, all these areas form part of a patient’s concerns that need be consideredand factored into rehabilitation based on the patient-centered care model. This is awell-established component of the delivery of many health and rehabilitation interventions; yet,deand their communication partners. Evidence fromvideo-recorded audiology appointments indicates that family members have a strong interestin being involved and sharing their experiences of the patient’s hearing loss, but thatthey are typically discounted by the audiologist  describes leisure choices made byolder people. People above 65 years old tend to spend more time doing sedate activitiessuch as watching TV and listening to music . “SuccesOur frameworks identify what experiences of living with hearing loss are shared and whatare unique to each perspective. The included studies demonstrated considerable variationin the study population characteristics and in the degree and etiology of hearing lossamong participants. Many studies did not fully describe the hearing status of the peoplewith hearing loss and rarely if ever disclosed the hearing status of the communicationpartners. There may be cases where the communication partner may have an undiagnosedhearing loss themselves, and some of their own hearing difficulties might unknowinglycontribute to the experiences described here. These limitations make it difficult toinvestigate how hearing status might influence the type of experiences and difficultiesassociated with hearing loss, but they also make it difficult to draw generalizableconclusions. It is also important to note that we did not find any data pertaining tocommunication partners who were not spouses or partners; there is nothing in the presentliterature about impacts on other communication partners such as siblings, children,friends, relatives, colleagues, and carers . Given tWhile the derivation of these domains consisted of a rigorous synthesis of a large amountof data, the domains and subdomains only represent hearing loss complaints that werereported by persons with hearing loss or communication partners in the studies analyzed.Every domain or subdomain could therefore only have entered into the framework either bysomeone mentioning it in response to an open query or because there was a correspondingitem or subscale in a questionnaire. The questionnaires extracted in the review tappedinto the subdomains depicted in the frameworks to varying degrees. The breadth can beillustrated by three examples: all three supra-domains are represented in the HHIE , butonlThere were a number of advantages to taking a data-driven approach in developing theframeworks, the primary one being that the frameworks are representative of patient- andcommunication partner-reported complaints, with minimized researcher bias. This does mean,however, that any complaints of hearing loss that were not reported in the includedstudies do not appear in the frameworks. The constructs represented in the each frameworkwere not based on the frequency of reporting in the literature, other than the constructbeing mentioned at least once within the extracted data. All the domains and subdomains inthe framework are therefore equally weighted. What remains unknown is whether thatequivalence in weighting is a conceptual reality: Do people with hearing loss and theircommunication partners actually consider certain domains to play a more significant rolein the daily lives? And to what extent are those impacts common to most people? This is aquestion for further research.Currently, auditory rehabilitation primarily serves to address the auditory degradation ofhearing loss. However, the consequences of hearing loss are multifaceted and can extend tovarious aspects of life as well as on people close to those with hearing loss. The DoHL-Pand DoHL-CP frameworks translate and summarize the vast qualitative research evidence ofcomplaints of hearing loss into an evidence-based hierarchy. The data demonstrate that thesecomplaints are Auditory , Social, and Self . These frameworks highlight aspects of hearing lossthat are not currently addressed in currently aural rehabilitation plans, especially thefar-reaching effects of hearing loss that may extend beyond the patient, particularly theeffects on family members and their involvement the patient’s experience living with hearingloss. This is particularly important during the early stages of auditory assessment anddiagnosis in order to form complete a clinical profile of patients with hearing loss andfacilitate personalizing rehabilitation plans to consider the patient in the wider contextof their circumstances.


A new cell type, interstitial Cajal-like cell (ICLC), was recently described in different organs. The name was recently changed to telocytes (TCs), and their typical thin, long processes have been named telopodes (Tp). TCs regulate the contractile activity of smooth muscle cells and play a role in regulating vessel contractions. Although the placenta is not an innervated organ, we believe that TCs are present in the placenta. We studied placenta samples from physiological pregnancies and in different variants of preeclampsia (PE). We examined these samples using light microscopy of semi-thin sections, transmission electron microscopy, and immunohistochemistry. Immunohistochemical examination was performed with primary antibodies to CD34, CD117, SMA, and vimentin, and TMEM16a (DOG-1), the latter was used for the diagnosis of gastrointestinal stromal tumours (GIST) consisting of TCs. We have identified a heterogenetic population of ТСs in term placentas, as these cell types differed in their localization, immunophenotype and ultrastructural characteristics. We assume TMEM16a could be used as the marker for identification of TCs. In PE we have revealed telocyte-like cells with ultrastructural signs of fibrocytes (significant process thickening and the granular endoplasmic reticulum content was increased) and a loss of TMEM16a immunohistochemical staining. This condition is characterized by increased blood pressure >140/90 mm Hg, oedema and proteinuria >0.3 g/day. In accordance with when the symptoms occur, PE is defined as early-onset PE and late-onset PE 1. During early human pregnancy, extravillous cytotrophoblasts invade the wall of the uterus, and spiral arteries transform them into large vessels of low resistance. Failed trophoblast invasion and spiral artery transformation leading to hypoxia occur in preeclampsia, restricting foetal growth3. Placental blood vessels are not innervated, and regulation of their vascular tonus is controlled by molecules brought by the blood supply4. However, vasoconstriction of the placental villous tree may also be very important in PE4. Recent data show that cells with pacemaker activity, named telocytes (TCs), promote smooth muscle cell contractions by generating electrical pulses10, suggesting that they could be potential regulators of vascular tonus in placental villi.Preeclampsia (PE) is a multisystem pathological condition with clinical manifestations appearing after the 2014.To date, limited studies have observed TCs in the walls of villi vessels in physiological pregnancy and obstetric pathologiesThe aim of the study was to study the ultrastructural and immunohistochemical characteristics of telocytes from placental villi in early-onset and late-onset preeclampsia.The histological examination of placenta sections stained with haematoxylin and eosin from the physiological full-term pregnancies showed normally capillarized villous trees with a balance of mature intermediate and terminal villi. Immature intermediate villi were present as small accumulations. All stem villi were completely formed.Histological examination of placenta sections from the control group at 26–31 weeks of gestation revealed villous trees with mature and immature villi Fig. . DystropElectron microscopy of placenta samples from the patients with physiological pregnancy displayed several types of TCs in the villi stroma. TCs were 2.85 ± 0.6 µm in diameter with 0.23 ± 0.08 µm-thick Tps (I type) Fig.  and were17. However, considering their morphological characteristics (long thin processes with which the cells interact and form a network), these cells may be classified as TCs18. The formation of a network via Tps is one of the key characteristics that allow these cells to be defined as TCs19.These cells were previously defined as reticular cells or foetal fibroblastsOne population displayed spindle-shaped cells (type II TCs) located under the syncytiotrophoblast basement membrane. These cells were connected with processes and formed a continuous chain under the trophoblast basement membrane. Type II TCs contain elongated nuclei surrounded by a thin cytoplasmic rim Fig. . Granula18  had ultrastructural features of fibroblasts (cisterns of well-developed granular endoplasmic reticulum located in the perinuclear cytoplasm and in processes) and smooth muscle cells (myofibrils and dense bodies located at the cell periphery). These cells contained well-developed granular endoplasmic reticulum cisternae with thin fibrillae and so-called “dense bodies” located at the periphery of the cellMyofibroblasts were also found in the adventitia of large vessels in stem villi and in the perivascular area in large mature intermediate villi. Spindle-shaped and stellated myofibroblasts (2.98 ± 1.10 μm in diameter) had 2-3 processes per irregular-shaped nuclei (2.17 ± 1.40 μm in diameter) with 1-2 nucleoli. Process widths varied from 0.23 to 1.16 μm. These cells had similar ultrastructural features, including long thin processes and a small number of organelles in the cytoplasm around the nuclei and local varicosities of the processes. Depending on cell location and the degree of villous fibrosis, the number of processes varied, their widths tended to increase, and cytoplasm was enriched with organelles. Depending on differentiation, the cells similar to TCs with long thin processes (in immature intermediate villi) were replaced with cells with ultrastructural signs similar to fibroblasts and myofibroblasts (in stem villi). Neither telocyte-like cells nor myofibroblasts were detected in the terminal villi.Immature intermediate villi immunohistochemical analysis revealed CD117-positive staining Fig.  where TCMyofibroblasts in the stem villi were characterized by weak and irregular expression of CD117 and CD34, but they were stained strongly with vimentin (+++) and SMA (+++). They also showed weak and moderate immunohistochemical staining (+−++) for the telocyte-specific marker TMEM16a (DOG-1).). The TMEM16a positive control was intestine samples. Immunohistochemical study of the positive controls with primary antibodies to TMEM16a revealed moderate staining of TCs with long thin Tps that formed a network by interacting with one another  in the control groups. However, more significant expression was detected in myofibroblasts in the vascular stem villi walls during full-term pregnancy Fig. . The TMEher Fig. .Figure 5The histological examination results revealed that intermediate villi (mature and immature) with evident dystrophic changes and focal stromal fibrosis were prevalent in patients with early-onset preeclampsia. Only small stem villi were completely formed, while predominantly large stem villi were not.Capillaries in intermediate villi were irregularly located and avascular villi were also present. Multiple villous tree infarctions were detected Fig. . Stem viThe early- and late-onset PE analysis of semi-thin placental sections with methylene blue staining revealed fields of view with sludged erythrocytes in the blood vessels of placental intermediate villi Fig. . Clots a. This difference was associated with the fibroblast cell differentiation and with notable dystrophic changes .CD117 and CD34 expression was negative in stem and intermediate villi stroma in the EPE placenta samples Fig. 3E,3 and 4G,HSMA expression in some areas of stem villi was weaker in the EPE placental samples than in the control group , though there was negative staining in other areas. In LPE, there were SMA-positive smooth muscle cells with dystrophic changes and irregular expression in stem villi. SMA expression was irregular and less in stem villi smooth muscle cells in patients with LPE than in patients from the control group with corresponding gestational ages.).Immunohistochemical study staining of the stem villi stroma with primary antibodies to TMEM16a were negative in the EPE cases, and only were staining was observed in single cells among the LPE cases Table .20. This network is one of the key features of TCs17. The name of these cells has recently been changed to TCs, and their typical thin, long processes are called Tps. To date, TCs have been found in all gastrointestinal organs22, urinary and biliary tracts, blood and lymphatic vessel walls22, fallopian tubes, the myometrium8, mammary glands23 and the placenta14. To avoid confusion, ICC cells located in organs outside the intestine are denoted ICLCs, though Popescu and Faussone-Pellegrini16 called all these cells TCs. Although the placenta is not an innervated organ, we believe that TCs are present. We studied placenta samples during physiological pregnancy and different variants of preeclampsia (PE). We previously detected TCs in placenta villi18, which is in agreement with studies by Succiu et al.14. According to the literature, the identification (recognition) of TCs was performed in accordance with the “gold” and “platinum” standards17. We have shown that there are differences in the ultrastructure of these cells depending on the villi type. Our data showed that all TCs in the placenta have long thin processes with local dilatations, with a few organelles in the perinuclear zone and in the Tp dilatations.In 1893, neurophysiologist S.R. Cajal described cells in the wall of the small intestine, which subsequently were named after him as interstitial Cajal cells (ICC). TCs interact with one another and other cells via long prolongations and establish a three-dimensional network around blood vessels and nerve endings18.Electron microscopy examination of the term placenta samples showed TCs in different villi types. TC ultrastructural characteristics indicated their heterogeneity and the presence of at least three cell types. All TCs had long thin processes with local dilatations and a small number of organelles in the perinuclear zone. In immature intermediate villi stroma, the TCs formed a network with their Tps that delineated the localization of future blood vessels  (I type). In mature intermediate villi, TCs formed a network under the syncytiotrophoblast basement membrane (II type) and around the villous capillaries (under endothelium) (III type), though in the stem villi vascular walls myofibroblasts were presentIn placental samples from patients with EPE and LPE, the morphological finding on TCs in the stroma of intermediate villi suggest possible fibroblast differentiation based on their larger cytoplasmic volume, the increase in granular endoplasmic reticulum cisternae, and the activation of synthetic processes, as TCs were reduced or absent. In physiological pregnancy, the same morphological changes were observed but only in the single intermediate villi. In contrast, the abovementioned processes were more pronounced in preeclampsia.25. We previously showed a placenta from a foetus having congenital abnormalities, including ugly mesenchymal stroma cells with thick processes and different sizes and forms. Moreover, TCs were absent26.Advanced fibrosis of the stroma in intermediate villi in PE had been described previously27. TGF-β enhances myofibroblast differentiation, but activation of the FAK signalling pathway leads to the transformation of myofibroblasts into fibrocytes and to the development of fibrosis28.Dystrophic changes in myofibroblasts in different organs under the hypoxia have been shown in a few studies24.Moreover, the smooth muscle cells and myofibroblasts from stem villi showed dystrophic changes in preeclampsia, including increased cellular vacuolization and accumulation of glycogen granules in the cytoplasm and decreased SMA expression. In patients with LPE, the stem villi, including their stroma, did not correspond to the gestation term (they were predominantly retarded)17. For example, TCs have been described as both CD117-positive and CD117-negative depending on the organ affiliation and localization17. TCs are probably a heterogeneous cell population30. Antibodies to TMEM16a (DOG-1) are not TC-specific but are specific for bowel stromal tumours (GIST). Because Vennucchi et al. (2013) described different types of TC in the gut, the authors of the manuscript assumed that TMEM16a was specific for TCs33. TCs in the fallopian tube wall were positive for TMEM16a34. TMEM16a is involved in the functioning of calcium-activated chloride channels. We used bowel tissue as a positive control because it has been thoroughly studied as part of the digestive system.Traditionally, СD117, CD34, and vimentin are known to be TC markersTMEM16a expression is present in TCs in all types of villi  in the control groups, but displayed more significant expression in myofibroblasts in vascular stem villi walls during full-term pregnancy.35.The stem villus originates from an immature intermediate villus, whereby a stromal channel network is reduced, and only a single large vessel remains in the centre of the villus. Stem villi would have dense stroma for the frame of the villous tree. Thus, it is believed that mature intermediate villi stromal channels should gradually disappear after 34 weeks of gestationin vitro studies36. Zheng Y. et al. (2014)36 showed that TCs could produce angiogenic factors and cytokines that promote proliferation and the formation of endothelial cells. The study provided the evidence that human lung TCs could produce growth factors, such as VEGF and EGF. Bosco et al. also demonstrated that TCs were positive for VEGF12. Conditioned culture medium from TCs induced the proliferation of human pulmonary microvascular endothelial cells. Thus, the authors considered that TC might play an important role in angiogenesis36.T.Cs could potentially mediate the development of vessels and endothelium. The basis for this hypothesis on the mutual influence of TCs and angiogenesis was demonstrated in 37, but our results showed that the cells in the stem villi have features of myofibroblasts.Recent evidence shows that TC localized in stem villi regulate smooth muscle cell contraction and may be potential regulators of placental villi vascular tonus2 and increased concentrations of pro-inflammatory cytokines38, which may be factors contributing to the appearance fibrosis in the stroma. This leads to a disturbance in the villous tree blood supply, the progression of foetal hypoxia, development of placental insufficiency, and preeclampsia. The abovementioned disturbances are associated with more severe effects before the 34th week of gestation39. Fibrotic stroma is responsible for low bloodstream resistance, impaired compensatory abilities, a tendency to sludge and clot formation.Impaired intermediate villi TCs have been associated with hypoxia and oxidative stress42. It has been found that hypoxia induces the development of new vessels43; however, these incompletely formed capillaries and vessels are often too narrow and fail to provide adequate blood supply in the placenta. Up to 25% microclotting in intermediate villi has been detected by scanning electron microscopy and previously noted by other authors44. Based on NOS expression, the authors assumed that TCs produced NO47. The production of the vasodilator NO in presence of hypoxia is accompanied by the production of the superoxide anion, which leads to the production of the peroxynitrite radical, which is a potent pro-oxidant that is immunohistochemically observed in TCs from PE placentas12.Normally, the vascular endothelium contains a wide range of antithrombotic and anticoagulant properties. Erythrocyte sludging and microclotting can potentially occur in the damaged endothelium of incompletely formed capillaries54. TCs are probably related to stem cells49. The ability of TCs to differentiate into smooth muscle cells has already been described previously50.It cannot be ruled out that TCs potentially have other functions. TCs may perform different functions aside from their pacemaker activity, including immunomodulation and functions related to angiogenesis and fibrosis52. We suggest the loss and impairment of TCs in preeclampsia . It cannot be ruled out that TCs potentially have other functions aside from their pacemaker activity, including immunomodulation and functions related to angiogenesis and fibrosis50.In our study, we have found a heterogenetic population of ТСs in the term placenta; these cells differed in their localization, immunophenotype and ultrastructural characteristics. Probably TMEM16a could be used as the marker for identification of TCs. Previously this marker was used for the diagnosis of gastrointestinal stromal tumours (GIST) consisting of TCsWe confirm that all methods in this article were performed in accordance with the relevant guidelines and regulations. We confirmed that informed consent was obtained from patients who participated in the study. A statement has been attached.Placental samples from 37 pregnant patients who had undergone Caesarean section at 25–39 weeks of gestation were examined, including 12 patients diagnosed with early-onset preeclampsia (EPE) and 10 patients with late-onset preeclampsia (LPE). The control group included 15 patients, including 10 patients with physiological full-term pregnancies (late control group) and 5 patients with a Caesarean section at 26–31 weeks not associated with preeclampsia (early control group). Additionally, 15 samples were examined using a ≪Philips CM100≫ electron microscope , including 9 samples from the placentas of women with preeclampsia (5 with early-onset preeclampsia and 4 with late-onset preeclampsia) and 6 samples from women with physiological pregnancies.Criteria for inclusion in the control group meant that placentas were obtained from physiological pregnancies after a Caesarean section that was not performed for the following reasons: (1) a uterine scar, 2) related to obstetrics pathology , or 3) an anatomically narrow pelvis.1.Criteria for inclusion in the PE group were as follows: (1) blood pressure >140/90 mm Hg and (2) proteinuria >0.3 g/dayCriteria for exclusion from both groups including the following: (1) acute and chronic inflammatory diseases, (2) severe extragenital pathology, (3) organ transplantation history, (4) history of oncologic diseases, (5) diabetes, (6) severe foetal pathology, (7) foetal congenital malformations, or (8) spontaneous delivery. All patients signed an informed consent to participate in the study.52; the latter was used for the diagnosis of gastrointestinal stromal tumours (GIST) consisting of TCs. Secondary anti-mouse and anti-rabbit antibodies with streptavidin-biotin complexes were also used .After macroscopic examination, tissue fragments from the central zone of each placenta were dissected and fixed in a 10% neutral formalin solution. Paraffin-embedded placental tissue sections (4 µm thick) were used for histological (stained with haematoxylin and eosin) and immunohistochemical examination with primary monoclonal antibodies to CD117 , CD34 , vimentin , SMA (smooth muscle actin) , and the new telocyte marker TMEM16a (DOG-1) th week of gestation obtained at an autopsy. For transmission electron microscopy, 1-mm3 fragments were taken from deep areas in the placental disk 2–5 minutes after the caesarean section. The material was fixed in 2.5% glutaraldehyde solution and 1% paraformaldehyde solution in 0.1 M phosphate buffer (pH 7.4), and additionally fixed in 1.5% OsO4 solution. Soon afterwards, this material was dehydrated and embedded in araldite. Semi-thin sections were stained using the PAS method with additional methylene blue staining. Ultra-thin sections were contrasted with uranyl acetate and plumbum citrate and further examined with a “Philips CM100” electron microscope 55. Cell size and prolongation thickness were measured by transmission electron microscopy. Statistical data analysis was performed using the “Statistica for Windows v. 8” program package. A difference was considered significant when p ≤ 0.05.The positive reaction products were detected by brown staining of the cells. An evaluation was performed using a semiquantitative method with the following grades: (1) 1 point (+) for weak staining, (2) 2 points (++) for moderate staining, and (3) 3 points (+++) for strong staining. As a negative control, the samples from the studied sections underwent a standard immunohistochemical analysis without incubation with primary antibodies. As a positive control, immunohistochemical staining with TMEM16a primary antibodies was performed with the following samples: (1) colon sample of a five-day-old newborn child after a surgical procedure due to the surgical pathology and (2) a small intestine sample taken from an abortion at the 20Investigation of the patients’ biological materials was legally confirmed by the patients’ informed consent. The Ethics Commission on biological investigations from the Research Center for Obstetrics, Gynecology and Perinatology from the Ministry of Health of Russia approved the study (protocol of the Commission meeting №6 of 09.06.2016)All the women provided written informed consent.This study has been performed within the State Assignment on the topic “Investigation of diagnostic and prognostic roles of molecular-genetic, immunologic and epigenetic factors in preeclampsia development” №116-08-22-1-000-2.


Thigh compartment syndrome (TCS) is a rare condition caused by high pressures within the fascial compartments of the thigh, impeding capillary flow and leading to decreased perfusion, tissue hypoxia, and necrosis. TCS is most frequently associated with trauma and anticoagulation but has also rarely been associated with exercise-related injury. We present the case of a 19-year-old female who reported painful swelling in her thighs and darkening of her urine after participating in a spinning class. On physical examination, the patient was found to have tight, painful thigh compartments with extreme tenderness on passive motion. Labs revealed a marked elevation of creatine kinase and leukocytosis. The patient was diagnosed with TCS and underwent emergent decompression fasciotomy and aggressive IV fluids for protection against myoglobinuria. Due to high clinical suspicion, prompt diagnosis, and early surgery, the patient experienced excellent recovery without functional deficits.  Thigh compartment syndrome (TCS) is a medical emergency that occurs when high pressures within the fascial compartments of the upper leg impede the flow of blood in capillaries. The initial lack of perfusion leads to pain and increased swelling within the closed compartment, and tissue hypoxia and necrosis eventually ensue. Therefore, TCS can be limb-threatening, life-threatening, or cause permanent neuromuscular deficits if left untreated .TCS typically occurs in the context of high energy trauma. TCS caused by bleeding into the fascial compartment, resulting from anticoagulation, underlying coagulopathy, or spontaneous hematoma, has also been described . On rareMost cases of exertional TCS are chronic in nature, as the large width of the thigh allows significant swelling to occur before the onset of circulatory damage. By contrast, acute TCS is a much rarer clinical entity. In this case report, we describe a patient who developed acute bilateral TCS following a high-intensity spinning class involving a stationary bike. The patient was a previously healthy 19-year-old Caucasian female college student who presented to the hospital with pain and swelling of the thighs bilaterally. Three nights prior to admission, she had attended a spinning class at a local gym for the first time. She stated that the session was intense and lasted 45 minutes, and although she exercised daily, the intensity of this class was outside of her normal level of activity. After the class, she noted swelling and pain in her thighs but did not seek medical attention until three nights later when she noticed that her urine was dark in color. Alarmed by the changes in her urine, the patient presented to the student health center, where she was noted to have rapid progression of thigh swelling accompanied by increasing pain. As a result of these findings, the patient was immediately transferred to the emergency department for further evaluation.Initial assessment revealed marked elevation of creatine kinase (CK) of 53,000 IU/L and leukocytosis of 11,260 white blood cells per microliter. The admitting team ordered an MRI of the lower extremities, which showed swelling in the anterior compartment with a feathery, high-intensity, fluid-sensitive signal within the vastus intermedius, lateralis, and medialis bilaterally  for decompression fasciotomy. In the OR, pressures were measured again on both sides, and the patient was found to have anterior compartment pressures of 28 mmHg and 26 mmHg on the right and left sides, respectively. The middle compartment pressures were 15 mmHg on the right, 13 mmHg left, and the posterior compartment pressures were 8 mmHg on the right and 7 mmHg on the left. During the operation, a lateral approach was used to ensure entry and decompression of all three compartments  bilaterally, and no debridement was required. Wet to dry dressings were placed with a plan for interval wound checks and closure by Plastic Surgery. Postoperatively, her pain improved and her sensation of tingling and numbness resolved. Her repeat CK decreased to 83,000 IU/L, and she was placed on aggressive IV fluids for renal protection against myoglobinuria. Serial metabolic panels were checked while monitoring recovery from rhabdomyolysis. On postoperative day 3, her wounds were appropriate for closure and she was taken by Plastic and Reconstructive Surgery to the OR. Her wound appeared healthy (Figure Following closure, her recovery was uneventful. The patient recuperated in the rehabilitation facility and was discharged from the hospital approximately two weeks after admission.In our review of the literature, we found two additional cases that reported TCS of the anterior compartment following spinning -4. OtherIt is not entirely clear why our patient developed TCS among the millions of other individuals who attend spinning classes. The patient was a college student and stated that she was a social drinker, which makes it possible that acute alcohol use in the days surrounding her spinning class contributed to the development of TCS . AdditioAlthough an MRI was ordered by the admitting team in this case due to the ambiguity of the patient’s symptoms and rarity of acute exertional TCS, it is important to stress that TCS is a clinical diagnosis and imaging is not usually needed. In fact, unnecessary imaging can delay diagnosis and surgery, thus increasing the risk of preventable complications. Clinical findings of pain on passive motion, increasing pain at rest and tenderness, paresthesia, pallor, paralysis, pulselessness, poikilothermia, and new onset neurovascular signs all suggest the presence of compartment syndrome. The diagnosis can be confirmed by measuring compartment pressures, with pressures around 30 to 45 mmHg typically indicating TCS and necessitating surgical intervention [A distinguishing feature of TCS that makes accurate diagnosis difficult is the delayed presentation of symptoms. This delay may be explained by the fact that the thighs have a relatively large potential space within the compartments, allowing increased swelling to occur before reaching the threshold for developing compartment syndrome. These patients may present with a slow clinical course, and the baseline bulk of thigh musculature further makes diagnosing TCS difficult. Therefore, physicians should maintain a high clinical suspicion for TCS as a missed diagnosis can result in catastrophic permanent functional deficits and complications that include ischemic contracture, infection, and crush syndrome . AccuratFollowing prompt diagnosis and fasciotomy, recovery from thigh syndrome can be excellent. Our patient was diagnosed early based on clinical suspicion and underwent surgery within 24 hours of admission, resulting in recovery without complications and a return to normal activities following rehabilitation. This demonstrates that a high clinical suspicion for TCS and rapid surgery can prevent permanent functional deficits from TCS.We conclude that our patient developed TCS and rhabdomyolysis following a high-intensity spinning class. As these and other high-intensity exercises become more prevalent, clinicians should consider exercise-induced TCS when evaluating patients for pain and neurovascular symptoms in the thigh. Early diagnosis and surgical decompression are critical to prevent the development of irreversible muscle damage and associated functional deficits. However, diagnosis may be difficult due to the subtle and delayed clinical symptoms of TCS. Detailed knowledge of the clinical signs of TCS, a high index of suspicion in patients with thigh injuries, and effective decompression technique can result in complete recovery and good functional outcomes.


Like all otophysan fishes, serrasalmids (piranhas and relatives) possess a Weberian apparatus that improves their hearing capacities. We compared the hearing abilities among eight species of serrasalmids having different life-history traits: herbivorous vs. carnivorous and vocal vs. mute species. We also made 3D reconstructions of the auditory system to detect potential morphological variations associated with hearing ability. The hearing structures were similar in overall shape and position. All the species hear in the same frequency range and only slight differences were found in hearing thresholds. The eight species have their range of best hearing in the lower frequencies (50–900 Hz). In vocal serrasalmids, the range of best hearing covers the frequency spectrum of their sounds. However, the broad overlap in hearing thresholds among species having different life-history traits  suggests that hearing ability is likely not related to the capacity to emit acoustic signals or to the diet, i.e. the ability to detect sounds is not associated with a given kind of food. The inner ear appears to be highly conservative in this group suggesting that it is shaped by phylogenetic history or by other kinds of constraints such as predator avoidance. They possess hearing structures that enable them to detect and identify different kinds of sounds in their environment, including conspecific calls, sounds produced by prey and predators, or abiotic cues that convey information on the environment7.The ability to discriminate both biotic and abiotic sound sources is crucial for fishes8, all fish species possess inner ears for sound detection but not all species possess sound generating mechanisms. This suggests that acoustic communication is not the primary constraint acting on the evolution of inner ears. Other life-history traits  could correlate with differences in hearing ability. Serrasalmidae is an ideal family to examine co-variation in hearing and life-history traits because this family includes species that share different features: sound production vs. mute species and herbivorous vs. carnivorous species.According to Ladich (2014)13. The Weberian apparatus consists of paired symmetrical chains composed of one to four Weberian ossicles  that lie in a linear sequence from the swimbladder to the inner ear. Interossicular ligaments and articulation of the Weberian ossicles with their respective centra allow ossicles to hold in position on each side of the vertebral column14. Von Frisch (1938)15 demonstrated that these ossicular chains function in sound transmission. However, the claustrum is no longer considered as having an auditory function, at least in characiforms and siluriforms. In these groups, it has a protective function of the anterior portion of the neural canal17. When the swimbladder vibrates in a sound field, vibrations are transmitted to the ossicles and this motion is transmitted to the endolymphatic fluid causing sagitta (saccular otolith) displacements19.The Ostariophysan subgroup Otophysi is characterized by a series of modified elements , called the Weberian apparatus, that connect the swimbladder to the inner ears21. Ladich and Wysocki (2003)22 demonstrated that bilateral extirpation of the tripus in the otophysan Carassius auratus, goldfish, resulted in a hearing loss ranging from 7 dB at 100 Hz to 33 dB at 2 kHz. Yan et al. (2000)23 showed that the deflation of the swimbladder in the goldfish caused a drop in sensitivity by 33 to 55 dB depending on the frequency. However, otophysans do not exhibit a common morphology of the Weberian apparatus and swimbladder and this variation seems to have an impact on hearing ability. In catfishes, larger swimbladders and Weberian ossicles as well as greater numbers of ossicles are associated with improved hearing capacities24.Several studies have highlighted the important role of the Weberian apparatus in the improvement of hearing abilities, in terms of both hearing bandwidth and auditory sensitivity27: Piaractus brachypomus ; Metynnis lippincottianus ; Myloplus rubripinnis  and Myleus schomburgkii . The other four, Serrasalmus elongatus Kner, 1858; Serrasalmus spilopleura Kner, 1858; Pygocentrus nattereri Kner, 1858 and Pygocentrus piraya , are classified as carnivorous piranhas29. Moreover, we note that five of these species are known to produce sounds. Serrasalmus elongatus, S. spilopleura and Py. nattereri emit low frequency sounds by contracting fast sonic muscles attached to the anterior chamber of the swimbladder32. Piaractus brachypomus and Pygocentrus piraya are also able to produce low frequency calls (unpublished data). The first goal of our study was to determine hearing capacities of these eight species and to investigate co-variation in hearing abilities and life-history traits . The second objective was to compare the morphology of hearing structures among the species and to evaluate if morphological variation may explain differences in hearing abilities. Our hypothesis is that vocal species as well as carnivorous piranhas would have better hearing abilities to optimize intraspecific acoustic communication and to localize their living prey, respectively.Our study focuses on the hearing abilities of eight species in the family Serrasalmidae. Four species are classified as leaf, fruit, or seed-eatingAuditory evoked potentials (AEP) were obtained for the eight serrasalmid species. AEP traces were similar in shape within a given frequency across all individuals Fig. . WaveforSerrasalmus elongatus detects frequencies up to 3300 Hz, whereas the frequency range of Myloplus rubripinnis and Pygocentrus piraya extends up to 3000 Hz. The eight serrasalmid species were most sensitive at the lower frequencies tested, between about 50 and 900 Hz  revealed a significant species effect on AEP thresholds (Table Post hoc tests (Tukey’s HSD) showed that, even if significant differences in auditory thresholds exist among certain species at certain frequencies, there is no clear distinction among the species over the entire frequency range . At 1200 Hz, the difference of sensitivity exceeded 27 dB SPL  and carnivores  largely overlapped  and non-vocal species  also largely overlapped  were described by means of three-dimensional reconstructions based on CT scans. Two species, ity Figs  and 6.FiPy. nattereri, Pi. brachypomus, M. rubripinnis, M. schomburgkii, S. elongatus and M. lippincottianus). The asteriscus (lagenar otolith) was the most voluminous otolith, with a mean value between 58.7% and 85.7% of total otolith volume  and Pi. brachypomus the most voluminous . The overall shape and location of the three pairs of otoliths in the neurocranium were similar in the six species  and the ascending process is shorter in the piranha species (Py. nattereri and S. elongatus).The number of ossicles in the Weberian apparatus was the same in the six species Figs , 6. EachP = 0.0195; relative volume: R² = 0.739, P = 0.028; relative surface area: R² = 0.914, P = 0.003; see Supplementary Table For each hearing structure , linear regressions were performed between the averaged relative morphological measurements of the structure and hearing thresholds at each frequency tested. No clear relationship between the morphology of these structures and hearing sensitivity was observed in serrasalmids  followed by an increase in sensitivity in higher frequencies. Comparisons of AEP audiograms showed few differences in mean hearing thresholds among the studied species.The Weberian apparatus is well-known to improve hearing abilities of otophysan fishes, in terms of both hearing bandwidth and hearing sensitivity35 previously determined the hearing thresholds of Pygocentrus nattereri by means of a behavioral method. He found that the highest sensitivity was between 100 and 600 Hz. He noted that the range of best hearing covers the frequency spectrum of drumming sounds produced by this species. This is also the case for the vocal species investigated in our study. Sounds emitted by S. elongatus, S. spilopleura, Py. nattereri and Py. piraya have a fundamental frequency between 100 and 200 Hz and contain harmonics32, whereas sounds produced by Pi. brachypomus have a dominant frequency around 80 Hz without harmonics . We infer that vocal serrasalmid species used structures to produce sounds within the hearing range in which they hear best. However, the hearing thresholds of non-vocal species are similar to the hearing thresholds of vocal species 38 showed that three species of acanthomorph labyrinth fishes (gouramis) differing widely in their abilities to produce sounds possessed similar auditory sensitivity and similar sensory epithelia morphology.All the species had their best hearing sensitivity in the low frequencies (50–900 Hz). Stabentheiner (1988)ies Fig. . This in36. A splashing noise emitted by a hurt fish swimming near the water surface is known to attract carnivorous piranhas and to motivate them to attack39. Herbivorous serrasalmids primarily eat seeds and fruits27. Consequently one could expect that their hearing abilities, in addition to their lateral line system, might allow them to detect these items falling into the water. However, the range of hearing thresholds of herbivorous and carnivorous species strongly overlapped  in our study possessed the smallest sagittae. However, further studies using microphonics, an electrophysiological recording technique, are needed to test experimentally the influence of sagittal morphology on high frequencies sensitivity41. It is also worth mentioning that the majority of significant hearing differences among serrasalmids were observed in the frequency range 900–2400 Hz. These hearing differences were not correlated with observed morphological variation.The number of ossicles comprising the Weberian apparatus was the same in each species and the overall shape and position of these ossicles were quite comparable, despite slight morphological variation among the species. The otoliths were also similar in overall shape and position. We found an association between sagittal morphology and hearing sensitivity at the highest frequencies tested (2700–3600 Hz). Small sagittae are correlated with low hearing thresholds (i.e. better hearing abilities). Chardon and Vandewalle (1997)44. Unfortunately, these last structures were not visible on the µCT scans and were not reconstructed.The slight differences in hearing sensitivity among the species investigated in our study could be due to more subtle morphological variation. For example, the slight morphological disparities observed in tripus shape or in the length of ossicles processes could influence hearing sensitivity. Moreover, the orientation patterns of ciliary bundles of the sensory hair cells on the maculae (sensory epithelia associated with the otoliths) could also have an impact on the acoustic abilities in fishes45. Contrary to Cypriniformes, the diversity in swimbladder and Weberian apparatus morphology in the otophysan Siluriformes (catfishes) is high. Lechner and Ladich (2008)24 showed that larger swimbladders and ossicles as well as higher ossicle number (i.e. less evolutionary loss of ossicles) improve hearing at higher frequencies in catfishes. The relative lengths of swimbladders and of ossicular chains were correlated with hearing sensitivity above 1 and 2 kHz, respectively, whereas the number of ossicles affected hearing at 4 and 5 kHz. In our study, the number and the morphology of Weberian ossicles were conserved among the species and did not have an obvious impact on hearing abilities.Morphological variation of the Weberian apparatus within a single fish family has rarely been studied. Within the otophysan Cypriniformes, the morphology of the Weberian apparatus is generally diagnostic for each family and only slight interspecific variations of the Weberian apparatus have been documented. These modifications comprised subtle changes in shape and length of Weberian ossicle processes (e.g. Bird and Hernandez)21. In the goldfish Carassius auratus, the Weberian apparatus reaches the adult condition at 25 mm length46. In the African bullhead catfish Lophiobagrus cyclurus, Weberian ossicles and ligaments are fully developed at 24 mm SL21, whereas ossicles are fully developed at 15 mm TL in the air-breathing catfish, Clarias gariepinnus47. According to the size of serrasalmid specimens used in AEP experiments, they all have a completely developed Weberian apparatus meaning the ear is fully functional. Although it is not always the case48, hearing sensitivity could potentially change with fish size, as in the catfish L. cyclurus that showed significant frequency-dependent change in hearing thresholds with size but no change in the range of detectable frequencies.The size ranges of the different species indicate that the majority of specimens were juveniles or subadults. Hearing range and auditory sensitivity are known to increase along with the development of the auditory structures50. Some authors hypothesized that hearing in fish may be adapted to the ambient noise of the fish’s habitat51. Since rheophilic species live in noisy rapids, it would be quite interesting to determine if their hearing abilities and the morphology of their auditory system vary compared to serrasalmid species living in quieter freshwater habitats.It is worth mentioning that there are a number of rheophilic serrasalmid species which were not included in this studyIn serrasalmids, the broad overlap in hearing ability among species having different life-history traits  suggests that hearing capacities are likely not related to the ability to produce sounds nor to food detection. The morphology of otoliths and Weberian apparatus is largely similar in the species investigated and does not explain obviously differences in hearing abilities. The slight differences in hearing thresholds among the species could be related to more subtle differences in shape and functional performance of the different Weberian ossicles or to variations in the shape of maculae or in the orientation patterns of ciliary hair cells on sensory epithelia.Piaractus brachypomus (69 to 95 mm SL), 10 Metynnis lippincottianus (76 to 87 mm SL), 3 Myloplus rubripinnis (98 to 108 mm SL), 8 Myleus schomburgkii (55 to 61 mm SL), 5 Serrasalmus elongatus (66 to 79 mm SL), 7 Serrasalmus spilopleura (75 to 105 mm SL), 10 Pygocentrus nattereri (64 to 70 mm SL) and 5 Pygocentrus piraya (96 to 112 mm SL) were purchased in the aquarium trade. They were housed in freshwater aquaria at 26 ± 1 °C and were maintained on a 12 h light/dark cycle. The tanks were equipped with external filters, internal heaters and bubblers for aeration. Herbivorous fishes were fed granule food (JBL Novo Tab), whereas flesh-eating species were fed mussels, three times a week. All procedures were approved by the ethical commission of the University of Liège (ethics case 1532).Ten 53. This technique records summation of acoustically evoked neuronal responses along the ascending auditory pathways from peripheral hearing end organs to the brain region53. Presence or absence of a response to acoustic stimuli of different frequencies and intensities allows the determination of AEP thresholds. The experimental setup was similar to that used by Parmentier et al. (2009)54 and Colleye et al. (2016)55.Hearing thresholds were measured using the AEP techniqueNo anaesthetic was used to restrain the fish during the AEP recordings. However, each fish was immobilized in a custom-made harness in order to prevent body movements while allowing normal respiration. The harness was closed dorsally and caudally with clamps attached to a steel frame. Three subdermal stainless steel needle electrodes  were used to record electric signals. The recording electrode was inserted approximately 1 mm into the head, over the otic region. The reference electrode was placed into the fish’s epaxial musculature, whereas the ground electrode was in the water in close vicinity to the fish. Fish were suspended 10 cm below the water surface in a steel tube  closed at the bottom with a square steel plate (40 × 40 cm). The tube was oriented vertically and filled with freshwater of approximately 26 °C up to a height of 1.12 m. An underwater loudspeaker  was placed at the bottom of the steel tube. The entire setup was enclosed in a soundproof chamber.A Tucker-Davies Technologies  AEP workstation was used to generate sound stimuli and record AEP waveforms. TDT SigGen software was used to create sound stimuli with an RP2.1 enhanced real-time processor, a PA5 programmable attenuator to control sound level, and a power amplifier before being sent to and emitted by the underwater speaker. Sound stimuli were tone bursts of 50 ms in duration gated with a Hanning window. The phase of the tone was alternated between presentations to minimize electrical artefacts from the recordings. Fifteen frequencies were presented to each fish: 50, 150, 300, 600, 900, 1200, 1500, 1800, 2100, 2400, 2700, 3000, 3300, 3600 and 4000 Hz. At each frequency, sound levels were presented up to 164 dB re 1 µPa and were attenuated in 6 dB steps until a threshold level was determined. Evoked potentials recorded by the electrode were amplified , connected to an RP2.1 enhanced real-time processor, routed into the computer and averaged by BioSig software. At each frequency and for each sound level, the signal was presented 500 times. Sound levels of the acoustic stimuli were calibrated with a Brüel and Kjær 8101 hydrophone  placed in the steel tube at the previous position of the fish’s head. The hydrophone was connected to a calibrated Brüel and Kjær 2610 amplifier that gave the absolute pressure level of sound stimuli. Evoked responses were averaged and power spectra were calculated using a 4096-point Fast Fourier Transform (FFT). The spectra were analysed for the presence of peaks at twice the stimulus frequency with heights at least 3 dB above the background level. For each frequency, the lowest sound level at which such peaks were present was defined as the auditory threshold for that frequency. Dead specimens of the eight species were also tested to confirm that recorded AEP traces were not artefacts. No responses were recorded with dead fishes  and carnivorous species , as well as for vocal  and non-vocal species . To achieve this, we selected the highest and lowest hearing thresholds among the species of each group  at each frequency tested.In addition to the hearing curves of each species, we also traced the hearing curves of maximum and minimum sensitivity for herbivorous , three Piaractus brachypomus , two Myloplus rubripinnis (136 mm and 140 mm in SL), two Myleus schomburgkii (242 mm and 252 mm in SL), four Serrasalmus elongatus , and four Metynnis lippincottianus  was investigated using computed tomography (CT). These specimens were scanned at the National Museum of Natural History in Paris with a µCT scanner . The imaging system was set at 70 kV and specimens were scanned at an isotropic voxel size between 37.1 and 99.9 µm. Volume and surface rendering was performed with AMIRA 6.3 and AVIZO . Morphological measurements were performed with AVIZO. For each specimen, we measured the volume of the three Weberian ossicles having an auditory function , the three otoliths  and the anterior part of the swimbladder. We also measured the surface area of each otolith as well as the length of the sagitta. In addition, the ratios of the volume of each Weberian ossicle to the total Weberian ossicle volume were calculated. The ratios of the volume (and surface) of each otolith to the total otolith volume  were also calculated, as well as the ratio of the volume of the anterior part of the swimbladder to the total swimbladder volume. Finally, we determined the ratio of the sagitta length to the fish standard length.The morphology of the auditory system of three All procedures and all methods were approved by the ethical commission of the University of Liège (ethics case 1532). All experiments were performed in accordance with the relevant guidelines and regulations.post hoc tests were performed to compare sound pressure thresholds between species at each frequency.The variables were tested for the assumption of normality using Kolmogorov-Smirnov tests. A nested ANOVA was performed to compare sound pressure thresholds (dependent variable) obtained at the different frequencies tested. Species and frequency were selected as fixed factors. Tukey’s HSD Linear regressions were used to examine the relationship between fish size and auditory sensitivity at the different frequencies and also to determine if there was a relationship between the averaged morphological measurements of the different auditory structures and auditory sensitivity at the different frequencies. We also looked for trends between morphological features and auditory sensitivity at three larger frequency bands .P < 0.05.Statistical analyses were performed with Statistica 13 and GraphPad Prism 5.0. Significance level was determined at The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.Supplementary information