Aeq = 67.6 dB) and 656 in the control one (LAeq = 53.4 dB). Respondents completed a validated ICBEN 5-grade scale “noise annoyance questionnaire”. In the exposed group a significant source of annoyance was road traffic noise (p < 0.001), noise from entertainment facilities (p < 0.001), industrial noise (p < 0.001), and noise from neighboring flats (p = 0.003). The exposure to PMPs was objectified by the conversion of the subjective evaluation of the volume setting and duration. With the cooperation of the Ear, Nose and Throat (ENT)specialist, we arranged audiometric examinations on the pilot sample of 41 volunteers. Results: From the total sample of respondents, 79.2% reported the use of a PMP in the course of the last week, and the average time was 285 min. There was a significant difference in PMP use between the road traffic noise-exposed (85.6%) and the control group (75.8%) (p = 0.01). Among PMP users 30.7% exceeded the lower action value (LAV) for industry . On a pilot sample of volunteers (n = 41), audiometry testing was performed indicating a hearing threshold shift at higher frequencies in 22% of subjects. Conclusions: The results of the study on a sample of young healthy individuals showed the importance of exposure to social noise as well as to road traffic noise and the need for prevention and intervention.Purpose: Social noise exposure is currently an emerging problem in adolescents and young adults. Various leisure time activities may be responsible for hearing impairment (temporary or permanent hearing threshold shift or hearing loss). The study aimed to quantify environmental noise from various sources—voluntary  noise , high-intensity noise exposure events), and road traffic noise and to detect hearing disorders in relation to individual listening to PMPs in the sample of young adults living and studying in Bratislava, the capital city of Slovakia. Methods: The study included 1003 university students  living in Bratislava for 4 or more years; 347 lived in the student housing facility exposed to road traffic noise (L Noise has attracted widespread attention as a significant environmental and occupational health concern. The environmental noise has often been referred to as the “forgotten pollutant” but is now recognized as an environmental and public health issue that needs to be addressed in modern society. It concerns mainly the population living in urban areas but also in rural areas with the main sources being road, rail and air traffic, industries, construction and public work, and the neighborhood ,2,3,4. AIn addition to exposure to environmental noise, voluntary or social noise exposure is very important nowadays. This type of exposure is currently a major problem, especially among adolescents and young adults. Various leisure time activities  and regular visiting of events with high noise exposure intensity) can be responsible for auditory  or non-auditory effects on individuals  ,3,7,13.Hearing loss due to noise exposure is the most common cause of deafness and hearing impairment. Although genetics and advanced age are the main risk factors, temporary and permanent hearing impairment is increasingly common among young adults and children, mainly due to increased exposure from personal music players and noisy leisure time activities .Excessive listening to personal music players often leads to the shift of hearing thresholds. The development of new technologies brings to market devices that can produce sound at high levels and thus cause considerable damage to the hearing organ. Hearing damage is the most common in people who listen to music on PMPs for more than 5 years. In Europe, there are 2.5 to 10 million listeners of PMPs, most often children and young people. Estimated unit sales ranged between 184–246 million for all portable audio devices and between 124–165 million for MP3 players . Listening to loud music from portable music players at least five hours a week exceeds the noise standards to which workers may be exposed in the noisiest industrial enterprises. By inserting the earphones into the ear, the hearing canal closes, and the intensity of the sound/music increases. These characteristics become increasingly detrimental with longer and louder PMP listening. This relates not only to music but also to any sound with a high volume ,16.The study aimed to quantify environmental noise from various sources—voluntary  noise , high-intensity noise exposure events) and road traffic noise and to detect hearing disorders in relation to individuals listening to PMPs in the sample of young healthy Slovak individuals—university students living and studying in Bratislava, the capital city of Slovakia.The research was conducted at the Institute of Hygiene Faculty of Medicine Comenius University in Bratislava, Slovakia. We used the method of subjective evaluation using a standardized anonymous questionnaire and methods of objectification by direct measurement of noise levels using the hand-held sound analyzer with the frequency analysis software. For screening of hearing, we used threshold tonal audiometry, which determines the hearing threshold for pure tones for both left and right ears.n = 1500) was composed of students enrolled at the Faculty of Medicine, Comenius University. The respondents represented a homogenous sample of young healthy individuals of comparable age, education, and lifestyle (n = 1100). Subjects from the exposed group stayed at the college dormitory Druzba at Comenius University (CU). This group included 347 (34.6%) subjects. Subjects from the control group stayed at the college dormitory of Ludovit Stur, Stare Grunty, CU. This group included 656 (65%) university students. The exposed housing facility and student dormitory is situated near the major transportation route, the main thoroughfare with railway transport; the control housing facility and student dormitory in a quiet area with surrounding greenery. Subjects significantly differed by age, by traffic noise exposure, flat location in relation to noise exposure, position of flat in floor height, length of stay in the given area, orientation and the type of windows, satisfaction with flat surrounding, and the use of personal music players (PMPs)  females and 306 (31%) males. The average age was 23.13 ± 2 years. The source population (ifestyle . The ress (PMPs) .We used a validated methodology for subjective evaluation of noise annoyance and interference with various activities. We assessed the quality of sleep and psychosocial well-being in relation to noise exposure. Respondents filled in validated “noise annoyance questionnaires”, using a standard 5-point scale  developed and recommended by experts from the The International Commission on the Biological Effects of Noise (ICBEN) team ,18. We fWe used the method of objectification by direct measurement of noise levels using a hand-held sound analyzer Brüel–Kjaer 2250 with the frequency analysis software. We measured equivalent sound levels in the exposed and also in the control area. All measurements were recorded according to the valid Slovak legislation during the time intervals from 17:00–18:00 and from 20:00–21:00 in the exposed and at the same time in the control area. [In the estimation of exposure from PMPs, we used the methodology of Portnuff et al. ,20. RespThe hearing examination was provided for volunteers in the cooperation with Ear, Nose and Throat (ENT) specialists in the out-patient Department for Otorhinolaryngology in Bratislava. The students were examined by a subjective audiometric method in which the hearing was checked by the electro-acoustic device-audiometry. The basic examination of the threshold tone audiometry established a threshold hearing for pure tones (lowest stimulus intensity). For determining the hearing threshold for pure tones, the diagnostic audiometer MAICO MA 52 was used. This audiometer allows investigating overhead lines at frequencies of 250–6000 Hz and bone conduction at frequencies 500, 1000, 2000, and 4000 Hz. During the examination, the subjects were set into a quiet chamber, the audiometric cabin in the medical office. The chamber is used for audiometric testing tone audiometry, in particular for determining the hearing threshold for pure tones and meeting the essential parameters, especially, the desired attenuation of the intensity of sounds. It corresponds to ISO 266: 1997 and ISO 389–3: 2016.To evaluate the results, we used the methods of descriptive and analytical statistics to identify mutual associations between lifestyle factors, psychosocial, biological, behavioral and environmental factors using statistical package Epi InfoTM software, version 7.1.5.0, Atlanta, GA, USA, and SPSS, version 24 .Aeq = 67.6; 64.7 dB). Sound levels in the control area were significantly lower (p < 0.001)  [The monitoring of sound levels in the exposed area showed levels above the national and international limits in the afternoon and in the evening time interval 17:00–18:00 and 20:00–21:00 (L54.3 dB) ,18. The In addition to monitoring the noise levels, we evaluated the traffic flow . In the NDE = 66 ± 2 dB vs. LNDE = 56 ± 4 dB (p < 0.05) (http://www.laermkarten.de/bratislava/) [The indicators estimated from Bratislava strategic noise map were Lislava/) .p < 0.001), noise from entertainment facilities (p < 0.001), industrial noise (p < 0.001), and noise from neighboring flats (p = 0.003) , noise from entertainment facilities , industrial noise , neighboring flats noise  and railway noise . There was no significant difference concerning noise annoyance risks from house constructions and aircraft noise.When comparing the results of noise annoyance from different sources between the exposed group to road traffic noise and the control group, a significant source of annoyance was road traffic noise (= 0.003) . Based op = 0.0002) and also in the duration of listening to PMPs in minutes (286 ± 367 vs. 292 ± 367) (p < 0.55), but it was not significant between genders (p = 0.08).From the total sample of respondents, 794 (79%) students reported listening to PMPs in the last week for an average time of 285 min. There was a significant difference in PMP use between the exposed group to road traffic noise (86%) and the control group (76%) (About 16% of students listen to the music on the loudness level 4 (they cannot hear the speech or even the traffic), and 86.5% use earbuds. There was no significant difference between the loudness level of PMP or in the duration of time spent at most events with high noise exposure between the exposed and control group.p = 0.01). Earbud-inserted phone types are more harmful according to SCENIHR (2008) and increase the sound level by 7–9 dB [p = 0.23). The presence of subjective hearing impairment of PMP users was not significantly higher (24%), compared to the non-PMP users (20.7%) (p = 0.4).The significant difference was in the type of headphones; earbuds are more often used by students from the exposed area (more than 90% of students) (y 7–9 dB . Based on = 756), where they spent on average 481 min. The second most frequent activity was visiting a cinema (n = 514), where students spent on average 227 min per month. The third one was visiting discotheques (n = 437), where they spent on average 544 min per month. The fourth most preferred activity was visiting sports events (n = 242) where they spent on average 538 min per month. Students spent 556 min playing a musical instrument on average (n = 215). Many subjects (n = 206) attended rock, pop, or jazz concerts, where they spent on average 302 min per month. Students spent 203 min visiting classical music concerts (n = 90) and 191 min in sport shooting (n = 18)  .p = 0.08). There was no significant difference between the duration of time spent at the other events with high noise exposure between the exposed and the control group.Students from the exposed group to road traffic noise spent in the cinema about 244 min per month, compared with subjects in the control group who spent in the cinema on average 170 min per month (n = 41), in which we found indicated hearing threshold shift in higher frequencies in 22% of subjects.In cooperation with the ENT specialist, we performed audiometric testing on a pilot sample of volunteers, university students  subjects on the right ear and five (12%) subjects on the left ear .We also calculated hearing threshold averages: Low-frequency average  for the left and right ear, high-frequency average on the  for the left and right ear. Normal hearing was defined as 0–15 dB, slight loss 16–25 dB, mild loss 26–40 dB, moderate loss 41–65 dB, and severe loss 66–95 dB. Due to the small group of young healthy volunteers, the results of audiometric tests were only in the first three categories .The examined subjects had impaired hearing in the higher frequencies of 6000 Hz and 8000 Hz bilaterally and also in the lower frequencies of 1000 Hz in the right ear and 2000 Hz bilaterally .Of those volunteers who attended audiometric testing, more than 43% exceeded the lower action value (LAV) for industry (80 dB) by listening to their PMPs. In this group, the decreased hearing in 28% of subjects was found .p < 0.001. We can conclude that, apart from traffic noise, the students from the exposed area were also exposed to social noise from personal music players. However, the length and intensity of PMP listening did not differ significantly between these two groups.In our study on 1003 university students from Bratislava, up to 794 students (79.2%) reported listening to personal music players (PMPs) in the last week, with an average listening time of 285 min, and 26% of PMP users exceeded LAV . In the exposed group, 85.6% of respondents were listening to PMPs compared to the control group, where PMPs were used by 75.8% of students. This difference was significant on On the basis of audiometric tests in a pilot sample of 41 volunteers, we found a threshold shift at high frequencies in 22% of examined subjects. This vulnerable group of young people from the age of 20–30 years is exposed to many noise sources . This group requires special attention in the prevention of hearing impairment. Leisure time activities with high-intensity noise exposure are comparable to occupational noise . VariousIn the most recent studies, the authors recommended the use of high-frequency audiometry and evoked otoacoustic emissions (EOAE) (namely transient-evoked otoacoustic emissions (TEOAEs) and distortion-product OAEs (DPOAEs)) . OAEs (on = 1843), the prevalence of audiometric notches was only 2.4% and indicated the need to follow this sample longitudinally or focus on the older age groups, such as university students [Our results are consistent with the results of other studies, reporting 88–90% of adolescents and young adults listening to PMPs through headphones, especially earplugs. Recent publications evaluated increased risk of hearing disorders in relation to the listening to PMPs and the current incidence of hearing loss and tinnitus. The prevalence of hearing impairment caused by noise in adolescents and young adults was 17% and 29% in Europe . In a lastudents .Students in the group exposed to road traffic noise were listening to PMPs more often than students in the control group . However, the volume of listening to PMPs was not significantly different between those two groups, and neither was the duration of time spent at leisure time events or other activities with high noise exposure. These results are difficult to compare with the other relevant literature because we are not aware of any studies that have recently researched such associations.However, the study has several limitations. The design is cross-sectional, taking place at a single point in time and does not allow the discovery of a causal relationship between cause and effect. Longitudinal design controls the time factor and the influence on the development of the analyzed facts, so undertaking this type of study in the future should be considered. Recall bias resulting from overestimating or underestimating the level of subjective noise annoyance reported by respondents may occur. Interviewer bias might occur because questionnaires were administered by persons involved in the study and familiar with the topic. Aircraft noise is not an issue in this study because the study population lived far from the airport, located on the other side of the city. The pilot sample of 41 volunteers undergoing audiometric examination is too low, and we would like to enlarge the sample to 100 subjects in the future.Hearing loss due to noise is the most common cause of deafness and hearing impairment. Although treatment options are limited, for most people suffering from noise-induced hearing loss, several modifiable health habits should start to grow from childhood and can prevent or delay the onset of hearing impairment.The studies on hearing loss of youth and the identification of causes of hearing loss in adolescents are very important in order to develop additional precautions. It is also important to determine which groups of those young and healthy individuals are particularly vulnerable to effectively target preventive measures.The results of the study on a sample of young healthy individuals showed the importance of exposure to social noise as well as to road traffic noise and the need for prevention and intervention.Personal music players are now available to everyone in the form of MP3 players or smartphones, and listening to music through PMPs is extremely popular, especially among young adults. Leisure time activities with high-intensity noise exposure are also very popular  as well as sports activities and household or garden work. All these activities may be responsible for an early hearing impairment  starting at a younger age. After the finalization of the study results, we would like to formulate the proposals and interventional procedures and effectively target the preventive measures  in the vulnerable groups of teenagers and young adults and their parents and teachers as well.


In cochlear implantation (CI), the two factors that are determined by the surgeon with a potential significant impact on the position of the electrode within the cochlea and the potential outcome, are the surgical technique and electrode type. The objective of this prospective study was to evaluate the position of the slim, perimodiolar electrode (SPE), and to study the influence of the SPE position on CI outcome.Twenty-three consecutively implanted, adult SPE candidates were included in this prospective cohort study conducted between December 2016 and April 2019. Mean age at surgery was 59.5 years. Mean preoperative residual hearing was 92.2 dB. Intra-operative fluoroscopy and high-resolution computed tomography scans were performed to evaluate electrode position after insertion using a cochleostomy (CS) approach. Follow-up was 12 months after implantation; residual hearing (6–8 weeks) and speech perception (6–8 weeks and 12 months) were evaluated in relation to the intracochlear SPE position.scala tympani residual hearing was preserved [mean absolute increase in PTA of 4.4 dB and 77.2% relative hearing preservation (RHP%)]. Translocation into the scala vestibuli occurred in 36% of the insertions, resulting in a mean absolute increase in PTA of 17.9 dB, and a RHP% of 19.2%. Participants with a translocation had poorer speech perception scores at 12-month follow-up.In most patients in whom the SPE was positioned in the Given the incidence of CS-associated translocations with the SPE and the negative effect on outcome, it is advised to insert the SPE using the (extended) round window approach. The cochlear implant (CI) electrode array is the fundamental component of the CI system, as it provides the interface to the auditory system of the patient. Current CI electrodes are designed as either “precurved” or “straight”. Precurved electrodes are designed to curl around the medial wall and to assume a midscalar or perimodiolar position close to the modiolus, while straight electrodes assume a more lateral position, following the lateral wall of the cochlea . SeveralTo reduce intracochlear trauma during insertion, the slim perimodiolar electrode (SPE) was introduced in 2016. This electrode is 60% thinner and more flexible than the previous generation perimodiolar electrode produced by the same manufacturer . The SPEThe scalar position of the SPE, visualized in high-resolution imaging, was evaluated in 96 out of these 257 participants , 15, 16.This paper reports on a prospective study with the SPE implanted using the CS approach in 23 participants. In a previous temporal bone study , as wellIn this prospective study, 23 consecutive patients were implanted with the slim, perimodiolar electrode  between December 2016 and February 2018. All included patients were indicated for cochlear implantation, based on the Dutch CI indication criteria. As a part of the CI indication procedure, existing hearing aid fitting was optimalized, including fitting new hearing aids if deemed necessary. A CI was indicated if results in terms of aided speech performance with hearing aids is insufficient. Patients with functional residual hearing were informed about the study and invited to participate. Both patients with early and late onset of hearing loss were included in this study. Early onset of hearing loss was defined as an onset of hearing loss within the first 5 years of life. Patients with an early onset of deafness—i.e. prelingual onset of deafness—were excluded from this study.Demographic data, history of hearing and pre-operative audiologic measurements were collected pre-operatively. All surgeries were performed in a hybrid operating theater equipped with a high-resolution, rotational cone beam CT scan . Intra-operative fluoroscopy and high-resolution rotational CT scans were performed after insertion. Post-operative residual hearing thresholds were measured at 2-month follow-up and speech perception, with electric stimulation only, was measured at 2- and 12-month follow-up. Approval was obtained from the Institutional Medical Research Ethics Committee (NL57456.091.16) and participants signed informed consent before participating.The SPE is precurved with an active length of 14 mm and a diameter of 0.35 mm × 0.4 mm at the tip, and 0.45 mm × 0.5 mm at the base. It is designed to provide full ST position with all common surgical approaches, including RW, eRW, or CS . The SPEThe Artis Zeego system , a multi-axis system for interventional imaging with a flat-panel detector, was used for intra-operative 3D imaging. Immediately after insertion, the surgeon used fluoroscopy imaging to rule out the presence of a tip fold-over. Post-operatively, the position of the SPE was evaluated on the CT images by an experienced Head- and Neck Radiologist (BV). For each of the 22 electrode contacts, the location was determined as either an ST or SV position.Pre- and post-operative (2 months) unaided pure-tone thresholds at 125 Hz, 250 Hz, 500 Hz, 1000 Hz and 2000 Hz were measured both of the CI ear and the contralateral (CL) ear using a headphone in a soundproof room according to standard audiometric procedures. If the air-conduction threshold of the CI ear was 45 dB or higher—i.e. worse  word lists, was used . The avet tests (IBM SPSS Statistics 25.0) with the significance level set at 0.05  years old and the mean preoperative residual hearing was 92.2 dB.Intra-operatively, no tip fold-over was identified on fluoroscopy imaging. However, post-operative evaluation of the CT-images showed a tip fold-over of the 4 most apical electrode contacts in 1 of the 23 participants (4.3%). In retrospect, this tip fold-over was present on intra-operative fluoroscopy imaging, which was not recognized intra-operatively. Additionally, the SPE of this participant translocated from the ST into the SV at the location of this tip fold-over  had all electrode contacts positioned inside the ST. In eight participants (36.4%), the SPE translocated to the SV. All eight translocations occurred in near proximity of the CS. In 7 participants, all 22 electrode contacts were placed in the SV. In one participant, the first two contacts were located in the ST, before the SPE translocated to the SV. In Fig. n = 18). Table p = 0.001). Moreover, RHP in participants with and without translocation showed a similar statistically significant difference (p = 0.01); RHP% was 19.7% (SD 16) in patients with translocation and 77.2% (SD 45) when there was no translocation. With respect to the categorical relative HP, there was no participant of the translocation group (n = 7) with complete HP; one showed partial HP and six minimal HP. In the group of participants without translocation (n = 11), five showed complete HP, five had partial HP and in one patient, there was minimal HP.Table The speech perception scores of participants with an early onset of hearing loss at 2 and 12 months post-implantation was lower  compared to the scores of participants with a late onset of hearing loss . As shown in Table The primary objective of this study was to evaluate the position of the SPE inside the cochlea after implantation using the CS approach. Intra-operative fluoroscopy and cone beam CT-scan were used for, evaluation of tip fold-over and scalar position, respectively. The secondary objective was to investigate the relationship between the position of the SPE and residual HP.One tip fold-over was found in this study (4.3%). In two other studies that investigated the SPE, 4.5% and 7.7% tip fold-over was reported , 15. Forn = 22), all electrode contacts were placed inside the ST, whereas 36.4% showed a translocation from the ST to the SV. In comparison, Shaul et al. [n = 116 and n = 220); in which it was reported that a CS approach, compared to an (extended) RW approach, is associated with higher risk of translocation, independent of the implanted conventional electrode type. Our study is the first to report CS associated translocations for the slim, perimodiolar electrode, which was designed to be non-traumatic with any surgical approach [In 63.6% of the participants without a tip fold-over  translocations in present paper remains unclear, based on the findings of this study and the reports in the literature, in favor of the RW approach rather than the CS , 24, it In this study, it was shown that the SPE can provide preservation of residual hearing (defined as RHL% > 75%), however, only if inserted non-traumatically. Participants with full ST position of the SPE array had an average loss of low frequency residual hearing (PTA3lowDiff) of 4.4 dB and RHP% of 77.2%. Yet, in participants with a translocation to the SV, we found a statistically significant higher average loss of low frequency residual hearing of 17.9 dB and low RHP of only 19.7%. The only other study that described translocations in the SPE  did not In this study, it was observed that participants with translocation to the SV had lower speech perception scores compared to participants with a ST position. These differences indicate that scalar position might not only be of importance for residual hearing, but also for speech perception results. While the finding that ST position is important for the best speech perception is in line with similar findings in literature , 11, 31;In this prospective study, it was confirmed that the SPE carries a risk of tip fold-over—underscoring the need for intra-operative control. The slim perimodiolar electrode, once positioned in the ST can provide preservation of residual hearing. However, CS-associated translocation to the scala vestibuli occurred in more than one-third of the participants and was shown to be detrimental for residual hearing thresholds. Based on the results of the present study and evaluation of literature, if the anatomical situation allows it, we advise to insert the slim perimodiolar electrodes using the eRW approach.


Our findings suggest the recommended intake levels of riboflavin, niacin, and retinol may help reduce ARHL in the elderly.Because age-related hearing loss (ARHL) is irreversible, prevention is very important. Thus, investigating modifying factors that help prevent ARHL is critical for the elderly. Nutritional status or nutritional factors for the elderly are known to be associated with many problems related to aging. Emerging studies suggest that there was the interaction between nutrition and ARHL. We aimed to investigate the possible impact of dietary nutrients on ARHL using data from the fifth Korean National Health and Nutrition Examination Survey (KNHANES) which included 4742 subjects aged ≥ 65 years from 2010 to 2012. All participants underwent an otologic examination, audiologic evaluation, and nutritional survey. The associations between ARHL and nutrient intake were analyzed using simple and multiple regression models with complex sampling adjusted for confounding factors, such as BMI, smoking status, alcohol consumption, and history of hypertension and diabetes. Higher intake groups of riboflavin, niacin and retinol was inversely associated with ARHL prevalence (riboflavin aOR, 0.71; 95% CI, 0.54–0.94;  Age-related hearing loss (ARHL) or presbycusis is a progressive, bilateral, symmetrical sensorineural hearing loss. ARHL is a result of degeneration of the cochlea or associated structures of the inner ear. Approximately ARHL affects 23% to 40% of the population older than 65 years of age . Disabling hearing loss was defined as better ear hearing more than 40 dB  in adults according to the World Health Organization (WHO) ,4,5, whiNutritional status or nutritional factors for the elderly are known to cause many problems related to aging, and emerging studies have suggested that there are interaction effects between nutrition and ARHL ,8. MetabTo reduce the risk of ARHL, it is important to identify protective nutritional factors. The Korean National Health and Nutrition Examination Survey (KNHANES) may be useful for determining whether or not there are protective nutritional factors. This survey collects comprehensive information, such as health status and nutritional condition from the general Korean population, using a complex, stratified, multistage, probability-clustered sampling method based on national census data.Two studies have investigated the relationship between nutrition and hearing loss using KNHANES data. One study reported a relationship between vitamins and ARHL, but there was a limitation of research to the population included for one year only in 2011, rather than the total of 3 years from 2010 to 2012 in the fifth KNHANES [In this study, we aimed to investigate the possible impact of dietary nutrients on ARHL using data from the fifth KNHANES, which includes cross-sectional surveys of subjects from 2010 through 2012. The population of overall disabling hearing loss included 10% of total participants, and up to 40% in the population older than 65 years . BecauseThis study used data from the fifth KNHANES. The KNHANES is an ongoing nationwide epidemiological study conducted by the Korea Centers for Disease Control and Prevention of the Ministry of Health and Welfare. The institutional review board (IRB) of the Korean Centers for Disease Control and Prevention review and approve the KNHANES survey annually. All participants are randomly chosen from randomly assigned districts of cities and provinces in South Korea. Among the selected candidate population, 80.8% of individuals participated in the fifth KNHANES from 2010 to 2012. Written informed consent was obtained from all participants before commencement of the study.During the period of the Fifth KNHANES, the total number of participants was 36,067 individuals. Among those 36,067 participants, 4742 subjects aged ≥ 65 years were included. Among the 4742 participants aged ≥ 65, 1022 participants were excluded because they had not received an audiometric test or had an abnormal tympanic membrane or had not completed the nutritional survey or had incomplete data . During the fifth KNHANES survey, otologic examinations were conducted by ENT doctors by using a 4-mm, 0° endoscope  to evaluate any abnormalities related to the tympanic membrane or middle ear including retraction, otitis media with effusion and cholesteatoma. Audiologic evaluation was performed by using a diagnostic audiometer  at 0.5, 1, 2, 3, 4 and 6 kHz in a soundproof booth. Hearing impairment was defined as a hearing level exceeding an average of 40 dB on pure-tone audiometry at 0.5, 1, 2 and 4 kHz. Hearing impairment was divided into bilateral hearing loss, considered ARHL, and unilateral hearing loss. Nutrition intake was surveyed using the complete 24-h recall method. All participants were instructed to continue their ordinary diets before the 24-h recall evaluation. Answers were not excluded by any certain days such as holidays or weekends. Nutrient intake was calculated by referencing the nutrient concentrations of foods described in the Korean Food Composition Table, which was devised by the Korean National Rural Resources Development Institute . As showt-tests for continuous parameters and χ2 tests for categorical parameters. Nutrient intakes were analyzed by quartiles, and the lowest quartile was the reference group. Univariate analyses of each nutrient were performed to investigate factors associated with hearing impairment. Multivariate logistic regression analyses were used to confirm the association between hearing impairment and each nutritional factor. The analyses were adjusted for the following potential confounders: age, sex, body mass index (BMI), smoking status, alcohol consumption, hypertension, and diabetes mellitus. The results of the logistic regression analyses are expressed as odds ratios (OR) with 95% confidence intervals (CI). p values of <0.05 were considered statistically significant. All statistical analyses were performed using SPSS software .The general characteristics between subjects with and without bilateral hearing impairment were compared using Student’s n = 665) in the over 65 years of age group. The average age of subjects with bilateral hearing loss was 75.4 ± 5.99 years old. Subjects with bilateral hearing impairment were older (p < 0.001), weighed less (p < 0.001), and were more likely to smoke (p = 0.0023) and to drink alcohol (p < 0.001) than those without bilateral hearing impairment.Participant demographics are shown in Quartile values of each nutrient intake are shown in p < 0.05) in the univariate analyses. In univariate analyses, the third quartiles (50th–75th percentile) of intake of fat (p = 0.007), protein (p = 0.005), ASH (p = 0.001), calcium (p = 0.007), iron (p = 0.011), retinol (p = 0.006), thiamin (p = 0.001), riboflavin (p = 0.006), niacin (p = 0.032), potassium (p = 0.005), and phosphorus (p = 0.001) showed significantly lower risk for bilateral hearing impairment. The second quartiles (25th–50th percentile) of carbohydrate (p = 0.024), fat (p = 0.037), calcium (p = 0.012), and iron (p = 0.040) showed significantly lower risk for bilateral hearing impairment in the univariate analyses. Multivariate analyses adjusted for age, sex, BMI, smoking status, alcohol consumption, hypertension, and diabetes mellitus showed significantly lower risk for bilateral hearing impairment in the highest quartiles of intake for riboflavin , niacin , and retinol .p = 0.042), retinol (p = 0.015), iron (p = 0.003), calcium (p = 0.022) and sodium (p = 0.003) showed significantly lower risk for unilateral hearing impairment. In addition, the highest quartiles of intake of β-carotene (p = 0.031), and vitamin A (p = 0.048) showed an inverse correlation with the prevalence of unilateral hearing loss. However, in multivariate analyses adjusted for age, sex, BMI, smoking status, alcohol consumption, hypertension, and diabetes mellitus showed no correlation between nutrient intake and unilateral hearing impairment. We found that dietary intake of riboflavin, niacin and retinol were associated with bilateral hearing impairment in elderly KNHANES participants, but there was no correlation between nutrient intake and unilateral hearing impairment. Overall, higher intake of riboflavin, niacin and retinol were related to a lower risk of ARHL.According to the Dietary Reference Intakes for Koreans 2015, the average requirement of riboflavin is 1.0 mg/day and the recommended intake is 1.2 mg/day for ages 65 years and older . In thisIncreased serum level or dietary intake of retinol decreased the incidence of ARHL in various countries such as Asian and Western countries ,18,19. HThere have been several studies evaluating the relationship between retinol and hearing, as retinol has a high concentration in the inner ear , and retRiboflavin also has an antioxidant role, especially with respect to lipid peroxidation and reperfusion oxidative injury . RiboflaARHL is caused by irreversible loss of sensory hair cells and/or degeneration of spiral ganglion neurons. Niacin increases synaptic plasticity and axonal growth in a rat model of stroke . Such fiThis report may be the first community-based epidemiological study to reveal that higher intake of niacin is inversely associated with the prevalence of ARHL in the elderly. However, we could not find the causal mechanism of the higher intake of niacin and ARHL. Therefore, measurement of serum of niacin in ARHL patients or animal study with niacin deficiency model will be needed to understand the relationship between niacin and hearing.In multivariate analysis, higher dietary intake of riboflavin, niacin and retinol had a strong correlation with ARHL. These results were fairly consistent with previous studies in which a healthy diet including multivitamins influenced better hearing ,35. VitaRetinol and riboflavin are mainly found in foods of animal origin like milk, cheese, and eggs. Niacin is found in a variety of whole and processed foods, including meat from various animal sources, seafood, and spices. The principal aspects of traditional Korean diet include proportionally high consumption of rice and vegetables, moderate consumption of fish, and low consumption of red meat and dairy products . Over thAs a cross-sectional epidemiological survey, our study cannot establish causal relationships between the nutritional intake and ARHL. To identify causal relationships, an experimental study using human or animal would be needed. Furthermore, nutritional intake was estimated based on the 24-h recall method of the subjects. Although trained staff interviewed the participants, responses were dependent on the subjects’ memory, leaving open the possibility of measurement error. Serum analysis of nutrient levels could be useful when investigating their direct association with ARHL, because nutritional bioavailability may vary from person to person. In conclusion, our study found that higher intake of riboflavin, niacin and retinol were inversely correlated with prevalence of bilateral hearing loss in the ≥65 years age group. ARHL was lower in the groups taking niacin and riboflavin above the recommended intake. Although Koreans have continued to adopt more western diets, traditional grain- and vegetable-oriented eating habits are still maintained by the elderly. Therefore, encouraging the consumption of dairy products and meat to ensure sufficient supplies of riboflavin, niacin, and retinol in older populations may help reduce the incidence of ARHL.


By generating a sound and then listening to how the sound waves bounce off of objects around them, these animals are able to “see” using sound. Although a number of species engage in some form of echolocation, including some birds, shrews, and even humans, the echolocation systems of bats and toothed whales  are exquisitely sophisticated. Echolocation evolved independently in these animals  under coThe interpretation of sound begins in the cochlea, a hollow, spiral-shaped bone in the inner ear that converts sound vibrations into nerve impulses. Because gaining the ability to hear very high frequencies likely required changes to the cochlea, the authors of the study, led by Keping Sun and Jiang Feng from Northeast Normal University and Jilin Agricultural University in China, analyzed the genes expressed in the cochleae of three bat species that use different forms of echolocation: constant-frequency, frequency-modulated, and tongue-click echolocation . They then compared these gene sequences to those from 16 other mammals, including other echolocating and nonecholocating bats, as well as both echolocating and nonecholocating whales. According to Sun and Feng, this allowed them to “provide for the first time a comprehensive understanding of the genetic basis underlying high-frequency hearing in the cochleae of echolocating bats and whales.”Through their comparative analysis, the researchers identified 34 genes involved in hearing or auditory perception that showed evidence for positive selection in echolocating species. This included 12 genes involved in bone formation that may help regulate the bone density of the cochlea to enable high-frequency hearing. It also included several genes with antioxidant activity that may help protect the ear from damage and hearing loss caused by chronic exposure to high-intensity noise. The study also revealed large numbers of parallel or convergent mutations between pairs of echolocating mammals, where the same genetic changes had occurred independently in distantly related echolocators. Interestingly, there were significantly more of these parallel/convergent mutations between pairs of echolocators than when comparing an echolocator to an equally distant nonecholocator . This suAs noted by Sun and Feng, confirming such a hypothesis requires functional assays for each candidate gene that may underlie echolocation, such as suppressing the expression of hearing genes using RNA interference technology. Unfortunately, such experiments can be costly and both time- and labor-intensive . Despite the remaining challenges, however, the authors are grateful that comprehensive studies like theirs are now possible thanks to recent advances in genomic technologies. “This is an exciting time for the study of adaptive evolution of echolocation in mammals. More and more genetic and genomic data sets have been published, providing insights into the evolutionary basis of echolocation.”


Neurodevelopmental changes occur with asymmetric hearing loss, limiting binaural/spatial hearing and putting children at risk for social and educational challenges. These deficits may be mitigated by providing bilateral hearing in children through auditory prostheses. Effects on speech perception and spatial hearing were measured in a large cohort of >450 children who were deaf and used bilateral cochlear implants or bimodal devices . Results revealed an advantage of bilateral over unilateral device use but this advantage decreased as hearing in the two ears became increasingly asymmetric. Delayed implantation of an ear with severe to profound deafness allowed asymmetric hearing, creating aural preference for the better hearing ear. These findings indicate that bilateral input with the most appropriate device for each ear should be provided early and without delay during development. On the other hand, access to sound in only one ear results in impaired binaural hearing8 which is the foundation for sound localization. Without access to spatial hearing, children with asymmetric hearing are at risk for social and educational deficits10. We thus sought to promote bilateral hearing development by providing the most appropriate device in each ear to our large cohort of children with deafness7. A cochlear implant was provided in ears with severe/profound deafness; children with bilateral deafness received two cochlear implants whereas children with better hearing in one ear received one cochlear implant and a hearing aid in the other, better hearing, ear 13.Cochlear implantation has become standard treatment for childhood deafness. One cochlear implant promotes significant gains in speech understanding16 in which they listen to sounds coming from multiple directions. Binaural hearing supports detection of these sounds and the ability to distinguish one sound from another by differences in their spatial locations. To do this, the auditory system largely depends on comparing the level and timing of sounds arriving at the two ears17. Even if this binaural coding is impaired18, children can take advantage of hearing with both ears by taking advantage of the ear with the better signal-to-noise ratio. This ear has best access to the sound of interest relative to other sounds. The ear closest to the sound source is often relied upon because intensity at the further ear is attenuated by the head (head shadow). Intact binaural hearing further improves listening compared to only listening with the ear that has the better signal-to-noise ratio  and provides better audibility by combining the signals from each ear 19. Restoring children’s access to spatial/binaural cues is thus important to aid hearing in most common situations. Spatial/binaural hearing is clinically evaluated by measuring speech recognition in difficult noise conditions (speech-in-noise) or while listening with both ears over each ear alone , and measuring the ability to better detect or understand speech when noise comes from different directions than the target speech . Asymmetric hearing may distort binaural cues, rendering poorer binaural benefit for speech perception and skewed abilities to detect speech in the presence of other sounds/noise.Limited or distorted binaural hearing challenges development. Children spend much of their time interacting and learning in dynamic environments, such as the playground and classroom25 as well as cortical areas involved in spatial attention and awareness26 but treating only one of two ears with hearing loss leaves the second ear deprived of sound. This creates a new problem termed the “aural preference syndrome”7. When hearing is asymmetric, the developing brain reorganizes to preferentially respond to the better hearing ear31, compromising the ability to process bilateral input32. This reorganization occurs within 2–3 years of unilateral hearing and persists even if bilateral input through bilateral cochlear implants or bimodal devices is provided thereafter and used for several years28. Recent studies suggest that the aural preference syndrome can be reversed if symmetric/balanced bilateral input is provided during early developmental periods28.The timing of bilateral cochlear implantation is important. As revealed by electrophysiological and functional imaging studies, delaying access to sound in early childhood allows cortical cross-modal plasticity to reorganize auditory areas39. These studies revealed better speech thresholds in noise while using two implants, but asymmetric preference for a better signal at the first implanted ear in several children who underwent sequential implantation. Studies with larger cohorts of children (≥50) suggest that children benefit from bilateral implants despite delays to bilateral input but longer delays impair performance in the second hearing ear, creating asymmetric abilities between the two ears for understanding speech43 and for spatial hearing45. One of the main problems of relating existing speech perception data to electrophysiological findings is that only a few of the behavioural studies include children with very short or no delays to bilateral implantation45, leaving a question about the most appropriate timing of bilateral input to prevent behavioural consequences of aural preference.In the present study, we examined whether this neurophysiological support for limited delays to cochlear implantation in children is consistent with functional  outcomes. Several groups have reported spatial hearing from small cohorts (<20) of children who received bilateral cochlear implants sequentially after fairly limited (<2 years) bilateral auditory experience28, previous studies largely focused on children who have significant (severe/profound) hearing loss in their non-implanted ear50. These studies reveal benefits of bimodal hearing over the use of a cochlear implant alone but continued challenges for listening to speech in noise. Data from some children with better hearing in the non-implanted ear also reveal bimodal improvements in speech perception and spatial hearing that depend on duration of deafness in the poorer ear and access to consistent sound in the better ear51. The cohorts of bimodal users represent a very diverse population of implant users54. Asymmetric hearing loss in children appears to have an increased incidence of auditory nerve hypoplasia, enlarged vestibular aqueducts and positive cytomegalovirus54. These etiologies are associated with acquired and/or progressive hearing loss which could mitigate some of the deleterious effects of inter-implant delay42. Accordingly, the present study aimed to characterize the pre-implantation hearing histories of all children and to use this information to predict lasting effects of the degree and duration of asymmetric hearing on speech perception and spatial hearing.Behavioural data from children using bimodal devices are also needed. Whereas electrophysiological data included children with a range of hearing in the non-implanted ear Given the importance of bilateral input during development, we asked whether providing bilateral input through bilateral cochlear implants or bimodal devices worked to promote symmetric functional outcomes in a large diverse cohort of children, thereby preventing functional aural preference. We hypothesized that the benefit of bilateral input: (1) increases with earlier access to bilateral input; (2) does not need to be restricted to one mode of stimulation/hearing and (3) is related to hearing experience/demographic information. Results suggest that listening through bilateral devices is better than with one device, and the best timing for intervention is to provide bilateral devices as early as possible. Decisions regarding the type of device should consider the degree of residual hearing and asymmetry between ears.p < 0.0001; Table Group demographic details are described in Table To describe what each PC encompassed, variables which contributed proportionally more to the PC than expected from equal contributions were considered . The first two components together explained 57.7% of the variance in hearing history and both included variables associated with asymmetric hearing experience prior to implantation . However, additional variables that differed between the two components contributed to different aspects of the asymmetric hearing experience. The first component (PC1) individually explained 42.6% of the variance in hearing history and, along with the 3 variables that were related to pre-implant asymmetric hearing experience, included variables associated with the duration and degree of residual acoustic hearing (duration of pre-acoustic hearing and unaided PTA in the CI2/HA ear). This first component thus reflected the contribution of the better ear to the asymmetric hearing experience. The second component included the unaided PTA in the CI1 ear, reflecting the contribution of residual hearing or deafness in the poorer hearing ear to the asymmetric hearing experience. This second component explained 15.1% of the variance. A third component, explaining 11.4% of the variance, was associated with any deafness or poor residual hearing pre- or post-implantation . The relationships between components are plotted in Fig. p < 0.001). PC1 differentiated groups with varying degrees and durations of asymmetry: bimodal users experienced the most asymmetry  followed by bimodal sequential users (Tukey HSD post-hoc test p = 0.008). The older simultaneous group had significantly less asymmetric hearing than both bimodal groups (both p < 0.001) but significantly more than the bilateral simultaneous (p < 0.001) and sequential (p < 0.001) groups, who both experienced very minimal asymmetry (p = 0.17). This is consistent with the number of children categorized as having (a)symmetric hearing loss and the mean PTA asymmetries in Table p < 0.001) compared to the minimal hearing experience of sequential and simultaneous bilateral CI users prior to implantation (PC2 scores around zero). This contrasts with the negative PC2 scores of bimodal groups  who had better residual hearing in the CI1 ear (p < 0.001), later implantation (p < 0.001), and longer durations of asymmetric hearing (p < 0.001) and unilateral deafness (p < 0.001). Although bimodal and bimodal sequential groups significantly differed by asymmetric (PC1 p = 0.008) but not deafness-related PC scores , bimodal sequential users had worse residual hearing in the ear that kept the HA while waiting to receive a second implant (p = 0.001), and consequently tended to have less hearing asymmetry pre-CI2 (p = 0.069).Groups were clearly identified by differences in PC scores (Table Pediatric Ranked Order Speech Perception (PROSPER) score was similar for both ears and condition at each test time. Representative data for CI1 in quiet are plotted Fig. χ2(1) = 219.4, p < 0.001; Fig. χ2(3) = 3.9, p = 0.274) but the intercept varied by group (χ2(3) = 63.9, p < 0.001). Simultaneous users began their bilateral hearing with easier tasks  and sequential users started with a harder task  than the bimodal and older simultaneous groups, which partly reflected their older age at bilateral device use  but all groups used both devices for at least 2 years . The simultaneous group used bilateral devices the longest  = 14.6, p < 0.001; noise: F = 2.9, p = 0.038). Speech perception was more asymmetric for bimodal and sequential groups than the simultaneous group in quiet (both Tukey HSD post-hoc p < 0.001), and for sequential compared to simultaneous bilateral CI users in noise (Tukey HSD post-hoc p = 0.032).Mean ± SE asymmetry Fig.  favouredχ2(1) = 5.4, p = 0.02; see Supplemental Fig. χ2(4) = 5.6, p = 0.20) but, consistent with Fig. χ2(4) = 66.8, p < 0.001): bimodal and sequential groups had the greatest asymmetry (25.7 and 22.0 RAU respectively) and simultaneous groups had the least (−1.1–1.3 RAU).Longitudinal measures in both ears were available in 307 (69.9%) children and revealed little change in asymmetry using linear mixed effects regression  = 5.4, p < 0.001) and condition  = 349.9, p < 0.001) but there was no interaction between group and condition  = 1.3, p = 0.26). Bilateral accuracy was 8.8 ± 2.4 RAU greater for quiet than noise , and simultaneous bilateral CI users were 10.7 ± 3.5 RAU more accurate than bimodal users .Mean ± SE bilateral speech perception scores in quiet and in noise are presented in Fig. t-test µ = 0, p < 0.05) but the degree of benefit significantly differed by ear and group  = 14.7, p < 0.001). In simultaneous and bimodal sequential groups, the bilateral advantage was equal regardless of which ear was the unilateral reference (Tukey HSD post-hoc p > 0.05). Conversely, bilateral benefit was asymmetric with more benefit relative to the overall poorer ear alone for bimodal  and sequential bilateral CI users . Figure F = 1.8, p = 0.13), bilateral advantage was 4.2 ± 1.7 RAU greater in noise than in quiet  = 6.3, p = 0.013) which highlights the importance of bilateral input in challenging acoustic conditions. Bilateral advantage over CI2/HA in noise is plotted in Supplemental Fig. The potential advantage of bilateral CI and bimodal device use relative to each ear was examined. As shown in Fig. R = −0.34 to −0.73, p < 0.05). A similarly negative relationship was found between the advantage of adding bilateral hearing to hearing with CI1 alone and asymmetry in speech perception .Figure F = 41.4, p < 0.001): simultaneous users were younger than bimodal users (Tukey HSD post-hoc p < 0.001), and both of these groups were younger than the other three groups (p < 0.05). Furthermore, simultaneous and sequential groups had longer bilateral device use than the other groups  = 9.3, p < 0.001) but not group  = 2.2, p = 0.07) or age  = 3.3, p = 0.07). Speech detection thresholds improved (more negative) when noise was spatially separated from the speech   and was best when noise was directed towards CI2/HA .Mean ± SE detection thresholds for speech in noise are presented in Fig. F = 0.01, p = 0.92) but there was a significant interaction between group and direction of noise  = 2.6, p = 0.035), whereby bimodal users experienced (mean ± SD) 2.5 ± 0.6 dB greater spatial unmasking with noise directed to the HA than CI1 . Overall, the groups experienced 3.3 ± 3.6 dB  to 3.9 ± 2.1 dB (older simultaneous group) spatial unmasking.To further explore these differences, the degree of spatial unmasking  was calculated for each noise direction Fig. . Most chF = 2.5, p = 0.041) regardless of age  = 0.5, p = 0.48). Bimodal  = −3.9, FDR-adjusted p = 0.002) and sequential bilateral CI users  = −2.2, FDR-adjusted p = 0.088) had notable asymmetries. Asymmetry in the bimodal group, in particular, was greater than the test step-size of 2 dB and greater than the asymmetry in simultaneous users . This preference for a better signal-to-noise ratio in CI1 occurred in the sequential users despite similar access to sound in each ear as measured by aided pure-tone average thresholds; the difference between the two CIs in this group was 4.1 ± 4.1 dB which was less than the test step-size of 5 dB . Yet, both measures revealed consistent preference for CI1  also underwent spatial unmasking testing. Each test was completed within 4.7 ± 13.5 months of each other. Outcomes for each test are compared in Fig. % also unCI1 Fig.  in bimodCI1 Fig. . In contF = 0.7, p = 0.58) or spatial unmasking asymmetry  = 0.2, p = 0.94)  and higher PC3 scores (any deafness) . Effects of individual variables comprising significant PCA associations were then assessed, along with the following variables not included in the PCA: delay to bimodal/bilateral implantation and bilateral device use at time of testing. Considering all children irrespective of group, speech perception asymmetry increased with delay to bimodal/bilateral implantation  and unaided hearing thresholds  in the HA/CI2 ear .Etiology did not predict speech perception asymmetry  = 41.2, p < 0.001, R2adjusted = 0.20), and increased by 9.5 RAU/10 dB worsening of hearing thresholds in the HA ear of bimodal users  = 25.6, p < 0.001, R2adjusted = 0.26). This means that bimodal users had similar symmetry to simultaneous users (±1 SD = ±15 RAU) when they had mild to moderately-severe hearing loss in the non-implanted ear (35–65 dB HL unaided PTA). Otherwise, they showed aural preference for CI1 when the non-implanted ear had severe/profound loss (PTA > 70 dB HL) or aural preference for the HA ear when that ear had near-normal hearing (PTA < 35 dB HL). Sequential bilateral CI users developed an asymmetry >15 RAU when delay to bilateral implantation exceeded 3.5 years.These relationships were particularly strong in sequential bilateral CI users and bimodal users respectively. Bivariate regressions in each group suggested that asymmetry increased by 3.9 RAU/year delay to bilateral implantation in sequential bilateral CI users  speech recognition is better with bilateral than unilateral hearing; (2) bilateral hearing does not need to be restricted to one modality; and (3) asymmetric hearing creates functional aural preference for the better/first ear, limiting bilateral/spatial hearing. These findings are consistent with electrophysiological evidence of aural preference and support the recommendation to avoid this developmental change by providing access to sound with the most appropriate device in each ear as soon as possible.1 or short (<2 year) inter-implant delays41, but is larger than the 0–10% reported for children with longer (>2 years) inter-implant delays43 or who use bimodal devices46. Including more children with limited auditory deprivation and better residual hearing likely contributed to the greater bilateral benefit exhibited by the present cohorts of sequential bilateral CI and bimodal users. It is also worthwhile noting that the bilateral advantage was further pronounced in noise  when listening with two devices over one ear alone Figs  even wheise Fig. , highlig37 or bilateral CIs37. Measures of speech detection allowed testing of children with wide ranges of age, native language, and developmental ability but detection is an easier task than speech recognition. This explains the lower SNR levels in this study compared to the −9 to +7 dB SNR speech recognition thresholds reported for children with normal hearing56 or CIs57. The ability to detect and understand speech in noisy conditions improves with age56 as the auditory system matures. Changes continue into adulthood37 with the largest changes occurring over the first 5 years56. In the present cohort, detection thresholds did not change with age when accounting for group, perhaps because most children were older than 5 years and had early access to sound  do not perceive music or emotion better than their peers with bilateral implants68. The greatest bilateral advantage may be derived through the most appropriate bilateral devices for the hearing loss in each ear, regardless of modality.The advantage of bilateral hearing existed for both bimodal and bilateral CI hearing. This suggests that the auditory system can integrate, at least to some extent, two very different auditory signals to facilitate listening in quiet and noisy situations. Indeed, even limited input from a HA can work with a CI to help bimodal users perceive speech and develop languageCIs Figs  and 6a. one Fig.  supportsone Fig. , also su69 and increased cortical representation from the better than poorer hearing ear28.Although there was clear benefit of listening bilaterally rather than unilaterally with bilateral CIs and bimodal hearing, most children developed asymmetric function which limited this advantage Fig. . This wa43 and 0–5%1 asymmetry or 0.9–1.1 CI2/CI1 ratio42 in simultaneously implanted children). Also consistent with previous studies42, delay to bilateral input contributed to asymmetric speech perception  SNR on one side signals a potential for reduced speech perception in the condition when the “preferred” or better ear is masked by noise. Although asymmetries in spatial hearing can decrease after 3 years of bilateral input as the second ear’s performance improves45, the present cohort used their bilateral devices for 3–6 years, suggesting that these aural asymmetries are long-lasting.Aural preference also affected spatial hearing. All groups showed ~3–4 dB spatial unmasking which was poorer than the normal range of 5–10 dBear Figs . This asBilateral implants provided similar audibility from each device for all children Table  but only69. Brainstem asymmetries69 and cortical aural preference28 favouring the first hearing/implanted ear occur within 2–3 years of asymmetric input. Years of subsequent bilateral input cannot simply reverse aural preference28, unless bilateral input is provided quickly and at early ages27. Similarly, stronger aural preference occurs with reduced age of unilateral hearing in deaf white kittens24 and with temporary asymmetric hearing in ferrets and rats31. Persistently asymmetric speech perception occurs when bilateral input is delayed for ~3.5–4 years as shown in Fig. 45. The slightly staggered timelines suggest that underlying neurophysiological changes develop rapidly and take ~1–2 years longer to translate into measurable and consistent functional changes. This could reflect increasing complexity of speech perception, which requires temporal and spectral processing, relative to responses in temporal (auditory) cortices evoked by brief broadband click/pulse stimuli28.This study is the first to directly compare asymmetries measured by two different measures of speech perception. Children exhibiting asymmetric speech perception also showed asymmetric spatial hearing that favoured the first implanted ear Fig. . This ag73 without increased risk for complications or cumulative costs76. Moreover, simultaneous bilateral implant users develop symmetric representations of each ear in the auditory cortex21, symmetric speech perception  were collected from 461 children with bilateral devices: 80 (17.4%) children who used one cochlear implant (CI) and had normal hearing or used a hearing aid (HA) in the contralateral ear ; 18 (3.9%) bimodal users who received a second CI ; 170 (36.9%) children who received two CIs in sequential surgeries but did not wear a HA during the delay ; 193 children who received two CIs in the same surgery, 154 (33.4%) before age 4 years (“Simultaneous”) and 39 (8.5%) after age 4 years (“Older Simultaneous”). Most implants were from Cochlear Ltd, except for 5 children who received an Advanced Bionics array in their first implanted ear. Most children received a peri-modiolar CI24RE internal electrode array ; the type of array was unknown for 11 (2.4%) CI1 and 4 (1.1%) CI2  = 17.1, FDR-adjusted p = 0.005) or family history (Χ2(3) = 22.5 FDR-adjusted p = 0.001) etiology of deafness, whereas bimodal users and older simultaneous bilateral CI users had a higher rate of malformations which are often associated with progressive and/or asymmetric hearing loss , incomplete partition type II (IP-II/Mondini), Cock’s dysplasia  or cochlear hypoplasia connected with Pendred, CHARGE, Branchio Oto Renal (BOR), X-linked deafness with stapes gusher (Phelp’s) and Klippel Feil syndromes; Χ2(3) = 15.7, FDR-adjusted p = 0.005).The distribution of known and unknown etiologies of deafness is shown for each group in Fig. n = 7:2/23:2); Digit Identification ; Word Identification by Picture Identification ; Glendonald Auditory Screening Procedure ; Multisyllabic Lexical Neighbourhood Test ; and Phonemic Balanced Kindergarten test . Children responded by either pointing to a picture best representing the heard word from a group of pictures  or repeating the heard word . Because number of words varied across tests (12–25 words), percent correct scores were transformed to rationalized arsine units (RAU) and then corrected for guessing on closed-set tests81.Speech perception was evaluated in both quiet and co-located + 10 dB SNR noise using words presented at 60 dB SPL through a loudspeaker at 0° azimuth in a sound-treated audiometric booth. Speech perception tests were chosen based on language and developmental stage of the child. The following tests were used at the earliest and latest (or only) test dates, ordered by difficulty: Early Speech Perception test  were analyzed and confirmed that test administration differences did not affect speech perception in quiet , calculated asymmetry, or bilateral advantage across groups  were administered with recorded words and the associated multi-talker noise in most cases; sometimes speech-weighted noise was used due to technical difficulties, and for some children live voice was used because of their language, developmental stage or attention abilities. Quiet and noise PBK scores from a subset of children n = 181, 2.8% wereIn quiet, 439 children listened to words presented from the front while wearing one device at a time , and 374 (85.2%) of these children also listened to words while wearing both devices at the same time . Scores were also obtained in noise for CI1 and for the bilateral condition for 288 (65.6%) of the children. Of these, 94 (32.6%) children also had unilateral scores for both ears in noise. Repeated tests were available in quiet for both ears in 312/439 (71.1%) children and for bilateral scores in 309/374 (82.6%) children, as well as in noise for the better ear (CI1) and bilateral conditions in 272/288 (94.4%) children. Data were available for CI2 in noise for 89/94 (94.7%) children. A detailed breakdown of numbers by group, ears tested and condition is provided in Supplemental Table Pediatric Ranked Order Speech Perception (PROSPER) score82, which hierarchizes score  and type of test into one score that could be followed over time. This accounts for a possible change in score by virtue of moving from a simpler to more challenging test. If two tests were available on a given test date, the more difficult test for that child was chosen. The number of available test dates ranged from 2–11, with a median of 3 tests per child  of whom performed the test more than once. Most of these children were tested on 2 occasions  during testing. Speech detection thresholds (SDT) were measured as described previouslyPrinciple component analysis (PCA) was completed on demographic variables in the hearing history rather than step-wise regression because many variables were correlated and would introduce collinearity into a multiple linear regression model. This also allowed for variables that described a similar aspect of hearing history to be combined into one component that guided correlation analyses with outcome measures.ANOVA was used to assess group differences in a number of measures: demographic variables provided in Table 84, which assesses the extent to which points conform to the line of best fit (correlated) and how far that line is from the unity line (perfect agreement). This analysis quantified asymmetry in each child while accounting for the absolute accuracy of scores or unmasking in each ear, which is lost when only difference measures  are provided. This complexity and richness in information is important to retain while evaluating the wide variability in outcomes in diverse groups of bilateral device users. Furthermore, Lin’s coefficient accounts for differences/deviations from agreement  (whereas Pearson correlations do not). This way, the complexity and variability of the individual data across children who had poor scores and good scores could be highlighted alongside asymmetry. An ANOVA analysis assessed group differences in speech perception asymmetry, bilateral advantage and spatial unmasking asymmetry. The significance of these difference measures were analyzed compared to zero using independent t-tests with false-discovery rate (FDR) corrections to p-values for multiple comparisons85.The asymmetry in speech perception and in spatial unmasking was assessed in order to further highlight the capabilities and challenges of bilateral device users. Asymmetry between CI1 and CI2/HA was further assessed using Lin’s concordance correlation coefficientPearson correlations were used to assess the following relationships between the following outcome measures: bilateral advantage versus absolute asymmetry in speech perception; bilateral speech detection thresholds versus bilateral speech perception in noise coming from the front; and asymmetry in spatial unmasking versus asymmetry in speech perception. Correlations were also completed to identify relationships between asymmetry in speech perception and the PCA components of hearing history. These correlations guided which demographic variables were considered for regression analyses in order to predict changes in speech perception asymmetry.86 were used to assess changes speech perception asymmetry over time, as well as the progression from easier to harder tests of speech perception (PROSPER score) with bilateral device experience. These mixed regressions were used instead of simple linear regression in order to account for repeated measures per child and to allow for individual variation in the relationship over time. To do this, random effects of both intercept and slope for each child (1 + years|subject) were added to the regression model. A likelihood ratio test was used to determine significance of predictors in the regression. The equation of the full linear mixed effects model is given below:Linear mixed effects regression modelsVisual inspection of residual plots for each analysis did not reveal any obvious deviations from homoscedasticity or normality.Supplementary Material


The aim was to determine the relationship between bulbar conjunctival microcirculation and retinal microcirculation in a healthy population.A functional slit-lamp biomicroscope (FSLB) was used to measure blood flow velocity (BFV) and blood flow rate (BFR) in the conjunctiva while a retinal function imager (RFI) was used to measure macular BFV and BFR in the retina. One eye of each subject of 58 self-reported healthy subjects was imaged in the same session on the same day.P < 0.001) and retinal venules . The BFR in the conjunctiva (0.09 nl/s) was also significantly lower than that in the retina . The BFVs and BFRs were not related between the conjunctiva and retina .The mean BFV in the venules of the conjunctiva was 0.49 ± 0.13 mm/s, which was significantly slower than that in the retinal arterioles (3.71 ± 0.78 mm/s, The microcirculation in the retina appeared to be different from that in the conjunctiva. The eye and the brain share similar embryological origins, with similar structural and physiological characteristics. The eye is regarded as a window to the brain and helps monitor microcirculation and neurodegeneration. The conjunctival and retinal microvessels both originate from the internal carotid artery, and both include capillaries and pre-capillary arterioles and post-capillary venules. Using current techniques of ophthalmic imaging such as the functional slit-lamp microscopy (FSLB) –3 and reOur previous studies using advanced ophthalmic imaging on bulbar conjunctival microvasculature and microcirculation have indicated that the microcirculation of the conjunctiva declines with age in healthy subjects . Short-tThe study was approved by the ethics committee board of the University of Miami and conducted according to the tenets of the Declaration of Helsinki. All subjects were recruited and informed about the purposes and methods of the study and each volunteer signed a consent form.A total of 58 normal healthy subjects were recruited at the Bascom Palmer Eye Institute of the University of Miami. All subjects with a refractive error of no more than − 6.00 D and no greater than + 3.00 D were recruited. Only one eye of each subject was selected to be imaged. All subjects were imaged within 1 hour at the same visit during office hours (from 9 AM to 5 PM) . The excAll subjects underwent complete ophthalmologic examinations, including the slit-lamp biomicroscope, best-corrected visual acuity (BCVA) measurement, fundus examination, and intraocular pressure. The best-corrected visual acuity of all subjects was 20/20 or above. Their blood pressures, including systolic pressure and diastolic pressure, and heart rates were also measured, and personal medical histories were obtained from all subjects.FSLB settings and image procedures have been well described in previous studies , 8, and RFI is an advanced ophthalmic imaging modality based on a fundus camera. The system and its applications have been recently reviewed  and the P < 0.05 were regarded as statistically significant.All data are presented in the format of mean ± standard deviation (SD) and analyzed using Excel . The sample size was calculated by a software program  recommended by Faul et al.  and BoneP < 0.001, Fig. P < 0.001). The BFR in the conjunctiva was also significantly lower than that in the retina .The arteriolar and venular BFV in the retina had a significant positive correlation with age . In contrast, a group of 58 healthy subjects (age range: 17 to 56 years) was studied using RFI in the present study. Positive relationships between retinal BFVs in the arterioles and venules and age were found. This discrepancy appears to be due to the different study groups with different age ranges.The microcirculation in the arterioles in the retina appeared to be affected by systemic variables such as blood pressure. A positive correlation was found between systolic blood pressure and arteriolar BFV in the retina. The arterioles branch out from an artery and lead to capillaries, which may be more influenced by the systolic blood pressure. On the other hand, both BFV measurements of the venules in the conjunctiva and retina were not associated with blood pressure, indicating that both venules in the conjunctiva and retina may share some common characteristics whether anatomical or otherwise. As we did not include patients with hypertension, further studies will be needed to validate our hypothesis. Interestingly, positive correlations were found between age and BFVs in the retina, which was not in agreement with a previous study from our group . In thatOur study has some limitations. First, two different machines were used to access the microcirculation in the conjunctiva and retina because there was no single machine available that could access the microcirculation of both. Second, we did not image the microcirculation of the retina and conjunctiva simultaneously. Further studies using the same imaging device (if available) could overcome the drawback encountered in the present study. Third, although all subjects were imaged using both imaging devices within 1 h at the same visit during office hours, the diurnal changes during office hours may influence the comparison between the conjunctival and retinal vascular measurement; however, in our experience, this is unlikely. Nevertheless, further studies are needed to identify the impact of the diurnal changes on blood flow measurements and their relationship between the conjunctiva and retina. Fourth, the fields of view and pixel relationships are different between RFI and FSLB, depending on their cameras and magnifications. However, both systems are capable of tracking the movement of clusters of red blood cells so that the measurements of the BFV can be performed. Fifth, we only measured the venules in the conjunctiva and measured both arterioles and venules in the retina because the majority of the conjunctiva vessels are venules. We calculated the measurements of the venules as done previously .In conclusion, this is the first study revealing that the microcirculation in the retina appears to be different in BFV and BFR from that in the conjunctiva, which may be due to the lack vegetative nerve stimulation, autoregulation and possible intraocular pressure pulse on the retina. Future studies with a diseased population may further reveal the nuances in the microcirculation in the conjunctiva and retina.


NELFCD/CTSZ locus was significantly associated with progression of primary biliary cholangitis (PBC) to jaundice stage in the Japanese population. In this study, we investigated the role of cathepsin Z in the etiology and pathology of PBC. Serum cathepsin Z levels were measured using enzyme-linked immunosorbent assay. The expression and localization of cathepsin Z in liver specimens were analyzed by western blotting and immunohistochemistry. In PBC patients, serum cathepsin Z levels were significantly increased with disease progression. In addition, its levels were positively correlated with alanine transaminase, aspartate transaminase and total bilirubin, and were negatively correlated with platelet count and albumin. Cathepsin Z expression was markedly increased in hepatocytes at later stages of PBC, and its localization was altered from the peri-bile canaliculus to the cytoplasm, where a fraction was no longer colocalized with endosomal/lysosomal vesicles. Similar altered expression of cathepsin Z was observed in end-stage of other cholestatic liver diseases including sepsis, obstructive jaundice, and Alagille syndrome. Our results indicate that altered expression and localization of cathepsin Z in hepatocytes are characteristic features of PBC and other cholestatic liver diseases, and are implicated in the progression of PBC.Our recent genome-wide association study found that the  Although treatment with ursodeoxycholic acid (UDCA) alone or in combination with bezafibrate is very effective for normalizing liver function and preventing PBC progression3, approximately 10–30% of PBC patients are resistant to these treatments and progress to end-stage hepatic failure6. We previously reported that the pattern of PBC progression can be classified into three different types: (1) slowly progressive, which does not influence life expectancy; (2) progressive, which proceeds to liver cirrhosis and/or portal hypertension, but rarely to jaundice and end-stage hepatic failure; and (3) progressive, which proceeds to jaundice and end-stage hepatic failure4. Using this method of classification, we recently found that genetic polymorphisms (rs13720 and rs163800) at the NELFCD/CTSZ locus were associated with progression to jaundice stage in PBC in a Japanese population7.Primary biliary cholangitis (PBC), previously referred to as primary biliary cirrhosis, is a chronic liver disease characterized by the destruction of intrahepatic bile ducts and progressive cholestasis, leading to liver cirrhosis, jaundice, and end-stage hepatic failure8. Cathepsin Z is a member of the cysteine cathepsins located in the lysosome, and exhibits exopeptidase activity. Cathepsin Z cleaves the carboxyl-terminus of beta integrin receptor, macrophage antigen-1, lymphocyte function-associated 1, and C-X-C motif chemokine 12, and regulates the adhesion, migration, and maturation of immune cells9. In addition, cathepsin Z cleaves extended polyglutamine tracts10, huntingtin11, and γ-enolase12, and is involved in the development of neurodegenerative diseases such as Alzheimer’s disease13 and Huntington’s disease12. Cathepsin Z expression is locally increased in cancer15 and Helicobacter pylori–associated gastritis14, and systemically increased in severe trauma16.Cathepsin Z is ubiquitously expressed in tissues and is highly expressed in the liver, spleen, lung, and kidneyThe expression and functional role of cathepsin Z have not been fully characterized in liver diseases, including PBC. In this study, we investigated pathoetiological roles of cathepsin Z in disease progression in PBC.P = 0.03). Serum cathepsin Z levels were also significantly higher in jaundice-stage PBC (5.37 ± 3.46 ng/ml) as compared to early-stage PBC (P = 5.3 × 10−7) and late-stage PBC (P = 0.04). In contrast to these results, there was no significant difference in serum cathepsin Z levels between early-stage CHC and late-stage CHC (data not shown), and levels were significantly lower in CHC (0.80 ± 1.07 ng/ml) than in late-stage and jaundice-stage PBC . , but significantly lower than those of jaundice-stage PBC (P = 0.002).We measured serum cathepsin Z levels in three clinical stages of PBC , in two clinical stages of CHC , and in obstructive jaundice (n = 12) , aspartate aminotransferase  and total bilirubin , and were negatively correlated with platelet count  and albumin . On the other hand, its levels were not significantly correlated with alkaline phosphatase.We next investigated whether cathepsin Z levels correlated with other biochemical markers of liver function in PBC patients Fig. . Serum cP = 0.63; period of UDCA treatment was 52.3 ± 41.1 months) . The serum cathepsin Z concentration of jaundice-stage PBC patients was measured before transplantation (6.48 ± 2.77 ng/ml) and after transplantation (1.18 ± 2.32 ng/ml), with a post-transplant observation period of 16.7 ± 14.8 months  or jaundice-stage (n = 1), and 1 out of 7 at late-stage PBC patients progressed to jaundice-stage. Serum cathepsin Z levels were not significantly decreased after UDCA treatment  Fig. . Liver tths Fig. . These rP = 4.9 × 10−5, jaundice-stage v.s late-stage, P = 4.7 × 10−3)   Fig. . In addi01) Fig. . By cont01) Fig. .Figure 3We analyzed the intracellular distribution of cathepsin Z in the liver by immunofluorescence staining using bile canalicular marker MRP2 and lysosomal marker LAMP1. Cathepsin Z was predominantly localized adjacent to MRP2 in hepatocytes of NL Fig.  upper anP = 3.7 × 10−3)  Fig. . Under o−3) Fig. . These rTo investigate whether the increase in cathepsin Z protein was due to transcriptional regulation, transcript levels of cathepsin Z were measured in liver samples Fig. . There wTo investigate whether increased expression and altered localization of cathepsin Z is associated with cholestasis, the expression and localization of cathepsin Z was analyzed at the end stage of other cholestatic liver diseases. Cathepsin Z protein expression was markedly increased in hepatocytes at the end stage of sepsis, Alagille syndrome, and obstructive jaundice as compared to NL Fig. . In addiIn this study, we report several novel findings about the role of cathepsin Z in the pathogenesis of cholestatic liver diseases, particularly primary biliary cholangitis. First, Serum cathepsin Z was significantly increased with the progression of PBC. Second, its levels were significantly correlated with several biochemical markers for liver function including alanine transaminase, aspartate aminotransferase, platelet count, total bilirubin, and albumin. Third, cathepsin Z was markedly increased in hepatocytes from patients with later stages of PBC. Forth, in hepatocytes of patients with jaundice-stage PBC, cathepsin Z was translocated from the peri-bile canalicular membrane region to the cytoplasmic region, where it no longer colocalized with lysosomal marker LAMP1. Fifth, increased expression and altered localization of cathepsin Z were also observed in hepatocytes at the end stage of other cholestatic liver diseases, including Alagille syndrome, obstructive jaundice, and sepsis. Finally, in contrast to PBC and other cholestatic liver diseases, cathepsin Z was not increased in sea and liver tissues of CHC. Our results indicate that altered expression and localization of cathepsin Z is a phenomenon particular to cholestatic liver diseases such as PBC and is implicated in the progression of PBC.16 and in cancer patients with shorter overall survival17, indicating that cathepsin Z is a potential serum marker for inflammation or chemosensitivity. In this study, increased serum cathepsin Z levels with PBC progression were significantly correlated with several biochemical markers for liver function  and were normalized after liver transplantation. On the other hand, serum cathepsin Z levels were significantly lower in CHC than in PBC and obstructive jaundice. In addition, its levels were not correlated with alkaline phosphatase which is a major diagnostic maker of bile duct injury and cholestasis in PBC. These results indicate that cathepsin Z is a potential serum marker for severe progression with cholestasis in PBC.Increased serum cathepsin Z levels were reported in non-survivors after trauma18. The polarity of cells is a key factor in intracellular trafficking and lysosomal exocytosis, and loss of hepatocyte polarity is observed in intra-/extra-hepatic cholestatic liver diseases20. In this study, cathepsin Z was increased in liver tissues from later stages of PBC via non-transcriptional regulation, and its expression in hepatocytes was correlated with bile duct loss. It is speculated that the loss of apical–basal polarity induces dysfunction of lysosome exocytosis and therefore dysfunction of biliary excretion as well, leading to increased expression and altered localization of lysosomal enzymes, including cathepsin Z, in hepatocytes of patients with PBC. Previously, UDCA was reported to insert MRP2 into canalicular membranes and stimulate organic anion secretion by protein kinase C–dependent mechanisms in a rat model of cholestasis21. However, in the current study, MRP2 was mainly localized in the cytoplasm of hepatocytes in jaundice-stage PBC and serum cathepsin Z levels did not decrease after UDCA treatment, indicating that UDCA is not effective at restoring hepatocyte polarity and excretion of cathepsin Z.The lysosomal enzymes in hepatocytes are mainly excreted from the bile canaliculus via lysosomal exocytosis, which is an important process for cell homeostasis22. Cathepsin Z was reported to be involved in the apoptosis of neurons23. In the current study, cathepsin Z was no longer colocalized with lysosomes in hepatocytes of jaundice-stage PBC patients, suggesting that cathepsin Z may be involved in hepatocyte cell death. Thus, increased expression and altered localization of cathepsin Z are not only a result of cholestasis, but may also be a cause of hepatocyte cell death, leading to the progression of PBC. This idea is supported by evidence that cathepsin Z polymorphisms are associated with the progression to jaundice stage in PBC7, although the functional role of the polymorphism is still under investigation. In addition, other cathepsin enzymes such as cathepsin B and D have been reported to be involved in cell death via lysosomal membrane permeabilization22. Further studies are needed to dissect the precise mechanism of cathepsin-induced hepatocyte cell death in PBC. Previously, the dysregulated maturation of cathepsin D was reported to be associated with pathogenesis of Alzheimer’s disease24. However, in this study, mature-form of cathepsin Z but not the pro-form was increased in the liver tissues from jaundice-stage PBC, suggesting that dysregulated maturation of cathepsin Z is not associated with the progression to jaundice stage in PBC.It has been reported that enlargement of the lysosome causes permeabilization of the lysosomal membrane and subsequent release of lysosomal enzymes into the cytoplasm, eventually leading to cell deathIn conclusion, we established that cathepsin Z is a useful marker for monitoring the progression of PBC. It is possible that inhibitors of cathepsin Z may attenuate cell death triggered by lysosomal membrane permeabilization and the subsequent mislocalization of cathepsin Z in PBC.25. CHC patients were diagnosed based on detection of hepatitis C virus RNA and anti-hepatitis C virus antibody in sera and were classified into early- and late-stage based on the Metavir scoring system, as described previously26. Patients with a score of 0–1 were classified as early-stage and patients with a score of 2–4 were classified as late-stage. PBC patients were classified into three clinical stages based on clinical manifestations as previously described4. The classification criteria were as follows: early-stage patients showed no signs of portal hypertension or cirrhosis; late-stage patients had signs of portal hypertension or cirrhosis, but not jaundice ; and jaundice-stage patients had persistent jaundice . PBC patients with other concomitant liver diseases, including autoimmune hepatitis and viral hepatitis, were excluded from this study based on biochemical and histological findings.The study subjects included 90 patients with PBC, 31 with chronic hepatitis C (CHC), 17 with obstructive jaundice, two with sepsis, two with Alagille syndrome, and six with no significant histopathological change (used as non-diseased liver), all of whom had been registered at the National Hospital Organization (NHO) Nagasaki Medical Center and Kanazawa University from December 1991 to July 2014. PBC patients were diagnosed based on established criteriaSera were obtained at the time of recruitment in this study. A part of sera from PBC patients who have undergone liver transplantation were collected before and after the transplantation. Liver tissues for subjects were collected at the time of diagnosis and/or during observation period. Demographics and clinical characteristics of PBC, CHC, and obstructive jaundice at the time of enrollment were shown in Table 27. Immunohistochemistry was performed using a standardized two-step method with Histofine-Simple Stain . Goat anti–human cathepsin Z polyclonal antibody  was used as a primary antibody. HRP-conjugated rabbit anti–goat polymer (Simple Stain MAX PO (G); Nichirei Biosciences) was used as a secondary antibody. The reaction products were visualized using 3,3′-diaminobenzidine (Dako) and counterstained with Mayer’s hematoxylin (Dako). As a negative control, normal goat IgG (R&D Systems) was used as the primary antibody. The stained sections were evaluated by a blinded pathologist.Hematoxylin and eosin staining was performed on 4-μm-thick, formalin-fixed, paraffin-embedded sections. Histological evaluation was performed as described elsewhereFor immunofluorescence staining, blocking was performed with 3% bovine serum albumin in PBS for 30 minutes. Primary antibodies  were incubated overnight at 4 °C or for 1 hour at room temperature. Secondary antibody conjugated with Alexa 488  or Alexa 594  was incubated for 1 hour at room temperature. Nuclei were stained with VECTASHIELD Mounting Medium with DAPI . Washing between each step was performed using 0.05% Tween-20  in PBS.2  was used as a secondary antibody. Signal was detected with LAS 3000 . Densitometric analysis was performed using Multi Gauge ver2.0 software (Fujifilm).Liver tissues were homogenized with Protein Elution/HEPES buffer  supplemented with protease inhibitor cocktail , using the Cell/Tissue resin kit (Apro Science). Protein concentration was quantified using the BCA assay (Thermo Fisher Scientific). Twenty micrograms of denatured protein was electrophoresed on a 10% SDS-PAGE gel (Bio-Rad Laboratories) and transferred to a PVDF membrane . Rabbit anti–cathepsin Z  and rabbit anti-actin  were used as primary antibodies. Donkey anti–rabbit IgG HRP–linked F(ab’)Total RNA was isolated from liver tissues using ISOGEN . cDNA was synthesized using ReverTra Ace® qPCR RT Master Mix with gDNA Remover . Quantitative PCR was performed using SYBR Premix Ex Taq II  and a Light Cycler system . The following primers were used: human cathepsin Z, forward: 5′-GAATTCATGGGGTGAACCAT-3′, reverse: 5′-TCCTTATAGGTGCTGGTCACG-3′; GAPDH, forward: 5′-TGAACGGGAAGCTCACTGG-3′, reverse: 5′-TCCACCACCCTGTTGCTGTA-3′. All primers were synthesized by Sigma Genosys .4.Serum cathepsin Z was measured using a human cathepsin Z ELISA kit . In brief, standards (156 pg/ml – 10 ng/ml) or 1/4 diluted serum samples were added to wells pre-coated with mouse anti–human cathepsin Z antibody and incubated for 2 hours at 37 °C. After removing the standards and samples, biotin-conjugated rabbit anti–human cathepsin Z antibody was added and incubated for 1 hour at 37 °C. After washing three times, avidin-conjugated horseradish peroxidase (HRP) was added and incubated for 30 minutes at 37 °C. After washing five times, the wells were developed with 3,3’,5,5’-tetramethylbenzidine substrate solution for 20 minutes at 37 °C followed by addition of sulfuric acid solution. The absorbance was measured at 450 nm. The detection limit of cathepsin Z was 62 pg/ml. All samples were run in duplicate. Anti-mitochondrial, anti-gp210 and anti-centromere antibodies were measured as previously describedThis study was approved by the Ethics Board at the National Hospital Organization Nagasaki Medical Center and was conducted after obtaining informed consent from each subject for the use of sera and liver tissue samples. All experiments were performed in accordance with relevant guidelines and regulations.All data generated or analyzed during this study are included in this published article.Supplementary information


Hearing loss affects 38 million Americans and is associated with cognitive and physical decline. Moreover, hearing loss limits communication between patients and providers. In this study, we aimed to determine whether hearing loss diagnosis impacts healthcare cost and utilization. In the OptumLabs Data Warehouse insurance claims database  we identified cases of age-related hearing loss  in adults over the age of 50 years. Hearing loss cases were propensity-matched (nearest neighbor) to persons without hearing loss on multiple socioeconomic , demographic , and health status  indices. There were 154,414, 44,852, and 4,728 subjects, respectively, at the 2-, 5-, and 10-year follow-up time points. We used regression modeling to examine healthcare cost , inpatient hospitalization, length of inpatient stay, outpatient visits, 30-day readmissions, and emergency department visits. Among 4,728 matched adults, hearing loss was associated with $22,434  or 46% higher total healthcare costs over a 10-year period compared to those without hearing loss. Persons with hearing loss experienced more inpatient stays  and were at-risk (44% higher risk) for greater 30-day hospital readmission  at 10-years post index date. Similar trends were observed at 2- and 5-year time points across all measures. Importantly, these pathways may be amendable to hearing treatment via amplification which warrants future research preventative strategies.